THE BIOLOGICAL BULLETIN AUGUST 1999 Editor Associate Editors Section Editor Online Editors Editorial Board Editorial Office MICHAEL J. GREENBERG Louis E. BURNETT R. ANDREW CAMERON CHARLES D. DERBY MICHAEL LABARBERA SHINYA INDUE, Imaging and Microscopy JAMES A. BLAKE, Keys to Marine Invertebrates of the Woods Hole Region WILLIAM D. COHEN, Marine Models Electronic Record and Compendia PETER B. ARMSTRONG ERNEST S. CHANG THOMAS H. DIETZ RICHARD B. EMLET DAVID EPEL GREGORY HINKLE MAKOTO KOBAYASHI DONAL T. MANAHAN MARGARET MCFALL-NGAI MARK W. MILLER TATSUO MOTOKAWA YOSHITAKA NAGAHAMA SHERRY D. PAINTER J. HERBERT WAITE RICHARD K. ZIMMER PAMELA CLAPP HINKLE VICTORIA R. GIBSON CAROL SCHACHINGER PATRICIA BURNS The Whitney Laboratory, University of Florida Grice Marine Biological Laboratory. College of Charleston California Institute of Technology Georgia State University University of Chicago Marine Biological Laboratory ENSR Marine & Coastal Center, Woods Hole Hunter College, City University of New York University of California, Davis Bodega Marine Lab., University of California. Davis Louisiana State University Oregon Institute of Marine Biology, LIniv. of Oregon Hopkins Marine Station, Stanford University Cereon Genomics. Cambridge. Massachusetts Hiroshima University of Economics, Japan University of Southern California Kewalo Marine Laboratory, University of Hawaii Institute of Neurobiology, University of Puerto Rico Tokyo Institute of Technology, Japan National Institute for Basic Biology, Japan Marine Biomed. Inst.. Univ. of Texas Medical Branch University of California, Santa Barbara University of California, Los Angeles Managing Editor Staff Editor Editorial Assistant Subscription & Advertising Secretary Published by MARINE BIOLOGICAL LABORATORY WOODS HOLE, MASSACHUSETTS Cover The ascoglossan sea slug, Elysia chlorotica (Gould), shown on the cover (photograph by S. K. Pierce), seeks out and specifically eats a chromo- phytic alga, Vancheria litorea. Like certain other species of sea slugs, E. chlorotica has developed the ability to acquire the chloroplasts from its algal foodstuff and to utilize them for nutrition. The plastids are usually engulfed by particular epithelial cells in the digestive gland where they photosyn- thesize and, in some species, provide sufficient nu- trients to sustain life and reproduction — even when no other food is available. In E. chlorotica, the function of the captured chlo- roplasts is maintained for up to 8 months — a surpris- ingly long period, surpassing similar chloroplast sym- bioses by many months. Throughout this period, furthermore, plastid proteins are continuously synthe- sized, and some of the proteins appear to be encoded by the slug genome. Another remarkable feature of these slug populations is the abrupt end of the annual life cycle — all of the animals dying synchronously, whether in the laboratory or in the field. In this issue. Skip Pierce and his colleagues (p. 1 ) report a widespread viral infection of the slug pop- ulation; this phenomenon also occurs annually and is coincident with the mass mortality. The viruses (see inset) seem to be endogenous and have many characteristics in common with retroviruses. The report suggests that the viruses may not only be involved in the regulation of the slug's life cycle, but may be the means by which algal genes are transferred to the slug genome. CONTENTS VOLUME 197, No I: AUGUST 1999 RESEARCH NOTES Pierce, Sidney K., Timothy K. Maugel, Mary E. Rumpho, Jeffrey J. Hanteii, and William L. Mondy Annual viral expression in a sea slug population: life cycle control and symbiotic chloroplast maintenance . 1 Thomas, Florence I.M., Kristen A. Edwards, Toby F. Bolton, Mary A. Sewell, and Jill M. Zande Mechanical resistance to shear stress: the role of echinoderm egg extracellular layers 7 Rinkevich, B., S. Ben-Yakir, and R. Ben-Yakir Regeneration of amputated avian bone by a coral skeletal implant 11 ECOLOGY AND EVOLUTION Miner, Benjamin G., Eric Sanford, Richard R. Strath- mann, Bruno Fernet, and Richard B. Emlet Functional and evolutionary implications of opposed bands, big mouths, and extensive oral ciliation in larval opheliids and echiurids (Annelida) 14 Johnsen, Sonke, Elizabeth J. Balser, Erin C. Fisher, and Edith A. Widder Bioluminescence in the deep-sea cirrate octopod Staurotnithis syitensis Verrill (Mollusca: Cephalopoda) . 26 NEUROBIOLOGY AND BEHAVIOR Ganter, Geoffrey K., Ralf Heinrich, Richard P. Bunge, and Edward A. Kravitz Long-term culture of lobster central ganglia; expres- sion of foreign genes in identified neurons 40 Hanlon, Roger T., Michael R. Maxwell, Nadav Shashar, Ellis R. Loew, and Kim-Laura Boyle An ethogram of body patterning behavior in the biomedicallv and commercially valuable squid Loligo /mild off Cape Cod, Massachusetts 49 Bushmann, Paul J. Concurrent signals and behavioral plasticity in blue crab (Callinectr* uipidiu Rathbun) courtship 63 PHYSIOLOGY Engebretson, Hilary P., and Gisele Muller-Parker Translocation of photosynthetic carbon from two algal symbionts to the sea anemone Anthopleura elegantissima 72 DEVELOPMENT AND REPRODUCTION Grabowski, Gregory M., John G. Blackburn, and Eric R. Lacy Morphology and epithelial ion transport of the alka- line gland in the Atlantic stingray (Dasyatis sabina) ... 82 Krug, Patrick J., and Adriana E. Manzi Waterborne and surface-associated carbohydrates as settlement cues for larvae of the specialist marine herbivore Ahitri/i moritsta 94 Chaparro, O.R., R.J. Thompson, and C.J. Emerson The velar ciliature in the brooded larva of the Chil- ean oyster Ostrea cltiltnsis (Philippi, 1845) 104 Annual Report of the Marine Biological Laboratory .... 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Authors of articles in black and white (no color figures) receive their first 50 reprints (without covers) free of charge. Color reprints and additional black-and-white reprints may be purchased; authors will receive order forms. Reprints normally will be delivered about 2 to 3 months after the issue date. Authors (or delegates for foreign authors) will receive page proofs of articles shortly before publi- cation. They will be charged the current cost of printers' time for corrections to these (other than corrections of printers' or editors' errors). Other than these charges for authors' alterations. The Biological Bulletin does not have page charges. Reference: Biol. Bull. 197: 1-6. (August 1999) Annual Viral Expression in a Sea Slug Population: Life Cycle Control and Symbiotic Chloroplast Maintenance SIDNEY K. PIERCE1 *, TIMOTHY K. MAUGEL1. MARY E. RUMPHO2. JEFFREY J. HANTEN1, AND WILLIAM L. MONDY1 Department of Biologv. University of Man-land, College Park, Maryland 20742: and 2 Department of Horticultural Sciences, Texas A & M University, College Station, Texas 77843 In a few well-known cases, animal population dynamics are regulated by cyclical infections of protists, bacteria, or viruses. In most of these cases, the pathogen persists in the environment, where it continues to infect some percentage of successive generations of the host organism. This persis- tent re-infection causes a long-lived decline, in either pop- ulation size or cycle, to a level that depends upon pathogen density and infection level (1-4). We have discovered, on the basis of 9 years of observation, an annual viral expres- sion in Elysia chlorotica, an ascoglossan sea slug, that coincides with the yearly, synchronized death of all the adults in the population. This coincidence of viral expres- sion and mass death is ubiquitous, and it occurs in the laboratory as well as in the field. Our evidence also sug- gests that the viruses do not re-infect subsequent genera- tions from an external pathogen pool, but are endogenous to the slug. We are led, finally, to the hypothesis that the viruses may be involved in the maintenance of symbiotic chloroplasts within the molluscan cells. Populations of the ascoglossan sea slug Elysia chlorotica occur in salt marshes from the Chesapeake Bay to Nova Scotia. The life cycle of the slug lasts about 10 months. The hermaphroditic adults lay egg masses in the spring of each year, and all of the adults die shortly afterward (5, 6). A week or so after the egg deposition, veliger larvae hatch. These larvae spend a few weeks in the plankton and, it filaments of the chromophytic alga Vaucheria litorea are present, each veliger homes in on one of them and attaches to it. During the next 24 h, the larva metamorphoses into a Received 1 June 1999; accepted 17 June 1999. * To whom correspondence should be addressed: E-mail: sp30@ umail.umd.edu juvenile slug while still attached to the algal filament. If the algal filaments are not present, metamorphosis rarely oc- curs, at least not in laboratory cultures (5. 6). Vaucheria is the only alga that E. chlorotica eats, and it is the source of the symbiotic chloroplasts that are acquired during feeding. The juvenile slug immediately begins eating the algal fila- ments and taking on its first load of chloroplasts, which are sequestered by specialized cells in the epithelium lining the digestive diverticula (5, 6). During the next several months. the slugs continue to eat Vaucheria and grow, until winter temperatures cause them to become inactive. As the salt marshes warm in the spring, the slugs become active again, begin laying egg masses, and then die. By the time the egg masses have hatched in May. all the adults are gone. This mass mortality occurs synchronously in the laboratory as well as in the field and regardless of the time of year that the slugs were collected. Symbioses in which chloroplasts — usually from a partic- ular species of alga — are taken up and retained within the cytoplasm of an animal cell occur in several phyla, but they are most commonly encountered in molluscan sea slugs, particularly in the order Ascoglossa ( = Sacoglossa) (Opis- thobranchia). Certain of the molluscan cells can capture chloroplasts from algal food (usually from a specific species of either Rhodophyceae or Chlorophyceae), and these or- ganelles retain some degree of photosynthetic function for a time (e.g.. 7, 8, 9). Whether this intracellular association is a symbiosis in the strict sense is debatable; some authors prefer terms like chloroplast symbiosis, chloroplast reten- tion, or kleptoplasty (7. 9, 11, 12, 13), which indicate that the benefit of the association is entirely to the animal. The duration of the association between the molluscan cell and the algal plastid varies from species to species. S. K. PIERCE ET AL Some associations last less than a week [e.g., Hermaea hifida and Elysia hedgpethi (see 14)]; and although the plastids are initially functional, photosynthesis stops or is greatly reduced after a week of starvation. In contrast, plastid function continues for more than a week in starved specimens of several species, most belonging to the asco- glossan family Elysiidae (e.g., 7, 10, 15, 16). In the species with the longer duration symbioses, the algal chloroplasts within the molluscan cells fix I4CO:, and the I4C appears in a variety of compounds in the animal tissues ( 15, 17. 18, 19, 20, 21). Thus, there is no doubt that the captured chloro- plasts are photosynthetically active within the molluscan cell cytoplasm, and that the products of the synthesis are utilized by the host animal. Indeed, once the symbiosis is established, the species with the longer-lived chloroplast associations can be starved and, as long as light is provided, will maintain or actually gain body weight until the chlo- roplast function finally fails (7). In plant cells, continued photosynthetic activity requires continuous synthesis of chloroplast proteins because several proteins, including those used in light harvesting, are rap- idly turned-over during the process and must be replaced (22, 23, 24). But, several of these photosynthetic proteins (or their subunits) are encoded in the nucleus, so plastid protein synthesis requires the integration of two distinct genomes, that of the chloroplast and that of the plant cell nucleus (reviewed in 25). Because normal plastid photosyn- thetic function is dependent upon major nuclear and cyto- plasmic support, the ultimate failure of photosynthesis by the symbiotic chloroplasts within the molluscan cells is not surprising. But the symbiotic plastids of E. chlorotica stay photosynthetically functional for 8-9 months (5, 26) — many months longer than those of any other species yet described. This remarkable persistence of chloroplast func- tion during starvation indicates that replacement of at least the essential photosynthetic proteins must be occurring within the plastid while it is housed in the molluscan cyto- plasm; and indeed, synthesis of plastid proteins, primarily those associated with photosynthesis, occurs in the E. chlo- rotica plastids (26. 27. 28). The plastid proteins synthesized within the cells of E. chlorotica are of two pharmacologically distinguishable sorts: those whose synthesis is blocked by chloramphenicol, and those blocked by cycloheximide (26). Protein synthesis on pltistul ribosomes is blocked by chloramphenicol, whereas synthesis on cytoplasmic ribosomes, usually di- rected by nuclear genes, is blocked by cycloheximide. These results suggest strongly that some plastid proteins are synthesized upon slug cell ribosomes (26). If this is the case, the genetic information coding for these proteins must somehow be present in the molluscan DNA or must be acquired by all the slugs from the alga in every generation. Our findings presented below suggest a vehicle for this transfer of genetic information from the alga to the slug. In 1990, as part of an ongoing ultrastructural study of morphological changes in the fine structure of the dying slugs from a population on Martha's Vineyard, Massachu- setts, we discovered viruses in digestive cells and hemo- cytes (29; also Fig. 1). Every year since then, every animal examined near the end of the life cycle has had viruses present, whether it was fixed within hours of collection from the field or maintained for months in our aquarium. No evidence of viruses has been found in any slug earlier in the year, except occasionally within the confines of the plastid (Fig. IE, see below). Viruses in the spring slugs are present in the nucleus and cytoplasm of several cell types. They appear to be assem- bled in the nucleus, then move into the cytoplasm, and finally bud out either into the extracellular space or into a vacuole (Fig. 1A). The diameters of the icosahedral viral capsids in the nuclei average 89 nm (±1.0); but in the cytoplasm, after they have picked up an envelope, they are 109 nm (±1.6) (Fig. IB). The shapes and sizes of the capsids and envelopes are very similar to those in the Retroviridae, but other viral types have similar dimensions, and known retroviruses are not assembled in the nucleus (30, 31). In addition to these larger viruses, some chloro- plasts within the cells of spring slugs contain structures (diameter = 20 nm ± 0.4) that could either be smaller viruses or viral cores. These particles occur loosely col- lected in areas between the plastid membranes (Fig. 1C). They also occur as particle clumps in the cytoplasm (Fig. ID), sometimes near material that could be the remnants of chloroplasts. In some instances, in a few fall animals, these particles are present in crystalline arrays (Fig. IE). Ribu- lose-l,5-bisphosphate-carboxylase oxygenase (RuBisCO) occurs in crystalline form in some plant plastids fixed under hyperosmotic conditions (32, 33). However, the particles in such RuBisCO crystals are much smaller and usually lack a visual substructure (32, 33), whereas the array particles in the symbiotic plastids in Elysia clearly have a substructure (Fig. IE). The particle arrays in the E. chlorotica plastids are more reminiscent of mosaic viruses in plants (34), although almost nothing is known about such crystals in the viruses and plastids of algae. In summary, the morphology suggests that either more than one viral type is present in the slug cell and captured plastids, or we have found several stages of a single viral type. In addition to the morphology, we have some biochemi- cal information about the identity of the viruses. Using differential and sucrose-gradient centrifugation, we have isolated a fraction from slug homogenates at a density of 1.18 g/ml that has reverse transcriptase (RT) activity. This activity, which is considered diagnostic for retroviruses, is two orders of magnitude higher in spring slugs than in slugs tested in the fall (Fig. 2). In addition, the RT activity of fall animals, but not spring animals, is inhibited by rifampicin (Fig. 3). This inhibition indicates that the activity measured SEA SLUGS, PLASTIDS. AND VIRUSES Figure 1. Electron micrographs of viruses in Elysiu chlorulica. (A) Viral particles in various stages of maturation are present in the nucleus (n) and cytoplasm of a hemocyte from a dying slug (magnification = 33,750 X, scale bar = 1 /urn). (B) Higher magnification ( 85.000 x, scale bar = 0.? /j.m) of viruses budding into cytoplasmic vacuoles. Icosahedral shape and double envelopes of the mature virus are apparent. (C) Viral aggregates (arrows) within the symbiotic chloroplast of a spring Elysiu (magnification = 16,880, scale bar = 1.0 fim). (D) Viral particles very similar in appearance to those in (C) located in the cytoplasm of a chloroplast (cp)-containing digestive cell of a spring slug. The diffuse, gray areas in the cytoplasm are lipid produced by the plastid (magnification = 27,000, scale bar = 1 .0 /j.m). (E) Viral crystal contained in a symbiotic chloroplast from a fall slug (magnification = 54,000, scale bar = 0.5 jim). 3. E 15(1(1(111-1 100000- 50000- A-fall animals S. K. PIERCE ET AL. 150000- 100000- 50000- B-spring animals i i i I i i i i i i i i i i 1.12 1 12 I 14 118 I 18 1 18 I 19 1 19 1.19 I 20 1.20 1.21 121 1 22 ~1 I I I I I 1 I I I I I 1 I I 106 1.10 I 12 I 14 I 15 1 17 1 17 1 18 I 18 1 IS I 19 1.19 1.19 120 1.20 Gradient fractions (gm/ml) Figure 2. Comparison of reverse transcriptase activity in sucrose-gradient fractions from a typical extract of fall slugs (A) and spring slugs (B). The results are essentially the same whether the animals were freshly collected in the spring or collected in the fall and overwintered in aquaria. in the fall animals is due to DNA dependent-RNA polymer- ase (which utilizes the same substrates in the RT assay) rather than RT, and confirms the absence of viruses in the fall animals. Taken together, the morphology, the buoyancy, and the presence of RT all suggest that a retro-like virus is present in the cells of the dying slugs. 150-1 100- 3 50- O-1 Fall Spring Figure 3. Rilampicin inhibits reverse transcriptase activity. Enzyme activity was assayed in pooled gradient fractions with densities from 1.16 to 1.18 g/ml prepared from fall and spring slugs. The effect of the inhibitor is expressed as a percentage of control values. The relationships between the nuclear, cytoplasmic, and plastid viruses are not known at present. However, for the last 9 years, the viruses have been found in every dying slug examined. Since some of the slugs had been maintained in the laboratory, in aquaria containing artificial seawater and with no access to Vauclierui for 8 months before the viruses appeared, the infection is unlikely to have been opportunis- tic. Instead, the results suggest either that the demise of the entire population is caused by an endogenous virus or that the virus can be expressed only as the defense systems of the aging animals begin to fail. Furthermore, if the effect on the life cycle is due to a retrovirus, as our data suggest, then the viral genome is probably transmitted to the next gener- ation of slugs in the molluscan DNA. Infection of germ cells by retroviruses produces endogenous proviruses that are inherited as Mendelian genes (33). Alternatively, the viruses might enter all of the slugs via the sequestered chloroplasts. either as part of the plastid genome or as constituted viral particles. In either case, viral expression is coincident with increases in environmental temperature, at least in the field slugs. Both the onset of egg laying and the death of the population are associated with the rise of water tempera- tures in the spring. We can delay the demise of the slugs by maintaining them at very cold temperatures (2°-5°C), but eventually — 6-8 weeks after the warmer-maintained slugs have died — the cooled slugs also die with viruses in their cells, indicating that temperature is not the only expression stimulus. It will take some time to sort out both the types of viruses involved here and the molecular relationships between the SEA SLUGS. PLASTIDS. AND VIRUSES slugs, the algae, the plastids. and the viruses. Nevertheless, the nine-year, annual occurrence of the association between population demise and viral expression at the end of the E. chlorotica life cycle strongly suggests that the virus has a role in regulating this coincidence. Indeed, we speculate that the viral infection may have caused the transfer, from the alga to the slug, of algal genes that allow the molluscan cells to assist in plastid maintenance. Although the transfer, in- tegration, and expression of such a group of genes between species should succeed only rarely, those successes that did occur should have profound, immediate, heritable effects on the phenotype of the infected species. Such heritable effects must certainly be associated with the mechanism of the widely accepted endosymbiotic origin of intracellular or- ganelles such as mitochondria and chloroplasts; a variety of genes must have been transferred from the symbiont into the host cell nucleus to consummate such a relationship. In addition, a retrovirus as a gene transmission vehicle might have merit as a hypothesis to explain genetic similarities between distantly related or unrelated species (e.g., 35) and is the basis of some genetic therapies (36). If a successful interspecies gene transfer between an alga and a slug me- diated by an endogenous virus could be demonstrated in the case of E. chlorotica. then an exemplary mechanism for this process would have been provided. Methods Viral isolation The fractionation procedure was carried out using au- toclaved equipment. All reagents were molecular biolog- ical grade (DNAase-, RNAase-. and protease-free; from Sigma Chemicals, unless otherwise noted) and filtered (0.2 /xm pore). Approximately 3.0 g of E. chlorotica was homogenized in an ice-cold buffer (450 mM NaCl, 1.0 mM EDTA, 5.0 mM 3-[/V-morpholino]propane-sulfonic acid (MOPS), 2.3 yuM leupeptin. 1.0 mM dithiothreitol (DTT), 500 /xM phenylmethylsulfonyl fluoride (PMSF), pH 7.5) containing the mucolytic agent H-acetyl cysteine (500 mM), which is necessary to disperse the copious mucus produced by the slug (26). The homogenate was filtered through six layers of cheesecloth, then through one layer of Miracloth (Calbiochem), and finally through two layers of Miracloth. The filtrate was centrifuged for 5 min at 4300 x g (4°C), and the supernatant was centrifuged at 20,000 X g for 30 min (4°C). The super- natant from the second spin was layered over a 20% sucrose cushion and centrifuged at 180,000 X g for 2 h (4°C) in a swinging bucket rotor. The supernatant was discarded, and the pellet was resuspended in ice-cold homogenization buffer (without H-acetyl cysteine). This suspension was layered on the top of a 15%-50% con- tinuous sucrose gradient and centrifuged at 180,000 X g for 43 h (4°C). The gradients were then fractionated by piercing the bottom of the centrifuge tube and raising the gradient out of the tube with 65% sucrose. Twenty 600-/xl fractions were collected, and the density of each was determined by weigh- ing 50 /xl with an analytical balance. The sucrose in each fraction was then diluted with homogenization buffer (with- out /i-acetyl cysteine), and each fraction was centrifuged a final time at 180,000 X g for 2 h. The supernatants from this last spin were discarded, and RT assays (see below) and protein assays (37) were run on the pellets. Reverse transcriptase assav The final pellets (above) were treated with a detergent buffer (50 mM Tris-HCl, 5 mM KC1, 0.2 mM EDTA. and 0.02% Triton X-100, pH 8.2). Fifteen-microliter aliquots of this digest were added last to 50 /xl of buffer (100 mM Tris-HCl, 200 mM KC1. 10 mM MgCl2, pH 8.2), ft /xl 100 mM DTT, 9.75 LI! 100 mM thymidine triphosphate (TTP), 1.25 /xl RNA-guard (Pharmacia), 1.0 /xl poly(rA)-p(dT) (Pharmacia), and 10 /xCi of 32P-TTP (ICN, 10 /xCi//xl). The final volume was adjusted to 100 /xl with buffer, as neces- sary, to compensate for 12P half life. This solution was mixed and incubated at 37°C for 65 min on a shaker table. The reaction was terminated by adding 30 /xl of 10 mM EDTA in 5% TCA and placing the reaction mixture on ice for 20 min. DN A was then precipitated by the addition of 9 /xl of 72% TCA, and the precipitate was pelleted by cen- trifugation at 6610 X g for 10 min. The pellet was washed three times in 5% TCA, the final pellet dissolved in 0.1 N NaOH. and the radioactivity determined by scintillation counting. The protein concentration of a separate aliquot was determined, and RT activity was expressed as counts per minute per microgram (cpm//xg) protein (38). Electron microscopy Small pieces of tissue were prepared for microscopy by fixation in 2% glutaraldehyde in 0.15 M cacodylate-0.58 M sucrose buffer (pH 7.3) at 900 mosm. The tissue pieces were post-fixed in 1 .0% OsO4 in the same buffer followed by 2.0% aqueous uranyl acetate. The fixed tissue was dehy- drated in an ethanol series, infiltrated with propylene oxide, and embedded in Spurr's medium. Silver sections were cut with an ultramicrotome (Reichert), mounted on 75 X 300 mesh copper grids, and stained with uranyl acetate and lead citrate. The sections were viewed and photographed with a transmission electron microscope (Zeiss EM 10). Acknowledgments This work was supported by an NSF grant to SKP and MER. We thank Margaret Palmer, Ulrich Mueller, and Jeffery DeStefano for critically reading early versions of 6 S. K. PIERCE ET AL this paper. Contribution #91 from the Laboratory of Bio- logical Infrastructure. Literature Cited 1 D'Amico, V., J. S. Elkington. G. Dwyer, R. B. Willis, and M. E. Montgomery. 1998. Foliage damage does not affect within-season transmission of an insect virus. Ecology 79: 1 104-1 1 10. 2. Hawkins, B. A., H. V. Cornell, and M. E. Hochburg. 1997. Pred- ators. parasitoids and pathogens as mortality agents in phytophagous insect populations. Ecology 78: 2145-2152. 3. Kohler, S. L., and M. L. Wiley. 1997. Pathogen outbreaks reveal large-scale effects of competition in stream communities. Ecology 78: 2164-2176. 4. Rothman. L. D. 1997. Immediate and delayed effects of a viral pathogen and density on tent caterpillar performance. Ecology 78: 1481-1493. 5. West, H. H. 1979. Chloroplast symbiosis and development of the ascoglossan opisthohranch Elysia ch/ororicu. Ph.D. dissertation. Northeastern University, Boston. 161 pp. 6. West, H. H., J. F. Harrigan, and S. K. Pierce. 1984. Hybridization of two populations of a marine opisthohranch with different develop- mental patterns. Veli^er 26: 199-206. 7. Hinde, R., and D. C. Smith. 1974. "Chloroplast symbiosis" and the extent to which it occurs in Sacoglossa (Gastropoda: Mollusca). Biol. J. Linn. Soc. 6: 349-356. 8. Clark. K. B., and M. Busacca. 1978. Feeding specificity and chlo- roplast retention in four tropical Ascoglossa. with a discussion of the extent of Chloroplast symbiosis and the evolution of the order. J. Molluxcan Stud. 44: 272-282. 9. Clark, K. B., K. R. Jensen, and H. M. Strits. 1990. Survey for functional kleptoplasty among West Atlantic Ascoglossa (= Saco- glossa) (Mollusca: Opisthobranchia). Veliger 33: 339-345. Clark. K. B., K. R. Jensen, H. M. Strits, and C. Fermin. 1991. Chloroplast symbiosis in a non-elysiid mollusc, Costasicl/a lilianac Marcus (Hermaeidae: Ascoglossa (=Sacoglossa): effects of tempera- ture, light intensity and starvation on carbon fixation rate. Biol. Bull. 160: 43-54. Taylor, D. L. 1970. Chloroplasts as symbiotic organelles. Int. Re\: Cytol. 27: 29-64. Gilyarov, M. S. 1983. Appropriation of functioning organelles of food organisms by phytophagous and predatory opisthohranch mol- lusks as a specific category of food utilization. Zli. Ohxlich. Binl. 44: 614-620. 13. Waugh, G. R., and K. B. Clark. 1986. Seasonal and geographic variation in chlorophyll level of Elysia tuca (Ascoglossa: Opistho- branchia). Mar. Biol. 92: 483-488. 14. Greene, R. W. 1970. Symbiosis in sacoglossan opisthobranchs: functional capacity of symbiotic chloroplasts. Mar. Biol. 7: 138-142. 15. Greene, R. W., and L. Muscatine. 1972. Symbiosis in sacoglossan opisthobranchs: photosyntheUc products of animal-chloroplast associ- ations. Mar. Biol. 14: 253-259. 16 Graves, D. A., M. A. Gibson, and J. S. Bleakney. 1979. The digestive diverticula of Alderia inmlexiu and Elysia clilorotica. Veliger 21: 415-422. 17. Trench, R. K. 1969. Chloroplasts as functional endosymbionts in the mollusc Tridachiu crispalu (Bcrgh), (Opisthobranchia, Saco- glossa). Nature 222: 1071-1072. IX Trench, M. E., R. K. Trench, and L. Muscatine. 1970. Utilization ol photosynthetic products of symbiotic chloroplasts in mucous syn- 10. 11 12. thesis by Placobranchus iantlmharixiis (Gould). Opisthobranchia, Sacoglossa. Comp. Biochem. Physio/. 37: 113-117. 19. Greene, R. W. 1970. Symbiosis in sacoglossan opisthobranchs: translocation of photosynthetic products from Chloroplast to host tis- sue. Malacologia 10: 360-380. 20. Trench, R. K., J. E. Boyle, and D. C. Smith. 1973. The association between chloroplasts of Codium fragile and the mollusc, Elysia viridis II. Chloroplast ultrastructure and photosynthetic carbon fixation in £. )•//•/ 14 setigers) captured two large particles (50 jum in diameter) while videorecorded through a dissecting microscope (Fig. 6). When the swim- ming larva contacted a large particle in the vicinity of the mouth, the larva slowed and rotated so that the lower lip was aligned with the particle. The larva opened its mouth and ingested the particle, presumably using oral cilia or muscu- lature. Swimming Urechis caitpo larvae used the mouth for direct capture of particles that passed over the episphere. Such captures occurred simultaneously with opposed-band particle captures (Fig. 5). Many of the particles caught directly by the mouth were too large to fit between opposed prototroch and metatroch, as illustrated by the gut contents in Figure 7 and the particles being rejected in Figure 8. In some cases the mouth gaped to admit a large particle. The 9-day-old larva in Figure 4 opened its mouth to a gape of about 35 jum with a width of 95 ;j.m. The 17-day-old larva in Figure 8 opened its mouth to 70 to 95 /urn, and the mouth's width when closed was about 125 /u,m. Cilia on the mouth's lower lip (anterior to the shorter cilia of the neu- rotroch) appeared to aid the movement of large particles into the mouth. These cilia seemed to be continuous with the metatrochal band, which would account for the posterior- 0.00 • 0.13 Figure 5. Videorecorded capture ol l\u> 12-fxm spheres by a Urechis fiiiil>t> larva. Time is in seconds in the lower right-hand corner. The particle marked by an adjacent black bar has entered a dorsolateral part of the food groove at 0 s. moves along the food groove toward the mouth al 0.04 and 0.13 s, and is near the side of the mouth at 0.30 s. The second particle passes over the prototroch directly into Ihe mouth. It is near the protolro- chal cilia at 0 s, passes over the anterior edge of the mouth at 0.04 and 0. 1 3 s. and has entered the mouth at 0.30 s. The mouth is at the lower left; the anterior end toward the upper left. At 0 s the larva is 170-jLim wide at the base of the prototrochal cilia. 20 B. G. MINER ET AL 0.00 Figure 6. Videorccoided capture of a 50-/nm sphere h\ a tree swimming Anuttndiu hrevis lar\'a under a dissecting microscope. Time in seconds is in the upper left-hand comer. All images are at the same magnifi- cation A black line indicates the particle. The larva approaches the particle and then orients its mouth towards the particle, which is on the bottom of the dish. The particle is captured at the larva's mouth, presumably moved by the large oral compound cilia, and swallowed. At 0 s the larva is 85-/am wide at the center of the body. to-anterior current past these cilia. In sonic cases a panicle was brought into the mouth over the lower lip (Fig. 7). Larvae of U. cuiipo captured large particles from an early stage. Small 4-day-old larvae ingested Sephadex spheres almost as large as those ingested by 16-day-old larvae (Table I). Even a 3-day-old larva ingested a 42-by-35-|u,m mineral grain. Larger larvae did capture larger spheres, however. When early and later stage larvae were fed the sai suspension, as in the last two lines of Table I, the median sizes and the largest si/.es of ingested spheres were significantly greater for larger, older larvae (Mann-Whitney U tests, H, 1 0. a 2 = 5, P < 0.05). Objects larger than the spheres olio can he ingested. For example, a 49-day-old larva, 375 /u,m id :, ingested an unidentified object 366 /xm long by 40 |um wide When larvae of U. cn of different ages and sizes were offered smaller plastic sphctes, all 10 of the small, 3-day- old larvae caught fewer spheres of 29-fj,m than of 1 2-jum, and all 4 of the larger, 48-day-old larvae ingested more of the 29-/j,m spheres than of the 12-/xm spheres (Table II). Small, early-stage larvae did ingest 5- and 20- /xm spheres in about the same ratio as ingested by larger larvae (Table II). Estimates of the width of the food groove of a single 5-day-old larva ranged from 22 to 34 /xm, but the width of the food groove varies with contraction of the larva. The upper limit on the sizes of particles that could be transported in the food groove was not determined. Rejection of particles Larvae could actively reject particles. Particle rejection often occurred after a particle had been transported to the mouth and entered the esophagus. When a larva of Ariiuin- dia brevis expelled a particle, the metatrochal cilia around ANNELID LARVAL FEEDING MECHANISMS 21 0.15 Figure 7. Videorecorded capture of a 40-fxm sphere by a Urechis caupo larva. Time is in seconds in the lower left-hand comer. The sphere is near the metatrochal cilia at the posterior lip of the mouth at 0 s and moves over this band of cilia toward the mouth at 0.1 and 0.15 s. It is just entering the mouth at 0.25 s. The anterior end is toward the upper right. At 0 s the larva is 300-^im wide at the base of the prototrochal cilia. the mouth stopped beating as the particle moved posteriorly down the body (Fig. 3). Metatrochal cilia at the mouth of larvae of Urechis caupo must also have altered beat during particle rejection, because large particles moved posteriorly over the lower lip and down the neurotroch during rejection (Fig. 8). in contrast to their posterior-to-anterior path over the lip during ingestion (Fig. 7). For larvae ofArmandia h rev is. prototroch circumference and prototrochal eilium length increased with number of setigerous segments (Fig. 9A. B). Larval volume increased exponentially with number of setigers (Fig. 9C). Particle velocities increased slightly with number of se- tigers for larvae of A. hrevis with 6-7. 11-12. and 15-16 setigers (H = 9) (Fig. 10). Increased particle velocities and eilium lengths resulted in a 30% increase in the area of water per prototrochal slice moved per second between larvae with 6-7 and 11-12 setigers and a 22% increase between larvae with 11-12 and 15-16 setigers (Table III). Maximum particle velocities were within the distal third of the eilium length (estimated for each size class from Fig. 9B). consistent with our expectations (Emlet and Strath- mann. 1994). Although Strathmann et al. ( 1993) suggested that eilium lengths might be underestimated from videore- cordings, our results indicate that this was not the case. In addition, our measurements agree with the eilium length of approximately 35 /im reported by Hermans (1964) for a larva with an unspecified number of setigers. Although estimated maximum clearance rates increased with number of setigers, they did not increase proportion- ately to body volume (Table III). Late-stage larvae (15-16 setigers) had a maximum ratio of clearance to body volume that was less than half of that achieved earlier in develop- ment (6-7 setigers; Table III). Prototrochal circumference and eilium length both in- creased with larval growth to a greater extent for larvae of U. caupo than for larvae of A. brevis. over the stages measured (Tables I-III). The relative increase in body length was much less for U. caupo. Early-stage larvae were nearly spherical and elongated to the shape shown in Figure 2A at later stages. Data for particle velocities are lacking for Figure 8. Videorecorded rejection of previously ingested spheres up to 50 jum in diameter by a Urcchix ciiii/x) larva. Time is in seconds in the lower left-hand corner. At 0 and 0.3 s the mouth gapes at least l(IO-(nm wide, and the clump of spheres moves over the posterior lip of the mouth and down the midventral neurotroch. The larva in the last frame is 295-/nm wide at the base of the prototrochal cilia, and the mouth, now rotated toward the viewer, is closed and approximately 1 20-ju.m wide. B. G. MINER ET AL Table I Sizes of Sephadex spheres ingested by larvae «/ Urechis caupo differing in size and age Particle diameter (/Limit Age Prototrochal diameter Cilium length Number (days) (fan)* (/Mm) In suspension Ingested of larvae 4 159 45 45,26-73(50) 36, 14-53(51) 12 5 165 44 44. 30-74 (50) 36, 19-60(34) 10 16 318 65 44, 30-74 (50) 38,21-73(104) 5 * Diameter of the prototrochal band is diameter at the base of the prototrochal cilia. t Values are median, range, and (in parentheses) number of particles. U. caupo, but the increase in prototrochal area (cilium length times prototrochal circumference) relative to body volume was greater for this species than for A. brevis. Discussion Our observations add the Opheliidae and Echiuridae to those annelid families known to possess larvae with op- posed-band feeding. As in other opposed-band feeders, lar- vae of both Armandia brevis and Urechis caupo possess a ciliated food groove between two parallel ciliary bands, a postoral metatroch and a prototroch. Direct observations confirm that particles are captured in the food groove (Figs. 3-5), probably through the combined action of long com- pound cilia in the prototroch (which beat anterior to poste- rior) and shorter compound cilia in the metatroch (which beat posterior to anterior). Simple cilia of the food groove may aid in retention of particles as well as in transport. This system is very effective in capturing relatively small parti- cles (5-12 /urn), regardless of which part of the prototrochal circumference is contacted (ventral, lateral, or dorsal). How common this feeding method is in larvae of other opheliids or echiurids is not known, but larvae of at least one other echiurid bear opposed bands of cilia (Salensky, 1876; Hatschek, 1880). Larvae of both A. brevis and U. caupo also ingested particles larger than the space between prototrochal and metatrochal bands. For A. brevis, it was later stage (14-17 setiger) larvae that ingested large (50-ju.m) particles. These larvae approached large particles so that contact was di- rectly at the mouth. This behavior was not observed in larvae at earlier stages. In contrast, larvae of U. caupo ingested particles greater than 50 /urn at early stages. Larvae of U. caupo did not appear to change orientation as they approached large particles; however, their movements were constrained by mesh cages. Particles that were captured directly at the mouth entered either over the episphere and prototroch or over the extension of the metatroch on the lower lip. In both species the mouths were large, could be opened to a wide gape, and were heavily ciliated. The cilia bordering the lower lip of the mouth appear to be a contin- uation of the metatroch. The oral cilia of A. brevis may include additional compound cilia (Fig. 1). For both A. brevis and U. caupo, the large ciliated oral field and the large mouth aid in the capture of large particles. The combination of two ciliary feeding mechanisms in individual larvae suggests hypotheses for evolutionary tran- sitions among the feeding larvae of annelids. Some larvae, such as those of serpulids, appear to be restricted to captur- ing small particles between opposed bands; other larvae, like those of polynoids, lack opposed bands and appear to capture mostly large particles one by one, using complex oral ciliature (Phillips and Fernet, 1996). Our results dem- Tahle II Sizes of plastic spheres iiixcMctl h\ lamie oj Urechis caupo differing in size and age Particle diameter Age (days) Prototrochal diameter (fun)* Cilium length (fj.ni) Ratio in suspension (29:12 /Mm) Ratio ingested (29:12 jum) Number of larvae 3 151 46 1.43:1 39/146 = 0.27 10 4S 347 76 1.43:1 206/112 = 1.84 4 (20:5 /im) (20:5 /urn) 4 161 45 11 146/30 = 4.9 8 15 310 67 1:1 99/37 = 2.7 8 Diameter of the prototrochal band is diameter at the base of the prolotrochal cilia. ANNELID LARVAL FEEDING MECHANISMS 23 500 4 6 8 10 12 14 16 18 40-i U. 35- OfJ u I 30- 25H 15 R2 = 0.84 0 2 4 6 8 10 12 14 16 18 U T 0 2 4 6 8 10 12 14 16 18 # of Setigers Figure 9. Binomial regression of various larval parameters vs. number of setigers for Armandia brevis. For all equations. X = number of setigers. The R2 value is reported in the lower right-hand corner of each plot. (A) Inner prototroch perimeter (;i = 36 larvae); larval circumference = 178.36 + 23.24x - 0.44x2. (B) Cilium length (n = 22 larvae); cilium length = 18.67 + 2.16x - 0.07x:. (C) Larval volume (n = 36 larvae); larval volume = 105 48 + 008x. onstrate that in at least two families of annelids, both types of mechanisms can be employed simultaneously by the same larva. In addition, it appears that the oral ciliature of A. brevis and U. caupo, which is responsible for the capture of large particles, is continuous with the lateral and dorsal extensions of the metatroch and food groove. As an evolu- tionary transition, expansion of oral filiation might result in a food groove and metatroch paralleling the whole length of the prototroch to produce an opposed-band system. Alter- natively, enlargement of the mouth and elaboration of oral ciliation (with loss of the lateral and dorsal parts of the opposed-band system) could produce the variety of oral 4000-1 3000 - 2000 - 1000- o u C/3 "e ^i IT 'o _0 U > — o 4000-1 3000 - 2000 - 1000- 4000n 3000 - 2000 - 1000- A — i 1 1 1 1 — o 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70 C 0 10 20 30 40 50 60 70 Distance To Cilium Base (urn) Figure 10. Particle velocity vs. distance of particle from the base of the prototroch for Armandia brevis larvae with (A) 6-7, (B) 11-12, and (C) 15-16 setigers. The vertical dotted line shows the estimated cilium length taken from the binomial regression of Figure 9B. 24 B. G. MINER ET AL. Table III Estimated clearance rate and clearuin c rule per lamil volume fur three .w.-r ( /, <>/ lumie o) Armandia hrcvis Cilium Water area per Midpoint Larval Clearance #of lencth prototroch slice prototrochal Max. clearance rate volume rate/volume Seligers ( nm I per unit timet (junr/s) circumference (jumi IjunvVs)- 10" (ju,m')± (1/S) 6-7 29.9 32846 422 13.9 998309 13.9 11-12 34.8 42602 549 23.4 2526475 9.3 15-16 36.4 51449 642 33.4 5310250 6.3 * Calculated from the binomial regression in Fig. 10B. t Calculated from the areas under the curves in Fig. 1 1. bound by the origin and the estimated cilium length lor that size class. i Estimated from the binomial regression in Fig. IOC. ciliature found in the diverse feeding larvae of annelids. Continued modification of such cilia might result in such unusual and functionally important structures as the group of long compound cilia on the left side of the mouth of polynoid larvae. Estimated maximum clearance rates did not scale isomet- rically with body volume among the three size classes of A. brevis. Cilium length, prototroch circumference, and parti- cle velocities through a prototrochal slice all increased as body volume increased, but not enough for maximum clear- ance rate to increase in proportion to body volume — thus the volume of water swept by cilia decreases relative to body volume as the larva adds segments. An analogous situation has been described for the cyphonautes larva of bryozoans. in which ciliated band length does not increase proportionately to body volume during growth and devel- opment (McEdward and Strathmann. 1987). This allometry is potentially unfavorable to larger larvae. In asteroid, echi- noid, and bivalve larvae similar in size to A. brevis larvae, metabolic rates scale isometrically with body mass (Hoegh- Guldberg and Manahan. 1995). Further, in the larvae of an echinoid, metabolic demand scales isometrically with larval volume (McEdward, 1984). If these results can be general- ized to larvae of A. brevis, and if we make the reasonable assumption that the masses of these larvae are proportional to their volume, then the maximum clearance rates of A. brevis larvae decline relative to metabolic demand as the larvae increase in size. However, larger larvae of A. hrcvis (>12 setigers) can supplement the amount of small particles captured by opposed-band feeding by capturing larger par- ticles at the mouth. The increased size range of food may compensate, at least partly, for the decrease in clearance rate. This decrease in maximum clearance rate per larval volume may have selected for larvae that possess two types of feeding mechanisms. Do other annelid larvae share this potentially unfavor- able allometry of maximum clearance rate and body volume? Some annelid larvae resemble A. hrevis in ex- treme elongation of a segmented body during the larval stage (Bhaud and Cazaux, 1987). Some of these larvae (<'.,!,'.. spionids) possess feeding mechanisms other than the opposed prototrochal and metatrochal bands. Thus, evolutionary changes in the size range of particles cap- tured may have been favored in several groups of anne- lids as a result of a small head circumference and long larval body. Other possible solutions to this problem are opposed bands elongated on ciliated lobes, as reported for the rostraria larva of an annelid (Jagersten, 1972). or the sinuous opposed bands of mitraria larvae of oweniid annelids (Emlet and Strathmann, 1994). The larvae of U. cinipo and some other annelids probably do not face such an unfavorable allometry of maximum clearance rate to body volume, however. The larvae of U. cuit/w develop from nearly spherical trochophores (at 3 to 5 days) to forms with more elongate bodies (at several weeks), but the elongation is not as extreme (cf. Fig. 2 to Fig. 6). Also, these larvae capture relatively large particles from an early stage. Nevertheless, the circumferential cili- ary bands are shorter, relative to body size, than similar bands that are extended on the velar lobes of many gastro- pod larvae (Richter and Thorson. 1975). Feeding on an extended size range of particles and extension of opposed, ciliary bands on lobes may be alternative ways of increasing ingestion rates. Further analyses of larval feeding methods, as well as robust phylogenies, are required to understand the evolution and functional consequences of diverse larval feeding mechanisms in the Annelida. For example, why are opposed bands apparently used only in the capture of small particles? What functional constraints place an upper limit on the spacing of the prototroch and metatroch in opposed-band feeders? Such analyses may also reveal why some larvae (c.i>.. serpulids) use restricted opposed bands to feed on small particles, and others («'.#., polynoids) use complex oral ciliature to feed primarily on large particles instead of employing both methods, as do the opheliid and echiurid larvae described here. ANNELID LARVAL FEEDING MECHANISMS 25 Acknowledgments NSF grant OCE9633193. the Robert Fernald Fellowship endowment, and the Friday Harbor Laboratories of the University of Washington supported the research on Annan- din hrevis. NSF grant OCE9301665 and the Bodega Marine Laboratory of the University of California at Davis sup- ported the research on Urechis caupo. K. Uhlinger advised on collection of adults and culture of larvae of U. caupo. W. Borgeson provided algal medium and Isochrysis galbana. N. E. Phillips and C. 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The emission of blue-green bioluminescence (Am.ix = 470 nm) was observed from sucker-like structures arranged along the length of the arms of the citrate octopod Stauroteuthis syrtensis. Individual photophores either glowed dimly and continuously or flashed on and off more brightly with a period of 1-2 seconds. Examination of the anatomy and ultrastructure of the photophores confirmed that they are modified suckers. During handling, the photo- phores were unable to attach to surfaces, suggesting that, unlike typical octopod suckers, they have no adhesive func- tion. The oral position of the photophores and the wave- length of peak emission, coupled with the animals' primary postures, suggests that bioluminescence in S. syrtensis may function as a light lure to attract prey. Introduction Bioluminescence is a common and complex characteris- tic in coleoid cephalopods. A large percentage of these animals are bioluminescent, many possessing complicated light organs utilizing lenses, reflectors, irises, interference filters, pigment screens, and shutters (Harvey, 1952: Her- ring, 1988). The diversity of the morphologies and anatom- ical distributions of cephalopod photophores is unparalleled among invertebrate phyla (Voss, 1967; Herring, 1988). However, despite this extraordinary radiation, biolumines- cence appears to be rare among octopods. Although 63 of the 100 genera of squid and cuttlefish have bioluminescent species, only 2 of the 43 genera of octopods have species confirmed to be bioluminescent —the bolitaenids Japetella and Eledonella (Robison and Youn«. 1981; Herring el Address tor correspondence: Dr. Sonke Johnsen, MS #33, Woods Hole Oceanographic Institution, Woods Hole, MA 02543-1049. E-mail: sjohnsen@whoi.edu 1987; Herring, 1988). In these genera, the light organs are found only in breeding females (Robison and Young, 1981 ) and are restricted to tissues associated with the oral ring and the base of the arms (Herring et ai. 1987). In the case of citrate octopods, bioluminescence has been suggested but never confirmed (Aldred et ai, 1982, 1984; Vecchione. 1987). This study provides the first description of biolumines- cence in the cirrate octopod Stauroteuthis syrtensis. We also describe the anatomy and ultrastructure of the photophores in comparison with the morphology reported for cephalopod photophores (Herring et til.. 1987) and octopod suckers (Kier and Smith. 1990; Budelmann et ai, 1997). In addi- tion, we present a hypothesis to explain how the presence of light organs relates to the feeding behavior postulated for these animals. A preliminary account of this research has been presented by Johnsen et al. ( 1999). Materials and Methods Source and maintenance of animals Three specimens of Stauroteuthis s\rtensis were obtained during a cruise of the R.V. Edwin Link to Oceanographer Canyon (on the southern rim of Georges Bank, USA) in August and September 1997. The animals were collected at depth using the research submersible Johnson-Sea-Link out- fitted with acrylic collection cylinders (11-liter volume) with hydraulically activated, sliding lids. The three speci- mens were caught during daylight at depths of 755 m (225 m from bottom), 734 m (246 m from bottom), and 919 m (165 m from bottom) (dive numbers 2925 and 2927) and maintained for up to 2 days at 8°C in water collected at depth. 26 BIOLUMINESCENCE IN A DEEP-SEA OCTOPOD 27 Video ami photography Specimens were videotaped in two situations. First, the behavior of two animals was recorded from the submersible. Second, the captured animals were filmed aboard ship in the dark by using an intensified video camera (Inte vac's Nile- Mate 1305/1306 CCTV intensifier coupled to a Panasonic Charge Coupled Device). During shipboard filming, the animals were gently prodded to induce bioluminescence. Representative video frames were digitized (ITSCE capture board, Eyeview Software, Coreco Inc.). The animals were also placed in a plankton kreisel (Hamner. 1990) and pho- tographed with a Nikon SLR camera with Kodak Elite 100 color film. Data from a previously recorded i';i xitu video of a specimen of S. svrtensis from the slope waters near Cape Hatteras at 840 m (35 m from bottom; August 1996; R.V. Edwin Link; dive 2777) are also reported in this paper. Spectrophotometry Bioluminescent spectra were measured using an intensi- fied optical multichannel analyzer (OMA-detector model 1420. detector interface model 1461, EG&G Princeton Ap- plied Research) coupled to a 2-mm-diameter fiber optic cable. The detector was wavelength calibrated using a low- pressure mercury spectrum lamp (Model 6047. Oriel Inc.) and intensity calibrated using a NIST referenced low-inten- sity source (Model 310. Optronics Laboratories) intended for the calibration of detectors from 350 nm to 800 nm. For further details on the theory of operation and calibration of the OMA detector, see Widder et al. ( 1983). Three emission spectra were recorded from one animal, and an average spectrum was calculated. Microscopy of photophores The fixation, dehydration, and infiltration procedures were performed at room temperature aboard the R. V. Edwin Link. The animals were sacrificed by over-anesthesia with MS222 (Sigma Chemicals Inc.). One specimen was fixed in 10% formalin in seawater and dissected to confirm species identification. One arm of a different specimen was fixed in 2.5% glutaraldehyde in 0.2 M Millonig's buffer at pH 7.4, adjusted to an osmolarity of 1000 mOs with NaCl. After an initial 1-h fixation, several photophores were dis- sected from the arm and placed in fresh fixative for an additional 5.5 h. Postfixation of the dissected photophores in \c/c osmium tetroxide in Millonig's buffer for 70 min was followed by dehydration through a graded series of ethyl alcohols. Over a period of 6 days, the specimens were slowly infiltrated with propylene oxide and Polybed 812 (Polysciences) and then embedded in Polybed 812. Semithin ( 1 /urn) and thin sections of embedded material were cut with a diamond knife (Diatome) and a Sorvall MT2 rotary ultramicrotome. Semithin sections were stained with 2% toluidine blue in 1% sodium borate and photo- graphed with a Zeiss Photomicroscope II using Kodak Tmax 100 black-and-white film. Intact arms fixed in 10% formalin in seawater were photographed with a Tessovar photographic system. Thin sections for ultrastructural eval- uation were stained with aqueous 3% uranyl acetate and 0.3% lead citrate. Stained sections were viewed and photo- graphed with a Zeiss EM 9 transmission electron micro- scope. For scanning electron microscopy, an arm with suckers was fixed in 2.5% gluteraldehyde (as described above). dehydrated with ethyl alcohol, infiltrated with hexa- methyldisali/ane (Pellco), and air-dried. Micrographs were obtained with a JEOL JSM 5800LV scanning electron mi- croscope using Kodak Polapan 400 film. Results General description of animal and distribution of photophores Figures 1A and IB show the largest of the three captured specimens of S. syrtenxis. The appearance of the specimen is typical for the species (Vecchione and Young, 1997). The mantle length is about 9 cm (mantle lengths of other two specimens ~ 6 cm), suggesting that all three animals were immature (Collins, unpubl. data). The measurements are highly approximate because the mantle in the living animal is easily deformed. The primary webbing extends for about three-quarters of the length of the arms. The arms are oral to the primary web and attached to it by a secondary web. The photophores are arranged in a single row along the oral surface of each arm, situated between successive pairs of cirri (Fig. IB). Each arm supports about 40 photophores. The distance between photophores decreases from the base to the tip of the arm, with the greatest distance being 4 mm and the smallest less than O.I mm. The diameter and the degree of development of the photophores located at the tip are less than those located at the base of the arm. The fresh tissue of the entire animal had a gelatinous consistency typical of many deep-sea cephalopods (Voss, 1967). Al- though orange-red under the photo-floodlights, the color of the animal was closer to reddish-brown in daylight. Bioluminescence When mechanically stimulated, S. svrtensis emitted mod- erately bright, blue-green light (Amax = 470 nm) from the sucker-like photophores along the length of each arm (Fig. 2). With continuous stimulation, these photophores pro- duced light for up to 5 min, though the intensity of biolu- minescence decreased over time. Individual photophores either glowed dimly and continuously or blinked on and off brightly at 0.5 to I Hz. The blinking photophores cycled asynchronously, producing a twinkling effect. All suckers 28 S. JOHNSEN ET AL Outer epithelium Figure 1. Photographs under artificial light of the deep-sea finned octopod Stauroteuthis syrtenxis with the wehhed arms in swimming pos- ture (A) and spread (B) displaying the photophores/suckers (arrowheads) that appear as white spheres along the length of the inner surface of the arms. The posture shown in (B) may he one of extreme withdrawal intended to startle intruders with the sudden appearance of hioluminescent suckers, ar, arm; ey, eye; fi, fin; wb, webbing between arms. Scale bars = 4 cm. (except possibly the very small ones at the tips of the arms) appeared capable of luminescence. No other portion of the body was observed to emit light. Morphology of photophores Each photophore is a raised papilla-like structure partially embedded in the connective tissue of the arm. The photo- phores are composed of three layers of cells: an outer epithelium modified to form a collar, infundibulum, and acetabulum: a capsule-like mass of muscle and neural tissue beneath the epithelium; and a thin layer separating the capsule from the dermis of the arm (Figs. 3, 4, 5). The collar epithelium is continuous with the epidermis and is folded inward, forming a rim around the central portion of the photophore (Figs. 3B, C; 4A). In both formalin- and glut- araldehyde-preserved specimens, the photophores appear to be either everted above (Fig. 3B) or retracted below (Fig. 3C) the outer edge of the collar. The outer and inner folds of the collar epithelium are morphologically distinct and are different from the epider- mis covering the arm (Figs. 5, 6). The epidermis of the arm is squamous to cuboidal in character and consists of epithe- lial cells possessing scattered apical microvilli (Fig. 6A). The outer edge of the collar is composed of columnar cells with apical microvilli, numerous electron-lucent and elec- tron-dense vesicles, and large, apically placed, elongated nuclei (Fig. 6B). Like the epidermis, this region of the collar is not covered by a cuticle. The inner edge of the collar is similar in cellular mor- phology to the outer collar epithelium except that the mi- crovilli are more densely arranged and are covered by a cuticle (Figs. 6C, D). In this region, the cuticle is composed of at least three layers: an outer lamina 0.3 yum thick with irregular projections; a second electron-dense lamina, also 0.3 /am thick: and an inner layer approximately 1 /urn thick consisting of amorphous material. The epithelium and the overlying cuticle of the inner edge of the collar continue as the epithelium of a flat recessed region of the photophore corresponding to the infundibulum of typical octopod suckers (Figs. 3B; 4 A: 5 A, B). The outer edge of the infundibulum is ringed by hook-shaped den- ticles (Fig. 4B-D), which are elaborations of the cuticle (Fig. 6C, D). In addition to the presence of denticles, the cuticle covering the infundibular epithelium differs from that described for the inner part of the collar in that the outer layer contains more irregular projections and the innermost lamina is greatly expanded. The cuticle in this region is apparently secreted by the infundibulum and, as supported by Figure 6C and D, is periodically molted and replaced by a new, pre-formed cuticle. Subcuticular spaces were ob- served in association with what appear to be newly forming denticles. Three cell types — gland cells, columnar epithelial cells, and multiciliated cells — were observed in the infundibular epithelium. Gland cells with narrow apical necks and a reduced number of apical microvilli are situated between columnar epithelial cells, which are characterized by a brush border of branched microvilli, rounded apical nuclei, apical endocytic vesicles, and mitochondria (Fig. 7 A). Both co- lumnar cells and gland cells have a tine granular cytoplasm replete with Golgi bodies and electron-dense and electron- lucent vesicles of varying sizes (Fig. 7B. C). Electron-dense granules, not bounded by a membrane, were observed be- tween microvilli. These presumably originate from the in- fundibular cells and are incorporated into the cuticle (Fig. 7C). Multiciliated columnar cells were infrequently ob- served as part of the infundibulum. Cilia were not found in epidermal or collar cells. The cilia of the infundibular cells have two nearly parallel striated rootlets and appear to have reduced axonemes that do not project above the level of the BIOLUM1NESCENCE IN A DEEP-SEA OCTOPOD 29 Figure 2. Digitized frames from a video sequence of light emission (white spots) from photophores/suckers taken from video of an animal filmed in the dark using an intensified video camera (Inlevac's NiteMatc 1305/1306 CCTV Intensifier coupled to a Panasonic CCD). Two amis are shown. For scale, their closest approach is approximately 1 cm. microvilli (Fig. 7C). All three cell types are interconnected by apical adherens and subapical septate junctions (Fig. 7D). At the center of the light organ, the infundibular epithe- lium invaginates to form the acetabulum, which is seen externally as an opening, or pore (Figs. 3B, 4A). This central opening continues internally as a blind canal (Fig. 5C). The acetabular cells differ from those of the infundib- ulum primarily in the basal position of the nuclei, the highly interdigitated lateral membranes, and the diminution of the outer two layers of the cuticle (Figs. 5C: 8A. B). The infundibulum and the acetabulum rest on a basal lamina beneath which is located an expanded layer of con- nective tissue with a maximum thickness of 1.5 jam (Figs. 5C; 8C, D). Fibers, presumably collagen, although confir- mation of this is not provided by the data, are arranged in alternating directions in multiple layers, giving the tissue a herringbone appearance. Occasional breaks, traversed by nerve axons, were observed in this otherwise continuous connective tissue sheath. Muscle and neural tissue Beneath the connective tissue underlying the epithelium of the infundibulum and acetabulum is a mass of tissue consisting of muscle and neural cells; this surrounds and encapsulates the outer epithelium (Figs. 5; 8A, E; 9). The myofilaments. which include thick filaments (25 and 50 nm in diameter) and thin filaments consistent with the size of myosin and actin, are oriented in three planes — circular. radial, and longitudinal with respect to the axis of the photophore. Although all sections were taken in the longi- tudinal plane of the photophore. the precise plane of each section for these transmission electron micrographs was not known. Thus, the differentiation of the fibers seen in Figure 8C (shown in cross-section) and Figure 9A (horizontal fibers shown in longitudinal section) as circular or radial cannot be determined. Intermingled with the muscle cells are nerve cells char- acterized by electron-dense granules O.I jum in diameter. Nerve axons are located throughout the capsule and espe- cially in the basal region closest to the dermis (Fig 9B). Although a direct connection was not documented, fluores- cent images of the photophores indicate that axons originat- ing from the large branchial nerve traverse the dermis and connect to the photophore. The innermost layer of the photophores is an epithelium that separates the muscular capsule from the dermis of the arm. The cells of this layer have interdigitated lateral mem- branes and a cytoplasm that appears more granular than that of the outer epithelium. This layer is associated with extrin- sic (to the photophore) muscle cells (Fig. 9A) and a blood vessel located in the dermis (Fig 9B). In situ behavior Animal I (from Cape Hatteras) was first seen in a bell posture with its fins sculling (Fig. 10A). It then moved away from the submersible, using a slow medusoid locomotion. After one contraction/expansion cycle, the animal closed its 30 S. JOHNSEN ET AL CO ct Figure 3. (A) Photograph of part of an arm of Slauroteiilhis syrtensis with the webbing removed. Photophores (arrowheads) are arranged in a single row along the length of the arm and are unequally spaced with decreasing distance between light organs at the proximal tip of the arm. The positions of the photophores alternate with the positions of the cirri (cit. Scale bar = 0.5 cm. (B) Light micrograph of a fluorescent image of a single formalin-fixed photophore in the extended position. Like octopus suckers, the photophore is elevated above the epidermis (ep). is surrounded by a collar of epidermal cells (co), and consists of an infundihulum (in) and central acetabular canal (ac). (C) Light micrograph of a retracted photophore that has been bisected longitudinally. Internally, a capsule-like mass of tissue (ca) underlies the epithelium of the infundibulum and acetabulum. ct, dermal connective tissue of the arm. Scale bar for B and C = 0. 1 mm. web and assumed a highly distended balloon posture with motionless fins (Fig. 10B). After several minutes in this posture, the arms opened to a bell posture, and then closed to a considerably smaller balloon posture (Fig. IOC) re- ferred to as the "pumpkin posture" by Vecchione (pers. comm.). After 2 min, the fins began sculling and the animal made one more medusoid contraction and then again closed its web to the pumpkin posture with fins sculling. After a minute, the animal made about seven more medusoid con- tractions and then closed to the pumpkin posture with fins sculling and head down. Animal 2 (from Oceanographer Canyon) was first seen with its arms spread in the horizontal plane with the mouth oriented upwards (Fig. 10D). It underwent one medusoid contraction and then inflated to a highly distended balloon posture with fins motionless and cirri extended and pressed against the primary web. After several minutes, the fins began sculling and the animal simultaneously twisted its body and opened its arms (Fig. 10E). Animal 3 (from Oceanographer Canyon) was first seen in a bell posture. Then, using slow medusoid locomotion, moved away from the submersible. During the escape, its fin sculled continuously and sometimes vigorously. During expansion of the primary web, the cirri could be seen and were extended perpendicular to the arms and pressed against the primary web. Discussion Morphology of photophores and homology with octopus suckers Although the anatomical position and morphology of the light organs of S. svrtensis indicate their homology with octopod suckers, other aspects of their structure are consis- BIOLUMINESCENCE IN A DEEP-SEA OCTOPOD 31 Figure 4. Scanning electron micrographs of photophores. (A) Externally, each photophore has three main recognizable parts: outer wall or collar (co). infundibulum (in), and acetabulum (arrow indicates the opening to the acetabiilar canal). Scale bar = 100 /Mm. (B) The junction between the infundibulum and the infolded collar is ringed by a row of denticles (arrowheads). Scale bar = 10 ^im. (C-D) These hook-like denticles (de). which are atypical of octopod suckers, appear to be elaborations of the cuticle covering the infundibulum and acetabulum. Scale bars for C and D = 1 jum. A and B adapted from Johnsen el ill ( 1999). with permission from Nature, copyright 1999 Macmillan Magazines Ltd. tent with those reported for simple photophores in other cephalopods (Young and Arnold, 1982; Herring et al., 1987, 1994). Definitive structural characteristics of octopod suck- ers are given by Kier and Smith (1990) and Budelmann et al. (1997). Like the suckers of other citrate octopods, the photophores of S. syrtensis are arranged in a single row along the oral surface of the arm with the largest, most developed organs located at the base of the arm, nearest the mouth. Suckers and these photophores both consist of three layers of tissue: an outer epithelium, an intrinsic muscular layer, and an extrinsic layer associated with muscle cells. The outer epithelium is covered by a cuticle that, as in suckers, appears to be periodically molted. Moreover, the epidermis associated with the photophore is modified to form the columnar epithelial cells of the recessed infundib- ulum and the invaginated acetabulum. The arrangement of myofilaments in the muscular capsule are consistent with the three-dimensional array of contractile fibers typical of suckers. Although this may be an artifact of fixation, the morphology and the arrangement of myofilaments would allow for the retraction and extension, as well as a change in the diameter, of the photophore and may be important in regulating the intensity of the emitted light. Although denticles are not common in octopod suckers (Nixon and Dilly, 1977; Budelmann et al., 1997), hooks and denticles of various sizes are found in decapod cephalopods. The functional significance, especially with the apparent loss of an adhesive function for the suckers, of the denticles on the photophores of S. syrtensis is unknown. They may, however, be vestigial structures indicating an evolutionary connection to the decapods. Although definitive morphological characteristics are 32 S. JOHNSEN ET AL - **?•> Figure 5. Light micrographs of a series of semithin sections from the outer edge of the infundihulum (A), through the middle region of the inl'undihulum (B), lo the center of the acetabular canal (C). Each photo- phore consists of an outer epithelium that is recessed below the level of a supporting epidermal collar (co). This epithelium forms the infundihulum (inland the acetabulum (ac)and is covered by a cuticle (cu). A capsule-like mass of tissue (ca) is located below the outer epithelium and is separated from the connective tissue (ct) of the arm by a third layer of cells (tl). Arrowheads, denticles; arrow, putative reflector. Scale bars = 0.2 mm lacking for photocytes in general (Herring, 1988). the epi- thelium of the acetabulum (and possibly of the infundihu- lum) is presumed to be the bioluminescent region of the photophores in S. syrtensis. Characteristics that identify photocytes in the octopod Japetella diaphana ( Herring et ai 1987) and the squid Ahralia trigonura (Young and Arnold, 1982) and are also found in the photophores of S. syrtensis include the presence of an amorphous, finely granular cy- toplasmic ground substance containing numerous electron- dense vesicles, large basal nuclei, highly interdigitated lat- eral plasma membranes, ciliary rootlets, and abundant Golgi bodies. To some degree, this cellular morphology is found in the cells of both the infundihulum and the acetabulum. Since these ultrastructural traits are also typical of secretory epithelia. one hypothesis is that the infundibular epithelium secretes the cuticle, and the acetabular epithelium is in- volved in light production. Reflectors in cephalopod photophores are typically com- posed of collagen fibers arranged in layers beneath the photocytes (Young and Arnold, 1982; Herring et ' Figure 8. Transmission electron micrographs of the acetahular epithelium ( A-C). the putative reflectoi i l>i. and distally positioned cells in the capsule of tissue beneath the outer epithelium (E). (A) A montage showing the presumptive photocytic epithelium (ph), reflector (re), and the underlying capsule (ca) of muscle and nerve cells. Scale bar = 3 /xm. (B) Like photocytes of other cephalopods, the cells of the acetabulum have highly digitized lateral membranes (arrowheads) and a finely granular cytoplasm (cy). Scale bar = 0.5 fim. (C. D) A layer of connective tissue separates the acetabular epithelium from the underlying muscle (mu) and nerve cells. Breaks in the connective tissue layer are bridged by neurons (nv). Fibers (fi) in the connective tissue are arranged in layers with alternating orientation, giving the tissue a herringbone appearance. Scale bars = 0.5 /^m. (E) Presumptive neurons with electron-dense granules (gr) are found throughout the capsule. Scale bar = 0.5 fxm. have suggested that these are trapped within a mucous web produced by buccal secretory glands and handled by the elongated cirri (Vecchione, 1987; Vecchione and Young, 1997). Since all three genera appear to have nonfunctional suckers (Aldred ct «/.. 1983; Voss and Pearcy, 1990), this method of feeding seems likely. In all three genera, the 36 S. JOHNSEN ET AL A Figure 9. (A, B) Transmission electron micrographs ot Ihc medial region of the capsule and ot the third innermost cell layer of the photophore. The capsule, like the intrinsic musculature of suckers, consists, in part, ot cells with myolilaments arranged in three planes (also see inset). Longitudinal dm) and horizontal fibers (nui) alternate throughout the capsule. Inset shows thick and thin filaments consistent with the si/e and appearance of myosin and actin. Neural axons (nv) are intermingled with muscle cells. The capsule is separated from the connective tissue of the arm (ct)by a third layer of cells ( ' I. which is associated with extrinsic muscle cells (cm), bv. lumen ot a blood vessel; nu. nucleus. Scale bars = 1 fj.ni. shape of the primary web precludes any flow-through feed- ing current. Therefore, the prey cannot be filtered from the water column, but must somehow be attracted to the mucous web. Since many deep-sea crustaceans have well-devel- oped, sensitive eyes and are attracted to light sources (Morin ft nl.. 1475). the photophores of S. syrteiisis may provide the lure. With the exception of the twisting behavior following ballooning (observed only once), the in situ behaviors of the three specimens of S. syrtensis reported here are similar to BIOLUMINESCENCE IN A DEEP-SEA OCTOPOD 37 Figure 10. Digiti/ed frames of in situ video of Stauroteuthis syrtensis: (A) bell posture; (B) distended balloon posture; (C) pumpkin posture (distinguished from the balloon posture by the fact the web is not fully inflated); (D) inverted umbrella posture; (E) animal twisting and opening its arms after ballooning. The highlights on the mantle in A. C, and E are reflections from the submersible lights. those described from videos of two other specimens ob- served by Vecchione and Young (1997). When first ap- proached. S. syrtensis is generally found with its arms spread in an umbrella or bell posture, with the mouth oriented either upwards or downwards (Roper and Brund- age, 1972; Vecchione and Young, 1997; Villeneuva et a!.. 1997). Since the animal almost certainly detects the rela- tively large and well-lit submersible well before itself being captured on video, it is difficult to know whether this is the natural posture or a defensive reaction. Given the assump- tion that the animals are in an undisturbed, non-withdrawal state when first filmed, their posture and the location of their photophores are consistent with the use of bioluminescence as a lure. As mentioned above, the wavelength of peak emission approximates the wavelength of maximum light transmission. This suggests that the emission spectrum of the photophores has been selected for maximum visibility. Finally, because the intensity of upwelling light is only a small percentage of that of downwelling light (Demon, 1990), animals bioluminescing in the mouth-up posture would be highly visible to potential prey in shallower depths. Collectively, these observations give credence to the idea that 5. syrtensis uses photophores to attract prey. Existence and evolution of photophores in octopods Aside from the present study, the only other conclusive evidence of bioluminescence in octopods is restricted to the breeding females of the family Bolitaenidae (Herring. 1988). However, the existence of light organs within suck- ers (at the base of the peduncle) has been suggested in the cirrate octopod C. nuimivi (Chun. 1910, 1913). As in the photophores of 5. svrtensis, these organs have a bright white appearance due to reflection from a connective tissue layer and are found in suckers that have many reduced traits compared to typical octopod suckers (Aldred et ui, 1983). Unlike the photophores of S. syrtensis, the postulated light organs of C. mtirmyi are not found within the sucker itself. In addition, the connective tissue layer is situated such that the produced light would be reflected into the tissue of the arm. After a complex subsequent study (Aldred et ai, 1978, 1982, 1983), Aldred et nl. (1984) tentatively interpreted the organs as unusual nerve ganglia (see also Vecchione, 1987). Because photophores and photocytes have a bewildering variety of morphologies (Buck, 1978; Herring, 1988), con- clusive determination of the presence or absence of light organs (which often emit only dim light) requires observa- tion of a healthy specimen in near-total darkness by a thoroughly dark-adapted observer (i.e., in near-total dark- ness for a minimum of 10 min) (Widder et ai, 1983). Owing to the bright lights of submersibles and remotely operated vehicles, bioluminescence is seldom observed in situ. In addition, since most bioluminescent animals produce light when disturbed, deep-sea cephalopods collected in nets generally have exhausted their light production by the time they reach the surface. Finally, observations of spontaneous luminescence are rare; most bioluminescent animals must be physically stimulated (often for a considerable period of time) before light is observed (Widder et «/., 1983). The evolutionary history of photophores in any animal group is extremely difficult to determine because biolumi- nescence has no fossil record (Buck, 1978). The evolution of bioluminescence in the coleoid cephalopods is particu- larly intriguing because of the extraordinary diversity and complexity of photophores in deep-sea decapods and vampyromorphs and their apparent rarity and simplicity in deep-sea octopods (Herring, 1988). However, biolumines- cence in the deep-sea octopods may not be as rare as previously assumed. For the reasons given in the previous paragraph, bioluminescence may be under-reported in the deep-sea octopods. Cirrothuuimi innrniyi and Cirroteitthis 38 S. JOHNSEN ET AL are found at abyssal depths (except in polar regions, where they can be found at the surface) (Voss, 1988), making capture of healthy specimens extremely difficult. Opistoteu- this is found at shallower depths and has been maintained in aquaria (Pereyra, 1965). but it is not known whether it was observed under the specialized conditions necessary to de- tect bioluminescence. However, given that Opistoteuthis feeds on a variety of benthic prey that it apparently captures using functional suckers (Villaneuva and Guerra, 1991 ), if it is bioluminescent, its photophores are probably in a differ- ent site. Octopod bioluminescence may exist only in S. syrtensis and the bolitaenids. A cladistic analysis of the Octopoda involving 66 morphological characters places the citrates basal to the incirrates and the bolitaenids basal among the incirrates (Voight, 1997). This analysis also supports the monophyly of the bolitaenids and the two clades (Cirroteu- thidae and Stauroteuthidae) composing the genera Stauro- teitthis, Cirrothauma, and Cirroteuthis. The homology of the photophores in S. syrtensis and the bolitaenids is un- likely. The photophores of S. syrtensis appear to exhibit the rare trait of muscle derivation. The only other example of muscle-derived light organs has been found in the scopel- archid fish Benlhalbella infans (Johnston and Herring, 1985). Although the luminous circumoral ring in the boli- taenid Japetella diaphann is initially a muscular band, the great increase in the size of the ring in adult females apparently requires tie uoro synthesis of luminous tissue (Herring et ai, 1987). In addition, the light organs differ in almost all other anatomical and morphological characteris- tics. Finally, only mature female bolitaenids have light organs, which appear to have a sexual function, whereas the light organs of S. syrtensis are found in immature animals and may be involved in feeding. Multiple independent evo- lutions of photophores are common in decapods, at least at taxonomic ranks of subfamily or higher (Young and Ben- nett, 1988; Herring et ai, 1992. 1994). Therefore, despite the close evolutionary relationship between the cirrates and the bolitaenids. photophores in these two groups seem to have evolved independently. However, the monophyly of Stauroteuthis, Cirroteuthis, and Cirrothauma and the fact that they all have suckers with reduced traits suggest the possibility of light production by modified suckers in the latter two genera. Acknowledgments We thank the captain and crew of the R.V. Edwin Link and the Johnson-Sea-Link pilots, Phil Santos and Scott Olsen. for assistance with all aspects of animal collection. We also thank Dr. Tamara Frank for a critical reading of the manuscript. Dr. Michael Vecchione for aid with identifying the specimens, Dr. Janet Voight for helpful discussions on octopod evolution, and Dr. Martin Collins for use of un- published data. The authors are grateful to the Smithsonian Marine Station at Fort Pierce, Florida, for allowing the use of the photomicroscopes. Our thanks are also extended to Dr. William Jaeckle for his assistance with the scanning electron microscopy and to Julie Piriano for help with the transmission electron microscope. This work was funded by a grant from the National Oceanic and Atmospheric Ad- ministration (subgrant UCAP-95-02b, University of Con- necticut, Award No. NA76RU0060) to Drs. Tamara Frank and EAW, a grant from the National Science Foundation (OCE-93 13872) to Drs. Tamara Frank and EAW. and by a Harbor Branch Institution Postdoctoral Fellowship to SJ. 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Marine biolumines- cence spectra measured with an optical multichannel detection system. B/o/. Bull. 165: 791-810. Young, R. E., and J. M. Arnold. 1982. The functional morphology of a ventral photophore from the mesopelagic squid, Abralia trigonura. Ma/aco/ogia 23(1): 135-163. Young, R. E., and T. M. Bennett. 1988. Photophore structure and evolution within the Enoploteuthidae (Cephalopoda). Pp. 241-251 in The Mollusca Vol. XII, E. R. Trueman and M. R. Clar! eds. Aca- demic Press, New York. Reference: Biol. Bull 197: 40-48. (August 1999) Long-Term Culture of Lobster Central Ganglia: Expression of Foreign Genes in Identified Neurons GEOFFREY K. GANTER, RALF HEINRICH. RICHARD P. BUNGE1 *, AND EDWARD A. KRAVITZt DC/HI rtinent of Neurohiologv, Harvard Medical School, 220 Longwood Avenue, Boston, Massachusetts 021 15: and Miami Project to Cure Paralysis, University of Miami School of Medicine, Miami, Florida 33136 Abstract. The ventral nerve cords of lobsters (Homarus americamts) can be cultured /';; vitro for at least 7 weeks. Over this period, neurons maintain their normal electro- physiological features and continue, among other measures of neuronal health, to synthesize RNA and proteins. One application of this culture system is demonstrated: the ma- nipulation of gene expression in identified neurons. After intracellular injection of complementary RNA (cRNA) en- coding green fluorescent protein (GFP), the amount of pro- tein product measured by fluorescent confocal microscopy increases for 4 days and then decreases to background by day 10. Thus, translation of the injected message must have increased for 4 days before declining. Moreover, after in- jection of cRNA encoding j3-galactosidase, the levels of enzyme activity were measured using a fluorogenic sub- strate, revealing a peak of /3-galactosidase activity at 6 to 9 days: this activity was still detectable for at least 10 days after injection. Therefore, either GFP or /3-galactosidase can be used as an injectable marker, allowing HI vivo quantitation of ex- Received 16 December 1998; accepted 21 April 1999. * This paper is dedicated to the memory of a good colleague and friend. Dr. Richard P. Bunge. Dick died on September 10. 1996, of esophageal cancer at the age of sixty-four. He was in the midst of an important project, that of rebuilding the Miami Project to Cure Paralysis, and in 1989 became the scientific director of that project. One of us (EAK) had the good fortune to work with Dick from 1968 to 1969 during his sabbatical visit to our laboratory in Boston. The organ culture system was developed at that time, and although these earlier experiments never were published, they are an important part of our present and future research activities. It is typical of Dick and his studies that they often were far ahead of their time. We are honored to include him as an author on this paper. t To whom correspondence should be addressed. E-mail: edward_kravitz@ hms.harvard.edu pression in individual cells over time. We measured long- lasting expression of these proteins after a single injection, suggesting that it may be possible to manipulate the levels of expression of any gene of interest. Introduction The ventral nerve cord preparation has been a useful tool for exploring the physiology and pharmacology of central neurons in the lobster (see Otsuka et ai. 1967; Livingstone ct /v,v?(/ neurons. Proc. Nail. Acud. Sci. USA 89: 1 133-1 137. Kennedy. I)., A. I. Selverstcm, and M. P. Remler. 1969. Analysis of restricted neural networks. Science 164: 1488-1496. Kosslak, R. M., M. A. Chamberlin, R. G. Palmer, and B. A. Bowen. 1997. Programmed cell death in the root cortex of soybean root necrosis mutants. Plant ./. 11: 729-745. I .iliumc n . M., and C. I). Richardson. 1995. Production of recomhi- nant baculoviruses using rapid screening vectors that contain the gene for beta-galactosidase. Mali. Mol. Binl. 39: 161-177. Linnik, M. D., M. D. Hatfield, M. D. Swope. and N. K. Ahmed. 1993. Induction of programmed cell death in a dorsal root ganglia X neuro- hlasioma cell line. J. Netirohiol. 24: 433-446 Livingstone, M. S., R. M. Harris-Warrick, and E. A. Kravitz. 1980. Serotonin and octopamine produce opposite postures in lobsters. Sci- ence 208: 76-79. Ma, P. M., and W. A. Weiger. 1993. Serotonin-containing neurons in lobsters: the actions of y-aminobutyric acid, octopamine. serotonin, and proctolin on activity of a pair of identified neurons in the first abdom- inal ganglion. J. Nenropli\.\iol. 69: 2015-2029. Ma, P. M., B. S. Beltz, and E. A. Kravitz. 1992. Serotonin-containing neurons in lobsters: their role as "gain-setters" in postural control mechanisms. / Neiiniphyxiol. 68: 36-54. MacGregor, G. R., A. E. Mogg. J. F. Burke, and C. T. Caskey. 1987. Histochemkal staining of clonal mammalian cell lines expressing E. culi beta-galactosidase indicates heterogeneous expression of the bac- terial gene. Somatic Cell Mol. Genet. 13: 253-265. O'Brien, M. C., and VV. E. Bolton. 1995. Comparison of cell viability probes compatible with fixation and pemieabilization for combined surface and intracellular staining in flow cytometry. Cyitnnetry 19: 243-255. Otsuka, M., E. A. Kravitz. and D. D. Potter. 1967. The physiological and chemical architecture of a lobster ganglion with particular reference to gamma-aminobutyrate and glutamate. J. Neurophysiol. 30: 725-752. Pelham, H. R. 1982. A regulatory upstream promoter element in the DriKopliilu h.\p70 heat-shock gene. Cell 30: 517-528. Prasher, D. C., V. K. Eckenrode, VV. W. Ward, F. G. Prendergast, and M. J. Cormier. 1992. Primary structure of the Act/norm \-tctoria green-fluorescent protein. Gene 111: 229. Roberts. A., F. B. Krasne, G. Hagiwara. J. J. Wine, and A. P. Kramer. 1982. Segmental giant: evidence for a driver neuron interposed be- tween command and motor neurons in the crayfish escape system. J. Neiimnliyuol. 47: 761-781. Schneider, H.. B. Trimmer, J. Rapus, M. Eckert. D. Valentine, and E. A. Kravitz. 1993. Mapping of octopamine-immunoreactive neu- rons in the central nervous system of the lobster. ./. Com/'. Nenrol. 329: 129-142. Schwarz, T. L., G. M.-H. Lee, K. K. Siwicki, D. G. Standaert, and E. A. Kravitz. 19S4. Proctolin in the lobster: the distribution, release, and chemical characterization of a likely neurohormone. J. Ncurotci. 4: 1300-131 I. Thnmsen, D. R.. R. M. Stenberg, VV. F. Goins. and M. F. Stinski. 1984. Promoter-regulatory region of the major immediate early gene of human cytomegalovirus. Proc. Null. Aaul. Sci. L/SA 81: 659-663. Weiger, W. A., and P. M. Ma. 1993. Serotonin-containing neurons in lobsters: the actions of ganima-aniinobutync acid, octopamine. seroto- nin and proctolin on ncuronal activity. ./. iVcnro/iliy\iol 69: 2015-2029. Vao, K.-M.. and K. White. 1994. Neural specificity ol <7440 h) and under a variety of behavioral circumstances. The natural behavior of the squid was filmed on spawning grounds off Cape Cod (northwest- ern Atlantic), and behavioral trials in the laboratory were run in large tanks. The body pattern components — 34 chro- matic (including 4 polarization components). 5 postural, and 12 locomotor — are each described in detail. Eleven of the most common body patterns are also described. Four of them are chronic, or long-lasting, patterns for crypsis: an example is Banded Bottom Sitting, which produces disrup- tive coloration against the substrate. The remaining seven patterns are acute; they are mostly used in intraspecific communication among spawning squids. Two of these acute patterns — Lateral Display and Mate Guarding Pattern — are used during agonistic bouts and mate guarding; they are visually bright and conspicuous, which may subject the squids to predation; but we hypothesize that schooling and diurnal activity may offset the disadvantage presented by Received 1 February 1999; accepted 20 April 1999. E-mail: rhanlon@nihl.edu increased visibility to predators. The rapid changeability and the diversity of body patterns used for crypsis and communication are discussed in the context of the behav- ioral ecology of this species. Introduction Cephalopods have a highly developed system of visual communication that is expressed mainly through the skin. The distinguishing features of this remarkable chromato- phore system are its speed of change and the diversity of body patterns that each individual uses for either crypsis or communication (Hanlon and Messenger. 1996). A body pattern is defined as the total appearance of the animal at any given time, and includes the expression of the full complement of chromatic (i.e.. color or visual), textunil. postural, and locomotor components (see Packard and Hochberg. 1977; Hanlon and Messenger, 1996). Among the components of the body pattern, the most conspicuous are chromatic, although squids probably perceive intraspecific signals monochromatically because cephalopods are thought to be color blind (Hanlon and Messenger, 1996). These chromatic components are produced primarily by chromatophore organs and various reflective cells in the dennis, and they are discrete neural entities (just as postural, textural. and locomotor components are) because the chro- matophore organs are controlled by radial muscles under the direct control of the posterior chromatophore lobes in the brain (e.g.. Dubas et al.. 1986). Most of the reflective cells are also controlled by the squid (Cooper et al., 1990). This neural control enables the cephalopod to change its appear- 49 50 R. T. HANLON ET AL ance in a fraction of a second, depending upon the visual sensory input it receives during behavioral interactions. Few. if any. animals can match the speed of change and diversity of cephalopod signals, and the body patterns are used in most behavioral interactions, whether they be for competition for resources or mates, or interactions between predators and prey. We are documenting these diverse body patterns, focusing primarily on adult squids during their inshore migration every year. Squids, like other cephalopods, are sensitive to the partial polarization characteristics of light (Saidel et ai. 1983; Hanlon and Messenger. 1996; for a description of polarized light see Kattawar. 1994; Wolff and Andreou. 1995). Shashar and Hanlon (1997) described a few specific polar- ization components of squid and correlated these patterns with the distribution of iridophore cells in the animals' skin. In cuttlefish, partial polarization patterns have been associ- ated with communication (Shashar et ai, 1996). Since squids may use polarization patterns for intraspecific com- munication, and since polarization-sensitive predators may be looking for polarization contrasts to locate squid prey, we also document here some polarization components pre- sented by the squid. The long-tinned squid Loligo pealei Lesueur, 1821, is a renowned model in neuroscience research. The third-order giant axon. its attendant giant synapse, the complex eye, and several other organ systems in L. pealei have been studied in detail for over 50 years at the Marine Biological Labo- ratory (MBL) in Woods Hole (see Gilbert et til., 1990). Although a great deal is known about the peripheral nervous system of L. pen lei. little is known about the behaviors of this squid, which like most cephalopods, has an enormous brain relative to its body size. Loligo pealei is also a valuable commercial resource in the northeastern United States — worth about $30 million annually (McKiernan and Pierce. 1995; NEFSC, 1995). Curiously, little is known about the ecology, life history, and behavior of this species (e.g., Verrill. 1880; Drew. 191 1; Stevenson. 1934; Griswold and Prezioso, 1981; Summers, 1983; Gilbert et al. 1990; Brodziak and Macy. 1996). The present report is part of a broad-based study that focuses on sexual selection pro- cesses in L. pealei from two perspectives: as a test of sexual selection theory (e.g.. Hanlon. 1996; Hanlon et til.. 1997) and as a study of the role that reproductive behavior plays in the life history and population dynamics of the species (Hanlon. 1998). Materials and Methods The behavior of Loligo pealei can be observed both in a natural setting and in the laboratory because the squids habituate quickly to divers and to laboratory surroundings. Overall, 27.5 h of videotape were analyzed for body pat- terning and behavior. During the months of May 1996. May 1997, and May 1998. 103 scuba dives were made on squid spawning grounds by RTH and NS off the southern arm of Cape Cod, Massachusetts. Depths ranged from 3-10 m and most sites were within 2 km of shore between Hyannis and Chatham. Spawning squids were found mostly in or near commercial weir traps whose inner pocket dimensions (or capture arena) were roughly 20 m2; often there were many thousands of squids in these traps, with a proportion of them actively engaged in reproductive behavior. Water temperatures ranged from about 4° to 13°C. currents were often strong, and visibility was usually poor. On about one-third of the dives, conditions were suitable for video. In total. 16.5 h of dive video were recorded, using video cameras (either an- alog or digital) in underwater housings, and analyzed, with multi-motion playback machines and high-resolution mon- itors. Laboratory trials of mating behavior were performed from May through October in 1996, 1997. and 1998 in the Marine Resources Center of the MBL. Three large tanks were used, each measuring 3 m (diameter) by 1 m (height) and containing about 28,000 1 of seawater. Each tank had a substrate of mixed gravel and sand, and a continuous supply of ambient seawater. Animals were acquired by squid jig- ging (both at night and during the day) off the MBL re- search vessel Gemma in Vineyard and Nantucket Sounds. This method minimizes skin damage for maximal survival in captivity (see Hanlon el al., 1983). Squids were fed live fish (Fundiihis sp.) daily. Trials involved from three to eight squids in various combinations of males and females. One set of trials was performed in an outdoor pond, 20 m X 20 m X 1 m deep, at the Environmental Systems Laboratory of the Woods Hole Oceanographic Institution. The squids were observed for 440 h in captivity. 1 1 of which were recorded on video. All videos were reviewed multiple times, each time look- ing for only one category of component (i.e.. first viewing for chromatic components, second viewing for postural components, third viewing for locomotor components). In the laboratory, chromatic, postural, and locomotor compo- nents were recorded on separate data sheets each time they were seen. A chromatic component was recorded if it was expressed for at least 2 s; locomotor and postural compo- nents were recorded if they were performed for at least 3 s. All chromatic components were illustrated using a computer graphics program. Polarization components were recorded using a video polarimeter based on a standard three-tube ENG camera (JVC BY-110) that uses a dichroic prism block for color separation. The dichroic prism has been replaced with a custom-made neutral prismatic splitter (Richter Enterprises, SQUID BODY PATTERNING AND BEHAVIOR 51 Manhattan Beach, CA) such that each of the three video channels receives 1/3 of the broad-spectrum image input. Since this assembly lacks the color-trimming filters ce- mented to the original dichroic prism, magnification errors due to pathlength differences were corrected with small quartz discs of appropriate thickness. A small disc of sheet polarizer (Polaroid, HNP'B) was placed immediately in front of each camera tube to impart polarization sensitivity to the channels. The orientation of the polarizers was ad- justed so that the color channels now encoded 0°, 45°, and 90° polarization images. The camera electronics encode the three polarization channels as if they were color, making it possible to store all the data on a regular portable videocas- sette recorder and allowing for immediate viewing of a pseudocolor polarization image on a color monitor. Nonpo- larizing elements of the scene have no color, whereas po- larizing elements do. The signal in all three channels is identical, and the output of the tubes was adjusted to give white for a saturating faceplate intensity. A polarizer placed in front of the lens such that horizontally polarized light is freely transmitted produces the following normalized sig- nals in the three "color" channels: the R channel signal is 1, the G channel is 0.707, and the B channel is 0. Monochro- matic images of the same scene, taken from the three channels separately, were transferred through a frame grab- ber into the computer and their linear polarization charac- teristics were analyzed following procedures in Cronin et al. (1994). This camera is better suited than previously de- scribed polarimeters (Cronin et al., 1994; Wolff and An- dreou, 1995; Horvath and Varju, 1997) for recording the polarization patterns of moving animals, because it provides true instantaneous measurements. Technological limitations made it impossible to get the camera in an underwater housing; thus measurements were limited to the laboratory. Furthermore, the light conditions during measurements had to be precisely controlled, thereby allowing only 3 h of recorded footage. During these periods, the squids exhibited only a few behaviors that included fighting, mate guarding, and egg laying. Ethogram We constructed an ethogram for Loligo pealei on the basis of our field and laboratory observations. The compo- nents and body patterns identified (Table I) represent a segment of all behaviors, especially those related to repro- duction. In fact, because of the size of the sample, most of the patterning components of the species were probably identified. The more than 440 h of observation far exceed the observation periods in other published accounts of Lo- ligo spp. (e.g., Hanlon, 1982, 1988; Hanlon et al., 1983, 1994; Porteiro et al., 1990). The chromatic components of the ethogram are illustrated in Figures 1 and 3, and some of the postural components are shown in Figure 2. Unlike octopuses and cuttlefishes, loli- ginid squids do not show textural components in the skin. Table I, which lists all components, includes the number of times that we counted a component on videotape or from observation notes, giving an impression of how commonly it occurs. Unless otherwise indicated, all components and body patterns were shown by both sexes. Light ' components Chromatic components are produced mainly by the action of dermal chromatophore organs, which number in the hundreds of thousands in an adult squid. Loligo pealei has three color classes of chromatophores: yellow, red, and brown. Expansion of the chromatophores darks the skin, while retraction of the chromatophores (and the resultant expression of underlying iridophores) produces a lightening or even brightening effect. Intense darkness produced by maximal expansion and intense brightness produced by maximal retraction mark two ends of a chromatic contin- uum, and thus it is somewhat arbitrary to assign a compo- nent to light or dark. Some of these components are com- mon to other Loligo spp., as described by Hanlon ( 1982) for Loligo plei, by Porteiro et ul. ( 1990) for Loligo forbesi, and by Hanlon et al. ( 1994) for Loligo vulgaris reynaudii. Clear is retraction of all or most chromatophores, thus rendering the animal translucent in clear water or white in murky water. In clear water, when viewed against a sand bottom or laterally against the aquatic background (Fig. 2B), the translucence renders the squid cryptic, or camou- flaged, and often the Dorsal iridophore splotches are ex- pressed simultaneously. Internal organs, such as the red accessory nidamental gland in females, are often visible. In murky water. Clear appears bright white in most lighting circumstances (i.e., the brightness surpasses the albedo of the greenish water, producing a whitish color). In the im- mediate vicinity of egg beds, the white form of Clear seems to function as an intraspecific signal to repel other squids; a squid displaying this component is almost always engaged in mate guarding, egg laying, or agonistic bouts (see Fig. 2C). White arms/head results from variable retraction of chromatophores on the head and arms (three variations are illustrated in Fig. 1 ). This component sometimes preceded all white (or clear) in intraspecific encounters; thus, it ap- pears to be a milder signal of alarm or repellent to approach- ing squids (Fig. 2G). White head/arms is most common in paired females near eggs and is seen when unpaired males approach. White dorsal stripe is retraction of chromato- phores along a dorsal mantle that is otherwise dark; the stripe may be short or long (Fig. 1). It has been seen in 52 R. T. HANLON ET AL Table I Body patients and their components in the squid Loligo pealei; compare Figure 1 BODY PATTERNS Chronic (mm to hours) 1. Basic Amber Pattern 2. Clear Body Pattern 3. Countershading 4 Chronic AM Dark 5. Banded Bottom Sitting 6. Chronic Bright White Pattern Acute (seconds) 1. Very Dark 2. Blanch-Ink-Jet Maneuver 3. Lateral Display 4. Mate Guarding Pattern 5. Accentuated Testis COMPONENTS* Chromatic Light: 1 . Clear 2. White arms/head 3. White dorsal stripe 4. Accentuated testis (m) 5. Accentuated oviducal gland (f) Iridescent: 6. Dorsal mantle collar indophores 7. Iridescent sclera 8. Dorsal iridophore splotches 9. Iridescent arm stripes 10. Dorsal iridophore sheen Light polarization components: 1 . Polarized arms 2. Skin surface polarization 3. Polarized eyes 4. Polarized dorsal sheen (861) (769) (194) (1179) (183) (a 1000) (167) (500) (338) (32) Dark: 1. All dark 2. Dark arms/head 3. Dark head 4. Dark dorsal stripe 5. Ventral mantle stripe 6. Mantle margin stripe 7. Dark arm stripes 8. Fin spots 9. Arm spots 10. Intraocular spot 11. Bands 12. Shaded eye 13. Dark fins 14. Dark posterior mantle 15. Shaded testis (m) 16. Shaded oviducal gland (f) 17. Red accessory nidamental gland (f) 18. Lateral mantle spot (f) 19. Lateral blush If) 20. Weak lateral flame (m) (1440) (133) (853) (47) (369) (283) (38) (195) (672) (129) (153) (190) (31) (42) (11) (16) (-200) (147) (88) (13) Locomotor 1. Inking (12) 2. Jetting/fleeing (336) 3. Chasing (17) 4. Bottom sitting (45) 5. Egg touching (120) 6. Parallel positioning (435) 7. Jockeying and parrying (m) (62) X, Fin beating (in) (93) 9. Forward lunge/grab (m) (206) 10. Male-parallel mating (59) 1 1 . Head-to-head mating (24) 12. Oviposition ( = 300) Postural 1 Raised arms 1 1065) 2. Splayed arms (667) 3. Drooping arms (54) 4 Raised & splayed arms (560) 5. Flared arms (30) * Letters in parentheses indicate that the component is sex-specific: f = female; m = male. Numbers indicate how many times each component was observed on video or in laboratory trials. Clear SQUID BODY PATTERNING AND BEHAVIOR All dark 53 Accentuated oviducal eland (0 Dorsal mantle Iridescent sclera collar indophojs Dorsal iridophore splotches •v Iridescent arm stripes Dorsal iridophore sheen • Shaded oviducal gland (f) -~r^" llatcnil vicwt Mantle margin stripe Fin spots Arm spots Bands (with variations) Red accessory nidamental eland (f) Lateral mantle spot (f) Figure 1. Chromatic components of body patterning in the squid Loligo pealei. The arrangement generally follows Table I and the text. 54 R. T. HANLON ET AL. Figure 2. Underwater video images of selected components and body patterns of Laligo pealei. (A) The chronic Basic Amber Pattern. (B) The chronic Clear Body Pattern. (C) The chronic Bright White Pattern amidst other squids in Basie Amber. (D) The chronic All Dark pattern viewed against a sand substrate. (E) The Banded Bottom Sitting pattern showing disruptive coloration against a gravel substrate. (F) Acute Mate Guarding Pattern shown by a large consort male (female is just below him) showing the Splayed arm posture and the Accentuated testis chromatic component. (G) Raised arms postural component in a male that also shows the chromatic component of While arms/head; he is directing this signal to the lone male at upper left as he guards his female mate (barely visible behind him). Intensity SQUID BODY PATTERNING AND BEHAVIOR 55 Partial polarization Orientation of polarization B D 0 0.25 0.5 0.75 1.0 Figure 3. Selected images demonstrating the main sources of polarization components in adult squids. LEFT: Black-and-white images of the squid. CENTER: Partial polarization images in which black represents unpolarized light -0, and white represents full linear polarization -1. RIGHT: Orientation of polarization; horizontal polarization is coded into white or black, and vertical polarization into 50% grey. Special iridophores on the arms create the predominant components (A, B). where the partial polarization can exceed 0.75. The orientation of polarization can be equal on all arms (A) or it can vary between them (B, indicated by arrows). Structural reflection from the skin-water interface can produce a polarization pattern that changes with the animal's motion (C). The reflection from the sclera of the eye may be highly polarized (D, arrow). The top of the mantle of the squid occasionally reflects light that is partially polarized (E). This polarization may arise from structural reflection, as in C. or from reflection by the indophores on the squid's mantle or splotches (Shashar and Hanlon. 1997). 56 R. T. HANLON ET AL consort males when an intruder male approaches. Accentu- ated testis is u male-only component shown when the chromatophores directly above the testis are retracted while the squid mantle is otherwise dark, thus accentuating the whiteness of the organ (Fig. 2F). This component was seen frequently in single or mate-paired males when reproductive behavior was actively occurring in the school. Accentuated oviducal gland is a female-only component analogous in form and function to Accentuated testis in the male. This was often seen in females paired with consort males. All of these light components except White dorsal stripe have been seen commonly in other Loligo spp. Light iridescent chromatic components Each of the light iridescent chromatic components is common to Loligo spp., and comparable color images may be viewed in Hanlon (1982). Dorsal mantle collar iridophores are on the anteriormost portion of the man- tle, and they appear as bright yellow or pink iridescence; this component tends to produce disruptive coloration by breaking up the longitudinal aspect of the squid's body. It and the next component are usually seen on calm squids near the bottom in the Clear pattern. Dorsal iri- dophore splotches occur on the dorsal mantle and head. They are a distinctive yellow or golden color, and they help to produce general camouflage (Fig. 2E). Iridescent arm stripes extend most of the length of the first three pairs of arms. These are usually expressed lightly during camouflage in the Clear pattern, but during agonistic encounters they can be expressed very brightly (see color illustration in Hanlon. 1982). Iridescent sclera is the bright silver iridescence on the back (or sclera) of the eye; squids have the ability to obscure this with chro- matophores with the Shaded eye component. Dorsal iri- dophore sheen is somewhat rare and is only noticeable from the side. Its function is unclear but may aid cam- ouflage in open water by disrupting the body shape. None of these are unique to L. pealei but are shared by other Loligo spp. Light polarization chromatic components These linear polarization components are newly de- scribed for Loligo spp. Polarized arms are highly polarized reflections that create the most conspicuous component of polarization (Fig. 3A, B). This component often exceeds partial polarization of 0.75, which is noteworthy because Flamarique and Hawryshyn ( 1997) showed that the natural underwater light field rarely exhibits partial polarization as high as 0.67. The orientation of polarization can be equal in all arms (Fig. 3A), or it may differ between arms (Fig. 3B). Skin surface polarization results from the difference in refractive indexes between the squid's body and the water, so that light reflected from any area of the skin may be partially polarized (Fig. 3C). However, the partial polariza- tion in this case is mostly low, rarely reaching 0.5. Polar- ized eyes result from reflection by iridophore cells that surround the eye (Fig. 3D, arrow). The dorsal mantle occa- sionally reflects light that is partially polarized, resulting in Polarized dorsal sheen. The orientation of polarization can vary, reaching 20 degrees from horizontal. This polarization reflection corresponds to the area of the Dorsal iridophore sheen, although the two components do not always coincide in time. The source of this polarization component can be either reflection from iridophores on the mantle or Skin surface polarization. Owing to the limitations of the equip- ment used to record polarization patterns, these are probably not the only polarization components that squids can show. Dark chromatic components All dark is the opposite of Clear: all or most chromato- phores are expanded to some degree. The maximal expres- sion of All dark (Fig. 2D) produces an overall deep brown coloration; it is characteristic of alarmed squids. However, the chromatophores need not be maximally expanded, and thus there are ranges of darkness. Often squids are in a "normal" or "basic" coloration that is roughly between Clear and All dark, producing an overall amber body pattern (Fig. 2A). There is also a striking unilateral expression of All Dark (Fig. 1 ). Dark arms/head is variable in expression (see Fig. 1) and is opposite to White amis/head. It is seen typically in mating pairs and may represent a mild state of alarm. Dark head is expansion of all the chromatophores around the head of the animal (but not the arms), causing the head to appear almost black. This component is frequently seen in mate pairs near the egg mop and probably represents a low-grade alarm signal. Four striped components occur in L. pealei, one used for crypsis and three used during intraspecific agonistic con- tests. Dark dorsal stripe extends halfway or fully down the mid-dorsal mantle. Seen mainly on calm squids, it appar- ently aids camouflage because it covers some of the bright organs such as the testis, oviducal glands, and ink sac. Ventral mantle stripe is a thin, distinct line of fully ex- panded chromatophores. L. pealei. in contrast to L. plei but in common with L vulgnris reymnulii. L. vulgaris, and L. forbesi, shows no protrusile flap of skin when exhibiting this component (Hanlon, 1988; Hanlon ct ai, 1994). The func- tion of this component is uncertain, but it is seen commonly on mating pairs and on males during mate guarding. Males often swim just above females, and pairs are frequently approached by other squids from below, so the ventral SQUID BODY PATTERNING AND BEHAVIOR positioning of this visual signal may be useful. It is also possible that the stripe helps disrupt the body form when viewed from below by predators. Mantle margin stripe is a dark line running along the fin insertion. It was seen most often as a mild reaction to disturbance or alarm during agonistic bouts, and was usually expressed in conjunction with Ventral mantle stripe. Fin spots, and weak Lateral flame (see below). Dark arm stripes are variable, being expressed either along the third pair of arms or along pairs 1, 2, and 3. This uncommon component was seen on a female that also expressed Dark fins (also uncommon, see below) just before a male mated her, and as another mating pair bumped into them. Thus it seems to be an expression of alarm when all three arm pairs are darkened. The simulta- neous expression of stripes on three arm pairs has not been reported for squids. Three spotted components are expressed during alarm or threat situations, mainly intraspecifically, and can be shown unilaterally on the side towards the other squid. Fin spots are a collection of small circular and oval dark spots scat- tered across the fins. This component is seen mostly during agonistic bouts or rarely when an aggressive male comes close by. Arm spots are small and occur at the base of the third arms, the second arms, or both. This component is seen on males during mate guarding and at the early stages of agonistic encounters; it probably constitutes a low grade of alarm (see also Arnold, 1962, 1990). Intraocular spot appears directly in front of the eye and has variations, including a circular shape that looks like an eye ring. The avenue of achieving signals of "increasing alarm" appears to be Arm spots > Infraocular spot > expanded to eye ring > Dark head. Various other dark components include two for crypsis and four for intraspecific alarm situations. Bands are vari- able (see Figs. 1 and 2E) and may occur on the fins, head, or arms. First reported by Stevenson (1934), this component is seen typically in calm, bottom-sitting squids and func- tions as disruptive coloration to break up the longitudinal outline of the squid. Shaded eye is a transverse head bar of expanded chromatophores that may aid crypsis by covering the bright Iridescent sclera of the eyes. Dark fins occur when all fin chromatophores are expanded maximally; it is not common but has been seen on females that are alarmed. Dark posterior mantle is similar to Dark fins, but the mantle chromatophores are expanded; it may be the next stage of alarm after Dark fins. Several dark components associated with reproductive behavior complement the light components Accentuated testis and Accentuated oviducal gland. Shaded testis and Shaded oviducal gland are selective expansion of chro- matophores over the testis or oviducal gland. Both are often indistinct and serve to mask these bright white organs, thus aiding crypsis. However, the complementary "shading/ac- centuating" allows rapid signaling. The Red accessory ni- damental gland can be seen through the translucent mantle and occurs only in fully mature females, so it may be a part of communication even though it is internal. Since it turns red only upon attainment of full sexual maturity, it may be a sign of female sexual maturity or even receptivity. Lateral mantle spot is a female-only component expressed as a small intense dark spot of chromatophores near the anterior fin insertion. It coincides roughly with the position of the Red accessory nidamental gland, and the two may function together in some way. The Lateral mantle spot is seen only when the female is paired with a large consort male, and could indicate either receptivity or rejection. Lateral blush is a female-only component expressed unilaterally as a diffuse dark area on the lateral mantle. It may be compara- ble to a variety of similar components shown by female squids, and it may function as a repellent to courting males (Hanlon and Messenger, 1996: their fig. 6.21). Weak lateral flame is a male-only component produced by longitudinally oriented rows of partly expanded chro- matophores. It is seen during low-grade agonistic contests. There are several variations of this component in other Loligo spp., the most well developed and dramatic of which is in Loligo plci (Hanlon, 1982; DiMarco and Hanlon, 1997). In Loligo vulgaris, Loligo vulgaris reynaudii, and Loligo forbesi there are Lateral mantle streaks that are arranged a bit differently in the skin, but they all function to provide a lateral signal to an opposing male. Loligo pealei has perhaps the weakest expression of this component, while L. plei has the strongest. Postural components Five postural components are expressed through the arm positioning of Loligo pealei. They are generally comparable to postures seen in other Loligo spp. Raised arms (Fig. 2G) is the unilateral or bilateral raising of the first pair of arms, which may be light or dark, and is seen in both males and females on the mating grounds. This component appears to be a signal of alarm during agonistic contests. It was pre- viously reported by Arnold (1962, 1990). Splayed arms (Fig. 2F) is a posture in which all eight arms are spread and flattened on the horizontal plane. This posture is expressed by both sexes but is most common in males that use it to guard female mates they are escorting to egg mops. Raised and splayed arms are a combination of the previous pos- tures in which the arms are all splayed except for the first pair, which is raised; it is a strong signal of alarm used when a rival male approaches closely. Drooping arms in a swim- ming squid is a posture in which all the arms appear relaxed and hang downward, but its function is unknown. Flared arms is a rare posture in which all of the arms are held 58 R. T. HANLON ET AL stiffly outward in a radial manner; it is seen during highly aggressive agonistic encounters between two males, and during mate guarding. Locomotor components Inking is the expulsion of ink mixed with mucus, either in small puffs or as a large dense cloud (Hanlon ct \ of Unpredictability. 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A provisional generic classification of the family Loligin- Wolff, L. B., and A. G. Andreou. 1995. Polarization camera sensors. idae. Pp. 215-222 in Systematic! and Biogeography of Ceph- Image Vis. Comput. 16: 497-510. Reference: Biol. Bull. 197: 63-71. (August 1999) Concurrent Signals and Behavioral Plasticity in Blue Crab (Callinectes sapidus Rathbun) Courtship PAUL J. BUSHMANN* Smithsonian Environmental Research Center. 647 Coulee's Wharf Road, Edgewater, Maryland 21037 Abstract. Behavioral flexibility and behavioral regulation through courtship signals may both contribute to mating success. Blue crabs (Callinectes sapidus} form precopula- tory pairs after courtship periods that are influenced by female and perhaps male urine-based chemical signals. In this study, male and female crabs were observed in 1.5-in circular outdoor pools for 45 min while the occurrence and sequence of courtship behaviors and pairing outcomes were recorded. These results were then compared with trials in which males or females were blindfolded; lateral antennule (outer flagellum) ablated; blindfolded and lateral antennule ablated; or had received nephropore blocks. The relative importance of visual and chemical sensory systems during blue crab courtship were then determined and urine and non-urine based chemical signals for both males and fe- males were examined. Courtship behaviors varied consid- erably in occurrence and sequence; no measured behavior was necessary for pairing success. Male or female blind- folding had no effect on any measured behavior. Males and females required chemical information for normal courtship behaviors, yet blocking male or female urine release did not affect courtship behaviors. Males required chemical infor- mation to initiate pairing or to maintain stable pairs. Male urine release was necessary for stable pairing, suggesting that male urine signals may be involved in pair maintenance rather than pair formation. Females that could not receive chemical information paired faster and elicited fewer male agonistic behaviors. The results demonstrate a great vari- ability and flexibility in blue crab courtship, with no evi- dence for stereotyped behavioral sequences. However, these behaviors appear regulated by urine- and nonurine-based redundant chemical signals emanating from both males and females. Although urine-based signals play roles in blue Received 30 March 1998; accepted 1 June 1999. * Current address: Anne Arundel Community College, 101 College Parkway. Arnold. MD 21012. E-mail: pjbushman@mail.aacc.cc.md.us crab courtship, chemical signals from other sites appear to carry sufficient information to elicit a full range of behav- ioral responses in males and females. Introduction Courtship and mating success depend upon correct be- havioral responses by both males and females. One might expect a degree of plasticity in these behaviors (Hazlett, 1995). Because behavior can quickly track changes in en- vironmental conditions (West-Eberhard, 1989). flexibility in the occurrence and timing of reproductive behaviors might help insure successful mating. Many invertebrates do ex- hibit plasticity in their behaviors (Carlson and Copeland. 1978; Dejean, 1987; Elner and Beninger, 1995) and this variability may be the rule for most animal species (Lott, 1991). Conversely, one might also expect courtship and repro- ductive behaviors to be controlled and regulated by conspe- cific communication signals. By eliciting appropriate be- havioral responses, these signals could enhance mating success and help to prevent interspecies mating. Courtship and mating in a fluctuating environment could be aided by multiple or redundant signals, which would make the trans- mission of adequate and correct information more likely. Multiple or redundant signals have been found in both invertebrate and vertebrate species (van den Hurk and Lam- bert. 1983; Linn <•/ «/., 1984; Rand et «/., 1992). Chemical communication signals appear to be nearly universal in the animal world. For aquatic crustaceans, chemical communication signals have been well docu- mented in courtship and reproduction (Ryan, 1966; Atema and Engstrom, 1971; Bales, 1974; rev. in Dunham. 1978, 1988; Gleeson, 1980; Borowsky, 1984, 1985), while visual (Christy and Salmon, 1991) and acoustic (Salmon and Horch, 1972) signals have received less study. Recent stud- ies with a variety of animal taxa have begun to examine 63 64 P. J. BUSHMANN multiple signals and signal interactions (Hazlett, 1982; Waas and Colgan, 1992; Stauffer and Semlitsch, 1993; Hughes, 1996). Like many crustaceans (Hartnoll, 1969), the blue crab Callinectes sapidn.\ Rathbun practices a polygynous mating system involving a complex coordination of female ecdysis, maturation, and copulation. The mating process has been well described (Hay, 1905; Churchill, 1921; Van Engel, 1958; Gleeson, 1980). Immature females nearing their final maturational molt, termed prepubertal females, are ap- proached and courted by mature males. Pairing success results in females being held beneath males in a "cradle carry" posture for a period of precopulatory guarding. They are released for their molt, mated while still soft, and carried again for a period of postcopulatory guarding. This latter guarding protects the female while she is soft and prevents subsequent inseminations by other males (Jivoff, 1997a). Females are thought to receive only one copulation in their lifetime while males mate repeatedly (Van Engel, 1958), although multiple inseminations are possible and occur oc- casionally (Jivoff, 1997a). Blue crab courtship can be divided into three phases: mate attraction, pair formation, and pair maintenance. In each phase a precise signaling system would seem impor- tant to help insure mating success. The coupling of molt and reproductive condition requires individuals to ascertain the physiological state of prospective partners. Signals can function in the reduction of agonistic behaviors (Tinbergen, 1953; Bastock, 1967), and during mating female blue crabs must in some way guard against injury or death by aggres- sive, cannibalistic males. Reproductive behaviors and se- quences might, therefore, be tightly regulated by commu- nication signals, making appropriate responses more likely and increasing the eventual mating success of the partici- pants (Ryan, 1990; Reynolds, 1993). Chemoreception and vision are the two best studied sen- sory modalities in blue crab courtship. Teytaud (1971) re- ported a role for visual signals in male recognition by pre-pubertal females. However, Gleeson ( 1980) showed that males did not respond to female visual stimuli alone, and pairing could proceed in darkness. Chemical signals are important tor both male (Gleeson, 1980) and female (Teytaud, 1971; Gibbs, 1996) mate recognition. Some ma- ture males respond with a courtship display to chemical compounds in pre-pubertal female urine (Gleeson, 1980; Gleeson et al., 1984) and reception of these chemical sig- nals occurs via the aesthetasc sensilla on the lateral filament (outer flagellum) of the male antennules (Gleeson, 1982). This signaling theme appears common in crustaceans: urine carries chemical courtship signals (Ryan, 1966; Bushmann and Atema, 1997; Bamber and Naylor, 1997) and the an- tennules appear to be the site of distance chemoreception (Ache, 1975; Ameyaw-Akumfi and Hazlett, 1975; Devine and Atema, 1982; Cowan, 1991). The presence of a male chemical signal has not been firmly established, although Gleeson ( 1991 ) showed female attraction to water that con- tained males and Gibbs (1996) demonstrated disruption of pairing with male antennule ablation. In this study, the occurrence and variability of courtship behaviors observed during blue crab pair formation were examined. These behaviors were then compared with those generated by male and female pairs with vision, distance chemoreception, both senses, or urine release impaired. This allowed a determination of the relative importance of visual and chemical sensory systems during blue crab court- ship and an examination of urine- and nonurine-based chemical signals for both males and females. Materials and Methods Adult male crabs (125 mm-170 mm carapace width) were collected from the Rhode River, an upper Chesapeake Bay subestuary, with baited commercial crab traps. Premolt prepubertal females (96 mm-127 mm carapace width) were purchased from two local businesses which hold molting females for the soft crab industry. Females ranged in molt stage from late D0 to D3 (Drach. 1939). Animals were held in floating cages in the Rhode River or flow-through sea- water tanks for no more than 48 h before participation in the study. Behavioral interactions were observed in outdoor circular pools (150 cm d. X 20 cm h.) with three centimeters of washed river sand as substrate. Prior to a trial, pools were filled with 15 cm of new river water filtered through a felt bag with 10 /nm mesh. A trial began by randomly selecting a male crab and placing him into a pool. Ten minutes later, a randomly selected prepubertal female was placed into the middle of the pool, inside an opaque plastic cylinder de- signed to prevent interactions prior to the start of the trial. After 10 min acclimation, the cylinder was removed, allow- ing the animals to freely interact. Three pools were started and watched simultaneously, and the ensuing behaviors were recorded by hand for 45 min. Carapace width and molt stage were recorded for each animal. Prior to trials either a male or a female from each pair was subjected to an experimental treatment. They were as fol- lows: 1. Nephropore Occlusion: Blue crabs possess bilateral nephropores, located anteriorly and just ventral to the eye stalks. Each opening is found in a pit in the carapace. A chitinous flap opens to allow urine to exit. A modification of a successful cannulation technique was used to prevent urine release. Each pit was first dried by blotting and a drop of acetone, then filled with a viscous cyanoacrylate glue. The glue was im- mediately hardened with a catalytic accelerator. This sealed the nephropore flap shut. Animals were oc- cluded 30 min prior to a trial. The blocks were CONCURRENT SIGNALS IN BLUE CRABS 65 checked for a tight bond with the carapace immedi- ately before and after a trial, n = 12 males (M: URINE). 14 females (F:URINE). 3. Antennule Ablation: the distal lateral filament (outer flagellum), containing the aesthetasc sensilla, of both antennules was removed, n = 12 males (M: ANTENN), 12 females (FiANTENN). 4. Blindfolding: two strips of black plastic (50 X 10 mm) were fastened with cyanoacrylate glue to the dorsal and ventral carapace so that each wrapped over and covered an eye stalk, n = 13 males (M:BLIND), 12 females (F:BLIND). 5. Antennule ablation and blindfolding: animals received both antennule ablation and blindfolding treatments. n = 12 males (M: ANT-BLIND). 12 females (F: ANT- BLIND). 6. Sham treatment: both animals in a pair were subjected to sham operations. Antennules were held with for- ceps without ablation, nephropores were treated with acetone and accelerator but not glued, and blindfolds were attached similarly, but lateral to the eye stalks so that vision was not impaired, n = 10. 7. Intact: No treatments or sham operations were per- formed on either animal, n = 12. Blue crab reproductive and agonistic behaviors have been well described over the years (Churchill. 1921; Van Engel, 1958; Teytaud, 1971; Jachowski. 1974; Gleeson. 1980). This study analyzed one agonistic and five reproductive behaviors. These behaviors were common, unmistakable, and reliable indicators of the nature of the interaction oc- curring. They were: 1 . Male Strike: an agonistic behavior in which the male strikes or seizes any female body part with either chelae without subsequent attempts at cradle carry. 2. Male Displav: A courtship behavior in which the male raises high on his walking legs, spreads his chelae laterally, and raises and rotates his 5th walking legs (periopods) laterally. 3. Female Present: a courtship behavior in which the female faces away from the male and holds her body in a cradle carry posture, with or without spread chelae. 4. Female Rock: a courtship behavior in which the fe- male rocks her body from side to side. 5. Initiation of Pair Formation: the male seizes the fe- male and attempts to pull her into a cradle carry position. Females often resist, males may make many attempts, and pairing may or may not become estab- lished. 6. Stable Pair Formation: this was scored at the end of a trial. Pairs were in stable cradle carry if both female and male struggling had ceased, and the animals had been paired for at least 10 min. Comparisons of the intact and sham-treated groups showed no differences in the frequency of occurrence of any measured behavior or pairing outcome. These two groups thus appeared to represent samples of the same population and their data were pooled to yield 22 intact control trials. Behaviors of these pairs were examined to determine a normal range of behavioral variability and sequence. Be- haviors were scored once if they occurred in a given trial. The number of trials in which behaviors occurred for the intact control group was then compared with those gener- ated by the treatment groups. Overall differences between treatment and control groups were evaluated with a Chi- square test for multiple independent samples (Siegel and Castellan. 1988). Where significance was found, differences between specific treatment groups and the control were evaluated with a Fisher exact test (FAT) (Siegel and Cas- tellan, 1988). The mean times between trial start and both the first behavioral interaction and Initiation of Pair For- mation were also compared between the control and treat- ment groups. Overall differences were evaluated with anal- ysis of variance (Jaccard. 1983), while mean differences between specific treatments and the control were evaluated with a non-directional r-test (Jaccard, 1983). Results Male and female blue crabs in intact control pairs showed great variability in the occurrence of their behaviors. During courtship, no behavior occurred with a high frequency (Ta- ble I). Male Strike, Male Display, Female Present and Female Rock occurred in only 41. 41, 56, and 36 percent of intact control trials, respectively. Pairing was initiated at a high rate, however (82% of trials), with 50% of trials resulting in Stable Pair Formation. No single behavior more likely led to the initiation of pairing or stable pairing, nor did the exhibition of any behavior preclude these out- comes (Table I). There was no single sequence of behaviors Table I Fret/iiencv of coiin.\lui> uncl agonistic behaviors in intact blue crah pairs. The number of trials in which each behavior occurred is shown for all trials, those trials in which Initiation of Pair Formation occurred, and those trials in which a stable pair was fanned Trials (%) with Trials ('',', ) with Imitation of Stable Pair Occurrence in Pair Formation Formation Behaviors 22 trials (%) (n = 181 (n = 111 Male Strike 9(41) 6(33) 2(18) Male Display 9(41) 1(1(56) 3 (30) Female Present 12(56) 10(56) 4(36) Female Rock 8(36) 8(44) 3(27) Initiation of Pair Formation 18(82) — — Stable Pair Formation 11 (50) — — 66 P. J. BUSHMANN Figure 1. Flow chart showing behavioral pathways from first encoun- ter, through courtship and/or male agonistic behavior, to stable pairing success or failure. The circled numbers represent the number of trials following that particular pathway. that predominated, nor any single sequence that invariably led to greater or lesser pairing success. Neither male or female courtship behaviors were correlated with female molt stage (early premolt D0 vs. late premolt D?) or the relative sizes of males and females. However, some general trends emerge from courtship sequences examined together with male agonistic behavior (Fig. 1 ). Most pairs ( 18 of 22) exhibited some sequence of courtship behaviors prior to pair formation (x2 = 8.91, P = 0.003). The presence of male agonistic behavior signifi- cantly reduced the likelihood of stable pairing (FAT, P = 0.040). Of the nine pairs in which males exhibited Male Strike, only two (22%) formed stable pairs. Of the remain- ing 13 pairs in which males did not exhibit Male Strike, nine (69%) formed stable pairs (Fig. 1 >. Examination of male agonistic and display behaviors revealed overall differences between treatment and control groups (x2 = 20.45. P < 0.05; r = '7.62, P < 0.05). The incidence of Male Strike was significantly diminished (FAT, P = 0.009) if females were antennule ablated (F: ANTENN) (Fig. 2A). Scores for females antennule ablated and blindfolded (F:ANT-BLIND) closely approached sig- nificance (FAT, P = 0.050). Male Display was significantly reduced when males were antennule ablated (M:ANTENN) (FAT, P = 0.009) or antennule ablated and blindfolded (M: ANT-BLIND) (FAT, P = 0.049), but were unaffected by female or male nephropore occlusion (F:URINE or M: URINE) (Fig. 2B). Blindfolding alone (M:BLIND and F:BLIND) had no effect on any measured behavior. When the behaviors Female Present and Female Rock were examined, there were significant overall differences between treatment and control groups (x~ = 45.78, P < 0.05; x2 = 20.2. P < 0.05). The incidence of Female Present was reduced when females were antennule ablated (FAT. P = 0.035) or antennule ablated and blindfolded (FAT, P = 0.009) (Fig. 2C). This behavior was also reduced by male antennule ablation (FAT, P = 0.001 ). Female Rock (Fig. 2D) was reduced in incidence when females were antennule ablated and blindfolded (FAT, P = 0.009): fe- male antennule ablation alone did not significantly reduce the occurrence of this behavior (P = 0.083). Female Rock also occurred less frequently when males were antennule ablated and blindfolded (FAT, P = 0.009). Male or female nephropore occlusions or blindfolding had no significant effect on either female courtship behavior. Initiation of Pair Formation occurred frequently (80% of trials) in the intact control group (Fig. 2E). There were significant overall differences between groups in the occur- rence of this behavior (x2 = 34.8, P < 0.05). It occurred significantly less often than the control group when males were antennule ablated (FAT. P = 0.007), while the reduc- tion for antennule ablated and blindfolded males ap- proached statistical significance (P = 0.062). Examination of stable pairing at the trials' conclusions showed significant overall differences between treatment groups (x2 = 31.36, 100 , 80 60 40 20 0 100 80 60 40 20 | 0 g 100 8 80 ° 60 § 40 £ 20 X o z 1°° I 80 g 60 1 "0 £ 20 j/i 0 2 100 >- 80 60 40 20 0 100 80 60 40 20 0 Male Display lUul I- Male Strike 1 1 !..•••• Female Present I. Female Rock Initiation of Pair Formation h.lillll Stable Pair Formation Illllllll Treatments Figure 2. The percentage of trials in which Male Strike. (2A). Male Display (2B), Female Present (2C), Female Rock (2D). Initiation of Pair Formation (2E), and Stable Pair Formation (2F) occurred for the intact control and treatment groups. Differences between intact control and treatment groups were evaluated with a Fisher exact test. Stars indicate statistical significance at a = 0.05. CONCURRENT SIGNALS IN BLUE CRABS 67 S 16 i ,. O 20 o 12 Figure 3. Mean lime to first observed behavior (3A) and Initiation of Pair Formation (3B) for the intact control and treatment groups. Bars represent mean standard error. Differences between intact control and treatment groups were evaluated with a non-directional t-test. Stars indicate statistical significance at a = 0.05. P < 0.05). Fewer pairs were stable (Fig. 2F) if the males were antennule ablated (FAT, P = 0.016) or antennule ablated and blindfolded (FAT, P = 0.002). The incidence of stable pairing was also reduced when male nephropores were occluded (FAT, P = 0.016). This was the only sig- nificant effect observed with any nephropore occlusion. An examination of the mean time between a trial's start and the first observed behavior (Fig. 3 A) showed significant differences between treatment groups (ANOVA F = 2.73, p = 0.009). The mean time to first behavior was signifi- cantly less than the control group when males were blind- folded (t = 2.97, P = 0.026), when males were blindfolded and antennule ablated (t = 2.28, P = 0.032), and when females were antennule ablated (t = 3.69, P = 0.001). Overall differences were found (ANOVA F = 2.29, P = 0.030) when the time between trial start and Initiation of Pair Formation was evaluated (Fig. 3B). In this comparison only the female antennule-ablated trials showed a signifi- cant reduction in time (t = 3.90, P = 0.001). Time differ- ences between the male blindfolded group and the intact controls closely approached significance (t = 2.01, P = 0.06), while those for the male blindfolded and antennule ablated group were not significant (t = 1.46, P = 0.170). Discussion Arthropod behavior has generally been considered ste- reotyped. Studies of some insects, such as many moth species, have demonstrated stereotypic courtship behavior: specific chemical signals elicit specific and predictable re- sponses (Kaissling, 1979; Charlton and Carde, 1990). Other insect species have shown greater flexibility, with individ- uals basing their behavioral responses upon current condi- tions and context (Carlson and Copeland, 1978; Dejean, 1987). Similarly, the behavior of many crustacean species is not based upon stereotyped responses but instead shows great plasticity and can be modified as context changes (Ra'anan and Cohen, 1984; Finer and Beninger, 1995; Hazlett, 1995). The current study demonstrates such flexibility in Calli- nectes sapidus courtship behavior. Courtship is variable in that no single behavior must occur, nor does any behavior invariably lead to successful pairing. No single behavior occurred more than approximately half the time, yet the odds of successful pairing remained high. This suggests that courtship follows multiple behavioral pathways, all poten- tially leading to successful pair formation. Such flexible courtship would be useful for both males and females in a species that mates in a fluctuating estuarine environment. With intense male competition for females (Jivoff, 1997b) and only one chance for females to receive sperm, it max- imizes the chances of an encounter producing pair forma- tion, with eventual mating and reproductive success. However, blue crab mating behavior is not without con- straints and regulation. In the intact control group most pairs displayed some courtship behaviors prior to pair formation, and male agonistic behavior reduced the likelihood of stable pairing. This demonstrates the importance of controlling male aggression during courtship and, together with the treatment trials, illustrates the role that communication sig- nals often serve in this regard (Tinbergen, 1953). For blue crabs, the most likely path to successful pairing, and there- fore successful reproduction, involves courtship and re- duced male aggression. The treatment trials suggest behavioral regulation through chemical communication signals and that both fe- male and male chemical signals play important roles in courtship and pairing. Males with ablated antennules showed reduced instances of Male Display, Initiation of Pair Formation and Stable Pair Formation. For the male, loss of distance chemoreception affected behavioral expres- sion and directly reduced courtship success. The relevant chemical information did not seem to reside solely in female urine, however, because females with occluded nephropores induced male behaviors at frequencies similar to intact controls. Although the results were less clear, females also appeared to exhibit fewer instances of courtship behaviors when their antennules were ablated, while pairing initiation or stability was unaffected. The physical act of pairing is initiated by the male, and evidently an antennule-ablated female is still attractive to males. However, an unreceptive female can likely flee and decline pairing in the wild. Blocking male urine release had no effect on female court- ship behaviors, again suggesting that the relevant chemical compounds are not restricted to urine. 68 P. J. BUSHMANN It is now generally recognized that many chemical signals are mixtures or blends and thus can serve as multiple or redundant signals (van den Hurk and Lambert. 1983; Vetter and Baker, 1983; Linn < t al. 1984). In blue crabs and other brachyurans, a chemical signal in female urine that induces male courtship behavior has been well described (Ryan. 1966; Gleeson. 1980; Seifert. 1982; Bamber and Naylor, 1997). The present study does not refute the existence of this signal, but rather suggests urine is only one source of courtship signals and is not obligatory for the initiation of male or female courtship behaviors. There appears to be chemical information from non-urine sources capable of eliciting the same behaviors when nephropores are oc- cluded. It is only when all chemical signals are lost through antennule ablation that behavior is negatively affected. These statements appear at odds with Ryan's (1966) work showing no male responses to seawater that had contained nephropore-blocked premolt Portiimis sanguinolentus fe- males. It may be that the relevant female P. sanguinolentus signal is sent only in urine. In addition, the females in Ryan's study were isolated in 8-1 buckets during signal release, while females in the current study were placed in larger tanks in the presence of a male. This more naturalistic behavioral context may have elicited female nonurine signal release and male responses not seen in the earlier study. Lastly. Ryan used molten paraffin rather than glue as blocks; this may have affected the animals differently from the blocks used here. These apparent interspecific differ- ences in behaviors and signals should be more closely examined. Blue crab courtship thus appears regulated by female and male concurrent chemical signals emanating from multiple sources. It is unknown if the concurrent signals demon- strated here are different compounds or if they are the same compound released at different sites. This knowledge awaits the purification and structural description of these chemical courtship signals. The release sites of the non-urine chem- ical compounds are likewise unknown. In lobsters (Hoimi- nis (imericanus), the gill current has been implicated as a method for transporting chemical signals to a receiver (Atema, 1985). Because blue crabs possess a similar cur- rent, it is possible that the gills themselves or structures within the gill cavity are sources of chemical signals. Teg- umental glands, found in blue crabs and other arthropods (Johnson, 1980; Talbot and Demers, 1993) have been sug- gested as chemical signal sources in several crustacean species (Berry, 1970; Kamiguchi, 1972; Bushmann and Atema, 1996) and also may play a role here. Loss of chemical signals in some instances had indirect effects on behavior. Males were less aggressive toward antennule-ablated females. Ablation evidently alters either female behavior or her signaling patterns in a way that affects male agonistic behavior. Similarly, female courtship behaviors were reduced when male chemical reception was impaired. Male antennule ablations must alter male behav- iors or communication signals in a way that makes them less attractive to females and less capable of inducing female courtship behavior. This is consistent with field work (Gibbs, 1996) demonstrating that antennule-ablated males in crab traps are less able to attract prepubertal females. There is evidence for an obligatory male urine-based signal involved in pair maintenance during precopulatory guarding. When male nephropores were occluded, initiation of pair formation was not affected yet there was reduced incidence of stable pairing. This was the only evidence for a urine-based signal in this study. However, female anten- nule ablation did not reduce the incidence of stable pair formation. It is possible that the direct contact involved in a cradle carry produces other avenues for signal reception, such as contact chemoreceptors on the dactyls or elsewhere on the exoskeleton (Fuzessery and Childress, 1975). Al- though the observed reduction in stable pairing could have resulted from some male trauma associated with the occlu- sion procedure, occluding females produces no such pattern and blue crabs and lobsters appear capable of suspending urine release for periods of several hours without ill effect (Bushmann. unpub. data, Breithaupt and Atema, 1993). Visual signals seem to play no role in influencing court- ship behaviors or outcomes. Blindfolded males and females courted, received courtship, and paired with success rates equal to the intact controls. This is consistent with previous observations for blue crabs and lobsters that visual signals are of secondary importance during social interactions (Gleeson, 1980; Snyder et al.. 1993: Kaplan et al.. 1993). Thus, the primary function of the male courtship display is likely not transmission of a visual signal. However, it may be an excellent method for transmitting both chemical and hydrodynamic signals to a potential partner. Rotation of the periopods causes a strong and highly turbulent flow of water directed forward of the animal (Gleeson, 1991; Bushmann, unpub. data). This flow would likely entrain any chemical signal emanating from the gills or nephropores. In addition, some crustaceans use hydrodynamic information during ag- onistic interactions and prey capture (Barron and Hazlett, 1989; Breithaupt ct al., 1995). The highly turbulent, di- rected flow generated by male paddle waving could provide directional or other information to females. Many aspects of the male courtship display remain un- clear. It must have some energetic cost and may draw attention by predators, yet it need not occur for successful pairing and occurred in less than half the observed encoun- ters. In this study its occurrence was not correlated with female premolt stage, the relative sizes of males and fe- males, or pairing success during the encounter. The function of this rather spectacular behavior and the stimuli leading to its initiation require further investigation. Loss of female chemoreception appeared to accelerate rather than retard pairing. When females were antennule- CONCURRENT SIGNALS IN BLUE CRABS 69 ablated, males showed little agonistic behavior, females exhibited fewer courtship behaviors, and pairs formed more quickly than in the intact control group (Fig. 3B) and they remained stable. This is at odds with Gibbs (1996), who found males to be more aggressive toward antennule- ablated females and the time required for pairing to be unaffected. The present study suggests that females use chemical information and courtship behaviors to lengthen courtship periods, perhaps as a way of better evaluating potential partners. Loss of chemical information through female antennule ablation would then result in less female evaluation and faster pairing. The significant reduction in time until first behavior seen in the male blindfolded group was probably a general be- havioral rather than specific communication effect. Blind- folded males, without visual stimuli, may have been less wary and more likely to begin moving about the pool after trial start. This male movement would result in more rapid encounters with females. The time until Initiation of Pair Formation was not significantly shortened, however (Fig. 3B), and blindfolding had no effect on any measured be- havior. Several studies have shown that lateral antennule ablation affects behavior by interfering with chemical reception (Ache, 1975; Ameyaw-Akumfi and Hazlett, 1975; Gleeson, 1980; Cowan, 1991). However, in any ablation experiment there is always a question of false-negative responses due to a general dampening of behavior caused by the procedure itself (Dunham, 1978). In the present study, while ablated males showed reduced reproductive behaviors, agonistic responses were unaltered. Antennule-ablated females, while not exhibiting many courtship behaviors, were nonetheless courted and carried by males. These ablations appeared to affect certain reproductive behaviors, presumably those de- pendent upon chemical signals, rather than causing a gen- eral reduction in behavioral responses. A second potential problem concerns the blocks applied to the nephropores to prevent urine release. Correct inter- pretation of results depends upon an effective block. Several lines of evidence suggest that these blocks prevented urine release. First, they are the initial step in the attachment of a urine cannula. This cannula can collect urine from blue crabs for several days without leaking (Bushmann, unpub. data). Second, three urine blocked animals were held after their trials. These individuals were swollen from fluid re- tention within 6 h and died within 12 h. Lastly, the water from four blocked animals held individually in 2-1 tanks showed reduced ammonia levels compared to water from four unblocked crabs (Bushmann, unpub. data). Ammonia levels from blocked crab water were not zero, because ammonia is also excreted across the gills (Mantel and Farmer, 1983). Taken together, these observations suggest that the blocks used in this experiment were effective in preventing urine release. In summary, Callincctes sapidus courtship illustrates both behavioral plasticity and the importance of behavioral regulation through a signaling system. The concurrent and seemingly redundant chemical signals discussed here may be different compounds or the same compound released from different sites. Chemical rather than visual signals from both male and female seem to play crucial roles in courtship and pairing. 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Ecol. 18(2): 2277-2284. West-Eberhard, M. J. 1989. Phenotypic plasticity and the origins of diversity. Annu. Rev. Ecol. Syst. 20: 249-278. Reference: BinL Bull 197: 72-81. (August 1999) Translocation of Photosynthetic Carbon From Two Algal Symbionts to the Sea Anemone Anthopleura elegantissima HILARY P. ENGEBRETSON AND GISELE MULLER-PARKER* Department of Biology and Shannon Point Marine Center, Western Washington University, Bellingham, Washington 98225-9160 Abstract. The intertidal sea anemone Anthopleura el- egantissima contains two symbiotic algae, zoochlorellae and zooxanthellae, in the Northern Puget Sound region. Possible nutritional advantages to hosting one algal symbi- ont over the other were explored by comparing the photo- synthetic and carbon translocation rates of both symbionts under different environmental conditions. Each alga trans- located 30% of photosynthetically fixed carbon in freshly collected anemones, although zoochlorellae fixed and trans- located less carbon than zooxanthellae. The total amount of carbon translocated to the host was equivalent because densities of zoochlorellae were two to three times greater than were densities of zooxanthellae. In A. elegantissima maintained under high and low irradiance ( 100 and 10 /xmol photons/rrr/s) at 20°C and 13°C for 21 days, both algae fixed and translocated carbon at greater rates at 20°C (trans- location rates: 0.38 pg C /zoochlorella/h; 1.12 pg C /zoo- xanthella/h) than at 13°C (translocation rates: 0.06 pg C /zoochlorella/h; 0.37 pg C /zooxanthella/h). However, zoochlorellate anemones received 3.5 times less carbon at 20°C than at 13°C because the higher temperature caused a significant reduction in the density of zoochlorellae. Envi- ronmental variables, like temperature, that influence the densities of the two symbionts will affect their relative nutritional contribution to the host. Whether these differ- ences in carbon translocation rates of the two algal symbi- onts affect the ecology of their anemone host awaits further investigation. Received 12 January 1998; accepted 3 June 1999. * To whom correspondence should be addressed. E-mail: gisele® biol.wwu.edu Introduction The temperate sea anemones Anthopleura elegantissima and Anthopleura xanthogrammica host both dinoflagellate zooxanthellae and green algae known only generally as zoochlorellae (Muscatine, 1971). Both algal symbionts pho- tosynthetically fix inorganic carbon and translocate some of the products to the animal host. Zooxanthellae in corals, as well as in A. elegantissima, translocate carbon to the host mainly as glycerol (Muscatine, 1967; Trench, 1971; Battey and Patton, 1987). Glycerol is used by the host to support its basal metabolism, while lipids that are also translocated by the algae are used to create lipid stores (Battey and Patton, 1987). We do not know what products are translocated by marine zoochlorellae to their host, although unpublished work by Minnick and McCloskey (cited in Verde and Mc- Closkey, 1996) indicates that zoochlorellae translocate sev- eral amino acids in addition to glycerol. For zoochlorellae in the freshwater green hydra, maltose is the principal form of translocated photosynthate (Mews and Smith, 1982). Further understanding of the nutritional relationship be- tween Anthopleura and the two algae may come from comparisons of the amount of carbon translocated from the algae to the host. Previous studies have suggested that zoochlorellae do not translocate as much carbon as zoo- xanthellae. Using I4C, O'Brien (1980) found that zoochlo- rellae in excised tentacles translocate from zero to 3.6% of the total carbon fixed by the algae to the epidermal tissues of Anthopleura xanthogrammica. Zooxanthellae in intact anemones translocate as much as 50% of the total I4C- labelled carbon fixed to the host fraction of A. elegantissima (Trench, 1971). Based on carbon budgets, Verde and Mc- Closkey (1996) calculate that zooxanthellae will have pho- tosynthetic products available to supply A. elegantissima 72 CARBON TRANSLOCATION IN ANEMONES 73 with 48% of its respiratory carbon requirement, while zoo- chlorellae will only he able to satisfy 9% of the anemone's respiratory needs. Verde and McCloskey conclude that the higher net photosynthesis and lower algal growth demand of zooxanthellae combine to provide more photosynthetic car- bon to a zooxanthellate host anemone than is the case for an anemone that contains zoochlorellae as its endosymbiont. These studies show that zooxanthellae appear to be the "better" symbiont with respect to carbon supplied to the host. It is important to directly compare carbon translocation rates of zoochlorellae and zooxanthellae under different temperatures and irradiance levels, because intertidal A. elegantissima are exposed to extreme seasonal fluctuations in these parameters (Dingman. 1998). Furthermore, both irradiance and temperature are thought to influence the distribution of these two algae within anemones. Field ob- servations of the distribution of Anthopleura xanthogram- mica in British Columbia, Canada, by O'Brien and Wytten- bach (1980) led the authors to suggest that zooxanthellae and zoochlorellae populations in anemones may be regu- lated by temperature. In the lower latitude, warmer regions of Anthopleura' s range zooxanthellae are the dominant symbiont, while zoochlorellae are more abundant in anem- ones in the higher latitude, colder regions of Anthopleura 's range (Secord, 1995). Are these distribution patterns related to differences in carbon translocation of the two algae? Saunders and Muller-Parker (1997) determined that in- creased temperature caused a reduction in the density of zoochlorellae in Anthopleura elegantissima tentacles over time. How do such changes in algal density affect the rate of carbon translocation to the host? This study compares carbon fixation and translocation rates of both zoochlorellate and zooxanthellate anemones collected from a single site and kept under different envi- ronmental conditions likely to be encountered in the field. The effects of irradiance and temperature on translocation of fixed carbon from zooxanthellae and zoochlorellae to A. elegantissima are examined by measuring the distribution of radioactively labelled carbon in the algae and in the animal host, and relating the carbon translocation rates to popula- tion densities of the respective algae. Materials and Methods Collection of anemones and determination of symbiont complement Anthopleura elegantissima was collected from a rocky intertidal area located on Anaco Beach, Fidalgo Island, Washington (48° 29'; 122° 42') in June and July of 1994. Ambient seawater temperature was 11°C. Both zooxanthel- late and zoochlorellate anemones were collected from the same large boulder, at one tidal height (+0.6 m). Nonsym- biotic (algae-free) anemones were collected from dark crev- ices in a nearby rock jetty. The anemones were placed in flow-through ambient seawater tables at Shannon Point Marine Center for one day before experiments began. The anemones were separated by color and excised ten- tacles from several anemones were examined microscopi- cally to verify that anemones that appeared brown in the field actually contained zooxanthellae, that green anemones contained zoochlorellae, and that white anemones were algae-free. The symbiont complement of all anemones was con- firmed by counting the number of zoochlorellae and zoo- xanthellae in homogenized anemone samples after 14C in- cubation. Zoochlorellate anemones from the field contained an average of 99.0% (±2.0 SD, ;; == 18) zoochlorellae, while zooxanthellate anemones contained an average of 97.3% (±3.2 SD. ;; = 18) zooxanthellae. Three field anem- ones that contained mixed populations of both symbionts contained from 40% to 60% of each alga (average = 53% zoochlorellae) within their tissues. Experimental treatments: symbiont. light, and temperature To examine the effects of irradiance and temperature on zooxanthellate and zoochlorellate anemones, a 2 X 2 X 2 factorial experiment was designed with factors of anemone symbiont type, irradiance level, and temperature. Two ex- periments were run sequentially in one incubator. For each, 28 anemones, consisting of 14 zoochlorellate anemones and 14 zooxanthellate anemones, were placed in individual 50-ml beakers containing 35 ml of 5 /xm-filtered seawater. For the first experiment the anemones were incubated at 20°C; for the second experiment the anemones were incu- bated at 13°C. The beakers containing the anemones were arranged randomly within the incubator under a bank of fluorescent lights providing a mean irradiance of 100 /j,mol photons/nr/s. For each experiment, half of each group of anemones was covered with mesh for the low irradiance treatment (10% of full irradiance; see Saunders and Muller- Parker. 1997, for details). The lights were set to a natural daylength cycle of 14 h:10 h (lighf.dark). The anemones were fed every three days with freshly hatched Anemia nauplii and were last fed two days prior to 14C incubation. The anemones were maintained under the experimental conditions for 21 days prior to measuring carbon fixation and translocation rates. Carbon fixation and translocation The amount of carbon photosynthetically fixed by the algal symbionts and translocated to the anemone host was measured using the I4C method (O'Brien, 1980; Battey and Patton, 1987), with some modifications. One hour prior to the I4C incubation period each anemone was transferred to an individual clear plastic vial (Nunc* tube). Exactly 10 ml 74 H P. ENGEBRETSON AND G. MULLER-PARKER of 5 /xm-filtered seawater was added to each vial and the anemones were returned to their treatment conditions. The I4C incubations were always begun at the same time of day (0900 h) to minimize variation due to any factors associated with the natural photoperiod of the anemone. The addition of 14C-bicarbonate to each vial was noted as time zero. After thorough mixing, 100 /j,l of the seawater was subsampled to determine the total activity of the seawater in the vial, which ranged from 13.6 to 21.3 juCi/anemone. Anemones in vials that were covered completely with foil to exclude light served as controls for each experiment. These controls were used to account for dark fixation of I4C by the algae and/or the animal under each set of conditions. Sep- arate controls were run for zoochlorellate and for zooxan- thellate anemones. All anemones were incubated with 14C for 1 .5 h under the appropriate temperature and irradiance conditions they had experienced for 21 days. After incuba- tion, the anemones were rinsed thoroughly with non-la- belled seawater, making sure that seawater retained in the coelenteron was also expelled. The seawater in the vials was replaced, and all of the vials were covered completely with foil. The vials were then returned to the appropriate incu- bation conditions for the dark chase period, which was 1.75 h for most experiments. Following the dark chase period, the anemones were rinsed again and individually homogenized in seawater with a motor-driven teflon tissue grinder (60 ml volume). Homogenate volume (= anemone) was measured and 1 ml of the homogenate was frozen for later protein analysis. A 0.5 ml sample of the homogenate was transferred to a 7-ml plastic scintillation vial and acid- ified with 0.3 ml 6 N HCI under a heat lamp in a fume hood to remove unincorporated inorganic I4C label. Assay of homogenate was used to determine the amount of I4C fixed by the whole anemone. The algae were separated from the host fraction to mea- sure the distribution of I4C in both fractions. Ten ml of the homogenate was centrifuged in a table top swinging bucket centrifuge for 10 min. The algal pellet was rinsed two times and the final algal pellet was resuspended in 5 ml of filtered seawater. The combined supernatant was the animal frac- tion of the homogenate and the resuspended pellet was the algal fraction. The final animal fraction volume was mea- sured and 1-nil samples of the animal and algal fraction were frozen for later analysis. Half-milliliter (0.5-ml) sam- ples of each fraction were acidified with 0.3 ml 6 N HCI, as described above. The acidified homogenate, animal, and algal samples in the scintillation vials were then neutralized with 0.3 ml 6 /V NaOH, 5 ml of Ecolume scintillation fluid was added, and disintegrations per minute (DPM) of each sample counted in a Packard TriCarb 1900TR liquid scin- tillation counter. To compare trunslocution of 14C by freshly collected field anemones to the anemones in the experimental treatments, anemones gathered from the field were subjected to I4C analysis the day after collection. These anemones were kept under a light bank of fluorescent lamps at a photosyntheti- cally saturating irradiance of 309 ^tmol photons/nr/s in a flow-through ambient seawater table ( 1 1°C) until I4C anal- ysis. Bioniciss parameters The protein content of the homogenate and animal frac- tions of each anemone was determined by the method of Lowry (Lowry el al., 1951). using bovine serum albumin (BSA) as a standard. Two replicates of both homogenate and animal fractions from each anemone were analyzed on a Hitachi 100-40 spectrophotometer. To ascertain the algal biomass and proportion of zoochlorellae and zooxanthellae in each anemone, cell counts were done on the frozen algal fractions. The number of each alga (zoochlorellae and zoo- xanthellae) in each sample was counted using a hemacy- tometer viewed under a compound microscope. Six repli- cate counts of algal numbers were done for each sample. The mean of the replicate counts was normalized to weight of anemone homogenate protein to provide an estimate of algal density in each anemone. Percent carbon translocation The percent of fixed I4C translocated to the host during the 1.75-h dark chase time was determined by dividing the DPM calculated for the whole animal fraction by DPM in the whole homogenate fraction. Any dark carbon fixation by the algae and host was accounted for by subtracting the mean DPM per nig protein of the dark control fractions for the appropriate symbiont type from the DPM per mg protein of each experimental anemone fraction (homogenate or animal) before calculating the percent translocation. For all symbiotic anemones, dark fixation accounted for less than 10% of the total carbon fixed by anemones in the light. For the nonsymbiotic anemones, dark fixation accounted for 86% of the total carbon fixed. Because the data were in the form of percentages, they were arcsine transformed for statistical analysis. Rates of carbon fixation anil translocation Although the percent of fixed carbon translocated to the host is important, it does not indicate the actual rate of carbon received by the anemone under different environ- mental conditions. For that information, the rates of carbon fixation and translocation must be examined. The specific activity of I4C in the seawater was used to calculate the actual amount of carbon fixed and translocated. The weight of carbon dioxide (all forms) present in the seawater was determined by the alkalinity method described in Parsons et al. ( 1984). The weight of the total inorganic carbon present in the seawater was then multiplied by the rate of uptake (or CARBON TRANSLOCATION IN ANEMONES 75 100 - c 80 - S | f 60 - ro O ! "0 -I c °- 20 - — I — 10 15 — 1 — 20 I 25 Time (h) Figure 1. The effects of symbiont type and dark chase period on the percent of carbon translocated to the host anemone, n = 2 for each group; ± 1 SD of the mean. translocation) of the labelled carbon in the sample, as de- termined by dividing DPM in the homogenate (or animal) fraction sample (corrected for DPM in the dark control) by the total activity (DPM) of the I4C added and the hours of incubation with I4C. The result is the rate of carbon fixation (or translocation), as amount of C fixed (or translocated) per hour. Carbon fixation and translocation rates can be expressed on the basis of both anemone biomass (protein) and on the basis of an individual algal cell. Comparison of rates nor- malized to these two parameters shows how algal density affects photosynthesis and translocation. The rate of carbon fixed by anemones was calculated by using the homogenate fractions in the above calculation and normalizing to either anemone protein biomass or to number of algae. The rate of carbon translocated to the animal was calculated by using the animal fractions in the above calculation. All analyses of variance and multiple range test statistics were examined with a significance level of 5%. Statistics were calculated using Statistix 4. 1 by Analytical Software. Results Percent C translocation over time A I4C pulse-chase time course experiment was conducted with field anemones to determine if and how the length of the dark chase time affected the percent of carbon translo- cated to the host by the two symbionts. A 2 X 6 factorial analysis of variance showed that symbiont type had a sig- nificant effect on percent translocation (P < 0.000). Over the entire chase time period, the percent of fixed carbon translocated to the host by zooxanthellae is significantly higher than the percent of fixed carbon translocated by zoochlorellae (Fig. 1). The length of the chase time period also significantly affected the percent of carbon translocated to the host anemone (P = 0.031). but there was no inter- action between symbiont type and chase period. Tukey's (HSD) multiple range test indicated that only chase time periods of 10.2 h and 22 h are significantly different from each other. To permit direct comparison of the effects of external factors (temperature and irradiance) on percent translocation. we used a short dark chase period ( 1.75 h) to compare C translocation of zoochlorellae and zooxanthellae in all subsequent experiments. Percent transl There was no significant difference in the percent of carbon translocated from the algae to the animal in zoo- chlorellate, zooxanthellate. and mixed anemones collected from the field and incubated under saturating irradiance and at ambient seawater temperature (comparison by ANOVA). Percent carbon translocated averaged 30% for all field anemones under these conditions (Fig. 2). The percent C translocated was higher for anemones maintained under the experimental treatments than for field anemones, and zoochlorellae translocated a greater percent of carbon (up to 65%; Fig. 2). Both temperature and sym- biont type are significant main effects on percent transloca- tion. Both symbionts translocated greater percentages of fixed carbon at 20°C than at 13°C (2X2X2 factorial analysis, P = 0.013). Additionally, zoochlorellae translo- cated a higher percent of fixed carbon than zooxanthellae (P = 0.036) at both temperatures. Irradiance was not a significant main effect on the percent of carbon translocated to the host (P = 0.437). No interaction effects were signif- icant. Although these results show that hosting zoochlorel- Field Zoochlorellate Zooxanthellate 100 n 80 - 60 - 40 - 20 - T I I 1 vv V A A Figure 2. Percent of carbon translocated to the anemone host after a 1.75 h dark chase period. Field anemones were incubated at 11°C and a light intensity of 309 jixmol photons/nr/s (for Zoochlorellate anemones. n = 4; lor zooxanthellate and mixed anemones, n = 2). Experimental Zoochlorellate and zooxanthellate anemones were incubated under their treatment conditions: high light (HL. 100 /j,mol/nr/s ) or low light (LL, 10 /j,mol/nr/s) at either 13 or 20"C (20 or 13). n = 5 for each group; ± 1 SD of the mean. 76 H. P. ENGEBRETSON AND G. MULLER-PARKER Zoochlorellate Zooxanthellate u.io - 'c AA f 0.10 - CL T O) "S 0.05 - 1 O 01 =3 n nn - r1] r*-\ -! B Figure 3. The rate of carbon fixation by zoochlorellate (D) and zoo- xanthellate (•) anemones incubated under their treatment conditions: high light (HL. KM) jamol/rrr/s) or low light (LL, 10 /xmol/nr/s) at either 13 or 20 C (20 or 13). it = 5 for each group; ± 1 SD of the mean. A. The rate of carbon fixation per mg anemone protein. B. The rate of carbon fixation per algal cell. lae at higher temperatures results in a greater percent of fixed carbon to the anemone, carbon translocation rates are needed to compare the actual amounts of carbon received by zoochlorellate and zooxanthellate anemones under field and experimental conditions. Rates of carbon fixation and translocation The rate of carbon fixation by zoochlorellate and zoo- xanthellate anemones maintained under high and low irra- diance at 13°C and 20°C for 21 days was significantly affected by an interaction between temperature and symhi- ont type (P = 0.009). While zooxanthellate anemones fixed carbon at the same rate at both temperatures, zoochlorellate anemones fixed about three times more carbon at 13°C than at 20"C for rates expressed on the basis of anemone biomass (Fig. 3a). Carbon fixation and translocation rates expressed on an algal cell basis are needed to compare these processes at the level of the individual algal cell with that of the symbiotic association. When the rate of carbon fixation is normalized to algal numbers instead of to anemone protein biomass, none of the interaction effects were significant and both algae fixed carbon at a lower rate at 13°C than at 20°C (2.3 times less and 3 times less, respectively; P = 0.004: Fig. 3b). The rate of carbon fixation per algal cell is signif- icantly greater under high irradiance than under low irradi- ance (P = 0.045), and at both temperatures the zoo.xanthel- lae fixed carbon at a significantly greater rate than did the zoochlorellae (P = 0.000). As shown in Figure 4a for carbon fixation rates normal- ized to anemone biomass, (he rate of carbon translocated to the host anemone is significantly affected by an interaction between temperature and symbiont type (P = 0.009). While zooxanthellate anemones experienced similar rates of carbon translocation at both temperatures, rates of translo- cation in zoochlorellate anemones were almost 3.5 times less at 20°C than at I3°C (Fig. 4a). At 13°C. rates of translocation are comparable for both zoochlorellate and zooxanthellate anemones, and these rates were higher at the high irradiance level at both temperatures (Fig. 4a). When carbon translocation rates are normalized to algal cell num- ber, a significant interaction between temperature and sym- biont type is again observed (P = 0.039; Fig. 4b). In this case, the rate of carbon translocation was also greater per Zoochlorellate Zooxanthellate 0 08 -, I o.oe H I "S ° °4 - i , — 1__ i o £ 0.02 - ro O _ n nn n 4 -i |3H 2> ro I 2 S ^2 1 1 O S 0 B Figure 4. The rate of carbon translocation by zoochlorellate (D) and zooxanthellate (•) anemones incubated under their treatment conditions: high light (HL, 100 /nmol/nr/s) or low light (LL. 10 /^mol/nr/s) at either 13 or 20"C (20 or 13). ;i = 5 for each group; ± I SD of the mean. A. The rate of carbon translocation per mg anemone protein. B. The rate ot carbon translocation per algal cell. CARBON TRANSLOCATION IN ANEMONES Table I Rales of carbon fixation and iranslocation by algae in zoochlorellate. zooxanthellate and mixed field anemones collected during summer, normalized to anemone protein biomass or to alga 77 ANEMONE TYPE CARBON FIXED CARBON TRANSLOCATED fj.g C fixed/mg protein/h pg C fixed/ alga/h /^g C translocated/mg protein/h pg C translocated/ alga/h Zoochlorellate Zooxanthellate Mixed Results of 1-way ANOVA 0.110 ± 0.03 0.145 ± 0.06 0.199 ± 0.02 NS 0.275 ±0.14 1.236 ± 1.13 0.684 ± 0.08 NS 0.034 ± 0.007" 0.038 ± 0.004" 0.065 ± 0.0 12b P = 0.014 0.091 ± .06 0.390 ± 0.042 0.221 ± 0.01 NS For zoochlorellate anemones, n = 4; for zooxanthellate and mixed anemones, n = 2. NS denotes the parameters (column headings) that are not significantly different among the three anemone types. Tukey's HSD Multiple Range Test indicated that both zoochlorellate and zooxanthellate anemones experienced similar rates of translocation per mg protein, while mixed anemones experienced a significantly greater rate of translocation per mg protein (a and b are used to indicate these differences among anemone types). zooxanthella than per zoochlorella at both 13°C and 20°C; however, while zooxanthellae translocated approximately 2.5 times less carbon at 13°C as at 20°C, zoochlorellae translocated almost 4 times less carbon at 13°C as at 20°C (comparisons between temperatures use pooled rates from both irradiance levels, because irradiance did not affect the rate of carbon translocation per algal cell). Although our sample size for field anemones is small, data obtained from these anemones provide a valuable com- parison to treatment anemones. When mixed anemones are included in the comparison of carbon fixation and translo- cation rates of field anemones, the carbon fixation rates of zoochlorellate, zooxanthellate, and mixed field anemones are not significantly different from each other, whether expressed on the basis of anemone protein biomass or algal cell (Table I). Although algal cell-based translocation is not significantly different, the rate of carbon translocation per mg protein in A. elegantissima is significantly affected by symbiont type (Table I). However, Tukey's HSD Multiple Range Test indicated that both zoochlorellate and zooxan- thellate anemones experienced similar rates of translocation per mg protein, while mixed anemones experienced a sig- nificantly greater rate of translocation per mg protein. Algal density in anemones Zoochlorellate field anemones contained significantly higher algal densities than did zooxanthellate field anemo- nes (Fig. 5; P = 0.000). Mixed anemones had algal densities between those of zooxanthellate and zoochlorellate anemo- nes; the density of algae in mixed anemones was not sig- nificantly different from the density of algae in either zoo- xanthellate or zoochlorellate anemones. A two-way ANOVA performed on the algal density within the anemones after 21 days under the experimental treatments showed that the interaction between temperature and symbiont type was significant (P = 0.001). All anem- ones held at 20°C contained similar densities of algae; however, at 13°C zooxanthellate anemones had signifi- cantly fewer algae per mg anemone protein than did zoochlorellate anemones (Fig. 5). Anemones held in the laboratory under all experimental treatments contained sig- nificantly fewer algae than did anemones freshly collected from the field (P = 0.000). Discussion Percent translocation and translocation rates In the field, zoochlorellate and zooxanthellate anemones receive the same amount of photosynthetic carbon from their symbionts during the summer in northern Puget Sound (Fig. 2, Table I). These results suggest that during summer 50 - C. 40 - Field Zoochlorellate Zooxanthellate 'o o. ID C o 30 - 20 - en 1 Figure 5. Density of algae in field anemones (n = 20, 17, and 3 for zoochlorellate. zooxanthellate, and mixed anemones respectively) and in zoochlorellate (D) and zooxanthellate • anemones after 21 days under high light (HL, 100 jumol/nr/s) or low light (LL. 10 /j.mol/nr/s) at either 13 or 20°C (20 or 13). n = 7 for each group; ± 1 SD of the mean. 78 H. P. ENGEBRETSON AND G. MULLER-PARKER there is no selective advantage, with respect to carbon, of hosting one symbiont over the other under saturating irra- diance levels and ambient temperature. However, under different environmental conditions imposed in a laboratory experiment, zoochlorellae translocated a greater percent of fixed carbon to the host than did zooxanthellae, and both algal symbionts translocated a significantly greater percent of the carbon they fixed at 20°C than at 13°C (Fig. 2). The implications of these results are discussed below. In our study, zoochlorellae translocated a much greater percent of the fixed carbon than shown by the previous studies of Muscatine ( 197 1 ), O'Brien (1980), and Verde and McCloskey (1996). However, the percent carbon translo- cated by both algae in A. elegantissima is comparable to values obtained for other temperate cnidarian symbioses (Sutton and Hoegh-Guldberg, 1990; Davy et at., 1997). Muscatine (1971), using I4C analysis, determined that zoo- chlorellae translocate only 1 .0% to 3.6% of the carbon they fix. However, Muscatine used only the tentacles and not whole anemones in his experiments; in addition, for some experiments the animal and algal fractions from tentacles were homogenized and separated before incubation with I4C. O'Brien (1980) found that zoochlorellae translocated 1.3% to 3.9% of the carbon they fixed. O'Brien also used only tentacles of A. xanthogrammica. He dissected the epidermis of the anemone from the algae-containing gastro- dermis after 14C incubation and used the epidermis as the animal fraction and the gastrodermis as the algal fraction for translocation calculations. Any labelled carbon that the al- gae had translocated to the gastrodermal tissues of the host was counted as fixed carbon retained by the algal fraction. In addition, any host mechanisms acting upon translocation would be lost due to the excision of the tentacle from the remainder of the anemone body. The 14C method employed in this study accounts only for short-term carbon products fixed and released by the algae from inorganic carbon supplied in the external environment. There is substantial evidence for zooxanthellae that recently fixed carbon is released to the host (Sutton and Hoegh- Guldberg, 1990; Wang and Douglas, 1997). In contrast, translocation of carbon based on the growth-rate method takes into account the daily carbon budget of the symbiotic algae (Muscatine et al, 1984). Because carbon required for algal growth may be supplied from the host animal (Trench, 1979), any contribution of host-derived carbon is wholly missed by the I4C method as applied here. This may explain the discrepancy between our results and those of Verde and McCloskey (1996), who found that zoochlorellae may have only minimal excess carbon available to translocate to the host. The algae may selectively translocate photosyntheti- cally fixed carbon while concurrently obtaining carbon for growth from the anemone host. This comparison also illus- trates the importance of defining the time scales used to assess carbon translocation. Zoochlorellate and zooxanthel- late anemones receive the same amount of translocated carbon during short-term (hours) I4C incubations (our re- sults), while growth rate comparisons based on longer time intervals (days to weeks) show that zoochlorellae translo- cate less carbon (Verde and McCloskey, 1996). The appro- priate time scale for comparisons of these two algae will depend on the metabolic fate of the translocated carbon and on the external supply of carbon derived from host feeding. Higher carbon fixation rates by both algae at the high irradiance level at both temperatures also resulted in greater carbon translocation rates (Figs. 3, 4). It appears that the symbiotic algae simply translocate fixed carbon at a higher rate under high irradiance because they have more photo- synthetic product available. These results indicate that, with similar algal densities, anemones located in areas exposed to high solar irradiance should receive larger amounts of fixed carbon from their symbionts than should anemones located in areas of low light. The same is true for temperature. Both zoochlorellae and zooxanthellae fixed and translocated car- bon at greater rates at 20°C. However, the advantage of greater carbon translocation at the higher temperature and irradiance level on an algal cell basis is offset by lower algal densities under these conditions, reducing the amount of carbon received by the anemone (see below). Algal density and carbon translocation in anemones Zoochlorellate anemones from the field contained ap- proximately two to three times the density of algae as did zooxanthellate anemones (Fig. 5), as has been found by others (Verde and McCloskey, 1996; Dingman, 1998). Thus, although an individual zoochlorella translocates car- bon to the host anemone at a lesser rate than does a zoo- xanthella (Table I; Fig. 4b), both anemone types receive fixed carbon at similar rates because of increased densities of zoochlorellae in field anemones (Fig. 5). Interestingly, although the zoochlorellae are numerically more abundant, volume comparisons indicate that they occupy the same "space" as the larger zooxanthellae within the anemones (unpub. data). Therefore, both anemone types in the field maintain similar ratios of algal to animal biomass and receive similar amounts of photosynthate. Anemones in all experimental treatments contained sig- nificantly fewer algae than did field anemones, and both types of anemone had lower algal densities at the higher temperature (Fig. 5). This may be related to differences in summer field conditions and laboratory incubator condi- tions. Although anemones were maintained at relatively low constant irradiances in the lab (an order of magnitude lower than noon irradiance levels in the field), they probably received more light on a daily basis than field anemones because of tide-related changes in water depth and rapid light extinction due to high plankton levels in summer. Field anemones also experienced pronounced daily changes in CARBON TRANSLOCATION IN ANEMONES 79 water temperature during periods of exposure to low tide. Changes in density of symbionts may result from differ- ences in both algal growth rate and algal expulsion rate under the experimental treatments. Although we did not measure these parameters in our study, zooxanthellate and zoochlorellate A. elegantissima have higher algal expulsion rates at 20°C than at 13°C (Saunders, 1995). McCloskey el al. ( 1996) also found that algal expulsion rates increase with increasing irradiance, and concluded that algal densities in A. elegantissima are regulated by expulsion of excess algae. In mixed anemones, the presence of the dominant symbiont is more likely due to that alga's ability to grow at a rate that meets or exceeds the rate of expulsion by the anemone and the growth rate of the other algal species. It is likely that greater numbers of algae were lost from zoochlorellate anemones than were lost from zooxanthellate anemones at 20°C since, as noted earlier, zoochlorellate anemones from the field contain higher densities of algae than do zooxan- thellate anemones. With respect to translocation of photosynthetic carbon, the relative abundance of zooxanthellae and zoochlorellae in A. elegantissima determines the amount of carbon trans- located within anemones. How does the advantage of greater carbon translocation at the higher temperature and irradiance level on an algal cell basis affect the amount of carbon received by anemones when these also contain lower algal densities (Fig. 5)7 A zoochlorellate anemone held at 13°C under high light receives 0.048 /xg C/mg protein/h from its algae (Fig. 4a). To maintain this rate of carbon translocation at 20°C. the anemone would require an algal density of only 9.6 X 104 algae/mg protein because indi- vidual zoochlorellae translocate 2.5 times more at the higher temperature. However, the density of zoochlorellae at 20°C was one-fourth (26%) of this density (Fig. 5), showing that the higher translocation rate per cell was not sufficient to compensate for the reduced density of zoochlorellae at the higher temperature. A similar calculation for a zooxanthel- late anemone shows that it needs 2.97 X 104 algae/mg protein at 20°C to maintain a translocation rate equivalent to that obtained at 13°C. However, zooxanthellate anemones held at 20°C contained 3.5 X 104 algae/mg protein (Fig. 5), about 18% more than required to maintain the translocation rate obtained at 13°C. This slightly elevated density of zooxanthellae was not sufficient to yield any significant difference in translocation rate (Fig. 4a). Using carbon translocation at 13°C as the basis of comparison, zoochlo- rellate anemones lost more algae than they should have at 20°C, and zooxanthellate anemones kept more algae than they needed to at this temperature. This comparison sug- gests that the nutritional contribution of the algae is not important to the host anemone and there is no regulation of algal densities to maintain certain carbon translocation rates. However, the cost to the host anemone of harboring symbionts at different densities is unknown. Should reduced algal densities lower the cost of maintaining the symbionts. then simply comparing carbon translocation rates is insuf- ficient for assessing benefit to the host. Application to the field The Anthopleura elegantissima-zoo\anthe\\a nutritional relationship has been examined by determining the percent contribution of translocated carbon to animal respiration (CZAR). Shick and Dykens (1984) indicated that CZAR was greater for low intertidal (34%) than for high intertidal anemones (18%) due to self-shading of the anemone during exposure to air. while Fitt el al. (1982) demonstrated that CZAR for fed anemones (13%') was less than that for starved anemones (45%). In the only study to compare CZAR of anemones harboring both symbionts, Verde and McCloskey (1996) showed that CZAR for zooxanthellate anemones was much greater than CZAR for zoochlorellate anemones. The use of CZAR as a tool of comparison hinges on the assumptions that the algae will translocate all un- needed fixed carbon, that the anemone will use all of the translocated carbon, and that the form in which the fixed carbon is translocated does not matter to the anemone. Some of these assumptions may not apply to temperate anemone symbioses. While there may be energetic advantages to the anemone to maintaining an algal population within its tissues, these advantages may be quite limited for temperate anemones (Davy el al.. 1997). Anthopleura elegantissima may not rely on carbon supplied by zooxanthellae for growth. Tsuchida and Potts (1994) demonstrated that A. elegantissima clones gained or lost weight in response to whether they were fed or not, regardless of whether they were kept in the light or dark, or whether they contained zooxanthellae or were al- gae-free. Similar results for zooxanthellate and zoochlorel- late anemones were obtained by Blevins (1991). The het- erotrophic supply of carbon appears to be the primary source of nutrition for these anemones. Indirect evidence for high rates of feeding under field conditions is provided by high ammonium concentrations in anemone-dominated tidepools (Jensen and Muller-Parker, 1994). Moreover, Davy el al. (1996) showed that reduced photosynthetic production of zooxanthellae in temperate anemones due to cloud cover, depth, and other environmental conditions could decrease the alga's translocatable carbon to just 0.7% of that fixed. Reliance on external carbon sources will be pronounced during seasonally low irradiance during the winter months. During such times the algae may represent a liability to the host, especially because algal densities in A. elegantissima during the winter season are the same as densities in midsummer (Dingman, 1998). In contrast with tropical symbiotic associations (Muscatine et al.. 1981; 1984; Davies, 1984), temperate symbiotic cnidarians like Anthopleura must often depend on sources outside of their 80 H. P. ENGEBRETSON AND G. MULLER-PARKER algal complement for their respiratory carbon requirements as well as their growth needs (Davy el ai. 1997). On the other hand, during warm and sunny periods, translocated photosynthate may be an important source of carbon. Clark and Jensen ( 1982) proposed that a period of high yield during such conditions may be sufficient for the anemone hosts to keep the symbionts year-round. Because their study of the anemone Aiptasia pallida showed that temperature also affects the nature of the translocated prod- ucts, it will be important to compare the metabolites trans- located by zoochlorellae and zooxanthellae under the range of environmental conditions experienced by anemones in the field. The nature of these metabolites, and the ability of the anemone host to use translocated compounds, may be more important than the amount of carbon translocated. Temperate symbioses exposed to pronounced seasonal vari- ations in environmental factors are ideal systems in which to explore variation in the nutritional contribution of algal symbionts to the host and the consequences for the associ- ation. The quantity of carbon translocated, as examined in this study, is only one factor in the symbiosis between zoochlo- rellae, zooxanthellae. and the anemone host in temperate regions. While this factor has justifiably received the great- est attention in tropical algal-cnidarian symbioses, it is not at all clear if provision of carbon is the most important benefit of the symbiosis to temperate A. elegantissima. If it was, our results suggest that zooxanthellae should predom- inate given their translocation potential under high temper- ature. Other selective advantages not directly related to carbon translocation must also be considered for this dual symbiosis. For example, there may be different energetic costs to hosting zooxanthellae and zoochlorellae associated with photooxidative stress resulting from photosynthesis, since host anemones must protect against toxic effects of reactive oxygen species (Shick, 1991). It would be interest- ing to compare antioxidant defenses in zooxanthellate and zoochlorellate anemones. There may be behavioral costs associated with harboring these two algae. If photosynthesis of zooxanthellae and zoochlorellae results in different ex- pansion and contraction behaviors of anemones in the field, these may affect primary productivity and feeding on zoo- plankton (Shick and Dykens, 1984), as well as gas and dissolved organic matter exchanges with the environment. Ecological consequences of harboring different symbionts must also be considered. For example, Augustine and Mul- ler-Parker (1998) have shown that selective predation on zooxanthellate anemones by a sculpin favors the survival and propagation of zoochlorellate anemones. Future studies should also focus on long-term comparisons of the growth and asexual reproduction of zooxanthellate and zoochlorel- late anemones under a variety of environmental conditions. Continuing studies of this dual symbiosis in a temperate environment should prove useful to researchers studying tropical symbioses as well. Acknowledgments We thank two anonymous reviewers for their helpful comments. This study was supported by a Project Develop- ment Award from Western Washington University to Gisele Muller-Parker. Literature Cited Augustine, L., and G. Muller-Parker. 1998. Selective predation by the mosshead sculpin Clinocottus globiceps on the sea anemone Antho- pleura elegantissima. and its two algal symbionts. Limnol. Oceanogr. 43: 711-71?. Batley, J. F., and J. S. Patton. 1987. Glycerol translocation in Condy- lactis gigantea. Mar. Biol. 95: 37 — \6. Blevins, J. K. 1991. Comparative growth and metabolism of zooxanthel- late and zoochlorellate Anthopletira elegantissima. Master's thesis. Western Washington University. 41 pp. Clark, K. B., and K. R. Jensen. 1982. Effects of temperature on carbon fixation and carbon budget partitioning in the zooxanthellal symbiosis of Aiptasia pallida (Verrill). / E.v/>. Mar. Biol. Ecol. 64: 215-230. Davies, P. S. 1984. The role of zooxanthellae in the nutritional energy requirements of Pocillopora eydoitxi. Coral Reefs 2: 181-186. Davy, S. 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Cove, and E. A. Verde. 1996. Symbiont expulsion from the anemone Anthopleiint elegantissima (Brandt) (Cni- dana; Anthozoa). J. E\p. Mai. Biol. Ecol. 195: 173-186. Mews, L. K., and D. C. Smith. 1982. The green hydra symbiosis. VI. What is the role of maltose transfer from alga to animal? Proc. R. Soc. Loiul. B 216: 347-413. Muscatine, L. 1967. Glycerol excretion by symbiotic algae from corals and Tridacna and its control by the host. Science 156: 516-519. Muscatine, L. 1971. Experiments on green algae coexistent with zoo- xanthellae in sea anemones. Pac. Sci. 25: 13-21. Muscatine, L.. L. R. McCloskey, and R. E. Marian. 1981. Estimating the daily contribution of carbon from zooxanthellae to coral animal respiration. Liuinoi. Oceanogr. 26: 601-61 1. Muscatine, L., P. G. Falkowski, J. W. Porter, and Z. Duhinsky. 1984. Fate of photosynthetic fixed carbon in light- and shade-adapted colo- nies of the symbiotic coral Stylophora pistilUitu. Proc. R Soc. Ltmd. B 222: 181-202. O'Brien, T. 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Biol. Ecol 211: 213-224. Secnrd, D. L. 1995. Host specificity and symbiotic interactions in sea anemones. Ph.D. dissertation. University of Washington, Seattle, WA. 88 pp. Shick, J. M. 1991. A Functional Biology of Sea Anemones. Chapman and Hall, London. Shick, J. M.. and J. A. Dykens. 1984. Photobiology of the symbiotic sea anemone Anihoplenra elexuntiviima: photosynthesis, respiration, and behavior under intertidal conditions. Biol. Bull. 166: 608-619. Sutton, D. C., and O. Hoegh-Guldberg. 1990. Host-zooxanthella inter- actions in four temperate marine invertebrate symbioses: assessment of effect of host extracts on symbionts. Biol. Bull. 178: 175-186. Trench, R. K. 1971. The physiology and biochemistry of zooxanthellae symbiotic with marine coelenterates. 1. Liberation of fixed 14C by zooxanthellae in vitro. Proc. floy. Sue. Loiul. B. Ill: 237-250. Trench, R. K. 1979. The cell biology of plant-animal symbiosis. Annu. Rev. Plant Physiol. 30: 485-53 1 . Tsuchida, C. B., and D. C. Potts. 1994. The effects of illumination, food and symbionts on growth of the sea anemone Anthopleura elcxan- tissima (Brandt, 1835). I. Ramet growth. J. Exp. Mar. Biol. Ecol. 183: 227-242. Verde, E. A., and L. R. McCloskey. 1996. Photosynthesis and respira- tion of two species of algal symbionts in the anemone Anthopleura elegantissima (Brandt) (Cnidaria; Anthozoa). J. Exp. Mar. Biol. Ecol. 195: 161-171. Wang, J.-T.. and A. E. Douglas. 1997. Nutrients, signals, and photo- synthate release by symbiotic algae. Plant Physiol. 114: 631-636. Reference: Biol. Bull 197: 82-93. (August 1999] Morphology and Epithelial Ion Transport of the Alkaline Gland in the Atlantic Stingray (Dasyatis sabina) GREGORY M. GRABOWSKI.1 JOHN G. BLACKBURN,2 AND ERIC R. LACY3'4 Department of Biology, University of Detroit Mercy, 4001 W. McNichols, P.O. Box 19900, Detroit, Michigan 48219; 2 Department of Physiology, 3 Department of Cell Biologv and Anatomy, Medical University of South Carolina, 171 Ashley Avenue, Charleston, South Carolina 29425; and 4 Marine Biomedicine and Environmental Sciences, Medical University of South Carolina, 221 Fort Johnson Road, Charleston, South Carolina 29412 Abstract. The alkaline glands are two fluid-filled sacs that lie on the ventral, posterior surface of each kidney in skates and rays. In this study, the morphology, transepithelial ion transport, fluid constituents, and histochemistry of the alka- line glands of the Atlantic stingray, Dasyatis sabina, were investigated. The duct from each gland joined the corre- sponding vas deferens and the resulting two common ducts emptied into the cloaca. Dark burgundy, aqueous fluid (pH 8.0-8.2) was secreted into the sacs by a simple columnar epithelium with extensive rough endoplasmic reticulum and large secondary lysosomes containing lipofuscin and mem- brane fragments. Zonulae occludentes were deep (—22 fibrils), reflecting an electrically tight epithelium (732 ohms/cm2). Carbonic anhydrase activity was localized his- tochemically within the intercellular spaces and less in- tensely in the mid-basal cytoplasm. In vitro electrophysiology showed that baseline short- circuit current (Isc, 29.1 /A A/cm2) was reduced 67.0% after Cl~ removal from the medium. Cl removal also com- pletely abolished luminal alkalinization (baseline 4.5 ± 0.7 /LtEq of acid/cnr/h). Luminal exposure to the chloride- bicarbonate exchange inhibitor, DIDS, reduced Isc by 38%. Simultaneous administration of DIDS and bumetanide (Na+/K + /Cl ~ cotransport inhibitor) to the serosal side of Received 12 April 1999; accepted 14 June 1999. Send correspondence to Eric R. Lacy. Marine Biomedicine and Envi- ronmental Sciences, Medical University of South Carolina, 221 Fort John- son Road. Charleston. SC 29412. A portion of this work was presented in abstract form (The FASEB Journal, Part I, #3024, 1992). the tissue caused the Isc to decrease >100%. Serosal expo- sure to ouabain (Na-K, ATPase inhibitor) decreased Isc 48%, whereas amiloride (sodium ion channel blocker) and acetazolamide (carbonic anhydrase inhibitor) had no statis- tically significant effect on Isc or alkalinization rates. Taken together the results suggest the presence of apical epithelial bicarbonate exchangers that are chloride or sodium depen- dent, basal sodium and HCO^ transport, and an Isc that is not totally dependent on Na+-K+ ATPase. Introduction Early anatomical studies of the male skate and stingray urogenital system reported a pair of blind-ended sacs, each of which opened into the cloaca. These structures were described initially as urinary bladders or sperm storage sacs (Borcea, 1906; Daniel, 1934), but the only evidence to support this functional nomenclature is the proximity of the sacs' openings to those of the ureters and vas deferens within the cloaca. The sacs secrete and store a watery fluid of high pH (8.0-9.2), thus their name, alkaline gland (Maren et al., 1963). On the basis of the high pH of the fluid, Smith ( 1929) speculated that it neutralized the potentially deleterious effects of acidic urine in the cloaca on the extruded sperm. As yet, however, no studies on the physiological function of the alkaline gland have been published. A few reports, from various skate species (little skate, Raja erinacea; barndoor skate, R. stabuliforis; big skate, R. ocellata), have described the gland's morphology and epi- thelial transport physiology (H.W. Smith. 1929; Maren et 82 STINGRAY ALKALINE GLAND 83 al.. 1963; Masur. 1984; P. L. Smith, 1981, 1985). These morphological accounts show that the gland lumen has mucosal "villar projections" lined by a simple columnar epithelium (Maren et al.. 1963; Masur, 1984). The mucosa generates and maintains a hundred-fold concentration gra- dient of OH ions and a 50-fold gradient of CO2 from plasma to gland lumen; these are some of the steepest alkaline gradients across any epithelium in nature (Maren el al., 1963). Given the unique epithelial transport properties of the alkaline gland, physiologic studies have focused on the mechanisms of fluid and bicarbonate secretion (Maren et al., 1963; Smith, 1981, 1985). Chloride and bicarbonate are the two main anions con- stituting alkaline gland fluid in the skate. In vitro experi- ments indicate that chloride secretion accounts for most, if not all, of the short-circuit current (Isc) (Maren et al.. 1963; Smith. 1981. 1985). These results led to speculation that chloride-dependent bicarbonate transport might be involved in fluid alkalinization. Although definitive evidence was lacking, secreted chloride was believed to recirculate into the epithelial cell by way of a Cr/HCOJ exchanger located at the apical plasma membrane (Maren et al., 1963; Smith, 1981. 1985). Carbonic anhydrase, an enzyme associated with many bicarbonate-secreting tissues, was identified bio- chemically in the alkaline gland of some but not all skate species studied (Maren et al., 1963). The concentration of carbonic anhydrase in the tissue was correlated with the pH of the alkaline gland fluid produced (Maren et al., 1963), suggesting that this enzyme has a role in bicarbonate secre- tion for some skate species. The present study uses transmission and scanning elec- tron microscopy and freeze fracture to elucidate the ultra- structural organization of the alkaline gland in a stingray species, Dasyatis sabina, the Atlantic stingray. The pres- ence and distribution of carbonic anhydrase activity, nerve fibers, and lipofusion were identified histochemically. These results are correlated with in vitro electrophysiological data and rates of fluid alkalinization. Some of the regulatory mechanism of ion transport were probed with various met- abolic inhibitors. The composition of the fluid removed from the alkaline glands was analyzed. Materials and Methods Sexually mature male Atlantic stingrays (Dasvatis sa- bina, wing span ~45 cm) purchased from Gulf Specimens Inc. (Panacea, FL) or captured along the coast of South Carolina were allowed to acclimate in a 16,000-1 holding tank for at least 5 days prior to experimentation. Water in the holding tank was drawn from Charleston (South Caro- lina) Harbor (650-850 mosm/1) and maintained at room temperature. Stingrays were fed shrimp twice a week and kept on a 12-h light/dark cycle. After acclimation, animals were anesthetized with MS222 (3-aminobenzoic acid ethyl ester, 0.5 g/1, Sigma Chemical Co.) and double pithed. The body cavity was opened by a ventral midline incision; the alkaline gland fluid was aspirated with a 25-gauge needle and saved at 4°C for further analysis; the alkaline gland was removed for use in morphology or electrophysiology exper- iments. Light and electron microscopy Fixative (2.5% paraformaldehyde, 5.0% glutaraldehyde, and 0.25% picric acid; Ito and Karnovsky. 1968) was in- jected into both sacs of the gland immediately after the fluid was removed. After 1 h the puboischiac bar was severed, and the alkaline gland was freed from surrounding tissue with fine forceps. Each gland was excised at its junction with the cloacal wall and placed in the same fixative for 24 h. The tissue was then rinsed, trimmed into 1-mnr pieces with a razor blade, and stored in 0.1 M sodium cacodylate buffer. Alkaline gland fluid was centrifuged at 200 X g for 10 min. The pellet was fixed for 4 h in the same fixative injected into the gland sacs (Ito and Karnovsky, 1968). Both pellet and pieces of fixed gland were then postfixed (1.0% osmium tetraoxide in 0.1 M sodium cacodylate buffer), dehydrated in graded ethanols, and embedded in Epon- Araldite. Sections were cut, stained (semithin sections stained with alkalinized toluidine blue and ultrathin sections with uranyl acetate and lead citrate), and examined using a light microscope or a JEOL 1 200 EX electron microscope. Additional gland tissue, fixed as described above but in aldehydes only, was cryoprotected in graded concentrations of glycerols to a final concentration of 30% glycerol for freeze fracture. The tissue was then frozen rapidly in liquid propane, followed by fracturing and replication in a Balzer 360 M device (Balzers, Fiirstentum Liechtenstein). Replicas were supported on 200-mesh copper grids and examined with the transmission electron microscope. Aldehyde-fixed tissue was also used for scanning electron microscopy. It was first postfixed in 1.0% osmium tetraox- ide in 0. 1 M sodium cacodylate buffer, followed by dehy- dration in graded ethanols, and then critical point dried using a Sorvall critical point dryer (Newtown, CT). Tissue was coated with gold/palladium for 3 min at 2.5 kV and 20 mA using an E5100 sputter coating unit (Polaron Instru- ments, Doylestown, PA) and examined with a JEOL 35C scanning electron microscope. Lipofuscin staining Alkaline gland tissue and paniculate matter from gland fluid of four stingrays were stained for lipofuscins using the Long Ziehl-Neelsen technique (Bancroft and Cook, 1984). The pellet, as described above, and gland tissue were fixed in Bouin's solution for 2 h, followed by dehydration in graded ethanols, clearing in xylene, and embedding in par- 84 G. M. GRABOWSKI ET AL aftin. Five-micrometer-thick sections were deparaffinized in xylene taken stepwise to water and stained in filtered carbol fuchsin for 1-3 h at 56°C. After staining, sections were washed in water, differentiated in 1% acid-alcohol, and counter stained in aqueous methylene blue. Slides were then rinsed in water, dehydrated, cleared in xylene. and mounted on glass slides. Lipofuscin appeared bright magenta, and nuclei stained blue against a pale magenta background. Silver staining of neural tissue Nerve fibers in alkaline glands were localized using the silver precipitate method of Sevier and Munger (1965). Five-micrometer-thick paraffin sections of Bouin's fixed tissue were incubated in 20% silver nitrate for 15 mm, washed with distilled water, and developed in ammoniacal silver (10% silver nitrate precipitated with 28%-30% am- monium hydroxide, plus 2% formalin). After a 2-min rinse in 5% sodium thiosulfate. slides were washed in distilled water, dehydrated, cleared in xylene, and mounted. Localization of carbonic anhydrase activity (CAM) Alkaline glands were fixed in a solution of 2.0% parafor- maldehyde, 2.5% glutaraldehyde, and 0.4% CaCK in 0.1 M sodium cacodylate buffer for localization of carbonic anhy- drase activity (CAH) using the Hansson's technique (Hans- son, 1967: Maren. 1980b: Sugai and Ito, 1980; Lacy, 1983b). Fixed tissue was frozen in 8% sucrose and sec- tioned at 10 M111 on an IEC CTF cryostat (International Equipment Company). Sections were floated on Hansson's medium (1.86 mM CoSO4. 55.9 mM H2SO4, 3.73 mM KH,PO4, and 158 mM NaHCO,) for 1-5 min. Sections were rinsed by floating on Sorensen's phosphate buffer (pH 8.0) for 1 min and then transferred onto 2% ammonium sulfide for 1-2 min. This was followed by rinsing sections on Sorensen's phosphate buffer at pH 5.0 and then mount- ing them in heated glycerin jelly on glass slides for obser- vation with a light microscope (Sugai and Ito, 1980; Lacy, 1983b). After the sections were incubated on 2% ammo- nium sulfide, low-pH buffers were used to prevent the black precipitate indicative of CAH activity from degrading. For electron microscopy, sections were postfixed in 1.0% os- mium tetraoxide in Sorensen's phosphate buffer (pH 5.0) for 30-45 min, stained en bloc with 1.0% uranyl acetate in maleate buffer (pH 5.2). dehydrated in graded ethanols, and embedded flat in epoxy resin. Ultrathin sections were stained and examined as described above. Acetazolamide ( 10~5 and 10~6 M) in Hansson's medium was used to inhibit CAH, thereby serving as a negative control. For evaluation of nonspecific activity, sections were incubated either in ammonium sulfide without prior incu- bation on Hansson's medium, or on bicarbonate-free Hans- son's medium. Morplioinctric analysis Ratios of basal cells to columnar cells were determined from counts made of cross-sectioned glands at the light microscopic level (epoxy resin sections, 50 X). The size and distribution of intramembranous particles observed in freeze fracture replicas were measured on electron micrographs using a scale magnification loupe (Baxter. Atlanta, GA). The luminal surface area of columnar epithelial cells was estimated by measuring the cell diameters of luminal plasma membranes from scanning electron micrographs. Constituents of alkaline gland fluid Fluid from the alkaline glands of five stingrays was pooled, cooled to 5°C, and centrifuged as described above. The supernatant was then frozen by placing the tube in dry ice and shipped overnight to Mayo Medical Laboratories (Rochester. MN) for analysis of its composition. Electrophysiology Each sac of the alkaline gland was freed in situ from suiTOunding connective tissue, excised, and placed in a petri dish of oxygenated elasmobranch Ringer (NaCl. 280.0 mM; KC1. 5.0 mM; MgCU 3.3 mM; CaCl2. 3.8 mM; NaHCOv 10.0 mM; urea, 350.0 mM; dextrose, 5.0 mM; 800 mOsm/1; pH 6.9). The Ringer was gassed with 95% O:/5% CO2, unless otherwise noted, and used at room temperature. Each sac was mounted between two halves of an Ussing chamber (4-mm diameter). Each half of the chamber was connected to a 20-ml circulation reservoir (Medical Re- search Apparatus, Clearwater, FL). The short-circuit current (Isc) and transepithelial potential difference (PD) were mea- sured using a voltage-current clamp (Physiological Instru- ments. San Diego, CA). Before tissue was mounted in the Ussing chamber, electrode polarization and fluid resistance was compensated with the VCC600 voltage-current clamp. Calomel electrodes (Fisher, Atlanta, GA) placed in a satu- rated KC1 solution were connected to the Ussing chamber via salt bridges (4% agar in elasmobranch Ringer) to mea- sure the PD. Platinum electrodes (Fisher. Atlanta, GA) were placed directly into the Ussing chamber to measure Isc. The PD and Isc were displayed on a Soltec 1242 strip chart recorder (Soltec Corp.. Sun Valley, CA). Transepithelial resistance was calculated using the open-circuit PD, and the closed-circuit Isc of the mounted tissue. All readings were in reference to the luminal medium. Transport inhibitors Once baseline electrophysiological parameters were es- tablished, the percent change of Isc was calculated after the tissue was exposed to the following transport inhibitors: ouabain. Na"/K+ ATPase inhibitor ( 10~4 M. serosal): bu- metanide, Na+/K+/Cl cotransport inhibitor (10 3 M. se- STINGRAY ALKALINE GLAND 85 rosal); DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, CT/HCO^ exchange inhibitor (10"' M, luminal); amiloride, sodium channel inhibitor, (10 3 M. luminal and serosal); acetazolamide, carbonic anhydrase inhibitor (10~5 M, luminal). Chloride was substituted in the medium with isomolar concentrations of gluconate. All reagents were purchased from Sigma Chemical Co.. St. Louis, MO. Alkalinization rates The alkalinization rate of the luminal medium was mea- sured using the pH stat technique on glands mounted in the Ussing chamber. Unbuffered (bicarbonate-free) Ringer bathing the luminal side of the gland was gassed with 100% oxygen during experiments and 30 min prior to tissue mounting. The serosal-bathing medium consisted of buff- ered elasmobranch Ringer, gassed with 95% oxygen/5% carbon dioxide. The rate of fluid alkalinization (/u,Eq of acid/cnr/h) was then determined via titration using 0.01 M sulfuric acid. The pH of the luminal medium was main- tained at pH 5.5 for at least six consecutive intervals of 5 min each. The pH was monitored using a pH microelectrode (Microelectrode, Londonderry, NH) connected to a Beck- man pH meter (Omega 40, Fullerton, CA). Two experiments were performed to determine the pres- ence of either chloride-dependent or sodium-dependent bi- carbonate transport. Alkalinization rates were measured af- ter each manipulation. Baseline values were made from tissues bathed on both sides with elasmobranch Ringer. The medium was changed on the luminal and serosal sides to iso-osmotic elasmobranch Ringer free of chloride or so- dium. In the first experiment, chloride-containing Ringer was added back to the luminal side; in the second experi- ment, sodium-containing Ringer was added back to the serosal side. After a new alkalinization rate was established, a bicarbonate transport inhibitor, SITS (4-acetamido-4'- isothiocyanatostilbene-2,2'-disulfonic acid, 10~3 M), was added to the luminal medium in both experiments. The buffering capacity of the various Ringers was deter- mined after each experiment, using the pH stat method. The buffering capacity of each medium was then subtracted from the alkalinization rate derived under the experimental conditions. Statistical analyses Statistical significance was evaluated using a two-tailed- paired t test, with the level of significance set at P < 0.05. Results Gross anatom\ The alkaline gland of the Atlantic stingray, Dasyatis sabina, consists of a pair of blind-ended, bladder-like sacs located within the pelvic girdle ventral to the posterior pole of the kidney and lateral to the vas deferens. In the animals we examined, the glands were retroperitoneal and symmet- rically aligned along the vertebral column. They were easily distinguished from surrounding tissue by their deep bur- gundy color. Each sac of the gland held a maximum of 4-5 ml of fluid. The mediocaudal portion of each gland nar- rowed to a single duct, which joined the respective sperm duct (vas deferens) on the same side of the animal. The resultant common duct for sperm and alkaline gland fluid was about 3-mm long and pierced the body wall to open on the crest of the urinary papilla in the cloaca. Microscopv The mucosa of the alkaline gland was highly folded and lined by a simple columnar epithelium (Figs. 1, 2). A rich capillary network lay immediately beneath the basement membrane. Within each fold were an arteriole and venule and dense tracts of nerve fibers (Figs. 1-3). Two populations of epithelial cells were distinguished on the basis of their apical membrane exposure to the lumen (Fig. 1 ). The length of the long axis of the apical cell surface differed significantly in the two populations (P < 0.05, n = 141); in one (84.4% of the total cells) the long axis of the apical cell surface was 7.2 ± 0.14 ^m; in the other (15.6%), the long axis was about twice that length (14.92 ± 0.49 /u,m). All cells that contacted the lumen had the same ultrastructural organization, despite the difference in lu- menal membrane area. Columnar epithelial cells had a prominent, basally lo- cated pleomorphic nucleus and exceptionally large and abundant secondary lysosomes (Figs. 1, 2). The secondary lysosomes stained positively for lipofuscin (data not shown) and were dark green-brown in unstained sections. (Fig. 3). The smooth-surfaced endoplasmic reticulum was evenly distributed throughout the cytoplasm. Mitochondria bearing lamellar cristae were located in the upper two-thirds of the cell, and Golgi complexes were abundant in the perinuclear region (Fig. 2). Many membrane-bound vesicles were present in the Golgi region and adjacent to the apical plasma membrane. Some of these vesicles were seen fusing with larger vesicles as well as with the apical plasma membrane (Fig. 2). Basal cells were also present in the lower third of the epithelium (Fig. 4) in a ratio of about 1 basal cell to 20 columnar cells. These cells, which ranged from 1.4 to 2.6 /j,m in diameter, were not highly interdigitated with adjacent columnar cells and were not observed in contact with an- other basal cell. The cytoplasm of basal cells surrounded a proportionately large nucleus and contained only a few organelles, which were limited to the endoplasmic reticu- lum, and small vesicles containing material of various de- grees of electron density. The apical surface of the columnar cells was elaborated 86 G. M. GRABOWSKI ET AL Figure 1. Figure 3. . STINGRAY ALKALINE GLAND 87 into microplicae (Figs. 1, 2). The basolateral plasma mem- brane was relatively straight nearest the lumen, but closer to the basal lamina it was interdigitated with itself and adjacent cells (Fig. 2). Freeze fracture of the lateral plasma mem- brane revealed some areas consisting only of large in- tramembranous particles (99 A ± 0.1, n = 52) (Fig. 5) loosely arranged as single particles or in groups of up to 20 particles. Outside these areas was a mixture of large and small intramembranous particles. No rod-shaped particles were observed in either the apical or basolateral plasma membrane. The zonulae occludentes were deep ( 1 .4 ± 0.7 jam, n = 19 replicas) and composed of 21.8(±4.5) fibrils (Fig. 6). Most of the fibrils were parallel to the apical plasma membrane, with those constituting the basal one- fourth of the zonulae occludentes forming a loose anasto- mosing network (Fig. 6). Ultrastructural observations of the solids from alkaline gland fluid showed cellular debris including multivesicular bodies, spherical particles with electron-dense cores that stained positively for lipofuscin, membrane whorls, and a few necrotic spermatozoa (Fig. 7). Localization of carbonic anhydrase activity Carbonic anhydrase activity (CAH) was indicated by a black precipitate at both the light and electron microscopic level (Figs. 8, 9). A minimum of 2 min in the incubation medium was required for the precipitate to develop, at which time CAH appeared first within the intercellular space of columnar cells. In electron micrographs, CAH was localized in the intercellular space between columnar cells but excluded from the zonulae occludentes (Fig. 9). Adja- cent to the basement membrane, CAH was observed only within the intercellular space formed by invaginations of the plasma membrane or interdigitation of cytoplasmic folds (Fig. 9). Regions of the basolateral plasma membrane that contacted the basement membrane did not exhibit CAH. After 3-10 min of incubation, the precipitate appeared in the basal two-thirds of columnar cell cytoplasm (Fig. 8). Control sections incubated on bicarbonate-free Hansson's medium or on ammonium sulfide alone were similar to unstained sections that were rinsed only on Sorensen's phosphate buffer, and showed no positive staining (data not shown). Complete inhibition of CAH occurred at acetazol- amide concentrations of 10~5 M in Hansson's medium (data not shown). Lower concentrations of acetazolamide (10~6 M) failed to inhibit CAH activity for incubation periods longer than 2 min. Analysis of alkaline gland fluid (AGF) Table I shows the analyzed constituents of AGF. Sodium and chloride were the dominant ions, with K+, Mg+ + , Ca+ + , and Fe++ in detectable amounts. The osmolality was near that of plasma (750-875 mOsm), and significant con- centrations of protein and urea were measured. The pH varied between 8.0 and 8.2. Electrophysiology Baseline parameters. The baseline PD was 14.5 ± 1.9 mV, Isc was 29.1 ± 4.2 juA/cm2, and transepithelial resis- tance was calculated to be 732.4 ± 184.6 ohm • cnr (n = 18). Transport inhibitors. The effect of specific ion transport inhibitors on the baseline Isc is shown in Table II. The serosal addition of ouabain, a Na+/K+ ATPase inhibitor, resulted in an almost 48% decrease of Isc within 45 to 50 min. Bumetanide, a Na+/K+/CF cotransport inhibitor, de- creased the Isc approximately 70% within 30 min, and DIDS, a Cr/HCO3-exchange inhibitor, placed on the lumi- nal side of the epithelium decreased the Isc almost 38% within 30 to 40 min. The Isc was completely inhibited, and in fact was slightly reversed, after consecutive addition of bumetanide within 30 min of DIDS addition to the lumenal surface. The effect of luminal exposure to acetazolamide, a carbonic anhydrase inhibitor, on Isc was sporadic, and pro- duced only a 16% overall reduction of Isc. Amiloride, a sodium ion channel inhibitor, placed in either the luminal or serosal media had no significant effect on the Isc (data not shown). The removal of chloride from the bathing media on both sides of the tissue with the substitution of gluconate resulted in a 67% reduction in Isc by 45 min. Alkalinization rates. Two experiments investigating de- Figure 1. Scanning electron micrograph of a transected mucosal fold. The asterisk is located in the center of an arteriole. A network of capillaries (arrows) lies directly beneath the epithelium, which has prominent secondary lysosomes (arrowheads). Bar = 50 /im. Figure 2. Transmission electron micrograph (TEM ) of the simple columnar epithelium of the alkaline gland. Arrows indicate secondary lysosomes located in the supranuclear region. Arrowheads indicate a nerve fiber closely adjacent to the epithelium. Note the numerous vesicles in the apical cytoplasm. Bar = 2 fj.m. Figure 3. Light micrograph (LM) of nerve fibers (arrows) in the subepithelial lamina propna stained black using the Sevier Munger silver technique. Nerve libers were closely associated wilh blood vessels (asterisks) and the epithelium (e). Note the multiple darkly staining secondary lysosomes in the apical cytoplasm of the epithelium. Bar = 4 /j.m. Figure 4. TEM of a basal cell (BO located between adjacent columnar cells (CC). Note the large proportion of the nucleus relative to the BC cytoplasm. Basal lamina (BL). Bar = 2 /tim. Figure 5. G. M. GRABOWSKI ET AL Figure 7. Table I Analyzed constituents of alkaline gland fluid STINGRAY ALKALINE GLAND Table II Effects of ion transport inhibitors on the short-circuit current (Isc) Component Concentration Na + 286 mA/ K+ 3.7 mA-/ Cl 113.0mA/ Mg+ + 1 .68 mA-/ Ca+ + 0.84 mA/ Cu + + 0.58 mA/ Zn+ + 0.87 mAf Fef* 0.61 mM Urea 271 mA/ Progesterone 0.012 ju,A/ Estradiol 0.16 pM Norepinephrine ND Epinephnne ND Dopamine ND Testosterone 4.3 tiM Protein 5.9 mg/ml Osmolality 875 mosm mosm = milliosmoles. ml = milliliter, mg = milligram. pM = pico- moles. fj.M = micromoles, mM = millimoles. ND = none detected. pendent and independent bicarbonate transport mechanisms are shown in Table III. The baseline alkalinization rate of control tissues varied from about 4 to 7.5 /uEq of acid/cnr/h depending upon the animal used. Chloride-dependent bicarbonate secretion was demon- strated by a significant decrease in alkalinization rate when both sides of the gland were exposed to chloride-free Ringer (Table III, Experiment 1 ). Alkalinization returned to control levels when chloride was added back to the luminal side of the tissue. SITS ( 10~3 M), a bicarbonate transport inhibitor, when applied to the luminal medium, had no statistically significant effect on the alkalinization rate after luminal exposure to chloride (Table III). However, the results varied widely from tissue to tissue. In the second experiment, the fluid alkalinization rate decreased significantly, 55%, after luminal and serosal ex- % Decrease Treatment of Isc n Ouabain (ICT-1 A/). S 47.8 ± 2.9 4 Bumetanide (10~3 Ml S 69.7 ± 5.5 8 DIDS (10"' A/1. L 37.9 ± 5.9 6 DIDS <10~' A/1, L + Bumetanide (10~3 A/), S 105.9 ± 12.2 5 Acetazolanude (1()~5 A/1. L 16.0 ± 9.0 3 Values are means ± SE, L = Luminal, S = Serosal. n = number of mounted glands. DIDS = 4,4'-diisothiocyanatostilbene-2,2'-disulfomc acid. posure to sodium-free media (Table III). The alkalinization rate increased immediately with the readdition of serosal sodium-containing Ringer. Addition of the bicarbonate transport inhibitor, SITS ( 10~3 A/), to the luminal medium caused a significant decrease (24%) in the alkalinization rate compared to the values after sodium readdition (Table III). Discussion Results of this study extend the presence of alkaline glands in the Elasmobranchii to include stingrays. Our gross anatomical explorations of several species of shark — spiny dogfish, Squalus aciintlmix; black tip, Carcharhinns lini- batus; smooth dogfish, Miistelus canis; scalloped hammer- head, Sph\rna lewini, and Atlantic sharpnose. Rhizoprion- odon terraenovae — did not reveal the presence of alkaline glands in these elasmobranchs. This finding is consistent with the notion that alkaline glands are present only in skates and rays and not in sharks. Furthermore, this study is the first to elucidate the morphology, ion transport mecha- nisms, enzyme histochetnistry, and fluid composition of the alkaline gland in a species of stingray. The gross anatomy of the Atlantic stingray alkaline gland is similar to that described for several species of skates Figure 5. Transmission electron micrograph (TEM) of freeze fracture replica of a loose cluster of large intramembranous particles (arrows) found on the P fracture face of the lateral plasma membrane. Bar = 270 nm. Figure 6. TEM of freeze fracture replica of the zonula occludens between two columnar cells. Note that numerous strands are arranged in a parallel array near the gland lumen (asterisk), but the more basal strands form an anastomosing network (P fracture face). Bar = 200 nm. Figure 7. TEM of solid constituents from centrifuged alkaline gland fluid. Arrows indicate degenerate sperm with outer plasma membrane separated from the sperm head. Arrowheads indicate masses ot membranes. Asterisks show roughly globular particles that composed the greatest part of the alkaline gland paniculate matter. Bar = 2 /j.m. Figure 8. Light micrograph of carbonic anhydrase activity in epithelial cells lining the alkaline gland. Typical staining pattern in sections incubated for 3-10 mm on Hansson's medium. Enzyme activity was strongly present in the intercellular spaces (arrows), as well as in the mid to basal cytoplasm of columnar cells. Bar = 7 jiiii. Figure 9. TEM of carbonic anhydrase activity in sections incubated for 2 min on Hansson's medium. Enzyme activity appears as electron-dense precipitate (arrows) confined to the intercellular space. N = nuclei of columnar cells. Note the absence of CAH activity along the basal lamina (BL). Bar = 2 ;am. 90 G. M. GRABOWSKI ET AL Table III In \ilro alkalini-alion mles cj ' alkiilinc ^ln Experimental conditions Alkalinization rate of acid/cnr/h) Experiment I Elasmobranch Ringer (Control) 3.88 ± 0.63 Chloride-free Ringer, L&S -1.93 ± 1.89* Chloride readdition. L 3.65 ± 1.17** Addition of SITS ( 1 m/W), L 1.45 ± 2.22 Experiment 2 Elasmohranch Ringer (Control ) 7.65 ± 0.67 Sodium-free Ringer. L&S 3.49 ± 0.41* Sodium readdition. S 5.64 ± 0.62** Addition of SITS ( 1 nW). L 4.30 ± 0.45** Values are means ± SE; n = 5 lor each experiment. L = luminal. S = serosal. SITS == 4-acetamido-4'-isothiocyanatostilbene-2.2'-disulfonic acid. * Significant difference compared to control, ** .significant difference compared to respective chloride or sodium free conditions, *** significant difference compared to respective chloride or sodium readdition. P < 0.05. (Maren et ai, 1963). However, one significant difference is the relationship between the alkaline gland duct and the sperm duct. In skates, Maren et al. (1963) reported that the alkaline gland ducts and sperm ducts have separate open- ings onto the urinary papilla. In the stingray, the alkaline gland duct joins the sperm duct, and the resultant common duct then opens onto the urogenital papilla. This anatomical arrangement in the Atlantic stingray allows mixing of sperm and alkaline gland fluid (AGF), suggesting that AGF may facilitate successful fertilization by its actions on spermato- zoa. Furthermore, the confluence of the two ducts in the Atlantic stingray may explain the presence of some necrotic sperm and cell membranes in AGF, because residual sperm in the common duct would have retrograde access to the alkaline gland lumen. The absence of spermatozoa in the AGF supports the contention that the gland is not a hona fide sperm storage organ, thus contradicting reports by early anatomists (Borcea, 1906; Daniel. 1934). Morphological features of columnar cells composing the epithelium of the alkaline gland of the Atlantic stingray are generally consistent with preliminary reports of the alkaline gland of the little skate (Maren et al.. 1963; Masur. 1984). Those cells exhibited a well-developed Golgi apparatus and endoplasmic reticulum, suggesting a high degree of active protein synthesis. The many vesicles we observed in the cytoplasm, especially those budding from the Golgi appa- ratus and fusing with larger vesicles or with the apical plasma membrane, support that idea. Although basal cells were morphologically distinct from columnar cells, we are uncertain whether they are a separate population of mature cells or are immature columnar cells. A striking microscopic feature of stingray alkaline gland epithelial cells was large secondary lysosomes that imparted a dark green-black color to the gland and were distinctive in unstained tissue sections. An accumulation of myelin fig- ures and lipofuscin granules in these secondary lysosomes was strongly suggestive of increased lysosomal processing of lipid membrane (Reed et ai, 1965; Harman, 1990). Interestingly, such features were also observed in epithelial cells of mammalian male reproductive organs such as the epididymis and seminal vesicle (Pappenheimer and Victor, 1954; Nicander, 1958; Mitchinson et al. 1975). Mitchinson et ill. ( 1975) suggested that the spermatozoa in the lumen of those organs may be the source of the intracellular lipofus- cin granules, whereby epithelial cells perform a "salvaging" function and store insoluble fatty acids as lipofuscin. A similar process may occur in alkaline gland epithelial cells: the necrotic sperm and cell debris observed in the lumen of the gland would be the extracellular source of the intracel- lular lipofuscin granules. The composition of stingray AGF differs from that pre- viously reported for three species of skates (Maren et ai, 1963) in several ways. Stingray AGF is a deep burgundy color and nearly opaque; in contrast, skate AGF is clear to slightly yellow. Stingray AGF has significant amounts of protein and urea; skate AGF is reported to lack protein and have only about one-third the concentration of urea found in the Atlantic stingray (Maren et ai, 1963). Furthermore, the ionic concentration was different: stingray AGF had one- half the concentration of K+ and Cl~ reported for skate AGF but 4 times more Mg + + and Ca+ + . The present study is the first to show AGF with immunodetectable steroid hormones. However, the immunological methods used an- tibodies to human hormones, which raises the possibility that the results may be due to nonspecific binding. A recent study (Biillesbach et ai, 1997) probed the pos- sibility that AGF contained relaxin, a peptide hormone found in mammalian reproductive tissues and secreted flu- ids. The fact that relaxin in mammalian seminal fluid stim- ulates sperm motility (Essig et ai, 1982; Weiss. 1989) was the basis for the investigation in the stingray. Biillesbach and colleagues (1997) showed that stingray AGF contains a unique relaxin-like molecule with an apparent molecular mass of 1 3 kDa formed by two polypeptide chains of 4 and 9 kDa. This molecule is the only member of the relaxin family known to be glycosylated. The relaxin-like molecule of stingray AGF did not alter stingray sperm motility in vitro (Biillesbach et ai, 1997), but this finding does not rule out the possibility that the AGF relaxin-like molecule acts on a different aspect of sperm function such as capacitation or that it functions in the female reproductive tract. The lumen of the stingray alkaline gland was not lined by the villar projections described in the skate (Maren et ai. 1963). but it did have mucosal folds, each of which con- tained a major arteriole and venule. The apical plasma membrane of the columnar epithelial cells was elaborated into microvilli characteristic of a secreting epithelium. STINGRAY ALKALINE GLAND 91 Freeze fracture replicas showed that the only distinguishing intramembranous particles were in the basolateral plasma membrane. The size and distribution of the particles form- ing these clusters was comparable to those forming gap junctions in mammalian cells. Apical and basolateral plasma membranes did not reveal any rod-shaped particles that would suggest proton transport (Brown and Montesano, 1980). The zonulae occludentes of columnar cells consisted of about 22 strands, suggesting that the epithelium is electri- cally tight and imparts a high transepithelial resistance (Claude and Goodenough. 1973; Claude, 1978). Our in vitro electrophysiological data showed that the transepithelial resistance was 732 ohm • cm2, confirming the tight junction morphology. The presence of "very tight" zonulae occlu- dentes and a high transepithelial resistance suggests that there is little paracellular solute transport across the epithe- lium of the alkaline gland (Bowman el al, 1992; Byers and Marc-Pelletier. 1992). Therefore, regulation of ion transport appears to occur primarily across the plasma membrane. Maren et al. (1963) and Smith (1981. 1985) have dem- onstrated that both bicarbonate and chloride are secreted in the little skate alkaline gland and that chloride is the main anion responsible for most of the Isc. This finding was extended to the stingray alkaline gland in the present study in which Isc decreased almost 70% when chloride was removed from the bathing medium. Using intracellular mi- croelectrodes. Smith (1981, 1985) showed that the apical plasma membrane was dominated by a large chloride con- ductance, whereas the basolateral plasma membrane con- tained a barium-sensitive potassium channel. However, the mechanisms involved in the alkalinization process have never been clearly established in this gland, despite specu- lation that a Cr/HCO3 exchanger may exist in the apical or basolateral plasma membrane or in both membranes (Maren et al.. 1963; Smith. 1981. 1985). In the present study, the marked reduction in Isc after serosal addition of bumetanide. an inhibitor of Na+/K+/Cl~ cotransport. suggests that this transporter is located in the basolateral plasma membrane. If so. it may be the main conductive pathway for chloride entry into the cell. The remaining Isc could be due to the secretion of intracellular chloride or another anion. such as bicarbonate. To test this latter possibility we added the stilbene, DIDS, which effec- tively inhibits bicarbonate cotransporters (Wiederholt et til.. 1985; Melvin and Turner. 1992) as well as chloride chan- nels (Bretag. 1987) to the luminal side of the epithelium. The resultant 38% decrease of Isc. and its further reduction to nominal levels after the consecutive addition of serosal bumetanide. substantiates this assumption. Furthermore, complete reduction of the Isc by consecutive addition of DIDS and bumetanide suggests a pathway for chloride secretion across the epithelial cells via a Na+/K+/CP co- transporter at the basolateral plasma membrane, and a chin- ride channel at the apical plasma membrane. Chloride movement across the epithelial basolateral plasma membrane, via a putative Na+/K+/CF cotrans- porter in epithelial cells in stingray alkaline gland, appears to be driven in part by Na^/K+ ATPase, as shown by the serosal addition of ouabain, which decreased the Isc by 48%. In contrast, ouabain completely abolished chloride secretion and Isc in the little skate alkaline gland (Smith. 1985). The lack of significant alkalinization rates after the tissue was exposed to medium free of chloride and sodium sug- gests that there is little independent transport of bicarbonate. If a significant portion of the alkalinization process involves an apical Cl /HCO, exchanger — as our results suggest — the absence of luminal chloride could impede that process, resulting in the accumulation of intracellular bicarbonate. Such a scenario has been observed in the rat parotid acini: SITS, an inhibitor of bicarbonate transport, increased intra- cellular pH and was thought to stimulate bicarbonate secre- tion via anion channels (Pirani et al.. 1987; Melvin and Turner. 1992). Chloride channels in a number of different epithelia. including pancreatic duct, sweat gland duct, and respiratory epithelia, have been shown to transport bicar- bonate (Gray et al.. 1989; Tabcharani et at.. 1989; Kunzel- mann et a I.. 1991 ) at a conductance as high as 50% of the conductance of chloride. The remaining Isc may be accounted for by a Na+/HCOJ symport, as demonstrated in this study by using pH stat methodology. Such mechanisms for bicarbonate transport have been demonstrated in renal proximal tubule (Yoshi- tomi et til.. 1985), corneal endothelial cells (Wiederholt et ill.. 1985), and gastric oxyntic cells (Curci et al.. 1987). Alkalinization of the luminal medium in the present study was dependent on the presence of both apical chloride and serosal sodium. The changes attributed to the absence and readdition of sodium suggests the presence of a Na+/HCO^ symport. The alkalinization rate attributed to the readdition of serosal sodium, and its reduction by luminal SITS, is indirect evidence that a Na 4 /HCO^ symport may be located at the apical plasma membrane. The stilbene. SITS, blocks not only bicarbonate transport via Na'/HCO, symporters (Curci et al.. 1987; Fitz et al., 1989; Wiederholt et al.. 1985), but also Cl /HCO, exchangers (Stewart et al.. 1989). Maren and co-workers (1963) demonstrated a possible relationship between CAH and higher pH levels in AGF of various skate species. They showed that inhibition of CAH /;; vivo reduced the pH of newly formed fluid to levels found in species that did not have glandular CAH. This was accomplished using intravenous injections of acetazolamide at least 10 times higher than the dose we used. In a study of rat distal colon, the need for high (millimolar) concentra- tions of acetazolamide to inhibit bicarbonate transport was 92 G. M. GRABOWSKI ET AL attributed to the drug's poor cellular penetration, the distri- bution of CAH within the cell, and the requirement of 99% inhibition of CAH for a significant decrease of Isc to occur (Feldman et al., 1988). The effectiveness of acetazolamide in reducing the Isc of the stingray alkaline gland is ques- tionable because of the erratic results from tissue to tissue. However, concentrations of acetazolamide greater than 10~4 M were not used in the present study, because reports have indicated that the drug interferes with other ion trans- port mechanisms (Nellens et al.. 1975; Weiner and Mudge. 1985). Because the response to acetazolamide in our exper- iments was not consistent, we conclude that, in the stingray alkaline gland, either higher concentrations of acetazol- amide are required to reduce the Isc, or bicarbonate secre- tion is not completely dependent on the presence of CAH. We chose Hansson's technique (Hansson, 1968) to local- ize CAH after indirect immunoperoxidase staining methods failed. Antibodies to mammalian carbonic anhydrase I and II failed to recognize stingray carbonic anhydrase, which has significant structural and kinetic differences from forms found in higher vertebrates (Maynard and Coleman, 1971; Maren, 1980b). The presence of CAH in the intercellular space of epi- thelial cells has been demonstrated not only in the alkaline gland in the present study, but also in other tissues such as the gall bladder, duodenum, and sweat gland (Hansson, 1968), as well as in the teleost opercular epithelium (Lacy, 1983b) and the elasmobranch rectal gland (Lacy. 1983a). This subcellular site may indicate the presence of either a membrane-bound or soluble form of CAH (Maren. 1980a). The exclusion of CAH activity from portions of the plasma membrane that contact the basement membrane suggests that its function is important in areas of cell-cell contact. Another possibility is that a soluble form of CAH exists in the intercellular space. The mechanisms that would prevent its diffusion along the basal aspect of the cell are unknown. In any case, CAH in intercellular spaces suggests that a bicarbonate reservoir may exist between epithelial cells (Lacy, 1983a) or that membrane-bound CAH may transport carbon dioxide, protons, or bicarbonate into or out of the cell (Enns. 1967; Wistrand. 1984). The exclusion of CAH from the apical region of the alkaline gland epithelial cells shown in the present study has been demonstrated in mitochondria-rich cells of the turtle bladder and interfoveolar epithelial cells of the rat stomach, both of which are thought to subserve bicarbonate secretion (Sugai and Ho. 1980; Fritsche et al.. I991a). A pattern similar to that seen in the alkaline gland was displayed in microvillated cells and microplicated cells under conditions inhibiting acid secretion (Fritsche et al., I991b). The difference in distribution pattern and stain develop- ment of CAH in the alkaline gland may reflect the presence of at least two carbonic anhydrase isozymes (Carter and Parsons. 1971 ). The appearance of CAH in the intercellular space after relatively short incubation periods may indicate a high-affinity membrane-bound carbonic anhydrase isozyme. A low-affinity cytoplasmic form of carbonic an- hydrase in the stingray alkaline gland is suggested by the longer incubation periods necessary for intracellular stain development. Acknowledgments This work was supported, in part, by the Slocum-Lunz Foundation (GMG), National Science Foundation (ERL # DCB 8903369), and the University Research Council, Med- ical University of South Carolina. Literature Cited Bancroft, J. D., and H. C. Cook. 1984. Pigments. Pages 144-158 in Manual of Histnlogical Techniques. Churchill Livingstone, New York. Borcea, I. 1906. Recherches sur la systeme urogenital des Elasmo- branches. Arch. Zoo/. Exper. 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KRUG1 * AND ADRIAN A E. MANZI2 ' Department of Biology, University1 of California, Box 95 J 606, Los Angeles, California 90095- ] 606; and 2 Cytel Corp., 9393 Towne Center Drive, San Diego, California 92121-3016 Abstract. Larvae of the specialist marine herbivore Alde- ria modesta (Opisthobranchia: Ascoglossa) metamorphose in response to a chemical settlement cue from the alga Vaucheria longicaulis, the obligate adult prey. Bioactivity coeluted with both high and low molecular weight carbo- hydrates in solution, and with insoluble high molecular weight carbohydrates associated with the algal cell wall. Larvae metamorphosed in response to water conditioned by V. longicaulis, as well as to frozen and homogenized algal tissue. The inducer was efficiently extracted from the algae with boiling water, but after all soluble activity was ex- tracted, residual tissue still induced larval settlement. Etha- nol precipitation of a boiled-water extract followed by gel filtration chromatography showed that the precipitate con- tained carbohydrates of > 100, 000 Da molecular weight, while the supernatant contained only low molecular weight carbohydrates (<2,000 Da); in both cases all activity was associated with the carbohydrate peak. An aqueous-insolu- ble 4% NaOH extract was chromatographed in 7 M urea to yield a bioactive high molecular weight carbohydrate peak. Activity was not affected by proteinase K or mild acid hydrolysis, but was significantly decreased by periodate treatment. The results indicate that larvae of A. modesta metamorphose in response to both water-soluble and sur- face-associated carbohydrates of V. longicaulis, and that the soluble cue exists as both high and low molecular weight isoforms. Received 3 March 1999; accepted 1 June 1999. * To whom correspondence should be addressed. E-mail: pkrug@ biology.ucla.edu Abbreviations: BVE = boiled Vaiicheriu extract. Introduction Most marine invertebrate species produce free-swimming larvae that disperse in the plankton until becoming compe- tent to settle to the bottom and metamorphose into the adult form (Grahame and Branch, 1985; Levin and Bridges, 1995). Larval recruitment plays a critical role in benthic marine ecosystems, structuring communities and regulating population dynamics (Grosberg, 1982; Roughgarden et al., 1988; Underwood and Fairweather, 1989). Microscopic lar- vae are generally viewed as passive particles transported by flow to the benthos (Eckman, 1983. 1990; Butman, 1987). Following hydrodynamic delivery of larvae to the bottom, recruitment can be divided into settlement and metamor- phosis (Chia and Koss, 1988; Pawlik, 1992). Settlement is characterized by active behaviors with which larvae explore the physical and chemical characteristics of potential sub- strata (LeTourneux and Bourget, 1988; Rodriguez et al., 1993). Larvae may reject a substrate and resume swimming, becoming resuspended in the water column (Butman et al., 1988; Butman and Grassle, 1992). Alternatively, larvae may respond to surface-associated cues and commit to metamor- phosis, an irreversible developmental transformation into the adult stage of the organism (Burke, 1983; Pawlik et al., 1991; Roberts et al., 1991; Pawlik, 1992). Larvae are capa- ble of fine-scale discrimination among substrata both in the laboratory and in the field (Keough and Downes, 1982; Raimondi, 1988). Recent studies have demonstrated that both surface-asso- ciated and water-soluble chemical cues can trigger larval behavioral responses that greatly increase rates of settle- ment and metamorphosis. Still-water laboratory assays have demonstrated the importance of surface-associated chemical cues for inducing larval metamorphosis of barnacles (Maki 94 CARBOHYDRATE SETTLEMENT CUES 95 et til., 1990), bryozoans (Hurlbut, 1991), corals (Morse et al., 1988), gastropods (Morse et nl., 1984), and polychaetes (Kirchman et al.. 1982). Hydrodynamic conditions and the presence of a surface cue associated with adult conspecifics had an interactive effect on settlement of larvae of the reef-building polychaete Phragmatopoma califomica in flow (Pawlik et al., 1991). Waterborne chemical cues also affect larval settlement processes. Soluble cues secreted by the adult prey organisms induced settlement and metamor- phosis in the opisthobranchs Pliestilla sibogae and Adalaria proximo (Hadfield and Scheuer, 1985; Lambert and Todd, 1994). Larvae of the oyster Crassostrea virginica showed dramatic behavioral responses to a chemical cue secreted by adult conspecifics, increasing settlement in both still and moving water (Tamburri et al., 1992; Turner et al., 1994; Tamburri et al.. 1996). However, despite decades of re- search into the nature of larval chemical settlement cues, relatively little is known about the molecules that regulate this crucial aspect of the life history of most benthic marine invertebrates. A recent study of a population of the opisthobranch mollusc Alderia modesta revealed several unusual features that make A. modesta an ideal experimental system for investigating larval life history and settlement processes (Krug, 1998a, b). A. modesta is an ascoglossan found in temperate estuaries in association with its obligate food source, the yellow-green alga Vaucheria longicaulis (Xan- thophyta: Xanthophyceae) (Hartog and Swennen, 1952; Hartog, 1959; Trowbridge, 1993). In southern California, A. modesta exhibits a reproductive polymorphism that is ex- tremely rare among marine invertebrates; study populations contain specimens that produce planktotrophic larvae and other individuals that produce lecithotrophic larvae (Krug, 1998b). Most lecithotrophic spawn masses contain a mix- ture of sibling larvae, some of which metamorphose spon- taneously within 2 days of hatching; the remaining veligers delay metamorphosis until encountering a chemical cue derived exclusively from the adult host alga V. longicaulis (Krug, 1998a). The present work used a bioassay for larval metamorphosis to determine whether the inductive activity was soluble or surface-associated in nature, and for bioas- say-guided isolation of active fractions as a preliminary step in purifying the settlement cue. Materials and Methods Collection of organisms and lan'al bioassay Alderia modesta (Loven, 1844) and Vaucheria longi- caulis were collected from mudflats in the Kendall-Frost Marine Reserve and Northern Wildlife Preserve, and in the San Diego River Flood Control Channel, San Diego, Cali- fornia, U.S.A. All algae used in this study conformed to published descriptions of V. longicaulis from California (Abbott and Hollenberg, 1976). Patches of V. longicaulis were grown under continuous lighting in the laboratory, and blades of algae were pulled free of the sediment base and rinsed in seawater before use in assays. Adult specimens of A. modesta were maintained in petri dishes under 1 cm of seawater. and lecithotrophic egg masses were harvested daily for 3 days. Egg masses from each day were pooled and maintained in 0.45 jam-filtered seawater (FSW); water was changed every other day until hatching. Upon hatching, larvae were maintained in FSW for 2 days, to allow spon- taneous metamorphosis to occur in cue-independent larvae (Krug. 1998a). The remaining larvae were then subsampled for use in the bioassay. For each experimental treatment, 1 5 larvae were added to each of 3 replicate dishes containing 4 ml FSW. After 2 days, larvae were scored for metamorpho- sis. Each experiment included a FSW-only treatment as a negative control and live V. longicaulis as a positive control. The percentage of metamorphosis for each replicate was arcsine transformed, and treatments were compared using a 1-way ANOVA. Unplanned comparisons of means were done using the Scheffe procedure (Day and Quinn, 1989). Secretion of settlement cue An experiment was designed to determine whether the Vaucheria-denved settlement cue was surface-associated or secreted by the algae. Small patches (1 cm2) of V. longi- caulis were cut from a growing mat and left attached to the sediment base. Conditioned seawater (CSW) was made by placing a patch in 4 ml FSW for either 3 h or 24 h, after which the CSW was filtered through cotton and placed in a sterile petri dish; larvae were added directly to the CSW for the bioassay. Conditioned fresh water (CFW) was made by placing patches of V. longicaulis in 4 ml deionized water for 24 h. The CFW was filtered through cotton, dried on a rotary evaporator, and resuspended in an equivalent volume of FSW for use in the bioassay. The negative control was FSW aged 24 h and filtered through cotton in parallel with treatements; the positive control was live V. longicaulis tissue. To determine whether Vaucheria longicaulis must be alive to trigger metamorphosis, pieces of the algae were frozen at -20°C for 3 days. Frozen patches were thawed by immersion in FSW at room temperature for 1 h prior to use in the bioassay. To determine whether algal tissue must be physically intact, blades of live V. longicaulis were pulled free of a 2 cm2 sediment base and washed in FSW. The algae was manually homogenized in 10 ml deionized water for 20 min. and the suspension sonicated for another 10 min. The homogenate was centrifuged ( 10 min, 2000 RPM) and the supernatant removed. The soluble homogenate was as- sayed by adding 200 /Ltl (high concentration) or 30 ju.1 (low concentration) aliquots to 4 ml FSW for use in the bioassay. The negative control was FSW, and the positive control was live intact V. longicaulis tissue. 96 P. J. KRUG AND A E. MANZI Sequential extraction with boiling water Four 20 X 20 cm mats of Vaucheria longicaulis attached to the natural sediment base were field collected (March 1997) and grown in the laboratory under continuous light- ing, moistened daily with 50% seawater. After 2 weeks algal blades had grown 1-2 cm in height, and were har- vested by cutting with dissecting scissors just above the sediment base. The V. longicaulis tissue (1.34 g wet weight) was placed in a beaker containing 50 ml deionized water and boiled for 10 min. The solution of boiled Vaucheria extract (BVE) was filtered through 100 /j,m Nitex mesh to remove Vaucheria residue, and then through a 0.45 ;u,m filter membrane. The Vaucheria residue was collected off of the mesh filter, put in 50 ml of fresh deionized water, and again boiled for 10 min to generate a second extract. This process was repeated four more times, yielding a total of six sequential boiling water extracts. The Vaucheria residue remaining after the sixth extraction was collected from the filter; this residue was yellow-brown in coloration but the blades were still physically intact. Each of the six extracts was assayed by adding a 50 jid aliquot to 4 ml FSW per replicate assay dish. Pieces of live V. longicaulis were assayed as a positive control, and equivalently sized pieces of the V. longicaulis residue remaining after the six sequen- tial extractions were also assayed. Biochemical characterization of boiled Vaucheria longicaulis extract (BVE) The initial extract made by boiling Vaucheria longicaulis for 10 min (described above) was subjected to preliminary biochemical characterization. Six volumes of ethanol were added to 1 ml of BVE and the solution was precipitated overnight at 4°C. The precipitate was pelleted by centrifu- gation, the supernatant removed, and the precipitate washed with ethanol and repelleted. The supernatant and wash eth- anol were combined and dried on a rotary evaporator. The precipitate and supernatant residue were individually resus- pended in 1 ml of MilliQ-purified water, such that the material in each fraction was present in solution at the same concentration as in the original extract. Aliquots (100 ju,l) of the initial BVE and of the resus- pended solutions of supernatant and precipitate were used in subsequent assays to determine the dry weight, carbohy- drate content, protein content, and bioactivity of each sam- ple. Lyophilized aliquots were weighed to determine dry mass. Carbohydrate content was determined for duplicate aliquots from each sample using the phenol-sulphuric col- orimetric assay (DuBois et al.. 1956). Measurements were calibrated to a standard curve generated with known con- centrations of glucose. Protein content was determined us- ing the BCA colorimetric assay (Pierce Co.) calibrated to a standard curve generated with commercially supplied albu- min standards. Bioactivity was determined using the larval settlement bioassay. Another 3 ml of BVE was precipitated with 6 volumes of ethanol overnight, and the supernatant and precipitated ma- terial were separated as before. The carbohydrate elution profiles of both the supernatant and precipitate fractions were determined using a gel filtration column (90 cm X 1 cm) of Sephacryl S-200 resin (Pharmacia Co.). The column was calibrated for molecular weight using Blue Dextran to determine the void volume (V0) and glucose to determine the included volume (V,) for small molecules; size stan- dards were detected in fractions after collection visually (Blue Dextran) or by the phenol-sulphuric colorimetric as- say (glucose). The supernatant residue was dissolved in a minimal volume and loaded onto the column, eluting with MilliQ-purified water at a flow rate of 6 ml/h and collecting 0.5 ml fractions. Aliquots were taken from each fraction and analyzed for carbohydrate content by the phenol-sulphuric colorimetric assay and for protein content by the BCA assay; the detection limit for both colorimetric assays was 0.5 /j,g/ml. Based on the resulting carbohydrate elution profile, fractions representing every 8 ml were pooled and lyophilized to give 5 total fractions spanning the void vol- ume and included volume. Each pooled fraction was dis- solved in water and 150 ^il aliquots were bioassayed. The precipitated fraction was chromatographed in an identical manner and fractions were collected, assayed for carbohy- drate content, and pooled to give five total fractions. Each pooled fraction was dissolved in water and 75 jul aliquots were bioassayed. A positive control using live Vaucheria longicaulis induced 84 ± 10% metamorphosis, while a negative control using FSW gave 4 ± 4% background metamorphosis. Sequential extraction of Vaucheria longicaulis with solvents of increasing polarity To determine whether macromolecules associated with the algal cell wall were bioactive, Vaucheria longicaulis was sequentially extracted with solvents of increasing po- larity and harshness to extract molecules of increasing mo- lecular weight. Lyophilized Vaucheria longicaulis (500 mg) was homogenized into a fine powder and extracted with 80% aqueous ethanol (50 ml, 7 h, 75°C), cold water (50 ml, 4 d, 20°C), hot water (50 ml, 24 h, 65°C), and 4% sodium hydroxide (50 ml. 24 h, 20°C) (Cleare and Percival, 1972). The ethanol extract was partitioned into a water-soluble fraction and a water-insoluble organic fraction. The cold and hot water extracts were precipitated with ethanol as before to generate supernatant and precipitate fractions for each extract. Aliquots corresponding to 250 /ng dry weight were taken from the water-soluble ethanol extract and from the cold and hot water supernatant and precipitate fractions and were assayed directly for bioactivity. An aliquot of the CARBOHYDRATE SETTLEMENT CUES 97 organic-soluble material from the ethanol extract was dis- solved in methanol, added to a dry culture dish, the solvent evaporated, and 4 ml of FSW added prior to the bioassay. The 4% NaOH extract was exhaustively dialyzed against MilliQ-purified water and lyophilized, giving a dry material (44 mg) that was completely insoluble in water but dis- solved readily in 7 M urea. The S-200 Sephacryl column was equilibrated in 7 M urea and calibrated for V,, and V, as before. A portion of the 4% NaOH extract was dissolved in a minimal volume of 7 M urea and loaded onto the S-200 column. The sample was chromatographed and fractions were collected and assayed exactly as before, except the column was eluted with 7 M urea. Fractions comprising the high molecular weight carbohydrate peak were pooled and dialyzed exhaustively against water using 10.000 molecular weight cutoff dialysis tubing. The dialysate was reduced to a volume of 1 ml on a rotary evaporator and 100 ;ul aliquots were bioassayed. Treatment of EVE with proteinase K, sodium periodate, and mild acid hydrolysis Chemical and enzymatic treatments were performed to determine the biochemical nature of the settlement cue. A solution of sodium periodate (0.37 M, 100 jal) was added to 1 .0 ml of BVE, and the solution was incubated at 4°C in the dark (Hassid and Abraham, 1957). The reaction was quenched after 24 h by the addition of excess glycerol (20 ;u.l ). As a control, 1 .0 ml of B VE was incubated at 4°C in the dark for 24 h, after which excess glycerol (20 /il) was added followed immediately by periodate as in the treated sample. Both samples were incubated for 1 h to allow the consump- tion of excess periodate, and were then dialyzed exhaus- tively against deionized water for one week. Both treatment and control samples were lyophilized, dissolved in 300 p.\ FSW, and 100 jul aliquots used as replicate treatments in the larval settlement bioassay. Proteinase K (600 /j,g) was added to a sample of BVE (300 /ill) which had been adjusted to pH 7.8 and incubated at 50°C for 24 h. The proteinase was then inactivated by heating at 100°C for 15 min. A control was done by adding proteinase to BVE immediately prior to heating at 100°C for 15 min. Samples were split into three replicate 100 pil aliquots and tested in the larval bioassay. A mild acid hydrolysis was performed by adding concentrated TEA ( 1 .5 /j.1) to BVE (400 fil) to achieve a final concentration of 0. 1 M TFA. The solution was heated at 100°C for 75 min (Lahaye and Ray, 1996) and dried under vacuum to remove TFA. As control for the presence of residual TFA salts. BVE (400 jul) was heated in parallel at 100°C for 15 min. and concentrated TFA was added to BVE immediately prior to drying under vacuum. Samples were dissolved in 300 jiil FSW, and 100 ju.1 aliquots used as replicate treatments in the bioassay. Differences between treatment and control sam- ples were compared using an unpaired two-tailed t-test on arcsine-transformed percentages for each of the three treat- ments, as different quantities of BVE were treated and bioassayed in each case. Results Secreted and surface-associated forms of the larval settlement cue Previous work had demonstrated that Alderia modesta larvae metamorphosed specifically in response to living tissue of Vauchcrid Innxicunlis (Krug, 1998a). The initial aim of the present study was to determine whether the settlement cue was secreted into seawater by living algae, and whether dead or homogenized algal tissue could induce settlement. Water previously conditioned by the presence of V. longicaiilis was as active in promoting metamorphosis as was the living algae (Fig. 1A, and results of a 1-way ANOVA: df = 4. 22; F = 32.73; P < 0.0001). The 120, 100 BO 40 . r1 livc lanchena CSW(3hl CSW(24h) CFW(24h) live I auciiena dead intact homogenate homogenate FSW I titiL-lit'na (high cone ) (low cone) Figure 1. Induction of larval metamorphosis by live Vaiicheria longi- caiilis, dead tissue, and conditioned water. Percentages of larval metamor- phosis are given as means + SD (n = 3); arcsine-transformed percentages were compared with a 1-way ANOVA, with a post-hoc Scheffe test for unplanned comparisons. Live V. longicaiilis tissue was used as a positive control and filtered sea water (FSW) as a negative control A. Secretion of larval settlement cue by living V. longicaulis. Means are percentages of metamorphosis induced by exposure to Wwc/ima-conditioned seawater (CSW) or conditioned fresh water (CFW). Duration of conditioning pro- cess is given in parentheses- Means not joined by a horizontal line differed significantly (P < 0.001 ). B. Inductive effect of dead or homogenized V. l«n<;ictiulis. Previously frozen and thawed Vaucheria tissue, or aliquots of homogenized algal tissue, were assayed for inductive effect. Means not joined by a horizontal line differed significantly (P < 0.0?l. 98 P. J. KRUG AND A. E. MANZI conditioning process occurred rapidly in the laboratory, such that water conditioned for 3 h induced the same level of metamorphosis as water conditioned for 24 h. Fresh water was also conditioned by the presence of V. longicaulis (Fig. 1A). There was no statistical difference between the level of metamorphosis induced by the living algae and any of the conditioned water treatments, all of which differed significantly from the seawater-only control (Scheffe test. P < 0.001). Vaucheria longicaulis tissue that was frozen and thawed induced significant larval metamorphosis, indicating that the algae does not have to be alive to trigger settlement (Fig. IB. and results of a 1-way ANOVA: df = 4, 16; F = 61.55; P < 0.0001 ). Homogenates of algal tissue were also active, confirming that V. longicaulis tissue does not have to be alive or intact to induce metamorphosis (Fig. IB). Signifi- cantly higher levels of metamorphosis were induced by frozen V. longicaulis and the higher concentration of tissue homogenate than by the negative control (Scheffe test, P < 0.05). The lower concentration of homogenate did not in- duce significantly more metamorphosis than the negative control, indicating that the larvae may be dose-responsive to preparations of the cue; dilution experiments with condi- tioned seawater support this conclusion (data not shown). When Vaucheria longicaulis was extracted with boiling water, the resulting aqueous extract was as active as positive controls when assayed at an 80-fold dilution (Fig. 2, and results of a 1-way ANOVA: df = 8, 18; F = 20.45; P < 0.0001). Conditioned seawater had no effect at such a dilution, indicating that boiling water extracted the settle- ment cue more efficiently than did the conditioning process. When the V. longicaulis tissue was re-extracted with boiling water for a second time, the resulting extract induced a low level of metamorphosis, but not significantly more than the negative control when assayed at an 80-fold dilution (Fig. % metamorphosis Ml 0 - 1st - 2nd ' 2,5 5,0 7,5 H I—1 1 ^-.b.c sequential extract 3rd - 4th ' ]"< c 5th ' c ~~ 6th ' ]- extracted residue - 3 ,a,b FSW ' h« Figure 2. Serial extraction of Vaucheria longicaulis with boiling wa- ter. Means + SD (n = 3) are percentages of larval metamorphosis induced by aliquots of 6 sequential boiling water extracts, tested at an 80-fold dilution, along with the fully extracted algal residue. Live V. longicaulis was used as a positive control, and FSW as a negative control. Means not identified with the same letter differed significantly (P < 0.05, 1-way ANOVA with a post-hoc Scheffe comparison). 2). Four further extractions with boiling water yielded ex- tracts that contained no appreciable bioactivity, even when assayed at higher concentrations. These data indicate that all of the measurable bioactivity was extracted from V. longi- caulis in the first two boiling water treatments. The insolu- ble residue remaining after six sequential extractions had thus been exhaustively extracted. However, larvae exposed to this residue metamorphosed at a level comparable to those exposed to living V. longicaulis (Fig. 2). Significant bioactivity thus remained associated with the Vaucheria cell wall residue after all the soluble settlement cue had been extracted. High and low molecular weight forms of the soluble settlement cue Boiled Vaucheria extract (BVE) was fractionated by eth- anol precipitation into a supernatant and precipitate, each of which was diluted back up to the starting volume of BVE for comparison. Biochemical analysis revealed that the car- bohydrate content of BVE partitioned equally between the precipitate and supernatant, while the majority of the protein in the crude BVE went into the ethanol precipitate (Table I). There was no significant difference between the bioactivity in 100 ju.1 of precipitate, supernatant, and BVE (1-way ANOVA. P > 0.3), although the supernatant consistently displayed slightly lower activity at several concentrations tested. Both the ethanol precipitate and supernatant were further fractionated by gel filtration chromatography on a Sephacryl S-200 column. When column fractions were assayed for carbohydrate content, contrasting elution profiles were ob- tained for the two samples (Fig. 3). All detectable carbohy- drate from the supernatant fraction eluted in the included volume of the column, indicating a molecular weight of <2,000 Da. In contrast, when the precipitate was chromato- graphed, all detectable carbohydrate eluted as one peal; in the void volume, indicating molecules of > 100, 000 Da molecular weight. When fractions were pooled and bioas- sayed, there was significant variation in the bioactivity of different fractions (Fig. 3, and results of a 1-way ANOVA: df = 11, 24; F = 17.33; P < 0.0001). For the precipitate, a high level of metamorphosis (54 ± 23% SD) was induced by the pooled fractions containing the high molecular weight carbohydrate peak, and a lower level was induced by the adjacent fraction containing the trailing edge of the peak. The level of metamorphosis induced by the high molecular weight peak was not statistically different from that induced by the positive control (Scheffe test, P = 0.20) but was significantly higher than the negative control (Scheffe test, P < 0.05). No bioactivity significantly higher than the negative control (4 ± 4%) was detected in the low molecular weight fractions from the ethanol precipitate. The bioactivity profile of the ethanol supernatant gave the op- CARBOHYDRATE SETTLEMENT CUES 99 Table I Comparative dry weight, protein content, carbohydrate content, and bioactivity (±SD) of 100 /J aliquots of a standard solution of boiled Vaucheria extract (BVE) and the precipitate and supernatant resulting from ethanol treatment of BVE. The precipitate and supernatant were dissolved in the starting volume of extract and aliquots were removed for chemical assays (n = 2) and bioassays (n = 3) Dry Weight Carbohydrate Protein Bioactivity Sample C/ug) <^g) (Mg> (%) BVE 270 ± 10 6 ± 1 25 ± 1 82 ± 25 supernatant 110 ± 10 4 ± 1 6 ± 1 49 ± 4 precipitate 140 ± 10 3 ± 1 17 ± 1 77 ± 21 posite result. The low molecular weight fraction of the supernatant, which contained all the carbohydrate, induced a level of metamorphosis that was not significantly different from the high molecular weight carbohydrate peak from the precipitate (Scheffe test, P = 0.79). No other fraction from the supernatant induced significant metamorphosis. Bioac- tivity thus co-eluted with the major carbohydrate peak of both the supernatant and precipitate, although the active peak from the supernatant contained only low molecular weight molecules while that from the precipitate contained molecules of high molecular weight. Identical carbohydrate I? 01 supernatant \ precipitate v, t *Ay^ . A -A ?0 40 50 60 70 30 40 50 60 70 Figure 3. Gel filtration chromatography of the supernatant and pre- cipitate from ethanol precipitation of boiled Vaucheria extract (BVE). Fractions were independently chromatographed on a size-calibrated col- umn of Sephacryl S-200 gel eluting with water. Molecules of molecular weight > 100,000 Da elute in the void volume (V0), while those of <2,000 Da elute in the included volume (V,). Column fractions (0.5 ml) were assayed for carbohydrate content by the phenol-sulphuric colonmetric assay. Fractions were pooled as indicated, lyophilized, and bioassayed for induction of larval metamorphosis. Percentages of metamorphosis are means + SD (n = 3). peak profiles were obtained when sarpples were chromato- graphed using 7 M urea as a chaotropic agent to disrupt any potential aggregation of macromolecules, and no major protein peaks were evident for either sample (data not shown). Sequential extraction of Vaucheria longicaulis Lyophilized Vaucheria longicaulis was sequentially ex- tracted with solvents of increasing harshness to determine if bioactivity was persistently associated with molecules of increasing molecular weight and stronger association with the algal cell wall. Aqueous extracts were ethanol precipi- tated to yield supernatant and precipitate fractions, and all soluble extracts were bioassayed at the same concentration per unit dry weight. The material extracted with 4% NaOH was insoluble in water but dissolved readily in 7 M urea, a chaotropic agent routinely used to solubilize and chromato- graph high molecular weight polysaccharides. One major carbohydrate peak was detected in the void volume of the S-200 column when the 4% NaOH extract was chromato- graphed with 7 M urea as eluant (Fig. 4). This carbohydrate peak was exhaustively dialyzed, and the material which remained in aqueous solution was bioassayed. There was significant variation in the bioactivity of different extracts (Fig. 5, and results of a 1-way ANOVA: df = 8, 39; F = 4.64; P < 0.0005). The water-soluble partition of an ethanol extract of V. longicaulis induced significantly higher levels of metamorphosis than the water-insoluble organic layer and the negative control (Scheffe test, P < 0.05), indicating all bioactive molecules are highly polar. Bioactivity above the level of the negative control (8 ± 8%) was found in all water-soluble extracts as well as in the resolubilized 4% NaOH extract, indicating that molecules of increasing mo- ml eluted Figure 4. Carbohydrate elution profile of 4% NaOH extract of Vaucheria longicaulis powder. Aqueous-insoluble material from the basic extraction was eluted from Sephacryl S-200 gel with the chaotropic agent 7 M urea. Fractions containing the carbohydrate peak eluting in the void volume were pooled, dialyzed, and reduced in volume before being bio- assayed. 100 P. J. KRUG AND A. E. MANZI metamorphosis live Vaucheria twiga ac metamorphosed in response to water condi- tioned with the hard coral Porites compressa (Hadfield, 1977; Hadfield and Scheuer, 1985). Larvae of the dorid nudibranch Adalaria proxima metamorphosed in seawater conditioned by the preferred adult prey, the bryozoan Elec- tro pilosa (Lambert and Todd. 1994). However, metamor- phosis of A. proxima larvae could only be induced by live colonies of E. pilosa and not by dead colonies or homoge- nized extracts (Todd ct ai. 1991 ; Lambert and Todd. 1994). In contrast, dead and homogenized V. longicaulis tissue induced metamorphosis in A. modesta. Secreted settlement cues are also involved in gregarious settlement of some species. Larvae of the sand dollars Dendraster excentricus and Echinarachinus parma meta- morphosed in response to sand beds and seawater condi- tioned by the presence of adult conspecifics (Burke, 1984; Pearce and Scheibling, 1990). The most detailed studies on the effects of a secreted chemical settlement cue have fo- cused on the oyster Crassostrea virginica. Larvae altered their swimming speed and turning rate in response to small basic peptides secreted by adult conspecifics, significantly increasing settlement in both still and moving water in response to the dissolved cue (Tamburri et ai, 1992; Turner ct ill., 1994; Tamburri el Educational Programs Summer Courses R21 Special Topics Courses R25 Other Programs R31 Summer Research Programs Principal Investigators R33 Other Research Personnel R34 Library Readers R35 Institutions Represented R36 Year-Round Research Programs R41 Honors R53 Board of Trustees and Committees R60 Administrative Support Staff R64 Members of the Corporation Life Members R67 Members R68 Associate Members R78 Certificate of Organization R82 Articles of Amendment R82 Bylaws R82 Photo credits: Beth Armstrong, R4 (bottom), R5 (bottom). R7. R2I, R33. R67 Jelle Ateina, R45 Ken Foreman, R32 Linda Colder, R24, R64 Roger Hanlon, R60 Diedtra Henderson, R47 Jan Hinsch. R53 Richard Howard, R2(top), R4(top), R5(top) Alan Ku/.irian, R2(bottom), R34. R49. R50 Lisa Ken- Lobel, Rl Chris Pauk, R22 P.A. Shave, RS2 James Shreeve, R35 Sam Sweezy. R5 1 Report of the Director and Chief Executive Officer The Marine Biological Laboratory remains a remarkable place as we approach the end of the 20th Century. At every turn there are feelings of pride and satisfaction, of excitement, curiosity, determination and anticipation of things to be discovered. These feelings are shared by both resident and visiting scientists and by students for whom time spent at MBL is an experience never to be matched. That spirit of scientific adventure and achievement is alive and thriving here, as it has been for more than a century. The MBL continues to build on its solid history, to add programs in research and education, to recruit new scientists and to raise funds for vital improvements to this place that is like no other. After establishing research and education priorities, we were able to define funding requirements and a timeframe enabling us in August of 1997 to launch Discovery: The Campaign for Science at the Marine Biological Laboratory. The goal is to reach $25 million by December 31, 2000. We are gratified by the response to this fundraising effort and are grateful to many of you who have already made generous contributions to this Campaign. I'm pleased to say that, by the end of 1998, we had raised $20.6 million, which is good news indeed. Education at the MBL The MBL's education program is growing both in numbers of students and faculty and in courses offered. During the summer of 1998 we hosted 594 faculty for 416 students from around the world. We were able to award more than $600,000 in scholarship support for those students, making it possible for the best and the brightest to continue to come to the MBL. Even as we grow, we have retained the high quality, intensive courses that have long set the MBL apart from other educational institutions. As Purnell Choppin, president of the Howard Hughes Medical Institute, stated in announcing a $2.2 million award to support education at the MBL in April of 1999, "The Marine Biological Laboratory serves as an international schoolhouse for the biomedical research community. Young scientists and established researchers alike gather there to learn the latest developments at the cutting edges of their fields." In 1998 we continued to attract international students with over 307c of our applications from students from 68 different foreign countries. We take great pride in maintaining the quality and dynamics of the courses and continue to be responsive to the changing face of biological research as demonstrated by our ability and interest in adding new courses to our roster of exceptional offerings. Two new courses were introduced in 1998: Frontiers in Reproduction: Molecular and Cellular Concepts and Neural Development and Genetics of Zebrafish. These were in addition to the Molecular Mycology: Current Approaches to Fungal Pathogenesis course and the Semester in Environmental Sciences, both of which were offered for the first time in 1997. Not only have we added new courses, but we have continued to change our long-standing courses through the planned turnover in course directors. For example, in 1999 David Garbers and Randy Reed will be the co- directors of the over 100-year-old Physiology course. They will succeed Kerry Bloom and Mark Moosekar who did a superb job in leading the course for the past four years. Our Semester in Environmental Sciences program was a great success again this year. Undergraduate students selected from a consortium of 34 liberal arts colleges were in residence for 14 weeks during the fall to learn about environmental sciences. The curriculum covered aquatic and terrestrial ecosystems and included electives in computational modeling and microbial ecology. Students gained a basic understanding of ecosystem structure and dynamics through intensive hands-on fieldwork at two local sites on Cape Cod. Major biogeochemical processes were studied and general problems concerning the global carbon cycle, fossil fuel emissions, increased concentrations of greenhouse gases Rl R2 Annual Report in the atmosphere, estuarine eutrophication, deforestation, and over-exploitation of fisheries were considered. Special emphasis was given to how changes in biodiversity affect ecosystem function. The MBL's Science Writing Fellowships Program, now about to enter its fourteenth summer, added a new hands- on laboratory course in environmental science during the summer of 1998. Co-directed by John Hobble and Jerry Melillo of the Ecosystems Center, this new component of the program was a great success, attracting environment writers from around the country. Research at the MBL The Marine Resources Center While John Glenn was the most famous traveler in space late last fall, two other passengers aboard the shuttle were of considerable importance to scientific experiments conducted during that mission. Two oyster toad fish participated in an experiment overseen by Steve Highstein that was designed to provide a better understanding of the effects of microgravity on our balance system. The fish, collected from the waters off Woods Hole, traveled more than three million miles in what was a follow-up to studies conducted during the Neurolab space mission in April of 1998. Balance, location and movement are so crucial to animals that the vestibular system was one of the first sensory systems to evolve. The toadfish has become a well-known experimental model for learning more about balance disorders, such as Meniere's disease and vertigo. It also is a good model for studying motion sickness, including that experienced by astronauts during space flight. Thanks to a $1 million challenge grant, the MBL has an exciting opportunity to build on its existing strengths as a developer of aquatic models for biomedical research. The technologically sophisticated Marine Resources Center is an ideal venue for this program. And MRC Director Roger Hanlon's expertise in the culturing of marine organisms such as Hawaiian squid and cuttlefish provides a great foundation for the expansion of aquaculture activities at MBL. Dr. Hanlon contributed his expertise in this area as a member of a National Research Council/National Academy of Sciences committee that published in 1998 a report titled "Biomedical Models and Resources: Current Needs and Future Opportunities." This paper is expected to help the National Institutes of Health structure research funding for model organisms, including many aquatic ones. The MRC challenge grant, which stipulates that two dollars must be raised for every one dollar awarded, will enable the MBL to establish a scientific aquaculture program in the Marine Resources Center. This exceptional gift will allow scientists to develop novel research techniques and to address problems being faced by scientific and commercial aquaculture interests alike. Studies will address problems such as disease diagnosis and management, water quality requirements for specific life stages, nutrition research for optimal diets and numerous aspects of reproductive biology. For many Report of the Director and CKO R3 years, commercial aquaculture companies have sought the MBL's expertise in addressing all of these issues. In recent years, we successfully maintained 95,000 juvenile flounder bound for the Japanese sushi industry and raised seedling scallops for the local shellfish trade. Now we will be in an even better position to provide advice and develop appropriate aquaculture techniques in the future. The Ecos\stems Center The Ecosystems Center recently launched a new tropical ecology program that focuses on the consequences of land-cover and land-use changes in the tropics. The possibility of a new joint research project with Brazilian scientists is being explored. The program is based on a challenge put to ecologists: "Now that you think you know how ecosystems work, why don't you try to fix some broken ones?" Perhaps we can test our understanding of ecosystem structure by working to rebuild a damaged one. The joint project would focus on large tracts of coastal forests northeast of Sao Paulo. The Ecosystems Center also received the only Long- Term Ecological Research Site award made in 1998. The MBL is now the only place in the country responsible for the oversight of two LTER sites — the new one at Plum Island Sound, located north of Boston, and the long-time Arctic Toolik Lake site, located on the North Slope of the Brooks Range in Alaska — and which has major involvement in a third (Harvard Forest in Petersham. Massachusetts). All of this research activity has resulted in remarkable growth over the past few years. Since 1979, Center staff has increased sixfold. The resulting demand for additional laboratories, offices, and staging areas for equipment and supplies used in field research has led to a severe shortage of space. And the MBL's new Semester in Environmental Sciences program for undergraduate liberal arts students is putting an additional squeeze on the Center's already over-taxed facilities. In November, the MBL Board of Trustees approved the architectural plans for a new facility to house research and education activities of the Ecosystems Center. The proposed three-story building will provide a cutting-edge GIS (geographic information systems) facility, state-of- the-art laboratories for plant and soil sample analysis, a stable isotope laboratory, modern offices, teaching facilities and a classroom/conference room for the Semester in Environmental Science program, ample storage areas for diving gear, field samples, and equipment, and field staging areas. The 32,000-square- foot building is designed to meet the needs of Ecosystems Center scientists for many years to come, as well as serve the needs of the entire MBL research and education programs. Fundraising is now underway, with a much appreciated $1 million challenge grant from the Clowes Fund leading the way. With groundbreaking scheduled for the spring of 2000, this new state-of-the-art Environmental Sciences Building will be a fitting tribute to a quarter century of excellence in ecological research and provide the foundation for continued scientific achievements as the MBL enters the 21st Century. The Josephine Buy Paul Center Under the direction of MBL Senior Scientist Dr. Mitchell Sogin, the Josephine Bay Paul Center for Comparative Molecular Biology and Evolution, dedicated in August of 1998, is flourishing. The research pace at the Center escalated during the past year, thanks to the arrival of a number of scientists and the receipt of several important grant awards. Early in 1998, the Center received a major grant from the National Institutes of Health, to be used in an important research initiative to sequence the genome of the parasitic protist, Giurtlia Unnblia. Giardia is a waterborne human pathogen that attacks the intestinal tract and exacts a terrible toll on public health worldwide. The NIH grant will provide salary support for nine scientists and technicians and has allowed us to establish a new automated DNA sequencing facility. There was still more exciting news at the Bay Paul Center in 1998, when NASA selected MBL as a member of the new Virtual Astrobiology Institute. This program will bring together astrophysicists, biologists, chemists, physicists, planetologists, and geologists for interdisciplinary studies on life in the universe and its cosmic implications. The MBL was one of 1 1 institutions selected to participate from a field of nearly 70 applicants. Dr. Michael Cummings joined the Bay Paul Center in R4 Annual Report early 1998 as an Assistant Scientist. His work is in the field of molecular evolutionary genetics. The major focus of that research is using novel statistical methods to study relationships between genotype and phenotype. Current investigations examine how gene sequence data can be used to understand and predict drug resistance in tuberculosis, variation in color vision, and basic immune system functions at the molecular level. Dr. Cummings is also studying the evolution of pathogenic bacteria by examining species within the genus Mycobacterium. The analysis of Mycobacterium DNA sequence data will reveal evolutionary patterns that demonstrate the emergence of both new pathogens and drug resistant strains. This information will assist clinicians with diagnosis and treatment of diseases such as tuberculosis and leprosy. Other Research Initiatives The MBL is home not only to the above centers, but to a number of individual laboratories where, for example, the basis of bioluminescence is being investigated, the fluxes of ions from individual cells are being measured, the evolution of heme biosynthesis is being traced, new antibiotics are being sought, and microscopy is being developed and used to understand more about the cell. A remodeled and expanded laboratory is serving Dr. Carol Reinisch, a recently appointed Senior Scientist at the MBL and a new year-round resident. She investigates how environmental factors influence the prevalence of leukemia using soft shell clams as a research model. She also studies surf clams to better understand how toxins such as PCBs disrupt nerve development in embryos that later influences normal learning and behavior. Drs. Barbara and Bruce Furie have modernized their MBL laboratory to accommodate ongoing work on the study of hemophilia and other blood disorders using the venomous cone snail. The conotoxins produced by these invertebrate snails share an amino acid that is found in mammals. A protein containing this unusual amino acid, when linked to vitamin K. triggers blood-clotting mechanisms that are distributed widely throughout mammalian species. Summer Research The MBL — as it has for more than a century — will host hundreds of scientists from around the world who come each summer to the Laboratory to participate in a unique and intense research experience. Often using marine and aquatic model organisms, these investigators study basic processes in the life sciences. Their work spans research on the protein assemblies that achieve accurate chromosome segregation in cell division, on the neural processing of visual information in the brain, and on how hormones and Pharmaceuticals stimulate the secretion of insulin from the pancreas. The MBL's Fellowship Program is an important element of summer research activities. Nineteen scientists were awarded summer research fellowships at the MBL in 1998. Examples of research projects by neurobiologists Report of the Director and CEO R5 — • include studies by Dr. Elizabeth Jonas of Yale University School of Medicine on the intracellular channels that regulate synaptic function; studies by Dr. Matthew Halstead of the University of New Zealand on the processing of electrosensory information in the midbrain of the skate; and studies by Dr. James Zheng from the Robert Wood Johnson Medical School on the cellular mechanisms underlying the formation of nerve connections. Cell biologists included Dr. Mark Alliegro of Louisiana State University, who studied cells from sea urchins and other organisms to learn how cells differentiate, and Dr. John Eriksson of the Turku Center for Biotechnology in Finland, who studied the mitotic protein phosphatases in surf clam eggs. Fourteen other scientists whose research focused on topics ranging from global climate change to sensory physiology rounded out the group of 1998 fellows. Improvements Around Campus As always there is work to be done on the MBL's physical plant, all of it important, all of it requiring time. effort, and financial support. In 1998, renovations were made at the Loeb building and to the Neurobiology course laboratories. And the Lillie Auditorium got a new roof. I'm pleased to report that commitments are now in hand to install an air-conditioning system at the Library, and very soon the MBL will have a new emergency generator. One of the most the exciting changes to the MBL campus last summer was the creation of the new Robert W. Pierce Visitors Center, which shares 100 Water Street with the MBL and Satellite Clubs. This beautiful new facility, which is also home to the MBL Associates Gift Shop, was dedicated and opened in the summer of 1998. It has already introduced thousands of Woods Hole visitors to the Marine Biological Laboratory. MBL Trustees In 1998. the MBL Board of Trustees welcomed Dr. John E. Dowling as President of the Corporation. Dr. Dowling succeeded Dr. James D. Ebert, who retired after serving as President for seven years. Dr. Dowling is the Maria Moors Cabot Professor of Natural Science at Harvard University, as well as an MBL summer investigator and former MBL Trustee. His research focuses on the physiology of vision, especially the correlation between structure and function in the vertebrate retina. He also is interested in retinal development and uses the zebrafish as a model organism for these studies. Last year the MBL Board of Trustees elected Ronald P. O'Hanley, President of Dreyfus Institutional Investors in Boston, and Vincent J. Ryan, President, Chairman, and CEO of Schooner Capital Corporation, also of Boston, to membership in the Class of 2003. The Laboratory is most fortunate to welcome these dynamic and thoughtful individuals to help guide our progress over the next few years. Burton J. Lee, III, Laurie J. Landeau, Darcy Brisbane Kelley, and Jean Pierce were reappointed to the R6 Annual Report Board in November 1998 as members of the class of 2003. Directors Emeriti The Board of Trustees voted to name three former directors of the Marine Biological Laboratory "Directors Emeriti." James D. Ebert, Paul R. Gross, and Harlyn O. Halvorson were recognized for the contributions that each of these men made to the growth and strength of the Laboratory during their tenures as director. Each of these individuals has left a legacy of achievement that has earned the respect and gratitude of the MBL community. In closing, 1998 was an exciting time, and 1999 should be no less so. The Marine Biological Laboratory remains a wonderful gathering place for scientists and students from around the world. Anchored by a top-notch team of year-round investigators, enlivened by some of the best students anywhere, and stimulated by the summer influx of great researchers, the MBL continues to serve science in a unique and exciting way. — John E. Burris Report of the Treasurer During 1998 the Marine Biological Laboratory continued a favorable trend in operations. This progress was due to healthy increases in five of the six areas of Operating Support. Government grants increased 9.6% and now represent 42.5% of the total support and revenues. Double digit increases in Private Contracts (38.3%), Fees for Conferences and Services ( 10.7%) and Miscellaneous Revenues (22.3% ) powered the year's success story. While there was an easing in the present value of Contributions this was predictable at the midpoint of our very successful Discovery Campaign. As already noted in the Report of the Director and CEO, the campaign is ahead of schedule. Focusing on the change in Unrestricted Net Assets, we enjoyed a three-year favorable trend. The change before nonoperating activity has improved from a deficit of $1 million in 1996, to a deficit of $753 thousand in 1997, to a deficit of only $256 thousand this year. This is particularly auspicious when one realizes these figures are after approximately $1.5 million in depreciation each year. While the Change in Net Assets before nonoperating activity was only half of 1997 results, it was still a robust $1.1 million. Total Investment Income and Earnings of only 820 thousand dollars were unsatisfactory when compared to the multi-million dollar returns in previous years. This was a result of the volatile markets and a revamping of our endowment management philosophy. As a result. Net Assets increased for the fourth year in a row, but the Return on Average Net Assets was only 1.1%. A review of the 1998 Balance Sheet demonstrates our continued strong liquidity and low and improving leverage. Property Plant and Equipment showed a slight decline (2.4%). but this is the smallest decline in the past four years as we are in the process of upgrading the physical plant. Plans are underway to expand our capital maintenance efforts and to build a new Environmental Sciences Building. Ultimately, this will make the Laboratory an even more attractive facility to conduct science. In summary, the Laboratory continues to demonstrate the ability to attract funds from the federal government, foundations and individuals. Our housing and conferences continue to generate surplus cash. Successful completion of the Discovery Campaign and a return to our history of very successful endowment performance will guarantee the financial strength of the Marine Biological Laboratory for the 21st century. — Mary B. Conrad R7 Financial Statements PrrcewaterhouseCoopers LIP One Post Offic e Square Boston MA 0_> 1 09 Telephone (hi 7) 478 5000 F.ii simile (111 7) 478 5900 REPORT OF INDEPENDENT ACCOUNTANTS To the Board of Trustees of Marine Biological Laboratory Woods Hole. Massachusetts In our opinion, the accompanying balance sheet of Marine Biological Laboratory (the "Laboratory") as of December 31, 1998 and the related statements of activities and cash flows for the year then ended present fairly, in all material respects, the financial position of the Laboratory as of December 31, 1998, and the changes in its net assets and its cash flows for the year then ended in conformity with generally accepted accounting principles. These financial statements are the responsibility of the Laboratory's management; our responsibility is to express an opinion on these financial statements based on our audit. We conducted our audit in accordance with generally accepted auditing standards. Those standards require that we plan and perform the audit to obtain reasonable assurance about whether the financial statements are free of material misstatement. An audit includes examining, on a test basis, evidence supporting the amounts and disclosures in the financial statements. An audit also includes assessing the accounting principles used and significant estimates made by management, as well as evaluating the overall financial statement presentation. We believe that our audit provides a reasonable basis for the opinion expressed above. Our audit was conducted for the purpose of forming an opinion on the basic financial statements taken as a whole. The supplemental schedule of functional expenses for the year ended December 31, 1998 is presented for the purpose of additional analysis and is not a required part of the basic financial statements. Such information has been subjected to the auditing procedures applied in the audit of the basic financial statements and, in our opinion, is fairly stated, in all material respects, in relation to the basic financial statements taken as a whole. April 9, 1999 R8 MARINE BIOLOGICAL LABORATORY BALANCE SHEETS December 31, 1998 (with comparative totals as of December 31. 1997) ASSETS Cash and cash equivalents Short-term investments, at market (Note C) Accounts receivable, net of allowance for doubtful accounts of $34,195 in 1998 and $36.782 in 1997 Current portion of pledges receivable (Note H) Receivables due for costs incurred on grants and contracts Other assets Total current assets Long-term investments, at market (Notes C and Di Pledges receivable, net of current portion (Note H) Plant assets, net (Notes B. E and F) Total long-term assets Total assets 1998 S 1,187,954 3.561,544 1,242,530 1 ,607.664 1,531,083 557.908 9.688.683 37.054.120 2.855.352 19.536.171 59,445.643 $69.134,326 1997 $ 560.801 4.408.046 1,221,781 2,219,056 1.157.165 560.269 10.127.118 35,614,151 2,238,826 20.026.580 57.879.557 $68.006.675 LIABILITIES AND NET ASSETS Current portion of long-term debt (Note E) Accounts payable and accrued expenses Deferred income and advances on contracts Total current liabilities Annuities and unitrusts payable Long-term debt, net of current portion (Note E) Advances on contracts Total long-term liabilities Total liabilities Commitments and contingencies (Notes F and H) 243,274 2,057,741 462.873 2.763.888 1.412,200 2.324.096 1.272.390 5.008.686 7.772,574 229,657 1.494,948 384.258 2.108.863 1.213.583 2,567.370 1.433.208 5.214.161 7,323,024 Net assets: Unrestricted Temporarily restricted Permanently restricted Total net assets (Note Bl Total liabilities and net assets 18,451.865 25.635.237 17.274.650 61.361.752 $69,134.326 18.729.311 25,596.656 16.357.684 60.683.651 $68.006,675 The accompanving notes are an integral part of the financial statements. R9 MARINE BIOLOGICAL LABORATORY STATEMENTS OF ACTIVITIES for the year ended December 31,1 998 (with comparative totals for the year ended December 31, 1997) Operating support and revenues: Government grants Private contracts Laboratory rental income Tuition Fees for conferences and services Contributions Investment income Miscellaneous revenue Present value adjustment to annuities Net assets released from restrictions Total operating support and revenues Expenses: Research Instruction Conferences and services Other programs (Note B) Total expenses Change in net assets before nonoperating activity Nonoperating revenue: Total investment income and earnings Less: investment earnings used tor operations Reinvested (utili/ed) investment earnings Total change in net assets Net assets, beginning of year Net assets, end of year Temporarily Permanently 1998 1997 Unrestricted Restricted Restricted Total Total $10,943,239 $ $ $10,943,239 $ 9.986.800 1.629,283 — — 1.629,283 1.178,192 1.470.372 — — 1.470.372 1.478.757 489,726 — — 489.726 399.703 3.415,519 — — 3,415,519 3.085,616 1.264,235 3,420,615 653.152 5,338,002 6.441.429 490,474 1,465,261 — 1.955.735 1 ,709,983 405,633 — — 405.633 322,667 — (68,849) (7.853) (76.702) (164.447) 4.100,624 (4,138,622) 37,998 — — 24,209,105 678,405 683.297 25.570.807 24.438.700 12,666.746 12,666.746 11.031.914 4.433,789 — — 4,433,789 4,144,508 1 ,999,433 — — 1 ,999,433 1,487.705 5,365,530 — 5.365.530 5.440.808 24,465,498 — — 24,465.498 22.104.935 (256,393) 678,405 683,297 1,105,309 2.333,765 27.353 558.683 233,669 819.705 4,869,035 (48.406) (1.198.507) — (1.246.913) (1.056,211) (21,053) (639,824) 233.669 (427.208) 3,812,824 (277.446) 38,581 916,966 678.101 6,146,589 18.729,311 25,596,656 16,357,684 60.683.651 54,537.062 $18,451,865 $25.635 2^7 $17 274650 $61 361 75"1 $60683 651 The accompanying notes are an integral part of the financial statements. RIO MARINE BIOLOGICAL LABORATORY STATEMENTS OF CASH FLOWS for the year ended December 31, 1998 (with comparative totals for the year ended December 31, 1997) Cash flows from operating activities: Change in net assets Adjustments to reconcile change in net assets to net cash provided by (used in) operating activities: Depreciation Unrealized (gain) loss on investments Realized (gain) loss on investments Present value adjustment to annuities and unitrusts payable Contributions restricted for long-term investment and annuities Provision for bad debt Provision for uncollectible pledges Change in certain balance sheet accounts: Accounts receivable Pledges receivable Grants and contracts receivable Other assets Accounts payable and accrued expenses Deferred income and advances on contracts Annuities and unitrusts payable Advances on contracts Net cash provided by operating activities Cash flows from investing activities: Purchase of property and equipment Proceeds from sale of investments Purchase of investments Net cash used in investing activities Cash flows from financing activities: Payments on annuities and unitrusts payable Receipt of permanently restricted gifts Annuity and unitrusts donations received Loan proceeds Payments on long-term debt Net cash provided by financing activities Net increase in cash and cash equivalents Cash and cash equivalents at beginning of year Cash and cash equivalents at end of year 1W8 $ 67S.10I 1.505.696 2,755.079 (2.805.560) 76.702 (682.817) 15,771 250.000 (36.520) (255.134) (373.918) 2.361 562,793 78.615 163.700 (160,818) 1.774.051 (1.015.287) 18.935,050 (19.478.036) (1.558.273) (41.785) 653,152 29,665 (229.657) 411.375 627.153 560.801 S 1.187.954 6.146,589 1.483.203 (1.740,501) (1.728,792) 164.447 (1.390,609) 21,781 89.620 (480.702) (314.658) (43,083) (77.277) (71.564) 62,260 120.052 222.258 2,463,024 (814,159) 23,450,218 (26.321.432) (3.685.373) (30.430) 1,321,302 69.307 250.000 (218.557) 1,391.622 169.273 391,528 560.801 The accompanying notes are an integral part of the financial statements. Rll R12 Annual Report Marine Biological Laboratory Notes to Financial Statements A. Background: The Marine Biological Laboratory (the "Laboratory") is a private, independent not-for-profit research and educational institution dedicated to establishing and maintaining a laboratory or station for scientific study and investigation, and a school for instruction in biology and natural history. The Laboratory was founded in 1888 and is located in Woods Hole. Massachusetts. B. Significant Accounting Policies: Basis of Presentation The accompanying financial statements have been prepared on the accrual basis of accounting and in accordance with the principles outlined in the American Institute of Certified Public Accountants' Audit Guide. "Not-For-Profit Organizations." The financial statements include certain prior-year summari/.ed comparative information in total but not by net asset class. Such information does not include sufficient detail to constitute a presentation in conformity with generally accepted accounting principles. Accordingly, such information should be read in conjunction with the Laboratory's financial statements for the year ended December 31, 1997, from which the summarized information was derived. The Laboratory classifies net assets, revenues, and realized and unrealized gains and losses based on the existence or absence of donor-imposed restrictions and legal restrictions imposed under Massachusetts State law. Accordingly, net assets and changes therein are classified as follows: Unrestricted Unrestricted net assets are not subject to donor-imposed restrictions of a more specific nature than the furtherance of the Laboratory's mission. Revenues from sources other than contributions are generally reported as increases in unrestricted net assets. Expenses are reported as decreases in unrestricted net assets. Gains and losses on investments and other assets or liabilities are reported as increases or decreases in unrestricted net assets unless their use is restricted by explicit donor stipulations or law. Expirations of temporary restrictions on net assets, that is, the donor-imposed stipulated purpose has been accomplished and or the stipulated time period has elapsed, are reported as reclassifications between the applicable classes of net assets. Temporarily Restricted Temporarily restricted net assets are subject to legal or donor-imposed stipulations that will be satisfied either by the actions of the Laboratory, the passage of time, or both. These assets include gifts plus monies for which the specific, donor-imposed restrictions have not been met, and pledges, annuities, and unitrusts for which the ultimate purpose of the proceeds is not permanently restricted. As the restrictions are met, the assets are released to unrestricted net assets. Also, realized/unrealized gains/losses associated with permanently restricted gifts which are not required to be added to principal by the donor are classified as temporarily restricted but maintain the donor requirements for expenditure. Permanently Restricted Permanently restricted net assets are subject to donor-imposed stipulations that they be invested to provide a permanent source of income to the Laboratory. These assets include gifts, pledges and trusts which require that the corpus be invested in perpetuity and only the income be made available for program operations in accordance with donor restrictions. Nonoperating revenues include realized and unrealized gains on investments during the year as well as investment income on the master pooled investments. Investment income from short-term investments and investments held in trust by others are included in operating support and revenues. To the extent that nonoperating investment income and gains are used for operations as determined by the Laboratory's total return utilization policy (see below), they are reclassified from nonoperating to operating on the statement of activities as "Investment earnings used for operations." All other activity is classified as operating revenue. The Laboratory recorded net realized gains of $2,805,560, net unrealized losses of $2.755,079 and dividend and interest income of $1,478,046 in 1998. Cash and Cash Equivalents Cash equivalents consist of resources invested in overnight repurchase agreements and other highly liquid investments with original maturities ot three months or less. Financial instruments which potentially subject the Laboratory to concentrations of risk consist primarily of cash and investments. The Laboratory maintains cash accounts with one banking institution. Investments Investments purchased by the Laboratory are carried at market value. Donated investments are recorded at fair market value at the date of the gift. For determination of gain or loss upon disposal of investments, cost is determined based on the first-in, first-out method. Investments with an original maturity of three months to one year are classified as short-term. All other investments are considered long-term. Investments are maintained primarily with five institutions. In 1924, the Laboratory became the beneficiary of certain investments, included in permanently restricted net assets, which are held in trust by others. The Laboratory has the continuing rights to the income produced by these funds in perpetuity, subject to the contractual restrictions on the use of such funds. Accordingly, the trust has established a process to conduct a review every ten years by an independent committee to ensure the Laboratory Financial Statements R13 continues to perform valuable services in biological research in accordance with the restrictions placed on the funds by the agreement. The committee met in 1994 and determined that the Laboratory has continued to meet the contractual requirements. The market values of such investments are $7.673.828 and $7,440.158 at December 31. 1998 and 1997, respectively. The dividend and interest income on these investments totaled $260.805 and $254.898 in 1998 and 1997, respectively. Investment Income and Distribution For the master pooled investments, the Laboratory employs a total return utilization policy that establishes the amount of the investment return made available for spending each year. The Finance Committee has approved a standing policy that the withdrawal will be based on a percentage of the latest three-year average ending market values of the funds. The market value includes the principal plus reinvested income, realized and unrealized gains and losses. Spending rates in excess of 5%, but not exceeding 1%, can be utilized if approved in advance by the Finance Committee of the Board of Trustees. For fiscal 1998 and 1997. the Laboratory obtained approval to expend 6% of the latest three-year average ending market values of the investments. The net appreciation on permanently and temporarily restricted net assets is reported together with temporarily restricted net assets until such time as all or a portion of the appreciation is distributed for spending in accordance with the total return utilisation policy and applicable state law. Investment income on the pooled investment account is allocated to the participating funds using the market value unit method (Note D). Plant Assets Buildings and equipment are recorded at cost. Donated facility assets are recorded at fair market value at the date of the gift. Depreciation is computed using the straight-line method over the asset's esiimated useful life. Estimated useful lives are generally three to ten years for equipment and 20 to 40 years for buildings and improvements. Depreciation expense for the years ended December 31. 1998 and 1997 amounted to $1.505.696 and $1.483,203. respectively, and has been recorded in the statement of activities in the appropriate functionalized categories. When assets are sold or retired, the cost and accumulated depreciation are removed from the accounts and any resulting gain or loss is included in unrestricted income for the period. Annuities tint! Unitnists Pavable Amounts due to donors in connection with gift annuities and unitrusts are determined based on remainder value calculations, with varied assumptions of rates of return and payout terms. Deferred Income and Advances on Contracts Deferred income includes prepayments received on Laboratory publications and advances on contracts to be utilized within the next year. Advances on contracts includes funding received for grants and contracts before it is earned. In certain circumstances, long-term advances are invested in the master pooled account until they are expended. Revenue Recognition Revenue is recognized at the time it is earned. The sources of revenue include grant payments from governmental agencies, contracts from private organizations, and income from the rental of laboratories and classrooms for research and educational programs. The tuition income is net of student financial aid of $523. 1 90 and 5536,097 in 1 998 and 1 997, respectively. Fees for conferences and other services include the following activities: housing, dining, library, scientific journals, aquatic resources and research services. Contributions Contribution revenue includes gifts and pledges. Gifts are recognized as revenue upon receipt. Pledges are recognized as temporarily or permanently restricted revenue in the year pledged and are recorded at the present value of expected future cash flows, net of allowance for unfulfilled pledges. Gifts and pledges, other than cash, are recorded at fair market value at the date of contribution. Expenses Expenses are recognized when incurred and charged to the functions to which they are directly related. Expenses that relate to more than one function are allocated among functions using various methodologies. Other programs expense consists primarily of fundraising, year-round labs and library room rentals, costs associated with aquatic resource sales and scientific journals. Total fundraising expense for 1998 and 1997 is $1.037,495 and $1.226,360, respectively. Use of Estimates The preparation of financial statements in conformity with generally accepted accounting principles requires management to make estimates and assumptions that affect the reported amounts of assets and liabilities and disclosure of contingent assets and liabilities at the date of financial statements and the reported amounts of revenues and expenses during the reporting period. Actual results could differ from those estimates. Tax-Exempt Status The Laboratory is exempt from federal income tax under Section 501(c)(3) of the Internal Revenue Code. R14 Annual Report C. Investments: The following is a summary of the cost and market value of investments at Dec ember 31. 1998 and 1997: Market Cost 199S 1997 1998 1997 Certificates of deposit $ 40.000 S 40.000 $ 40.000 $ 40,000 Money market securities 1.052.276 2,168.958 1.052,276 2.168,958 U.S. Government securities 1,397,686 1.292.600 1,136.219 1.098.526 Corporate fixed income 2.504.507 2,587,861 2.472.653 2.472,653 Common stocks 5,033.704 5,279,266 4,290,581 4,271,853 Mutual funds 29,548,89! 23.223,812 26.225.214 19,317,499 Limited partnerships 1,038.600 5.429,700 958.982 3,309.994 Total investments $40,615,664 $40,022,197 $36,175,925 $32,679,483 Investment portfolios tor the years ended December 31, 1998 and 1997 are as f< allows: Mark el Cost 1998 1997 1998 7997 Short-Term Investments Certificates of deposit $ 40,000 $ 40.000 $ 40,000 $ 40,000 Money market 1784 Fund 559,314 1 .759.589 559.314 1 .759.589 Common stocks 6.241 551.780 6,241 530.936 Mutual funds 2,955,989 2,056.677 2,940.929 2,056.679 Total 3.561,544 4,408,046 3.546.484 4.387,204 Mark et Cost 1998 1997 1998 1997 Long-Term Investments Pooled investments: Master pooled investments $27.057.909 $26,163,702 $23.723.343 $20,201,962 Separately invested: General Chase trust 6,038,153 5,846.916 5,433,574 4,986.443 Library Chase trust 1,635,675 1,593.242 1.477,462 1.358.149 Annuity and unitrust investments 2.322.383 2,010,291 1.995,062 1,745,725 Total 37.054,120 35,614,151 32.629.441 28.292,279 Total investments $40.615.664 $40.022.197 $36.175.925 $32.679.483 Financial Statements KI5 D. Accounting for Pooled Investments: Certain net assets are pooled for investment purposes. Investment income from the pooled investment account is allocated on the market value unit basis, and each fund subscribes to or disposes of units on the basis of the market value per unit at the beginning of the calendar quarter within which the transaction takes place. The unit participation of the funds at December 31. 1998 and 1997 is as follows: Unrestricted Temporarily restricted Permanently restricted Advances on contracts 199N 4.001 44,455 65.016 6.437 119.909 1997 4.192 42,693 65,411 6.506 118,802 Pooled investment activity on a per-unit basis was as follows: Unit value at beginning of year Unit value at end of year Total return on pooled investments 1998 $ 220.30 225.51 $ 5.21 1997 $ 186.35 220.30 $ 33.95 E. Long-Term Dchj: Long-term debt consisted of the following at December 31 : Variable rate (5.15% at December 31, 1998) Massachusetts Industrial Finance Authority Series 1992A Bonds payable in annual installments through 2012 6.63% Massachusetts Industrial Finance Authority Series 1992B Bonds, payable in annual installments through 2012 5.8% The University Financing Foundation. Inc.. payable in monthly installments through 2000 5.8% The University Financing Foundation. Inc.. payable in monthly installments through 2002 I99N $ 925,000 1,230.000 226.024 186.346 $2.567.370 1997 $ 960,000 1.280.000 325.210 231.817 S2.797.027 The aggregate amount of principal due on long-term debt for each of the next five fiscal years and thereafter is as follows: 1999 2000 2001 2002 2003 Thereafter Less current portion of long-term debt Long-term debt net of current return $ 243.274 267.404 173,664 148,028 125.000 1.610.000 2.567.370 (243.274) $2.324.096 In 1992. the Laboratory issued $1.100.000 Massachusetts Industrial Finance Authority (MIFA) Series 1992A Bonds with a variable interest rate and SI. 500.000 MIFA Series 1992B with an interest rate of 6.63%. Interest expense debt totaled $136.340 tor the year ended December 31. 1998. The Series 1992 A and B Bonds mature on December 1. 2012 and are collateralized by a first mortgage on certain Laboratory property. On March 17. 1998, the Laboratory entered into a ten-year interest rate swap contract in connection with the Series 1992A Bonds. This contract effectively fixes the interest rate at 6.30% through December 17. 200S. R16 Annual Report The agreements related to these bonds subject the Laboratory to certain covenants and restrictions. Under the most restrictive covenant of this debt, the Laboratory's operating surplus, exclusive of interest expense and depreciation expense, must be greater than or equal to 1.2 times all debt service payments, as defined by the agreement. The Laboratory was in compliance with these covenants and restrictions at December 31, 1998. In 1996. the Laboratory borrowed $500,000 with an interest rate of 5.8% per annum from the University Financing Foundation. Inc. The interest expense for the year ended December 31. 1998 was $16,253. The loan matures in 2000 and is collaterali/ed by 50.000 shares of a fixed income fund with a fair value of $595.000 at December 31. 1998. In 1997, the MBL borrowed $250.000 with an interest rate of 5.8% per annum from the University Financing Foundation. Inc. The interest expense for the year ended December 31, 1998 was $12,249. This loan matures in 2002 and is collateralized by 19.440 shares of a fixed income mutual fund with a fair value of $231,336 at December 31. 1998. The Laboratory has a line of credit agreement with BankBoston from which it may draw up to $1.000.000. No amounts were outstanding under this agreement as of December 31. 1998 and 1997. F. Plant Assets: Plant assets consist of the following at December 3 1 : 1W8 1997 Land Buildings Equipment Total Less: Accumulated depreciation Plant assets, net $ 702.908 33,334,107 4,401,184 $ 702,908 32,419,072 4,300,932 38,438.199 (18,902,028) 37,422.912 (17,396,332) $19.536.171 $20,026.580 G. Retirement Plan: The Laboratory participates in the defined contribution pension plan of TIAA-CREF (the "Plan"). The Plan is available to permanent employees who have completed two years of service. Under the Plan, the Laboratory contributes 10%< of total compensation for each participant. Contributions amounted to $737,156 and $715,858 for the years ended December 31, 1998 and 1997. respectively. H. Pledges: Unconditional promises to give are included in the financial statements as pledges receivable and the related revenue is recorded in the appropriate net asset category. Unconditional promises to give are expected to be realized in the following periods: In one year or less Between one year and five years After five years /W,S $1,607.664 3,110,354 146,586 7997 $2,219,056 2.485,851 80.000 Total 4,864.604 4.784.907 Less: discount of $301,588 in 1998 and $227,025 in 1997 and allowance of $100,000 in 199S and $100.000 in 1 997 (401.5X8) (327,025) $4,463.016 $4.457.882 Financial Statements R17 Pledges receivable at December 31 have the following restrictions: Research and education Permanently restricted net assets $3,933,988 529,028 1997 $3.787,882 670.000 $4.463.016 $4,457.882 I. Postretirement Benefits: The Laboratory accounts for its postretiremen! benefits under Statement No. 106, "Employers' Accounting tor Postretiremen! Benefits Other than Pensions." which requires employers to accrue, during the years that the employee renders the necessary service, the expected cost of benefits to be provided during retirement. As permitted, the Laboratory has elected to amortize the transition obligation over 20 years commencing on January 1 . 1994. The Laboratory's policy is that all current retirees and certain eligible employees who retired prior to June 1 . 1994 will continue to receive postretirement health benefits. The remaining current employees will receive benefits; however, those benefits will be limited as defined by the Plan. Employees hired on or after January 1. 1995 will not be eligible to participate in the postretirement medical benefit plan. The following tables set forth the Plan's funded status as of December 31: Benefit obligation at December 31 Fair value of plan assets at December 3 1 Funded status Accrued benefit cost Weighted-average assumptions as of December 3 1 : Discount rate Expected return on plan assets Compensation increase rate Benefit cost Employer contribution Benefits paid 1998 $ 2.171.119 820.645 $(1.350.474) $ (26.654) 6.75% 7.25% N/A 210,339 192.082 109.404 1997 $ 1.919.865 701,140 $(1,218.725) $ (8.397) 7.50% 8.00% N/A 192,082 192.082 111.255 For measurement purposes a 7.5% annual rate of increase in the per capita cost of covered health care benefits was assumed for 1999. The rate was assumed to decrease by half of 1 .00% per year to 4.25% in 2006 and remain at that level thereafter. Pension plan assets consist of investments in a money market fund. 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ON \o n OO 3 2 s v. n-, r*-, — <-| sC IT, r^, -(- O r- r*~, r-' O O oq 2 = 3^ 1 CQ ^ G -J > *^ >O — Osr*-, — O — w-i— I-^OO — ' ON' r*"i O -t c: *T ~-f lO M w ^ ^2 z g -£ > a; 5 'S ^ Ori OsOO-t oc r~~ r~~ oon ^t r- ir> r^ co r~- r- ri oc w~, O r^l Wj OS -?t OO OO^ -t -± O r- ^^ ' ^ C! I^I p , -^ . Xi "•fOs'lm -? — oo — < \O JC -.' r-' oo f^i Os ri rj NO — n-, -t c/) S 0 torsion angles. Structure generation based on interproton distance restraints and torsion angle measurements yield convergent structures generated using distance geometry and simulated annealing methods. The goal of this project is to determine the structural role of y- carboxyglutamic acid in the Gla-containing conotoxins. Staff Barbara C. Furie, Scientist Bruce Furie, Scientist Johan Stenflo. Visiting Scientist Eva Czerwiec. Postdoctoral Fellow Gail Begley, Postdoctoral Fellow Alan Rigby. Postdoctoral Fellow Staff Hanlon. Roger, Senior Scientist Sussman, Raquel, Investigator Hatfield, Emma, Postdoctoral Fellow Maxwell, Michael, Postdoctoral Scientist Rurnmel, John, Visiting Scientist Shashar. Nadav. Postdoctoral Scientist Visiting Investigators Boal, Jean, Visiting Scientist Gabr, Howaida, Graduate Student. Suez Canal University. Egypt Cavanaugh, Joseph. Graduate Student, Boston University Marine Program Fern, Sophie, Graduate Student, Boston University Marine Program Wittenberg. Kim, Boston University Marine Program Laboratory of Shinya Inoue Scientists in this laboratory study the molecular mechanism and control of mitosis, cell division, cell motility, and cell morphogenesis, with emphasis on biophysical studies made directly on single living cells, especially developing eggs in marine invertebrates. Development of biophysical instrumentation and methodology, such as the centrifuge polarizing microscope, high-extinction polarization optical and video microscopy, digital image processing techniques, and exploration of their underlying theory are an integral part of the laboratory's efforts. Staff Inoue, Shinya. Distinguished Scientist Knudson. Robert. Instrument Development Engineer Maccaro, Jackie, Laboratory Assistant MacNeil, Jane. Executive Assistant Laboratory of Alan M. Kuzirian Research in the laboratory explores the functional morphology and infrastructure of various organ systems in molluscs. The program includes mariculture of the nudibranch. Hermissenda crassicnnus. with emphasis on developing reliable culture methods for rearing and maintaining the animal as a research resource. The process of metamorphic induction by natural and artificial inducers is being explored in an effort to understand the processes involved and as a R50 Annual Report video, and digital image processing for fast analysis of specimen birefringence over the entire viewing field. Examples of biological systems currently investigated with the Pol-Scope are: microtubule-based structures (asters, mitotic spindles, single microtubules); actin-based structures (acrosomal process, stress fibers, nerve growth cones); zona pellucida of vertebrate oocytes; and biopolymer liquid crystals. Staff Oldenbourg. Rudolf, Associate Scientist Katoh. Kaoru. Postdoctoral Research Associate Geer. Thomas. Research Assistant Knudson. Robert. Instrument Development Engineer Barahy, Diane. Laboratory Assistant means to increase the yield of cultured animals. Morphologic studies stress the ontogeny of neural and sensory structures associated with the photic and vestibular systems which have been the focus of learning and memory studies, as well as the spatial and temporal occurrence of regulatory and transmitter neurochemicals. Concurrent studies detailing the toxic effects of lead on Hermissenda learning and memory, feeding, and the physiology of cultured neurons are also being conducted. New studies include cytochemical investigations of the Ca:+/GTP binding protein, calexcitin. and its modulation with learning and lead exposure. Collaborative research includes histochemical investigations on strontium's role in initiating calcification in molluscan embryos (shell and statoliths). immunocytochemical labelling of cell-surface antigens, neurosecretory products, second messenger proteins involved with learning and memory, as well as intracellular transport organelles using mono- and polyclonal antibodies on squid (Loligo pealei) giant axons and Hermissenda sensory and neurosecretory neurons. Additional collaborations involve studying neuronal development and defects, as well as nerve regeneration and repair in phylogenetically conserved nervous systems. Additional collaborative research includes DNA fingerprinting using RAPD-PCR techniques in preparation for isogenic strain development of laboratory-reared Hermissenda and hatchery-produced bay scallops (Argopectin irradiana) with distinct phenotypic markers tor the rapid field identification. Systematic and taxonomic studies of nudibranch molluscs, to include molecular phylogenetics, are also of interest. Staff Ku/.irian, Alan M.. Associate Scientist Visiting Scientists Chikarmane, Hemant. Assistant Scientist, MBL Clay. John R.. NINDS/NIH Gould, Robert. NYS Institute of Basic Research Laboratory of Rudolf Oldenbourg Laboratory for Reproductive Medicine, Brown University and Women and Infants Hospital, Providence Work in this laboratory centers on the investigation of the underlying mechanisms behind female infertility. Particular emphasis is placed on the physiology of the oocyte or early embryo, with the aim of assessing developmental potential and mitochondria dysfunction arising from mtDNA deletions. The studies taking place at the MBL branch of the Brown Laboratory use some of the unique instrumentation available through the resident programs directed by Rudolf Oldenbourg and Peter J. S. Smith. Most particularly, non-invasive methods for oocyte and embryo study are being sought. Of several specific aims, one is to use the Pol-Scope to analyze the birefringence of the preimplantation mammalian zona pellucida — a structure most predictive of successful implantation. We also have used this instrument to examine meiotic spindles. An additional aim is to continue the studies on transmembrane ion transport using the non-invasive electro-physiological techniques available at the BioCurrents Research Center. Preliminary studies indicate that the calcium transport may form an accurate predictor of oocyte and embryo health. The newly developed oxygen probe also offers the possibility of looking directly at abnormalities in the mitochondria arising from accumulated mtDNA damage. Our laboratory has also focused on studying the mechanism underlying age-associated infertility in terms of oocyte quality, attempting to rescue the developmentally compromised oocytes or embryos through nuclear- cytoplasmic transfer technology. We have characterized oxidative stress- induced mitochondria! dysfunctions, developmental arrest and cell death in early embryos using animal models. Ultimately, in addition to investigating the mechanisms behind cellular aging underlying infertility, this laboratory aims to produce clinical methods for assessing preimplantation embryo viability, a development that will make a significant contribution to the health of women and children. Staff Keefe. David. Director Liu. Lin. Research Scientist Pepperell, John. Visiting Investigator Trimarchi. James. Postdoctoral Scientist The laboratory imotig.itcs the molecular architecture ot living cells and of biological model systems using optical methods for imaging and manipulating these structures. For imaging non-invasively and non- destructively cell architecture dynamically and at high resolution, we have developed a new polari/ed light microscope (Pol-Scope). The Pol- Scope combines microscope optics with new electro-optical components. Laboratory of Sensory Physiology Members of this laboratory have conducted research on various facets of vision since 1473. Current investigations use UV/V1S light microspectrophotometry on vertebrate retinal photoreceptors for the Yciir-Round Research R51 determination of visual pigment ahsorbance characteristics. One aim is to arrive at a better understanding of the method of spectral tuning that forms the chemical basis of color vision. Polarized light microscopic techniques are used to measure linear dichroism and linear birefringence aimed at revealing structure-function relationships and biophysical mechanisms. An area of interest is polarization discrimination, the mechanisms that could account for the ability of some fish species to detect the direction of polarization of light collected by their eyes. As a recent development, investigations are carried out on sickling in fish red blood cells due to hemoglobin polymerization, once again making extensive use of polarized light microscopic techniques. Staff Harosi, Ferenc I.. Senior Scientist, MBL. and Boston University School of Medicine Novales Flamarique. I., Postdoctoral Fellow Laboratory of Osainu Shinwmiira Biochemical mechanisms involved in the bioluminescence of various luminescent organisms are investigated. Based on the results obtained, various improved forms of bioluminescent and chemiluminescent probes are designed and produced for the measurements of intracellular free calcium and superoxide anion. Staff Shimomura, Osamu. Senior Scientist, MBL, and Boston University School of Medicine Shimomura, Akemi. Research Assistant Laboratory of Robert B. Silver The members of this laboratory study how living cells make decisions. The focus of the research, typically using marine models, is on two main areas: the role of calcium in the regulation of mitotic cell division (sea urchins, sand dollars, etc.) and structure and function relationships of hair cell stereociliary movements in vestibular physiology (oyster, toadfish). Other related areas of study, i.e. synaptic transmission (squid), are also, at times, pursued. Tools include video light microscopy, multispectral, subwavelength, and very high speed (sub-millisecond frame rate) photon counting video light microscopy, telemanipulation of living cells and tissues, and modeling of decision processes. A cornerstone of the laboratory's analytical efforts is high performance computational processing and analysis of video light microscopy images and modeling. With luminescent, fluorescent, and absorptive probes, both empirical observation and computational modeling of cellular, biochemical, and biophysical processes permit interpretation and mapping of space-time patterns of intracellular chemical reactions and calcium signaling in living cells. A variety of in vitro biochemical, biophysical, and immunological methods are used. In addition to fundamental biological studies, the staff designs and fabricates optical hardware, and designs software for large video image data processing, analysis, and modeling. Staff Silver. Robert. Associate Scientist Visiting Scientist Pearson, John, Los Alamos Nations Laboratory Interns King, Leslie A., REU Intern. Duke University Wise. Alyssa. REU Intern. Yale University Laboratory of Seymour Zigman This laboratory is investigating basic mechanisms of photooxidative stress to the ocular lens due to environmentally compatible UVA radiation. This type of oxidative stress contributes to human cataract formation. Other studies are the search for and use of chemical antioxidants to retard the damage that occurs. Cultured mammalian lens epithelial cells and whole lenses in vitro are exposed to environmentally compatible UVA radiation with or without previous antioxidant feeding. The following parameters of lens damage are examined: molecular excitation to singlet states via NADPH (the absorber); cell growth inhibition and cell death; calalase inactivation; cytoskeletal description (of actin. tubulin. integrins): and cell membrane damage (lipid oxidation, loss of gap junction integrity and intercellular chemical communications). Thus far. the most successful antioxidant to reduce these deficiencies is alpha-tocopherol (10 /j.g/ml) and tea polyphenols (especially from green tea). The preliminary phases of the research are usually carried out using marine animal eyes (i.e.: smooth dogfish) as models. Our goal is to provide information that will suggest means to retard human cataract formation. Staff Seymour Zigman. Laboratory Director, Professor of Ophthalmology. Boston University Medical School Keen Rafferty. Research Associate. Boston University Medical School Nancy S. Rafferty, Research Associate, Boston University Medical School Buiinie R. Zigman. Laboratory Manager, Boston University Medical School R52 Annual Report The Marine Resources Center The Marine Resources Center (MRC) is one of the world's most advanced facilities for maintaining and culturing aquatic organisms essential to advanced biological, biomedical, and ecological research. Service and education also play an important and complementary role in the modern. 32,000-square-foot facility. The MRC and its life support systems have already increased the ability of MBL scientists to conduct research and have inspired new concepts in scientific experiments. Vigorous research programs focusing on basic biological and biomedical aquatic models are currently being developed at the Center. The Program in Scientific Aquaculture was initiated in 1998. In addition to research, the MRC provides a variety of services to the MBl. community through its Aquatic Resources Division, the Water Quality and System Engineering Division, and the Administrative Division. Research and educational opportunities are available at the facility to established investigators, postdoctoral fellows, graduate, and undergraduate students. Investigators and students will tind that the MRC's unique life support and seawater engineering systems make this a favorable environment in which to conduct independent research and masters and doctoral theses using a variety of aquatic organisms and flexible tank space for customi/ed experimentation on live animals. Prospective investigators and students should contact the Director of the MRC for further information. Staff Hanlon, Roger. Director and Senior Scientist Sussman. Raquel. Investigator Ku/.irian, Alan, Associate Scientist Maxwell. Michael, Postdoctoral Scientist Shashar, Nadav. Postdoctoral Scientist Visiting Invextiguturx Adamo, Shelly, Dalhousie University. Canada Baker. Robert. New York University Boal. Jean. Visiting Scientist Cavanaugh, Joseph, Boston University Marine Program Gabr, Howaida. Graduate Student, Sue?- Canal University, Egypt Gilland, Edwin, Staff Scientist Kier, William. University of North Carolina Spotte, Stephen. University of Connecticut Wittenberg, Kim, Boston University Marine Program Honors Friday Evening Lectures June \<-> June 26 July 3 July 10 July 17 July 23, 24 July 31 August 7 August 14 Stephen L. Hajduk, School of Medicine and Dentistry, University of Alabama at Birmingham "Carriers of Death: Civil War and Tsetse Flies" Mary Lidstrom. College of Engineering, University of Washington, Seattle "Borrowing Genes to Create New Metabolism" (Glassman Lecture) David Garbers. Howard Hughes Medical Center, Dallas "From Sea Urchins To High Blood Pressure: Smell and Vision" Donald Brown. Department of Embryology, Carnegie Institution of Washington "How Tadpoles Turn Into Frogs" Irene Pepperberg, Department of Ecology and Evolutionary Biology, University of Arizona, Tucson "In Search of King Solomon's Ring: Studies on Cognitive and Communicative Abilities of Grey Parrots" (Lang Lecture) Nicolas Spitzer, Department of Biology, University of California, San Diego 1. "The Development of Electrical Excitability in Nerve and Muscle" 2. "Breaking the Code: Regulation of Differentiation by Patterns of Calcium Transients" (Forbes Lectures) Eric Kandel. Center for Neurobiology and Behavior, Howard Hughes Medical Institute, Columbia University "Genes, Synapses and Long-Term Memory" Peter Raven. Missouri Botanical Gardens, St. Louis "Biodiversity, the Global Environment, and the New Millennium" Matthew Meselson. Harvard University "Evolution Without Sexual Reproduction and Genetic Recombination" Fellowships and Scholarships Robert Day Allen Fellowship Fund Drs. Joseph and Jean Sanger MBL Associates Endowed Scholarship Fund MBL Associates Mr. and Mrs. Douglas P. Amon Dr. and Mrs. Leonard Laster Frank A. Brown, Jr. Memorial Readership Dr. and Mrs. Francis D. Carlson C. Lalor Burdick Scholarship Fund The Lalor Foundation Gary Nathan Calkins Scholarship Fund Ms. Sarah A. Calkins Charles R. Crane Fellowship Fund Friendship Fund Mr. Thomas S. Crane John O. Crane Fellowship Fund Friendship Fund Mr. Thomas S. Crane Jean and katsuma Dan Fellowship Fund Drs. Joseph and Jean Sanger Mrs. Eleanor Steinbach Bernard Davis Fellowship Fund Mrs. Elizabeth M. Davis E. E. Just Research Fellowship Fund Ayco Charitable Foundation Dr. Jewel Plummer Cobb Mr. and Mrs. Jonathan Conrad Fiduciary Trust Company International Daniel Grosch Scholarship Fund Ms. Alice C. Leech Ms. Lena T. Lord Ms. Enid K. Sichel Dr. Margaret W. Taft Aline D. Gross Scholarship Fund Dr. and Mrs. Benjamin Kaminer Technic, Inc. R53 R54 Annual Report Keffer Hartline Fellowship Fund Dr. Lloyd M. Beidler Dr. Lawrence Eisenberg Dr. Paul Rosen Mr. Robert L. Schoenfeld Dr. and Mrs. Jonathan D. Victor Dr. Earl Weidner Dr. Torsten Wiesel and Ms. Jean Stein Fred Karush Endowed Library Readership Dr. and Mrs. Laszlo Lorand Dr. and Mrs. Arthur M. Silverstein Stephen W. Kuffler Fellowship Fund Dr. and Mrs. Edward A. Kravitz Frank R. Lillie Fellowship and Scholarship Fund Dr. and Mrs. George H. Acheson Mr. and Mrs. John J. Valois Josiah Macy, Jr. Research Fellowship Fund Josiah Macy, Jr. Foundation James A. and Faith Miller Fellowship Fund Drs. David and Virginia Miller Frank Morrell Scholarship Fund Dr. and Mrs. Maynard M. Cohen Dr. Leyla de Toledo Morrell Mr. Paul Morrell Mountain Memorial Fund Dr. and Mrs. Dean C. Allard, Jr. Ms. Brenda J. Bodian Dr. and Mrs. Benjamin Kaminer Ms. Anne C. Kimball, Ph.D. Mr. and Mrs. Amos L. Roberts Mr. and Mrs. William B. Sanford Mr. and Mrs. Hans L. Schlesmger Dr. and Mrs. R. Walter Schlesinger Neural Systems & Behavior Scholarship Fund Dr. Ronald Calabrese and Dr. Christine Cozzen Dr. and Mrs. Alan Gelperin Dr. Warren M. Gnll Dr. Ronald Hoy and Dr. Margaret Nelson Drs. Darcy B. Kelley and Richard M. Bockman Dr. William Kristan and Dr. Kathleen French Dr. Richard and Mrs. Jane Levine Dr. Janis C. Weeks and Dr. William M. Roberts Nikon Fellowship Fund Nikon. Inc. The Ann Osterhout Edison/Theodore Miller Edison and Olga Osterhout Sears/Harold Bright Sears Endowed Scholarship Fund Mrs. Jean S. Holden Dr. Susan M. Plourde William Townsend Porter Scholarship Fund William Townsend Porter Foundation Phillip H. Presley Scholarship Fund Carl Zeiss, Inc. Ruth Sager Endowed Scholarship Dr. Arthur B. Pordee Science Writing Fellowships Program Association for Research in Vision and Ophthalmology American Society for Biochemistry and Molecular Biology American Society for Cell Biology American Society for Photobiology Charles A. Dana Foundation Federation of American Society for Experimental Biology Foundation for Microbiology Friendship Fund New York Times Foundation Nicholas B. Ottaway Foundation Society for Integrative and Comparative Biology The Times Mirror Foundation The Washington Post Company Milton L. Shifman Endowed Scholarship Milton L. Shifman Scholarship Trust The Evelyn and Melvin Spiegel Fellowship Fund Drs. Joseph and Jean Sanger Drs. Melvin and Evelyn Spiegel The Sprague Foundation H. B. Steinbach Fellowship Fund Mrs. Eleanor Steinbach Marjorie R. Stetten Scholarship Fund Ms. Pauline F. Blanchard Mr. and Mrs. John C. Campbell Cognos Corporation Cognos Inc. Ms. Ann P. B. Fit/.gerald Mr. and Mrs. Douglas W. Lucy Mr. and Mrs. William Morton Ms. Linda A. O'Donnel Mrs. Jane Lazarow Stetten Mrs. Janet L. Vanderweil Ms. Ann M. White Horace W. Stunkard Scholarship Fund Dr. Albert Stunkard and Dr. Margaret Maurin Walter L. Wilson Endowed Scholarship Dr. Paul N. Chervin Mr. and Mrs. Rexford A. English Dr. Jean R. Wilson Honors R55 Fellowships Awarded MBL Summer Research Fellows • Mark C. Alliegro, Ph.D.. Louisiana State University. Dr. Alliegro uses sea urchins, and a variety of mammalian cells in culture to study the mechanisms of cell differentiation. He was supported by the Frederik B. Bang Fellowship Fund, the James A. and Faith Miller Memorial Fund, and an MBL Associates Fellowship. • Brian D. Bovard, Ph.D., Duke University. Dr. Bovard worked at a field site located in Abisko, Sweden, this summer. He studies relations between plants and water as part of a climate change project being conducted by scientists at the MBL's Ecosystem Center. He was supported by the William Townsend Porter Fellowship for Minority Investigators. • Wei-Jun Cai, Ph.D.. University of Georgia. Dr. Cai is developing microelectrodes to aid in the study of benthic carbon recycling. He was supported by the Lucy B. Lemann Fellowship. • William Cohen, Ph.D.. Hunter College. Dr. Cohen uses blood clams in his studies of the formation and function of the cellular framework known as the cytoskeleton. He was supported by the Erik B. Fries Endowed Fellowship. • John Costello, Ph.D., Providence College. Dr. Costello studies the feeding behavior in the comb jelly, Mnemiopsis leidyi. He was supported by the Erik B. Fries Endowed Fellowship, the Lucy B. Lemann Fellowship, and an MBL Associates Fellowship. ' John E. Eriksson. Ph.D.. Turku Center for Biotechnology, Finland. Dr. Eriksson is studying mitotic protein phosphatases in the eggs of the surf clam, Spisula. He was a Herbert W. Rand Fellow. • Andrew F. Giusti. University of California. Santa Barbara. Mr. Giusti investigates the role of the SRC tyrosine kinase during egg activation at fertilization. He was supported by the Frederik B. Bang Fellowship Fund. • Matthew Halstead. Ph.D.. University of Auckland, New Zealand. Dr. Halstead studies sensory processing of electrosensory information in the midbrain of the little skate. Raja. He was supported by the M.G.F. Fuortes Fellowship, the Frank R. Lillie Fellowship, and an MBL Associates Fellowship. ' Jonathan J. Henry. Ph.D.. University of Illinois. Dr. Henry examines the cellular and molecular mechanisms involved in embryonic cell fate and axis determination using barnacles as his research model. He was supported by the Evelyn and Me/vin Spiegel Fellowship Fund and the NASA Life Science Program Fellowship. • Elizabeth Jonas, Ph.D.. Yale University School of Medicine. Dr. Jonas studies the intracellular channels that regulate synaptic function. She was supported by the Ann E. Kammer Memorial Fellowship Fund, the H. B. Steinbach Fellowship, an MBL Associates Fellowship, the Charles R. Crane Fellowship, and the John O. Crane Fellowship Fund. • Nicholas Lartillot, Universite Paris Sud. Mr. Lartillot conducted a molecular study of mesoderm specification in marine spiralians. He was an MBL Associates Fellow. • Guy Major, a Research Fellow from Lucent Technologies. Mr. Major took voltage-sensitive dye recordings from multiple parts of single brain cells. He was a Herbert W. Rand Fellow. • Mark Martindale, Ph.D., University of Chicago. Dr. Martindale studies the evolution of development, in particular axial specification and the role of the cleavage program in body plan evolution. He was a NASA Life Sciences Program Fellow. • Paul McNeil, Ph.D., Medical College of Georgia. Dr. McNeil hopes to discover how cells reseal tears in their outer covering, the plasma membrane, and to demonstrate that such membrane tears are physiological events. He uses sea urchins, starfish eggs, and squid in his studies. He was supported by a Robert Day Allen Fellowship and a NASA Life Science Program Fellowship, • Allen Mensinger. Ph.D.. Washington University School of Medicine. Dr. Mensinger is developing an acoustical transmitter tag for neural telemetry. He was a NASA Life Science Program Fellow and an MBL Associates Fellow. ' Inigo Novales Flamarique, Ph.D.. University of Victoria, Canada. Dr. Novales Flamarique studies the functional organization of visual pathways from the retina to the brain in fishes. He was a Herbert W. Rand Fellow and an MBL Associates Fellow. • Elaine C. Seaver, Ph.D., University of Texas, Austin. Dr. Seaver studies the mechanism of segmentation in polychaetes. She was supported by the Evelyn and Melvin Spiegel Fellowship Fund. • Matt Wachowiak, Ph.D.. University of California. Berkeley. Dr. Wachowiak studies the transmission of olfactory information from sensory cells to the central nervous system. He was supported by a Stephen W. Kuffler Fellowship and an MBL Associates Fellowship. • James Q. Zheng. Ph.D., Robert Wood Johnson Medical School. Dr. Zheng studies the cellular mechanisms underlying the formation of nerve connections. He was a Nikon Fellow. Grass Fellows • Pamela England, Ph.D.. California Institute of Technology. Project: Probing the role of the protein tyrosine kinase SRC in long-term potentiation. • Alexander Gimelbrant. Ph.D., University of Kentucky Medical Center. Project: Characterization of cDNAs specific to individual lobster olfactory receptor neurons. • Kathryn Jessen-Eller, Ph.D., Tufts University School of Veterinary Medicine. Project: Serotonergic growth and p53 expression in developing embryos. • Jane Roche King, Ph.D.. University of Arizona. Project: Vestibular contribution to escape turning and orientation to prey in the leopard frog. Rana pipiens. • Maria Fabiana Kubke. Ph.D., University of Maryland. Project: Analysis of early position as a function of best frequency in the hindbrain auditory nuclei of the chicken. • David P. Len/.i. Ph.D.. University of Oregon. Project: The role of the synaptic ribbon at sensory cell output synapses. • Andrey Loboda, University of Pennsylvania. Project: Elucidation of the role of the S4-S5 linker in gating of the shaker potassium channel by site-directed crosslinking and gating current measurements. • Matthias Lorez. University of Zurich. Project: The role of HRS- 2 in synaptic transmission in the giant synapse of the squid Loligo pealei. • Kimberly McAllister. Ph.D.. The Salk Institute for Biological Studies. Project: Properties of synapse formation between cultured cortical neurons. • Kristina S. Mead, Ph.D., University of California, Berkeley. Project: The biomechanics and neurobiology of chemoreception in stomatopods. • Hong-Sheng Wang, Ph.D., SUNY at Stony Brook. Project: Angiotensin modulation of transient outward current of cardiac myocytes. MBL Science Writing Fellowships Program Fellows Monica Allen. Bangor Daily News Kevin P. Carmody. Chicago Daily South/own R56 Annual Report Thomas Carney. Des Moines Register Randall J. Edwards. Columbus Dispatch Don Finley, Sun Antonio Express-News Joel Greenberg. The Los Angeles Times Ralph K. M. Haurwitz, Austin American-Statesman Diedtra Henderson. Seattle Times Edie Lau. Sacramento Bee Larry Proulx. The Washington Post Frank D. Roylanee, Baltimore Sun Angela Swafford, Mas Vida/CBS Diane Toomey. WUNC Radio Ulysses Torassa. Cleveland Plain Dealer Karin Vergoth. National Public Radio/Science Friday Joby S. Warrick. The Washington Post Philip Yam, Scientific American Program Directors Robert D. Goldman, Northwestern University Boyce Rensberger. Knight Science Journalism Program Htinds-On Laboratory Course Directors Rex Chisholm, Northwestern University (Biomedical) John Hobbie. Marine Biological Laboratory (Environment) Jerry Melillo. Marine Biological Laboratory (Environment) Robert Palazzo, University of Kansas (Biomedical) SPINES — Summer Program in Neuroscience Ethics and Survival SPINES is a month-long program directed by Joe L. Martinez, Jr., and James Townsel. The program is supported by grants from NIMH administered by the American Psychological Association and the Association of Neuroscience Departments and Programs. SPINES offers an introduction to the opportunities available at the MBL and in the field of neuroscience in general. Fellows are taught responsible conduct in research and other survival skills such as scientific writing, poster construction, presentations, grant mechanisms, and how to seek a postdoctoral or job position. Fellows Carlos Bolanos-Guzman Morry Brown Winfred Monica Bryan Damani Nabet-Yero Bryant Jameel Dennis Cynthia Gentry Karen Gilliums Caterina Maria Hernandez William Meilandt Silke Monn Nivia Perez Acevedo Osceola Whitney Scholarships Awarded Aline D. Gross Scholarship Fund Tao, Haiyang, Ohio University American Society for Cell Biology Minorities Affairs Committee Anderson, Tonya, University of California. Los Angeles Foster, Andrea, Stanford University Freeman, Antoinette, Boston University School of Medicine Hinojos, Cruz, University of Texas, Houston Tafari. Tsahai, University of California. San Diego Arthur Klorfein Scholarship and Fellowship Fund Garcia. Ana Anton, Universidad Miguel Hernandez. Spain Jacobson, Eyal. Technion, Israel Rossi. Francesco, Scuola Normale Superiore, Pisa Tarlera, Silvana, Universidad de la Republica of Uruguay Hubby. Bolyn, University of Georgia Lambert, Laurence, Universitat Miinchen Lovett, Jennie. Washington University Lyons, Emily, Indiana University Matuschewski, Kai, New York University Nde, Pius, Humboldt University, Germany Oli, Monica, Auburn University Paul, Kimberly, Princeton University Burroughs Wellcome Fund Frontiers In Reproduction Course Arechavaleta-Velasco. Fabian, National Institute of Nutrition, Mexico Beg, Mohd, National Institute of Immunology, India Chen, Chie-Pein, MacKay Memorial Hospital Moreno, Ricardo, Oregon Regional Primate Research Center Kumar, Ramasamy Sampath, University of Western Ontario Sanchez-Partida. Luis. University of Adelaide Santos. Joao, Oregon Regional Primate Research Center Biology Club of the College of the City of New York Belluscio, Leonardo, Columbia University Burroughs Wellcome Fund Biology of Parasitism Course Alves. Fabio. Fundaeao Oswaldo Cruz, Brazil Arevalo, Myriam, Universidad del Valle Cali Artis, David, University of Manchester Henze, Katrin. The Rockefeller University Burroughs Wellcome Fund Molecular Mycology Course Cisalpino. Patricia, Universidade Federal de Minas Gerais Edens, Heather, Montana State University Haycocks, Neil, University of Texas Medical Branch Lee, Samuel, Yale University School of Medicine Nagabhushan, Moolky, Loyola University, Chicago Santangelo, Rosaria. Public Health Research Institute, Italy Schaffrath, Raffael, University of Halle. Germany Sheppard. Don, McGill University Honors R57 C. Lalor Burdick Scholarship Shirasaki, Ryuichi, Osaka University Caswell Grave Scholarship Fund Champion, Mia, University of California, Davis Pollack, Anne, University of Arizona Tao, Haiyang, Ohio University Wagner, Eric, Duke University Charles Baker Metz and William Metz Scholarship Fund in Reproductive Biology Arechavaleta-Velasco, Fabian, National Institute of Nurtrition, Mexico Carabatsos, Mary Jo, Tufts University Euling, Susan, US Environmental Protection Agency Grazul-Bilska, Anna. North Dakota State University Halvorson, Lisa, Brigham and Women's Hospital Rulli, Susan, Hospital General de Ninos Ricardo Gutierrez Sanchez-Partida, Luis, University of Adelaide Daniel S. Grosch Scholarship Fund Castro, Hector. University of Florida Edwin Grant Conklin Memorial Fund Wagner. Eric, Duke University Frank R. Lillie Fellowship and Scholarship Fund Lee, Agnes, Yale University Strieker, Jesse, Duke University Vos. Johannes, University of Massachusetts Gary N. Calkins Memorial Scholarship Fund Bjornsson, Christopher, University of Manitoba Herbert W. Rand Fellowship and Scholarship Fund Azouz, Rony. University of California, Davis Battaglia, Francesco, SISSA, Italy Bi, Guoqiang, University of California. San Diego Brenner, Naama, NEC Research Institute Buschbeck, Elke. Cornell University Cai, Rick, University of California, Los Angeles d'Avella. Andrea, Massachusetts Institute of Technology Fairhall, Adrienne, Weizmann Institute. Israel Fellows, Matthew, Brown University Hartings. Jed, University of Pittsburgh Kepecs, Adam, Brandeis University Klug, Achim, University of Texas Lee, Ann, Brown University Machens, Christian, Humboldt University, Germany Majewska. Anna, Columbia University Pollack, Anne, University of Arizona Ruggiano, Stephanie, Boston University Shaub, Amy, University of North Carolina. Chapel Hill Scares, Daphne, University of Maryland Van Rossum. Mark, University of Pennsylvania Weber, Stacy, Ohio University Wright, Brian, University of California, San Francisco Zhang, Ying, Harvard Medical School Howard A. Schneiderman Endowed Scholarship Buschbeck, Elke, Cornell University Shirasaki, Ryuichi. Osaka University Tahmci, Emilios. Boston University Wonsettler, Angela, Marshall University School of Medicine Howard Hughes Medical Institute Educational Scholarship Funding Berggren, Kirsten, University of Vermont Bjornsson, Christopher, University of Manitoba Castro, Hector, University of Florida Chang, Sunghoe, University of Illinois Gladfelter, Amy, Duke University Jacobson. Eyal, Technion, Israel Locke, Emily, Johns Hopkins University Mansharamani, Malini, Texas Tech University Health Science Center Marchant. Jonathan. University of California. Irvine Ober, Elke. Max-Planck-Institut Tubingen Pappu. Kartik. Wesleyan University Roch. Fernando. Wellcome/CRC Institute. United Kingdom Rozowski. Marion, Wellcome/CRC Institute. United Kingdom Runt't. Linda, University of Connecticut Shaub, Amy, George Washington University Medical Center Strieker, Jesse, Duke University Wagner, Eric, Duke University Zhou. Ming. State University of New York. Buffalo Indo-U.S. Contraceptive and Reproductive Health Initiative Program Award Beg. Mohd. National Institute of Immunology. India International Brain Research Organization Scholarships Burzio, Veronica, University of Chile Concha, Miguel, University of Chile Gallo. Gianluca, University of Minnesota Miiller, Ferenc, IGBMC. Strasbourg St. Amant, Louis. McGill University Wang, Feng, Yale University Jacques Loeb Founders' Scholarship Fund Gladfelter, Amy. Duke University Marjorie W. Stetten Scholarship Fund Avila. Antonia. CINVESTAV-IPN, Mexico Pepi, Milva, University of Siena Speirs, Kendra, University of Pennsylvania Massachusetts Space Grant Consortium Awards Monteiro, Antonia, Harvard University Rocha-Olivares, Axayacatl. Scripps Institution of Oceanography Rosenthal, Benjamin. Harvard University R58 Annual Report MBL Pioneers Scholarship Fund Geraci, Fabiana, Dip di Biologia Cellulare e dello Sviluppo, Italy Lupo. Guiseppe. University of Pisa Ober, Hike, Max-Planck-Institut Tubingen Vonica, Alin, Cornell University Medical Center Merck & Company, Inc. Scholarships Locke, Emily, Johns Hopkins University Paul, Kimberly, Princeton University Runt't, Linda. University of Connecticut Saxowsky, Tina, Johns Hopkins School of Medicine Speirs, Kendra, University of Pennsylvania Waller, Ross, University of Melbourne Zaph, Colby, University of Victoria Billing. Susan. US Environmental Protection Agency Grazul-Bilska. Anna, North Dakota State University Halvorson, Lisa, Brigham and Women's Hospital McBnde. Helen. University of Utah McCauley, David. Pennsylvania State University Smith, Katherine, University of Virginia Stimson, Laura, University of Arizona Surdna Foundation Scholarship Ghazi, Arjuman, National Centre for Biological Sciences, India Lupo, Giuseppe, University of Pisa Pappu. Kartik. Wesleyan University Roch, Fernando, Wellcome/CRC Institute, United Kingdom Tahinci, Emilios, Boston University Mountain Memorial Fund Scholarship Chenevert, Janet, CNRS, France Deavours. Bettina, Virginia Tech Lam, Phoebe, Princeton University Lanntina. Samuel, Emory University Omara, Felix, Universite de Quebec Zaarour, Rania. Yale University Pfizer Inc. Endowed Scholarship Fund Locke, Emily. Johns Hopkins University Runft, Linda, University of Connecticut Phillip H. Presley Scholarship Award, Funded by Carl Zeiss, Inc. Kappler, Andreas, University of Konstanz Paemeleire. Koen, University of Ghent Paliulis. Leocadia. Duke University Pepi. Milva. University of Siena Rossi. Francesco, Scuola Normale Superiore, Pisa Takasu, Mari, Harvard University Planetary Biology Internship Awards Klappenbach. Joel, Michigan State University Spear. John. Colorado School of Mines Ruth Sager Memorial Scholarship Weber, Stacy. Ohio University S. O. Mast Memorial Fund d'Avella, Andrea, Massachusetts Institute of Technology Komarova, Svetlana. NASA Ames Research Center Society for Developmental Biology Scholarships Carabatsos. Mary Jo. Tufts University Chen. James, Harvard University Walter L. Wilson Endowed Scholarship Fund Mansharamani. Malini. Texas Tech University Health Science Center William F. and Irene C. Diller Memorial Scholarship Fund Champion. Mia. University of California. Davis William Morton Wheeler Family Founders' Scholarship Bjomsson. Christopher. University of Manitoba Soares. Daphne. University of Maryland Zhou, Ming, State University of New York. Buffalo William Randolph Hearst Foundation Scholarships Wang, Jing. Bell Laboratories Lee, Agnes, Yale University William Townsend Porter Fellowship and Scholarship Fund Anderson, Tonya. University of California, Los Angeles Foster. Andrea, Stanford University Freeman, Antoinette. Boston University School of Medicine Hinojos, Cruz, University of Texas, Houston McFarlane, Matthew, Stanford University McGiffert. Christine, University of California, San Diego Tafan, Tsahai, University of California, San Diego World Health Organization Scholarships Arechavaleta-Velasco, Fabian. National Institute of Nutrition, Mexico Cohen. Debora, IBYME, Argentina Rulli, Susan. Hospital General de Ninos. Argentina Honors R59 Post Course Research Awards Brinda Dass, Texas Tech University Health Sciences Center, Physiology Jonathan Marchant, University of California, Irvine. Physiology Bettina Deavours, Virginia Tech, Physiology David McCauley. Penn State University, Embryology James Hitt, SUNY Health Science Center. Syracuse, Neural Systems Linda Runft, University of Connecticut, Physiology and Behavior Tshai Tafari, University of California. San Diego, Physiology Adam Kepecs. Brandeis University. Neural Systems and Behavior Sinju Tauhata. Dep. de bioquimica FMRP/USP. Brazil. Shann Kim. University of Illinois. Chicago. Physiology Physiology Malini Mansharamani. Texas Tech University Health Science Center. Johannes Vos. University of Massachusetts. Physiology Physiology Board of Trustees and Committees Corporation Officers & Trustees Chairman of the Board of Trustees, Sheldon J. Segal. The Population Council Co-Vice Chair of the Board of Trustees, Frederick Bay, Josephine Bay Paul and C. Michael Paul Foundation Co-Vice Chair of the Board of Trustees, Mary J. Greer, New York. NY President of the Corporation, John E. Dowling. Harvard University Director and Chief Executive Officer, John E. Burris, Marine Biological Laboratory* Treasurer of the Corporation, Mary B. Conrad. Fiduciary Trust International* Clerk of the Corporation, Neil Jacobs, Hale and Dorr Chair of the Science Council, Kerry S. Bloom. University of North Carolina* Class of 2002 Class of 1999 Mary-Ellen Cunningham, Grosse Pointe Farms. MI Darcy Brisbane Kelley, Columbia University Laurie J. Landeau. Marinetics. Inc. Burton J. Lee, III, Vero Beach, FL Robert E. Mainer, The Boston Company Jean Pierce, Boca Grande. FL Class of 2000 Alexander W. Clowes, University of Washington School of Medicine Story C. Landis, Case Western Reserve University Irwin B. Levitan, Brandeis University G. William Miller. G. William Miller and Co.. Inc. Frank Press, The Washington Advisory Group Christopher M. Weld. Sullivan and Worcester Class of 2001 Porter Anderson. North Miami Beach. FL Frederick Bay, Josephine Bay Paul and C. Michael Paul Foundation, Inc. Martha W. Cox. Hobe Sound, FL Mary J. Greer. New York. NY William C. Steere, Jr.. Pnzer Inc. Gerald Weissmann. New York L'niversity School of Medicine *Ex officio Sydney M. Cone, III. Cleary. Gottlieb. Steen & Hamilton John R. Lakian. The Fort Hill Group. Inc. Joan V. Ruderman. Harvard Medical School Sheldon J. Segal, The Population Council William T. Speck, New York Presbyterian Hospital Alfred Zeien. The Gillette Company Honorary Trustees James D. Ebert. Baltimore. MD William T. Golden. New York. NY Ellen R. Grass. The Grass Foundation Trustees Emeriti Edward A. Adelberg, Yale University John B. Buck. Sykesville. MD Seymour S. Cohen, Woods Hole, MA Arthur L. Colwin, Key Biscayne, FL Laura Hunter Colwin, Key Biscayne, FL Donald Eugene Copeland, Woods Hole, MA Sears Crowell, Jr., Indiana Lmiversity Alexander T. Daignault. Falmouth. MA (deceased) Teru Hayashi, Woods Hole, MA Ruth Hubhard, Cambridge. MA Lewis Kleinholz, Reed College Maurice Krahl. Tucson, AZ C. Ladd Prosser. University of Illinois W.D. Russell-Hunter. Syracuse University John W. Saunders, Waquoit. MA D. Thomas Trigg. Wellesley, MA Walter S. Vincent, Woods Hole. MA Directors Emeriti James D. Ebert, Baltimore, MD Paul R. Gross, Falmouth, MA Harlyn O. Halvorson, Woods Hole, MA K60 Trustees and Committees R61 Executive Committee of the Board of Trustees Sheldon J. Segal. Chair Frederick Bay. Co-Vice Chair Mary J. Greer, Co- Vice Chair John E. Burris* Ronald L. Calabrese (1998) Kerry S. Bloom Mary B. Conrad Mary Ellen Cunningham Robert Mainer Joan V. Rudemian Gerald Weissmann Science Council Ronald L. Calabrese. Chair (8/98) Donald Abt (1999) Clay M. Armstrong (8/98-8/2000) Peter Armstrong (8/98-8/2000) Vincent E. Dionne (1999) John Dowling (8/98) Barbara Ehrlich (1999) Laurinda Jaffe (8/98-8/99) Charles Hopkinson (8/98-8/2000) Bruce J. Peterson (8/98) Mitchell Sogin (8/98-8/2000) Standing Committees of the Board of Trustees Development Mary Ellen Cunningham. Chair Porter W. Anderson Robert Barlow Fred Bay Mary B. Conrad Martha Cox James Ebert Philip Grant Neil Jacobs John Lakian Burton Lee G. William Miller Jean Pierce William Speck William Steere Christopher Weld Facilities & Capital Equipment Joan Ruderman, Chair Porter W. Anderson Frederick Bay Lawrence Cohen Neal Cornell Story Landis Irwin Levitan Jean Pierce Frank Press Christopher Weld Investment Robert Mainer. Chair Svdnev M. Cone Mary B. Conrad John R. Lakian G. William Miller Sheldon Segal Alfred Zeien Finance Robert Mainer. Chair Alexander Clowes Sydney M. Cone Mary B. Conrad Donald DeHart Neil Jacobs Darcy Kelley John R. Lakian Laurie Landeau Werner Loewenstem Robert Manz G. William Miller Ronald O'Hanley Alfred Zeien Nominating Gerald Weissmann Ronald L. Calabrese Alexander Clowes Martha Cox Mary Ellen Cunningham Mary Greer Story Landis Sheldon Segal William Steere Standing Committees of the Corporation and Science Council Buildings and Grounds Lawrence B. Cohen. Chair Barbara C. Boyer Alfred B. Chaet Richard Cutler* William R. Eckberg *Ex officio R62 Annual Report Barry Fleet* Ferenc Harosi Joe Hayes* Bruce J. Peterson Kenyon S. Tweedell Ivan Valiela Education Committee John Dowling, Chair Kerry S. Bloom Elaine Bearer Vincent Dione Paul Dunlap Rachel Fink Roger Hunlon Holger Jannasch George M. Langford Michael Mendelsohn Steve Zottoli Ron Calahrese* E.A. Dawidowicz* Dorianne Chrysler Mebane* LouAnn King* Robert P. Malchow Darrell R. Stokes Ann E. Stuart Janis C. Weeks MBL/WHOI Library Joint Advisory Committee David Shepro, Chair. MBL Judy Ashmore, MBL* David Dow, NMFS Daniel Fornan. WHOI G. Richard Harbison, WHOI John Hobbie, MBL Sylvia Kane, NMFS Mark Kurz, WHOI Colleen Hurter, WHOI* Cathy Norton, MBL* James Robb. USGS Birgit Rose, MBL Peter J.S. Smith. MBL Bruce Warren. WHOI Fellowships Thoru Pederson. Chair Linda Deegan Barbara Ehrlich George M. Langford Jose Lemos Cindy Lee VanDover E.A. Dawidowicz* Sandra Kautmann* Research Services and Space Housing, Food Service and Child Care Carole Browne, Chair Kerry S. Bloom Hans Laufer, Chair Peter B. Armstrong Neal Cornell Richard Cutler* E.A. Dawidowicz* Kenneth Foreman Louis M. Kerr* David Landowne Andrew Mattox* Merle Mizell Peter J.S. Smith Paul Steudler Ivan Valiela Discovery: The Campaign for Science at the Marine Biological Laboratory Steering Committee Frederick Bay, Campaign Chair William T. Golden. Honorary Chair Ellen R. Grass, Honorary Chair Alexander W. Clowes. Vice-Chair Martha W. Cox, Vice-Chair G. William Miller, Vice-Chair Gerald Weissmann, Vice-Chair Porter W. Anderson Robert B. Barlow. Jr. Norman Bernstein Jewell Plummer Cobb Mary B. Conrad Mai> I'llen Cunningham *Ex < John E. Dowling James D. Ebert Gerald D. Fischbach Robert D. Goldman Mary J. Greer M- Howard Jacobson Laurie J. Landeau George M. Langford Burton J. Lee, III Jean Pierce Robert A. Prcndergast David Shepro William T. Speck William C. Steere, Jr. Christopher M. Weld Alfred M. Zeien Trustees and Committees R63 Council of Visitors Norman B. Asher, Esq., Hale and Dorr, Boston. MA Mr. Donald J. Bainton. Chairman & CEO, Conlinental Can Co., Boca Raton, FL Mr. David Bakalar, Chestnut Hill. MA Mr. Charles A. Baker, The Liposome Company, Inc., Princeton, NJ Dr. George P. Baker, Massachusetts General Hospital, Boston, MA Dr. Sumner A. Barenberg. Bernard Technologies, Chicago, 1L Mr. Robert P. Beech. President/CEO. Component Software International. Inc., Mason, Ohio Mr. George Berkowitz. Chairman and Founder, Legal Sea Foods, Allston, MA Dr. Elkan R. Blout, Harvard Medical School, Boston. MA Mr. and Mrs. Philip Bogdanovitch. Lake Clear. New York Mr. Malcolm K. Brachman. Northwest Oil Company. Dallas, TX Dr. Goodwin M. Breinin. New York University Medical Center, New York, NY Mr. John Callahan, President, Carpenter. Sheperd & Warden, New London, NH Mrs. Elizabeth Campanella, West Falmouth, MA Thomas S. Crane, Esq., Mintz Levin Cohen Ferris Glovsky & Popeo, PC, Boston, MA Dr. Stephen D. Crocker. Chief Technology Officer, Cyber Cash Inc., Reston. VA Ms. Lynn W. Piasecki Cunningham, Film and Videomaker, Piasecki Productions, Brookline, MA Dr. Anthony J. Cutaia, Sr. Director, Office of Health Issues. Anheuser-Busch. Inc.. St. Louis, Missouri Dr. Georges de Menil. DM Foundation. New York, NY Mrs. Sara Greer Dent, Chevy Chase, MD Mr. D. H. Douglas-Hamilton. Vice President, Research and Development. Hamilton Thorne Research, Beverly. MA Mr. Benjamin F. Du Pont. Du Pont Company, Deepwater, New Jersey Dr. Sylvia A. Earle, Founder, Deep Ocean Engineering. Oakland. CA Mr. Anthony B. Evnin, General Partner, Venrock Associates, New York, NY Stuart Feiner, Esq., Vice President and Secretary, General Counsel. Inco Limited. Toronto, Ontario, Canada Mrs. Hadley Mack French, Consultant, Edsel & Eleanor Ford House, Grosse Pointe Farms. MI Mr. William J. Gilbane. Jr.. Gilbane Building Company, Providence. Rl Dr. Michael J. Goldblatt, Intelligent Biocides, Tewksbury. MA Mr. Maynard Goldman, President, Maynard Goldman & Associates, Boston, MA Ms. Charlotte I. Hall, Edgartown. MA Mr. Thomas J. Hynes, Jr.. President, Meredith & Grew, Inc., Boston, MA Mr. M. Howard Jacobson, Bankers Trust Westborough. MA Mrs. Elizabeth Ford Kontulis, New Canaan. CT Mr. and Mrs. Robert Lambrecht, Boca Grande. FL Dr. Catherine C. Lastavica. Tufts University School of Medicine. Boston. MA Mr. Joel A. Leavitt, Boston, MA Mr. Stephen W. Leibhol/. President. TechLabs. Inc.. Huntingdon. PA Mrs. Margaret Lilly, West Falmouth, MA Mr. George W. Logan, Chairman, Valley Financial Corp., Roanoke, VA Mr. Michael T. Martin, SportsMark, Inc.. New York, NY Mrs. Christy Swift Maxwell. Grosse Pointe Farms. MI Mr. Ambrose Monell. G. Unger Vetlesen Foundation, Palm Beach, FL Dr. Mark Novitch, Washington, DC Ms. Julie Packard, Executive Director, Monterey Bay Aquarium, Monterey, CA Mr. David R. Palmer, Founder & Managing Director, David Ross Palmer & Associates, Waquoit, MA Dr. Roderic B. Park, Richmond. CA Mr. Santo P. Pasqualucci, President/CEO Falmouth Co-Operative Bank. Falmouth. MA Mr. Robert Pierce, Jr., Pierce Aluminum Co., Canton, MA Mr. Richard Reston, Editor and Publisher, Vineyard Gazette, Edgartown, MA Mr. Marius Robinson, Managing Partner, Fundamental Investors Ltd., Key Biscayne, FL John W. Rowe, M.D., President, Mt. Sinai School of Medicine and Mt. Sinai Medical Center, New York, NY Mr. Edward Rowland, Tucker, Anthony, Inc.. Boston. MA Mr. Gregory A. Sandomirsky, Mintz Levin Cohen Ferris Glovsky & Popeo, PC, Boston, MA Mrs. Mary Schmidek. Marion. MA Dr. Cecily C. Selby. New York. NY Mr. Robert S. Shifman. St. Simon's Island, GA Mr. and Mrs. Gregory Skau, Grosse Pointe Farms. MI Mr. Malcolm B. Smith. Vice Chairman. General American Investors Co., New York, NY Mr. John C. Stegeman, Owner, Campus Rentals, Ann Arbor, MI Mr. Joseph T. Stewart. Jr.. Skillrnan. NJ Mr. John W. Stroh. Ill, Chief Executive Officer. The Stroh Brewery Company, Detroit. MI Mr. Gerard L. Swope. Washington. DC Mr. John F. Swope, Concord, NH Mr. and Mrs. Stephen E. Taylor, Boston, MA Mrs. Donna Vanden Bosch-FIynn, Spring Lake. NJ Mrs. Carolyn W. Verbeck, Vineyard Haven, MA Mr. Benjamin S. Warren, III, Grosse Pointe Farms, MI Nancy B. Weinstein, R.N., The Hospice, Inc., Glen Ridge, NJ Stephen S. Weinstein, Esq., Morristown, NJ Mr. Frederick J. Weyerhaeuser, Beverly, MA Mr. Tony L. White, The Perkin Elmer Corporation. Norwalk. CT Dr. Torsten N. Wiesel. President Emeritus, The Rockefeller University, New York, NY Administrative Support Staff1 Biological Bulletin Greenberg, Michael J., Editor-in-Chief Hinkle, Pamela Clapp. Managing Editor Burns, Patricia Gibson. Victoria R. Schachinger. Carol H. Financial Sen'ices Office Lane, Jr., Homer W., Chief Financial Officer Roddy, Timothy, Chief Financial Officer Bowman, Richard, Controller Arbnso, Janis Barry, Maureen Dwyer, Patricia E. Eidelman, Dana Hopkins, Ann E. Lancaster, Cindy Poravas. Maria Ranzinger. Laura Sprague, Patricia A. Stark, Judy M. Stellrecht, Lynette Slock Room Schorer, Timothy M., Supervisor Capano, Holly2 O'Connor-Lough. Susan Purchasing Hall Jr., Lionel E., Supervisor Shamon, Lynne R. Stone, Janice G.2 Director's Office Burris, John E.. Director and Chief Executive Officer Donovan, Marcia H. MacNeil, Jane L. External Affairs Carotenuto, Frank C., Director Butcher. Valerie 1 Including persons who joined or left the staff during 1998. : Summer or temporary. Callahan Jr., John L.: Faxon. Wendy P. Martin, Theresa H. Maxwell, Thanh L.2 Patch-Wing. Dolores Quigley. Barbara A. Scibek, John C. Shaw. Kathleen L. Associates Program Bohr. Kendall B. Brown. Shannon K.2 Gault, Miciah Bay2 Communications Office Hinkle, Pamela Clapp, Director Burton. Anne E. Flynn, Bridget Hinkle. Kristen" Joslin, Susan Liles. Beth R Housing and Conferences King. LouAnn D.. Director Barry, Maureen J. Grasso, Deborah Hanlon, Arlene K.2 Johnson-Herman. Frances N. Masse, Todd C. Perito, Diana Switchboard Baker, Ida M.: Ridley, Alberta W.2 Human Resources Goux. Susan P., Director Cox, Sarah2 Orange. Stacey B. Houser, Carmen Renaud, Nina L. Marine Resources Center Hanlon, Roger T.. Director Moni/., Priscilla R64 Administrative Support Staff R65 Aquatic Resources Department Enos, Jr., Edward G., Superintendent Bourque, Ryan M.2 Chappell, P. Dreux2 DeGiorgis, Joseph A.2 Grossman, William M. Gudas. Christopher N.2 Kilpamck. Brian2 Klimm III, Henry W. Luther. Herbert Mansfield, Darren P.2 Sexton, Andrew W. Smith, Gary2 Sullivan, Daniel A. Tassinari, Eugene MRC Life Support System Mebane. William N., Systems Operator Hanley, Janice S. Kuzirian, Alan Solbo, Jr.. Steven2 Stukey, Jetley M. Till, Geoffrey A. MBLAVHOI Library Norton, Catherine N., Director Ashmore, Judith A. Costa, Marguerite E. Crocker, Daniel2 Cullen. Cynthia M.2 Deveer, Joseph M. Duda, Laurel E. Farrar, Stephen R. L. Medeiros, Melissa Monahan. A. Jean Moniz, Kimberly L. Nelson. Heidi Riley, Jacqueline Swasey, Anne E.2 Copy Center Mountford, Rebecca J., Supervisor Abisla. Richard L.2 Clark, Tamara L. Delaney. Elizabeth S. (Suwijn)2 Kefeauver, Lee LaPlante, Robert F. Mancini. Mary E. Sorocco. Debra2 Wallace, Jennifer2 Warner, Kathleen2 Information Systems Division Smith, Adrian P., Assistant Director Berrios, Kelly2 Ennis, Douglas E.2 Gage, Timothy J.2 Katz, Corey2 Malchow, Robert2 Mountford, Rebecca J. Moynihan, James V. Remsen, David P. Renna, Denis J. Space. David B. Safety Sen'ices Mattox, Andrew H.. Environmental. Health, and Safety Manager Bradley. Margaret2 O'Neill, Maureen D.2 Sen'ice, Projects and Facilities Cutler. Richard D.. Director Enos, Joyce B. Apparatus Baptiste, Michael G. Barnes. Franklin D. Haskins, William A. Building Senices & Grounds Hayes, Joseph H., Superintendent Anderson. Lewis B. Atwood, Paul R. Baker, Harrison S. Barnes. Susan M. Berrios, Jessica L.2 Boucher, Richard L. Brenerman, Brian2 Brereton, Richard S.2 Callahan. John J. Cameron, Lawrence M.2 Collins. Paul J. Cowan, Matthew B.2 Cutler, Matthew D.2 Dirnond, Jay2 Dorris. John J. Eldridge, Myles2 Fernandez, Peter R.2 Gibbons, Roberto G. Gonsalves, Nelson Gray, Joshua2 Hannigan. Catherine Harrington. James D. Illgen. Robert F. Lawrence. Adam2 Ledwell. L. Patrick2 Luther, Herbert Lynch, Henry L. Maccaro. Jackie Mayock, Michael J.2 McNumara, Noreen M. McQuillan, Jeffrey2 Plant, Stephen W. Rattacasa, Frank" Ryan. Timothy A.: Sholkovitz.. David2 Silva. Cynthia C. Stites, Clint2 Tardif. Joseph G. R.2 Ware, Lynn M. Plant Operations anil Maintenance Fleet. Barry M.. Superintendent Cadose. James W., Maintenance Supervisor R66 Annual Report Barnes, John S. Blunt, Hugh F. Bourgoin. Lee E. Carini, Robert J. Carroll, James R. Deree. Dana J. Fish Jr., David L. Fuglister, Charles K. Goehl, George Gonsalves, Jr.. Walter W. Hathaway, Peter J. Henderson. Jon R Justason, C. Scott Langill. Richard Lochhead, William M. McAdarns III, Herbert M. McHugh. Michael O. Mills, Stephen A. Olive, Jr.. Charles W. Schoepf, Claude Settlemire, Donald Shepherd. Denise M. Sylvia, Frank E.2 Toner, Michael Wetzel, Ernest D.2 Photolab Nelson, Linda M. Research Administration & Educational Programs Dawidowicz, Eliezar A.. Director Hamel. Carol C. Kaufmann. Sandra J. Kefauver, Lee Iwaszko, Nicole2 Lynn, Rebecca Mebane. Dorianne C. Malmude-Davis, Anna2 Palmer, Pamela2 Patten. Brooke A.2 Stukey, Jetley Central Microscopy Facility and General Use Rooms Kerr, Louis M.. Supervisor DeProto, Jamin E.2 Luther, Herbert Peterson. Martha B. Josephine Bay Paul Center for Comparative Molecular Biology and Evolution Administrative Staff Harris, Marian Lim, Pauline Journal of Membrane Biology Loewenstein. Werner R., Editor Fay, Catherine H. Howard Isenberg. Linda L. Lynch, Kathleen F. Satellite/Periwinkle Children 's Programs Robinson. Paulina H.2 Browne, Jennifer L.2 Collins, Anne E.2 Curran, Kelly2 Douglas, Alicia D.2 Fitzelle. Annie2 Gallant, Carolyn A.2 Gallant, Cynthia2 Guiffrida, Beth2 Griffin, Courtney A.2 Jenkins, Michelle2 Laundy, Jennifer2 McCusker. Stephanie2 Robinson, Jayma L.2 NASA Center for Advanced Studies in the Space Life Sciences Dawidowicz. Eliezar A., Administrator Amit, Udem P. Ecosvstems Center Administrative Staff Berthel. Dorothy J. Donovan, Su/.anne J. Nunez, Guillermo Seifert, Mary Ann Members of the Corporation Life Members Acheson, George H., 25 Quissett Avenue. Woods Hole, MA 02543 Adelberg, Edward A., 204 Prospect Street. New Haven. CT 065 1 1 - 2107 Afzelius. Bjorn, University of Stockholm, Wenner-Gven Institute. Department of Ultrastructure Research. Stockholm, SWEDEN Amatniek, Ernest, address unknown Arnold, John M., 329 Sippewissett Road, Falmouth. MA 02540 Bang, Betsy G., 76 F. R. Lillie Road. Woods Hole, MA 02543 Bartlett. James H., University of Alabama. Department of Physics. Box 870324. Tuscaloosa. AL 35487-0324 Berne, Robert M., University of Virginia School of Medicine, Department of Physiology. Box 1116, MR4. Charlottesville, VA 22903 Bernheimer, Alan W., New York University Medical Center, Department of Microbiology, 550 First Avenue. New York. NY 10016 Bertholf, Lloyd M., Westminster Village. #2114, 2025 East Lincoln Street, Bloomington. IL 61701-5995 Bosch, Herman F., P.O. Box 353, Woods Hole, MA 02543 Buck, John B., Fairhaven C-020, 7200 Third Avenue. Sykesville, MD 21784 Burbanck, Madeline P., P.O. Box 15134. Atlanta. GA 30333 Burbanck. \\illiam D.. P.O. Box 15134. Atlanta. GA 30333 Carlson, Francis D., Johns Hopkins University. Biophysics Department Jenkins Hall, North Charles Street, Baltimore. MD 21218 (deceased) Clark, Arnold M., 53 Wilson Road, Woods Hole, MA 02543 Clark, James M., 258 Wells Road. Palm Beach. FL 33480-3625 Cohen, Seymour S., 10 Carrot Hill Road, Woods Hole, MA 02543- 1206 Colwin, Arthur L., 320 Woodcrest Road, Key Biscayne, FL 33149- 1322 Colwin, Laura Hunter, 320 Woodcrest Road. Key Biscayne, FL 33149-1322 Cooperstein. Sherwin J., University of Connecticut. School of Medicine, Department of Anatomy, Farmington. CT 06030-3405 Copeland. D. Eugene, Marine Biological Laboratory. Woods Hole. MA 02543 Corliss, John O., P.O. Box 2729, Bala Cynwyd, PA 19004-21 16 Costello, Helen M., Carolina Meadows, Villa 137, Chapel Hill, NC 27514-8512 Crouse, Helen, Rte. 3, Box 213. Hayesville. NC 28904 DeHaan, Robert I,., Emory University School of Medicine, Department of Anatomy & Cell Biology. 1648 Pierce Drive, Room 108. Atlanta. GA 30322 Dudley, Patricia L., 3200 Alki Avenue SW. #401. Seattle. WA 98116 Edwards, Charles, 3429 Winding Oaks Drive, Longboat Key, FL 34228 Elliott, Gerald F., The Open University Research Unit. Foxcombe Hall. Berkeley Road. Boars Hill. Oxford OX1 5HR, ENGLAND Failla, Patricia M., 2 1 49 Loblolly Lane, Johns Island, SC 29455 Ferguson, James K. W., 56 Clarkehaven Street. Thornhill, Ontario L4J 2B4, CANADA Glusman, Murray, New York State Psychiatric Institute. 722 W. 168th St.. Unit #70, New York, NY 10032 Goldman, David E., 140 Ter Heun Drive, Room 212. Falmouth. MA 02540 (deceased) Graham, Herbert, 36 Wilson Road, Woods Hole, MA 02543 Hamburger, Viktor, Washington University. Department of Biology. 740 Trinity Avenue. St. Louis. MO 63 1 30 Hamilton. Howard L., University of Virginia, Department of Biology, 238 Gilmer Hall, Charlottesville. VA 22901 Harding, Jr., Clifford V., 54 Two Ponds Road, Falmouth. MA 02540 Haschemeyer, Audrey E. V'., 21 Glendon Road, Woods Hole, MA 02543-1406 Hauschka, Theodore S., 333 Fogler Road, Bremen, ME 0455 1 Hayashi, Teru, 1 5 Gardiner Road, Woods Hole, MA 02543- 1113 Hisaw, Frederick L.. 1 765 SW Tamarack Street, Apt 11, McMinnville, OR 97128-7416 Hoskin, Francis C. G., c/o Dr. John E. Walker, U.S. Army Natick RD&E Center. SAT NC-YSM, Kansas Street, Natick, MA 01760- 5020 Humes, Arthur G., Marine Biological Laboratory. Boston University Marine Program, Woods Hole. MA 02543 Hunter, W. Bruce, 305 Old Sharon Road, Peterborough, NH 03458- 1736 R67 R6S Annual Report Hurwitz, Charles, Stratum VA Medical Center. Research Service. Albany. NY 1220S Kalz, George, Merck. Sharp and Dohme. Fundamental & Experimental Research Laboratory. PO Bo\ 2000, Rahway, NJ 07065 Kingsbury, John M., Cornell University, Department of Plant Biology. Plant Science Building. Ithaca. NY 14853 Kleinholz. Lewis, Reed College. Department of Biology, 3203 SE Woodstock Boulevard. Portland, OR 97202 Kusano, Kiyoshi, National Institutes of Health, Building 36, Room 4D- 20, Bethesda, MD 20892 Laderman, Ezra, Yale University, New Haven. CT 06520 LaMarche, Paul H., Eastern Maine Medical Center, 489 State Street, Bangor, ME 04401 Lauffer, Max A., Penn State University Medical Center, Department of Biophysics & Physiology. Hershey. PA 1 7033 LeFevre, Paul G., 1 5 Agassiz Road, Woods Hole. MA 02543 (deceased) Lochhead, John H., 49 Woodlawn Road, London SW6 6PS. UK Loevvus, Frank A., Washington State University, Institute of Biological Chemistry, Pullman, WA 99164 Loftfield, Robert B., University of New Mexico, School of Medicine. Albuquerque, NM 87131 Malkiel, Saul, 174 Queen Street, #9A, Falmouth. MA 02540 Marsh, Julian B., 9 Eliot Street. Chestnut Hill. MA 02467-1407 Martin, Lowell V., 10 Buzzards Bay Avenue, Woods Hole, MA 02543 Mathews, Rita W., East Hill Road, P.O. Box 237. Southfield. MA 01259-0237 Moore, John A., University of California. Department of Biology. Riverside, CA 92521 Moscona, Aron A., University of Chicago, Department Molecular Genetics & Cell Biology, Chicago. IL 60637 Musacchia, X. J., P.O. Box 5054, Bella Vista, AR 72714-0054 Nasatir, Maimon, P.O. Box 379, Ojai, CA 93024 Passano, Leonard M., University of Wisconsin, Department of Zoology. Birge Hall. Madison. WI 53706 Prosser, C. Ladd, University of Illinois, Department of Physiology, 524 Burrill Hall, Urbana. IL 61801 Prytz, Margaret McDonald, address unknown Ratner, Sarah, Public Health Research Institute, Department ol Biochemistry. 455 First Avenue, New York. NY 10016 Renn, Charles E., address unknown Reynolds, George T., Princeton University, Department of Physics, Jadwin Hall. Princeton, NJ 08544 Rice, Robert V., 30 Burnham Drive, Falmouth, MA 02540 Rockstein, Morris, 600 Biltmore Way. Apt. 805, Coral Gables. FL 33134 Ronkin, Raphael R., 3212 McKinley Street. NW. Washington. DC 20015-1635 Sanders, Howard L., Woods Hole Occanographic Institution, Woods Hole, MA 02543 Sato, Hidemi, Nagova University, 3-24-101. Oakinishi Machi, Toha Mie 517-0023, JAPAN Saz, Arthur K., Cieorgi •)<:•• n University Medical School, Department of Immunology, Washington. DC 20007 Schlesinger, R. Walter, 7 Langley Road, Falmoulh, MA 02540-1809 Scott, Allan C., Colby College, Waterville. ME 04901 Silverstein, Arthur M.. Johns Hopkins University. Institute of the History of Medicine. 1900 E. Monument Street, Baltimore, MD 21205 Sjodin, Raymond A., University of Maryland. Department of Biophysics. Baltimore. MD 21201 Smith. Paul F., P.O. Box 264. Woods Hole. MA 02543-0264 Speer, John VV., 293 West Main Road, Portsmouth, RI 02871 Sperelakis, Nicholas, University of Cincinnati, Department of Physiology/Biophysics, 231 Bethesda Avenue, Cincinnati, OH 45267- 0576 Spiegel, Evelyn, Dartmouth College, Department of Biological Sciences, 204 Oilman, Hanover. NH 03755 Spiegel, Melvin, Dartmouth College, Department of Biological Sciences. 204 Gilman, Hanover, NH 03755 Steinhardt, Jacinto, 1508 Spruce Street, Berkeley, CA 94709 (deceased) Stephens, Grover C., University of California, School of Biological Sciences, Department of Ecolocy and Evolution/Biology, Irvine, CA 92717 Strehler, Bernard L., 2310 N. Laguna Circle Drive, Agoura, CA 9130I-2SS4 Sussman, Maurice, 72 Carey Lane, Falmouth, MA 02540 Sussman, Raquel B., Marine Biological Laboratory. Woods Hole, MA 02543 Szent-Gyorgyi, Gwen P., 45 Nobska Road, Woods Hole, MA 02543 Taylor, Robert E., 339 Gifford Street. Apt. 303, Falmouth, MA 02540 (deceased) Thorndike, W. Nicholas, Wellington Management Company, 200 State Street, Boston, MA 02104 Trager, William, The Rockefeller University, 1230 York Avenue. New York. NY 10021-6399 Trinkaus, J. Philip, 870 Moose Hill Road, Guilford, CT 06437 Villee, Jr., Claude A., Harvard Medical School, Carrel L, Countway Library. 10 Shattuck Street. Boston, MA 021 15 Vincent, Walter S., 16 F.R. Lillie Road, Woods Hole, MA 02543 Wald, Ruth, Harvard University. Biological Laboratories. Cambridge, MA 02138 Waterman. Talbot H., Yale University, Box 208103, 912 KBT Biology Department, New Haven, CT 06520-8103 Wigley, Roland L., 35 Wilson Road, Woods Hole, MA 02543 Wilber, Charles G., Colorado State University, Department of Biology, Forensic Science Laboratory, Fort Collins. CO 80523 (deceased) Members Aht, Donald A., Marine Biological Laboratory, Laboratory of Aquatic Animal Medicine and Pathology. Woods Hole, MA 02543 Adams, James A., 348 1 Paces Ferry Road. Tallahassee. FL 32308 Adelman, William J., 160 Locust Street. Falmoulh. MA 02540 Alkon, Daniel L., N1H Laboratory of Adaptive Systems. 36 Convent Drive. MSC 4124, 36/4A2I, Bethesda, MD 20892-4124 Allen, Garland E., Washington University. Department of Biology, Box 1 137, One Brookings Drive, Street Louis, MO 63130-4899 Allen, Nina S., North Carolina State University, Department of Botany, Box 7612, Raleigh. NC 27695 Alliegro, Mark C., Louisiana State University Medical Center. Department of Cell Biology and Anatomy. 1901 Perdido Street, New Orleans. LA 701 12 Anderson, Everett, Harvard Medical School, Department of Cell Biology, 240 Longwood Avenue, Boston, MA 021 15-6092 Anderson, John M., 110 Roat Street, Ithaca, NY 14850 Members of the Corporation R6M Anderson, Porter W., 100 Bayview Drive #2224. North Miami Beach, FL 33160 Armett-Kihel, Christine, University of Massachusetts. Dean of Science Faculty. Boston. MA 02125 Armstrong, Clay M., University of Pennsylvania School of Medicine. B701 Richards Building. Department of Physiology. 3700 Hamilton Walk. Philadelphia. PA 19104-6085 Armstrong, Ellen Prosser, 57 Milltield Street. Woods Hole, MA 02543 Arnold, William A., Oak Ridge National Laboratory. Biology Division. 102 Balsalm Road. Oak Ridge. TN 37830 Ashton, Robert W., Bay Foundation, 1 7 West 94th Street. New York, NY 10025 Atema, Jelle, Boston University Marine Program. Marine Biological Laboratory. Woods Hole. MA 02543 Baccetti, Baccio, University of Sienna. Institute of Zoology, 53100 Siena. ITALY Baker. Robert G., New York University Medical Center, Department Physiology and Biophysics, 550 First Avenue, New York, NY 10016 Baldwin, Thomas O., Texas A and M University. Department of Biochemistry and Biophysics. College Station, TX 77843-2128 Baltimore, David, California Institute of Technology, 204-31. Pasadena, CA 91125 Barlow, Robert B., SUNY Health Science Center at Syracuse, 750 East Adams Street, Center for Vision Research, 3258 Weiskotten Hall. Syracuse, NY 13210 Barry, Daniel T., 2415 Fairwmd Drive, Houston, TX 77062-4756 Barry. Susan R., Mount Holyoke College. Department of Biological Sciences. South Hadley, MA 01075 Bass. Andrew H., Cornell University, Department of Neurobiology and Behavior. Seely Mudd Hall, Ithaca, NY 14853 Battelle, Barbara-Anne, University of Florida, Whitney Laboratory, 9505 Ocean Shore Boulevard, Street Augustine. FL 32086 Bay, Frederick, Bay Foundation. 17 W. 94th Street. First Floor. New York. NY 10025-7116 Baylor, Martha B., P.O. Box 93. Woods Hole. MA 02543 Bearer, Elaine L., Brown University. Division of Biology and Medicine. Department of Pathology. Box G. Providence. RI 02912 Beatty, John M., University of Minnesota. Department of Ecology and Behavioral Biology, 1987 Gortner, Street Paul, MN 55108 Beauge, Luis Alberto, Instituto de Investigacion Medica. Department of Biophysics. Casilla de Correo 389. 5000 Cordoba, ARGENTINA Begenisich, Ted, University of Rochester, Medical Center, Box 642, 601 Elmwood Avenue. Rochester, NY 14642 Begg, David A.. University of Alberta, Faculty of Medicine, Department of Cell Biology and Anatomy. Edmonton. Alberta T6G 2H7, CANADA Bell, Eugene, Tissue Engineering, Inc.. 451 D Street, Boston, MA 02210 Benjamin, Thomas L., Harvard Medical School, Pathology. D2-230, 200 Longwood Avenue, Boston. MA 021 15 Bennett. Michael V. L., Albert Einstein College of Medicine. Department of Neuroscience, 1300 Morris Park Avenue, Bronx. NY 10461 Bennett, Miriam F., Colby College, Department of Biology. Waterville, ME 04901 Berg, Carl J., P.O. Box 681. Kilauea. Kauai. HI 96754-0681 Berlin, Suzanne T., 5 Highland Street, Gloucester. MA 01930 Bernstein, Norman, Columbia Realty Venture. 5301 Wisconsin Avenue, NW. #600. Washington. DC 20015-2015 Bezanilla, Francisco, Health Science Center, Department of Physiology, 405 Hilgard Avenue, Los Angeles, CA 90024 Biggers, John D., Harvard Medical School. Department of Physiology. Boston. MA 02115 Bishop. Stephen H., Iowa State University, Department of Zoology, Ames, IA 50010 Blaustein, Mordecai P., University of Maryland, School of Medicine, Department of Physiology, Baltimore. MD 21201 Blennemann, Dieter, 1117 East Putnam Avenue, Apt. #174. Riverside, CT 06878-1333 Bloom, George S., The University of Texas Southwestern Medical Center. Department of Cell Biology and Neuroscience. 5323 Harry Hmes Boulevard. Dallas. TX 75235-9039 Bloom, Kerry S., University of North Carolina. Department of Biology. 623 Fordham Hall CB#3280. Chapel Hill, NC 27599-3280 Bodznick, David A., Wesleyan University, Department of Biology, Lawn Avenue. Middletown. CT 06497-0170 Boettiger, Edward G.. 17 Eastwood Road, Storrs, CT 06268-2401 Boolootian, Richard A., Science Software Systems. Inc.. 3576 Woodcliff Road. Sherman Oaks. CA 91403 Borgese, Thomas A., Lehman College, CUNY. Department of Biology. Bedford Park Boulevard. West. Bronx. NY 10468 Borst, David W., Illinois State University. Department of Biological Sciences, Normal, 1L 61790-4120 Bowles, Francis P., Marine Biological Laboratory, Ecosystems Center, Woods Hole. MA 02543 Boyer, Barbara C., Union College, Biology Department, Schenectady. NY 12308 Brandhorst, Bruce P., Simon Fraser University, Institute of Molecular Biology/Biochemistry, Barnaby. B.C. V5A 1S6. CANADA Brinley, F. J., NINCDS/NIH. Neurological Disorders Program. Room 812 Federal Building, Bethesda, MD 20892 Bronner-Fraser, Marianne, California Institute of Technology, Beckman Institute Division of Biology. 139-74. Pasadena. CA 91125 Brown, Stephen C., SUNY. Department of Biological Sciences. Albany. NY 12222 Brown, William L., 80 Black Oak Road. Weston, MA 02193 Browne, Carole L., Wake Forest University. Department of Biology. Box 7325 Reynolds Station, Winston-Salem. NC 27109 Browne, Robert A., Wake Forest University. Department of Biology, Box 7325. Winston-Salem. NC 27109 Bucklin, Anne C., University of New Hampshire, Ocean Process Analysis Laboratory. 142 Morse Hall, Durham. NH 03824 Bullis, Robert A., Manne Biological Laboratory. 7 MBL Street, Woods Hole. MA 02543 Burger, Max M., Friedrich Miescher Institute. P.O. Box 2543. CH 4002 Basel, SWITZERLAND Burgess, David R., Boston College. Academic Vice President and Dean of Facilities, Bourneuf House, 84 College Road, Chestnut Hill. MA 02467-3838 Burgos, Mario H., IHEM Medical School, UNC Conicet, Casilla de Correo 56, 5500 Mendoza, ARGENTINA Burky, Albert, University of Dayton. Department of Biology. Dayton, OH 45469 Burris, John E., Marine Biological Laboratory, 7 MBL Street, Woods Hole, MA 02543 Burstyn, Harold Lewis, United States Air Force. Air Force Materiel Command, Rome Research Site RL/JA, 26 Electronic Parkway, Rome, NY 13441-4514 Bursztajn. Sherry, LSU Medical Center, 1501 Kings Highway, Building BR1F 6-13. Shreveport, LA 71130 Calabrese, Ronald L., Emory University, Department of Biology. 1510 Clifton Road. Atlanta. GA 30322 Callaway, Joseph C., New York Medical College. Department of Physiology. Basic Sciences Building. Valhalla. NY 10595 Cameron, R. Andrew, California Institute of Technology. Division of Biology 156-29, Pasadena, CA 91 125 R70 Annual Report Campbell, Richard H., Bang-Campbell Associates, Eel Pond Place, Box 402. Woods Hole, MA 02543 Candelas, Graciela C., University of Puerto Rico, Department of Biology. P.O. Box 23360, UPR Station. San Juan. PR 00931-3360 Cariello, Lucio, Stazione Zoologica "A. Dohm", Villa Comunale. 80121 Naples, ITALY Case, James F., University of California. Marine Science Institute. Santa Barbara. CA 93106 Cassidy, Joseph D., Providence College, Priory of Street Thomas Aquinas. Providence, Rl 02918-11001 Cavanaugh, Colleen M., Harvard University, Biological Laboratories, 16 Divinity Avenue, Cambridge, MA 02138 Chaet, Alfred B., University of West Florida, Department of Cell and Molecular Biology, 1 1000 University Parkway, Pensacola, FL 32514 Chambers, Edward L., University of Miami School of Medicine. Department of Physiology and Biophys., P.O. Box 016430, Miami. FL 33101 Chang, Donald C., Hong Kong University, Science and Technology, Department of Biology, Clear Water Bay, Kowloon, HONG KONG Chappell, Richard L., Hunter College, CUNY. Department of Biological Sciences. Box 210. 695 Park Avenue. New York, NY 10021 Child III, Frank M., 28 Lawrence Farm Road, Woods Hole. MA 02543-1416 Chisholm, Rex Leslie, Northwestern University, Medical School, Department of Cell Biology, Chicago, IL 6061 1 Citkowitz, Elena, Hospital of Street Raphael, Lipid Disorders Clinic, 1450 Chapel Street, New Haven, CT 0651 1 Clark, Eloise E., Bowling Green State University, Biological Sciences Department. Bowling Green, OH 43403 Clark, Hays, 150 Gomez Road, Hobe Sound, FL 33455 Clark, Wallis H., 12705 NW I 12th Avenue, Alachua, FL 32615 Claude, Philippa, University of Wisconsin, Department of Zoology, Zoology Research Building 125. 1 1 17 W Johnson Street, Madison, Wl 53706 Clay, John R., NIH-NINDS. Building 36. Room 2-CO2. Bethesda. MD 20892 Clowes, Alexander W., University of Washington, School of Medicine. Department of Surgery, Box 356410. Seattle. WA 98195-6410 Cobb, Jewel Plummer, California State University, 5151 University Drive, Health Center 205, Los Angeles, CA 90032-8500 Cohen, Carolyn, Brandeis University. Rosenstiel Basic Medical, Sciences Research Center, Waltham, MA 02254 Cohen, Lawrence B., Yale University School of Medicine, Department of Physiology. 333 Cedar Street, New Haven. CT 06520 Cohen, Maynard M., Rush Medical College, Department of Neurological Sciences, 600 South Paulina, Chicago, IL 60612 Cohen, William D., Hunter College. Department Biological Sciences, New York, NY 10021 Coleman, Annette W., Brown University, Division of Biology and Medicine, Providence. Rl 02912 Colinvaux, Paul, Smithsonian Tropical Research Institute. Unit 0948. Apo AA 34002-0948, USA Collier. Jack R., 3431 Highway. #107. P.O. Box 139. Effie. LA 71331 Collier, Marjorie McCann, 3431 Highway 107. P.O. Box 139. Effie, LA 71331 Cook, Joseph A., Edna McConnell Clark Foundation, 250 Park Avenue, New York. NY 10177-0026 Cornell, Neal W., Marine Biological Laboratory, Woods Hole. MA 02543 Cornwall, Melvin C., Boston University School of Medicine, Department of Physiology L714, Boston. MA 021 18 Corson, D. Wesley, Storm Eye Institute, Room 537, 171 Ashley Avenue. Charleston, SC 29425 Corwin, Jeffrey T., University of Virginia, School of Medicine, Department Otolaryngology and Neuroscience, Box 396. Charlottesville, VA 22908 Couch, Ernest F., Texas Christian University. Department of Biology, TCU Box 298930, Fort Worth. TX 76129 Cox, Rachel Llanelly, Woods Hole Oceanographic Institute, Biology Department. Woods Hole. MA 02543 Crane, Sylvia E., 438 Wendover Drive, Princeton, NJ 08540 Cremer-Bartels, Gertrud, Universitats Augenklinik, 44 Munster, GERMANY Crow, Terry J., University of Texas Medical School, Department of Neurobiology and Anatomy. Houston, TX 77225 Crowell, Sears, Indiana University, Department of Biology. Bloomington. IN 47405 Crowther, Robert J., Shriners Hospitals for Children. 51 Blossom Street. Boston, MA 021 14 Cunningham, Mary-Ellen, 62 Cloverly Road, Grosse Pointe Farms, MI 48236-3313 Cutler. Richard D., Marine Biological Laboratory. Woods Hole. MA 02543 Daignault, Alexander T., Edgewood #6308. 575 Osgood Street, North Andover, MA 01845 (deceased) Davidson, Eric H., California Institute of Technology. Division of Biology. 156-29. 391 South Holliston. Pasadena. CA 91 125 Davison, Daniel B., Bristol-Myers Squibb PR1. Biomformatics Department, 5 Research Parkway, Wallingford. CT 06492 Daw, Nigel W., 5 Old Pawson Road, Branford, CT 06405 Dawidowicz, Eliezar A., Marine Biological Laboratory. Office of Research Administration and Education, Woods Hole, MA 02543 De Weer, Paul J., University of Pennsylvania, B400 Richards Building. Department of Physiology, 3700 Hamilton Walk, Philadelphia. PA 19104-6085 Deegan, Linda A., Marine Biological Laboratory, The Ecosystems Center, Woods Hole, MA 02543 DeGroof, Robert C., 145 Water Crest Drive. Doylestown, PA 18901- 3267 Denckla, Martha Bridge, Johns Hopkins University. School of Medicine. Kennedy-Krieger Institute. 707 North Broadway, Baltimore. MD 21205 DePhillips, Henry A., Trinity College, Department of Chemistry, 300 Summit Street. Hartford, CT 06106 DeSimone, Douglas \V., University of Virginia, Department of Cell Biology. Box 439, Health Sciences Center. Charlottesville, VA 22908 Dettbarn, Wolf-Dietrich, Vanderbilt University. School of Medicine, Department of Pharmacology, Nashville. TN 37232 Dionne, Vincent E., Boston University Marine Program, Marine Biological Laboratory. Woods Hole. MA 02543 Dowling, John E., Harvard University, Biological Laboratories, 16 Divinity Street, Cambridge, MA 02138 Drapeau, Pierre, Montreal General Hospital, Department of Neurology, 1650 Cedar Avenue. Montreal. Que. H3G 1A4. CANADA DuBois, Arthur Brooks, John B. Pierce Foundation Laboratory. 290 Congress Avenue, New Haven. CT 06519 Duncan, Thomas K., Nichols College, Environmental Sciences Department. Dudley, MA 01571 Dunham, Philip B., Syracuse University, Department of Biology. 1 30 College Place. Syracuse. NY 13244-1220 Dunlap, Paul V., University of Maryland Biotechnology Institute. Center of Marine Biotechnology. Columbus Center. Suite 236, 701 East Pratt Street, Baltimore, MD 21202 Ebert, James I)., The Johns Hopkins University. Department of Members of the Corporation R71 Biology. Homewood, 3400 North Charles Street. Baltimore. MD 21218-2685 Eckberg, William R., Howard University. Department of Biology, P.O. Box 887. Administration Building. Washington, DC 20059 Edds, Kenneth T., R & D Systems, Inc., Hematology Division. 614 McKinley Place, NE, Minneapolis, MN 55413 Eder, Howard A., Albert Einstein College of Medicine, 1300 Morris Park Avenue. Bronx, NY 10461 Edstrom, Joan, 53 Two Ponds Road, Falmouth, MA 02540 Egyud, Laszlo G., Cell Research Corporation, P.O. Box 67209, Chestnut Hill. MA 02167-0209 Ehrlich, Barbara E., Yale University Medical School, B207 SHM. New Haven, CT 06473 Eisen, Arthur Z., Washington University, Division of Dermatology, Street Louis, MO 63110 Eisen, Herman N., Massachusetts Institute of Technology. Center for Cancer Research, El 7- 128, 77 Massachusetts Avenue, Cambridge, MA 02139-4307 Elder, Hugh Young, University of Glasgow, Institute of Physiology, Glasgow G12 8QQ, SCOTLAND Englund, Paul T., Johns Hopkins Medical School, Department of Biological Chemistry. 725 North Wolfe Street. Baltimore, MD 21205 Epel, David, Stanford University, Hopkins Marine Station, Ocean View Boulevard, Pacific Grove, CA 93950 Epstein, Herman T., 18 Lawrence Farm Road, Woods Hole, MA 02543 Epstein, Ray L., 1602 W. Olympia Street, Hernando. FL 34442 Farb, David H., Boston University School of Medicine, Department of Pharmacology L603, 80 East Concord Street, Boston. MA 02 1 1 8 Farmanfarmaian, A. Verdi, Rutgers University. Department of Biological Sciences. Nelson Biology Laboratory FOB 1059, Piscataway. NJ 08855 Feldman, Susan C., University of Medicine and Dentistry, New Jersey Medical School, 100 Bergen Street. Newark, NJ 07103 Festoff, Barry William, VA Medical Center, Neurology Service (151), 4801 Linwood Boulevard. Kansas City. MO 64128 Fink, Rachel D., Mount Holvoke College, Department of Biological Sciences, Clapp Laboratories, South Hadley, MA 01075 Finkelstein, Alan, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461 Fischbach, Gerald D., National Institute of Health, Neurological Disorders and Strokes, 31 Center Drive. MSC 2540, Bldg 31, Rm 8A03. Bethesda, MD 20892-2540 Fishman, Harvey M., University of Texas Medical Branch, Department of Physiology and Biophysics, 301 University Boulevard, Galveston, TX 77555-0641 Flanagan, Dennis, 12 Gay Street, New York, NY 10014 Fluck, Richard Allen, Franklin and Marshall College, Department of Biology, Box 3003, Lancaster, PA 17604-3003 Foreman, Kenneth H., Marine Biological Laboratory, Woods Hole, MA 02543 Fox, Thomas Oren, Harvard Medical School, Division of Medical Sciences. MEC 435. 260 Longwood Avenue, Boston, MA 021 15 Franzini-Armstrong, Clara, University of Pennsylvania, School of Medicine. 330 South 46th Street, Philadelphia, PA 19143 Fraser, Scott, California Institute of Technology, Beckman Institute 139-74, 1201 East California Boulevard, Pasadena, CA 91 125 Frazier, Donald T., University of Kentucky Medical Center, Department of Physiology and Biophysics, MS501 Chandler Medical Center. Lexington, KY 40536 French, Robert J., University of Calgary, Health Sciences Centre, Alberta, T2N 4NI, CANADA Fulton, Chandler M., Brandeis University, Department of Biology, MS 008, Waltham, MA 02454-91 10 Furie, Barbara C., Beth Israel Deaconess Medical Center. BIDMC Cancer Center, Kirstein 1, 330 Brookline Avenue, Boston, MA 02215 Furie, Bruce, Beth Israel Deaconess Medical Center, BIDMC Cancer Center, Kirstein 1, 330 Brookline Avenue, Boston, MA 02215 Furshpan, Edwin J., Harvard Medical School. Department of Neurobiology. 220 Longwood Avenue. Boston, MV\ 021 15 Futrelle, Robert P., Northeastern University, College of Computer Science, 360 Huntmgton Avenue, Boston, MA 021 15 Gabr, Howaida, Sue/. Canal University, Department of Marine Science. Faculty of Science, Ismailia, EGYPT Gabriel, Mordecai L., Brooklyn College. Department of Biology, 2900 Bedford Avenue, Brooklyn, NY 11210 Gadsby, David C., The Rockefeller University, Laboratory of Cardiac Physiology, 1230 York Avenue, New York, NY 10021-6399 Gainer, Harold, NIH. NINDS.BNP.DIR, Neurochemistry. Building 36, Room 4D20, Bethesda. MD 20892-4130 Galatzer-Levy, Robert M., 180 North Michigan Avenue. Suite 2401, Chicago, IL 60601 Gall, Joseph G., Carnegie Institution, 1 15 West University Parkway, Baltimore, MD 21210 Garber. Sarah S., Allegheny University of the Health Sciences, Department of Physiology, 2900 Queen Lane, Philadelphia, PA 19124 Gascoyne, Peter, University of Texas. M. D. Anderson Cancer Center, Experimental Pathology. Box 89, Houston, TX 77030 Gelperin, Alan, Bell Labs Lucent, Department Biology Comp., Rm 1C464, 600 Mountain Avenue, Murray Hill, NJ 07974 German, James L., The New York Blood Center, Laboratory of Human Genetics, 310 East 67th Street, New York, NY 10021 Gibbs, Martin, Brandeis University, Institute for Photobiology of Cells and Organelles, Waltham. MA 02254 Giblin, Anne E., Marine Biological Laboratory. The Ecosystems Center. Woods Hole. MA 02543 Gibson, A. Jane, Cornell University, Department of Biochemistry. Biotech. Building. Ithaca, NY 14850 Gifford, Prosser, 540 North Street, SW. Apt. #S-903. Washington, DC 20024-4557 Gilbert, Daniel L., National Institutes of Health, Biophysics Sec., BNP, Building 36, Room 5A-27, Bethesda, MD 20892 Giudice, Giovanni, Universita di Palermo, Dipartimento di Biologia, Cellulare e Dello Sviluppo, 1-90123 Palermo. ITALY Giuditta, Antonio, University of Naples, Department of General Physiology, Via Mezzocannone 8, Naples, 80134, ITALY Glynn, Paul, P.O. Box 6083, Brunswick, ME 04011-6083 Golden, William T., Chairman Emeritus, American Museum of Natural History, Rm. 4201. 40 Wall Street, New York, NY 10005 Goldman, Robert D., Northwestern University Medical School, Department of Cell and Molecular Biology. 303 E. Chicago Avenue, Chicago, IL 60611-3008 Goldsmith, Paul K., National Institutes of Health. Building 10, Room 9C-101. Bethesda. MD 20892 Goldsmith. Timothy H., Yale University, Department of Biology, New Haven, CT 06510 Goldstein, Moise H., The Johns Hopkins University, ECE Department, Barton Hall, Baltimore. MD 21218 Goodman, Lesley Jean (deceased) Gould, Robert Michael, NYS Institute of Basic Research, 1050 Forest Hill Road, Staten Island, NY 10314-6399 Govind, C. K., Scarborough College, Life Sciences Division, 1265 Military Trail, West Hill, Ontario MIC 1A4, CANADA Grace, Dick, Doreen Grace Fund, The Brain Center, Promontory Point, New Seabury, MA 02649 Graf, Werner M., College of France. 1 1 Place Marcelin Berthelot. 75231 Paris Cedex 05, FRANCE R72 Annual Report Grant, Philip, National Institutes of Health. NINDS.BN.DIR.Neurochemistry. Building 36. Room 4D20. Bethesda. MD 20892-4130 Grass, Ellen R., The Grass Foundation, 77 Reservoir Road. Quincy. MA 02 170-3610 Grassle, Judith P., Rutgers University, Institute of Marine and Coastal Studies, Box 23 1 , New Brunswick, NJ 08903 Graubard, Katherine G., University of Washington, Department of Zoology, NJ-15. Box 351800, Seattle, WA 98195-1800 Greenberg, Everett Peter, University of Iowa. College of Medicine. Department of Microbiology. Iowa City, IA 52242 Greenberg, Michael J., University of Florida, The Whitney Laboratory, 9505 Ocean Shore Boulevard. St. Augustine, FL 32086-8623 Greer, Mary J., 176 West 87th Street, #12A, New York. NY 10024- 2402 Griffin, Donald R., Harvard University. Concord Field Station, Old Causeway Road, Bedford, MA 01730 Gross, Paul R., 1 1 1 Perkins Street. Apt. 45. Jamaica Plain, MA 02130- 4320 Grossman, Albert, New York University Medical Center, 550 First Avenue. New York, NY 10016 Grossman, Lawrence, The Johns Hopkins University. Department of Biochemistry. 615 North Wolfe Street. Baltimore, MD 21205 Gruner, John A., Cephalon, Inc.. 145 Brandyw'ine Parkway, West Chester, PA 19380-4245 Gunning. A. Robert, P.O. Box 165. Falmouth. MA 02541 Gwilliam, G. F., Reed College, Department of Biology, Portland, OR 97202 Haimo, Leah T., University of California. Department of Biology. Riverside. CA 92521 Hajduk, Stephen L., University of Alabama. School of Medicine/Dentistry, Department of Biochemistry/Molecular Genetics. University Station. Birmingham. AL 35294 Hall, Linda M., SUNY. Department of Biochemstry Pharmacology, 329 Huchstetter Hall, Buffalo, NY 14260-1200 Hall, /;K h YV., University of California, Department Physiology, San Francisco. CA 94114 Halvorson, Harlyn O., University of Massachusetts, Policy Center for Marine Biosciences and Technology. 100 Morrissey Boulevard. Boston. MA 02125-3393 Haneji, Tatsuji. Kyushu Dental College. Department of Anatomy, 2-6- 1, Mana/.uru. Kokurakita-Ku, Kitakyushu 803. JAPAN Hanlon, Roger T., Marine Biological Laboratory. Woods Hole, MA 02543 Harosi, Ferenc, New College of the USF, Division of Natural Sciences, 5700 North Tamiami Trail. Sarasota, FL 34243-2197 Harrigan, June F., 7415 Makaa Place, Honolulu. HI 96825 Harrington, Glenn W., Weber State University, Department of Microbiology, Ogden. UT 84408 Harrison, Stephen C., Harvard University, Department of Molecular and Cell Biology, 7 Divinity Avenue. Cambridge. MA 02138 Haselkorn, Robert, University of Chicago. Department of Molecular Genetics and Cell Biology, Chicago, IL 60637 Hastings, J. Woodland, Harvard University. The Biological Laboratories. 16 Divinity Avenue, Cambridge. MA 02138-2020 Huydnn-Baillie, Wensley G., Porton Institute, 2 Lowndes Place, I ondon SW1X 8Dd, ENGLAND Hayes, Raymond L., Howard University, College of Medicine. 520 W Street. NW, Washington. DC 20059 Heck. Diane E.. F.OHSI. Department of Pharmacology/Toxicology, 681 Frelinghuysen Road, Piscataway, NJ 08855 Henry, Jonathan Joseph, University of Illinois, Department of Cell and Struct. Biology. 601 South Goodwin Avenue #BI07, Urbana, II. 61801-3709 Hepler. Peter K., University of Massachusetts. Department of Biology. Morrill III. Amherst, MA 01003 Herndon, Walter R., University of Tennessee. Department of Botany, Knoxville, TN 37996-1 100 Herskovits, Theodore T.. Fordham University. Department of Chemistry. John Mulcahy Hall. Room 638. Bronx. NY 1045S Hiatt. Howard H., Bngham and Women's Hospital. Department of Medicine. 75 Francis Street, Boston, MA 021 15 Highstein, Stephen M., Washington University. Department of Otolaryngology. Box 8115. 4566 Scott Avenue, Street Louis, MO 63110 Hildehrand, John G., University of Arizona, ARL Division of Neurobiology. P.O. Box 210077. Tucson, AZ 85721-0077 Hill, Richard W., Michigan State University, Department of Zoology, East Lansing. MI 48824 Hill, Susan D., Michigan State University. Department of Zoology, East Lansing. MI 48824 Hillis, Llewellya W., Smithsonian Tropical Research Institute. Unit 0948. APO. AA 34002-0948 Hinkle, Gregory J., Bioinformatics Group, Cereon Genomics, One Kendall Square, Building 200. Cambridge. MA 02139 Hinsch, Gertrude W., University of South Florida. Department of Biology. Tampa, FL 33620 Hinsch, Jan, Leica. Inc.. 1 10 Commerce Drive, Allendale. NJ 07401 Hobhie, John E., Marine Biological Laboratory. The Ecosystems Center. Woods Hole. MA 02543 Hodge, Alan J., 3843 Mount Blackburn Avenue. San Diego, CA 921 1 1 Hoffman, Joseph F., Yale University School of Medicine, Cellular and Molecular Physiology. 333 Cedar Street. New Haven. CT 06520-8026 Hollyfield, Joe G. address unknown Holz IV, George G., New York University Medical Center, Medical Sciences Building Room 442, 550 First Avenue. New York. NY 10016 Hopkinson, Charles S., Marine Biological Laboratory, Woods Hole, MA 02543 Houk, James C., Northwestern University Medical School, 303 East Chicago Avenue, Ward 5-315. Chicago. IL 6061 1-3008 Hoy, Ronald R., Cornell University. Section of Neurobiology and Behavior, 215 Mudd Hall. Ithaca, NY 14853 Huang, Alice S., California Institute of Technology. Mail Code 1-9. Pasadena, CA 91125 Hufnagel-Zackroff. Linda A., University of Rhode Island, Department of Microbiology, Kingston. RI 02881 Hummon, William D., Ohio University. Department of Biological Sciences. Athens. OH 45701 Humphreys, Susie H., Food and Drug Administration, HFS-308, 200 C Street. SW, Washington. DC 20204-0001 Humphreys, Tom, University of Hawaii. Kewalo Marine Laboratory. 41 Ahui Street. Honolulu, HI 96813 Hunt, Richard T., ICRF, Clare Hall Laboratories. South Minims Potter's Bar, Herb EN6-3LD. ENGLAND Hunter, Robert D., Oakland University, Department of Biological Sciences. Rochester. MI 48309-4401 Huxley, Hugh E., Brandeis University, Rosenstiel Center, Biology Department. Waltham. MA 02154 1 1. HI. Joseph, Case Western Reserve University, School of Medicine. Department of Anatomy, Cleveland. OH 44106 Ingoglia, Nicholas A., New Jersey Medical School, Department of Pharmacology/Physiology, 185 South Orange Avenue. Newark. NJ 07103 Inoue, Saduyki, McGill University. Department of Anatomy, 3640 University Street, Montreal.PQ H3A 2B2, CANADA Inoue, Shinya, Marine Biological Laboratory. Woods Hole, MA 02543 Members of the Corporation R73 Isselbacher, Kurt J., Massachusetts Genera] Hospital Cancer Center. Charlestown. MA 02129 Issidorides, Marietta Radovic, University of Athens, Department of Psychiatry, Monis Petraki 8. Athens, 140, GREECE Izzard, Colin S., SUNY-Albany. Department of Biological Sciences, 1400 Washington Avenue. Albany, NY 12222 Jacobs, Neil, Hale and Dorr, 60 State Street, Boston, MA 02109 Jaffe, Laurinda A., University of Connecticut Health Center, Department of Physiology. Farmington Avenue. Farmington, CT 06032 Jaffe, Lionel, Marine Biological Laboratory, Woods Hole, MA 02543 Jannasch. Holger W., Woods Hole Oceanographic Institute. Department of Biology. Woods Hole, MA 02543 (deceased) Jeffery, William R., University of Maryland. Department of Biology. College Park. MD 20742 Johnston, Daniel, Baylor College of Medicine. Division of Neuroscience, Baylor Plaza. Houston. TX 77030 Josephson, Robert K., University of California, School of Biological Science. Department of Psychobiology, Irvine. CA 92697 Kaczmarek, Leonard K., Yale University School of Medicine, Department of Pharmacology, 333 Cedar Street. New Haven, CT 06520 Kaley, Gabor, New York Medical College. Department of Physiology. Basic Sciences Building. Valhalla. NY 10595 Kaltenbach, Jane, Mount Holyoke College. Department Biological Sciences, South Hadley, MA 01075 Kaminer, Benjamin, Boston University Medical School, Physiology Department, 80 East Concord Street, Boston, MA 021 18 Kaneshiro, Edna S., University of Cincinnati, Biological Sciences Department, JL 006. Cincinnati. OH 45221-0006 Kaplan, Ehud, 450 E 63rd Street. New York. NY 10021-7928 Karakashian, Stephen J., Apartment 16-F. 165 West 91st Street. New York. NY 1 0024 Karlin, Arthur, Columbia University, Center for Molecular Recognition, 630 West 168th Street, Room 1 1-401. New York, NY 10032 Keller, Hartmut Ernst, Carl Zeiss. Inc.. One Zeiss Drive. Thomwood. NY 10594 Kelley, Darcy B., Columbia University. Department of Biological Sciences. 911 Fairchild. Mailcode 2432. New York. NY 10027 Kelly, Robert E., 5 Little Harbor Road, Woods Hole, MA 02543 Kemp, Norman E., University of Michigan, Department of Biology, Ann Arbor. Ml 48109 Kendall, John P., Faneuil Hall Associates, 176 Federal Street, 2nd Floor. Boston. MA 02110 Kerr, Louis M.. Marine Biological Laboratory. Woods Hole, MA 02543 Keynan, Alexander, Israel Academy of Science and Humanity. P.O. Box 4040, Jerusalem. ISRAEL Khan, Shahid M.M., Albert Einstein College of Medicine. Department of Physiology and Biophysics. 1300 Morris Park Avenue. Room U273. Bronx, NY 10461 Khodakhah, Kamran, University of Colorado School of Medicine. Department of Physiology and Biophysics, 4200 East 9th Avenue. C-240, Denver, CO 80262 Kiehart, Daniel P., Duke University Medical Center. Department of Cell Biology. Box 3709, 308 Nanalme Duke Building. Durham. NC 27710 Kleinfeld, David, University of California. Department of Physics. 0319 9500 Oilman Drive. La Jolla. CA 92093 Klessen, Rainer, Address unknown. Klotz, Irving M., Northwestern University. Department of Chemistry. Evanston. II. WI20I Knudson, Robert A., Marine Biological Laboratory. Woods Hole. MA 02543 Koide, Samuel S., The Rockefeller University. The Population Council. 1230 York Avenue. New York. NY 10021 Kornberg, Hans, Boston University. The University Professors, 745 Commonweath Avenue, Boston. MA 02215 Kosower, Edward M., Tel-Aviv University, Department of Chemistry. Ramat-Aviv. Tel Aviv, 69978, ISRAEL Krahl. Maurice E., 27X3 West Casas Circle, Tucson, AZ 85741 Krane. Stephen M., Massachusetts General Hospital. Arthritis Unit, Fruit Street, Boston. MA 021 14 Krauss, Robert, P.O. Box 291. Demon. MD 21629 Kravitz, Edward A., Harvard Medical School. Department of Neurobiology, 220 Longwood Avenue, Boston. MA 02 1 1 5 Kriebel, Mahlon E., SUNY Health Science Center, Department of Physiology. Syracuse. NY 13210 Kristan Jr., William B., University of California. Department of Biology 0357, 9500 Oilman Drive, La Jolla, CA 92093-0357 Kropinski, Andrew M., Queen's University, Department of Microbiology/Immunology, Kingston. Ontario K7L 3N6. CANADA Kuffler. I). inn, 11 P., Institute of Neurobiology. 201 Boulevard del Valle. San Juan 00901. PR Kuhns. William J., Hospital for Sick Children, Biochemistry Research, 555 University Avenue. Toronto, Ontario M5G 1X8, CANADA Kunkel, Joseph G., University of Massachusetts, Department of Biology, Amherst, MA 01003 Kuzirian, Alan M., Marine Biological Laboratory, Woods Hole, MA 02543-1015 Laderman, Aimlee D., Yale University. School of Forestry and Environmental Studies. 370 Prospect Street, New Haven. CT 065 1 1 Landeau. Laurie J., Listowel. Inc.. 2 Park Avenue. Suite 1525. New York. NY 10016 I .nulls. Dennis M.D., University Hospital of Cleveland, Department Neurology. I 1 100 Euclid Avenue, Cleveland. OH 44106 Landis, Story C., National Institutes of Health, Building 36. Room 5A05, 36 Convent Drive. Bethesda. MD 20892-4150 Landowne, David, University of Miami Medical School, Department of Physiology, P.O. Box 016430. Miami. FL 33101 Langford, George M., Dartmouth College. Department of Biological Sciences. 6044 Oilman Laboratory. Hanover, NH 03755 Laskin, Jeffrey, University of Medical and Dentistry of New Jersey, Robert Wood Johnson Medical School, 675 Hoes Lane. Piscataway, NJ 08854 Lasser-Ross. Nechama, New York Medical College. Department of Physiology. Valhalla. NY 10595 Laster, Leonard. University of Massachusetts Medical School. 55 Lake Avenue. North, Worcester. MA 01655 Laties, Alan, Scheie Eye Institute, Myrin Circle, 51 North 39th Street, Philadelphia, PA 19104 Laufer, Hans, University of Connecticut. Department of Molecular and Cell Biology. U-125. 75 North Eagleville Road Storrs. CT 06269- 3125 Lazarow, Paul B., Mount Sinai School of Medicine. Department of Cell Biology and Anatomy, 1190 Fifth Avenue. Box 1007, New York, NY 10029-6574 Lazarus, Maurice, Federated Department Stores, Sears Crescent, City Hall Pla/a. Boston. MA 02108 Leadhetter, Edward R., University of Connecticut, Department of Molecular and Cell Biology, U-131. Beach Hall. Room 249. 354 Mansfield Road. Storrs. CT 06269-2 1 3 1 Lederberg, Joshua. The Rockefeller University. 1230 York Avenue. New York, NY 10021 R74 Annual Report Lee, John J.. City College of CUNY. Department of Biology, Convent Avenue and 138th Street. New York, NY 10031 Lehy, Donald B., 35 Willow Field Drive, North Falmouth. MA 02556 Leibovitz, Louis, 3 Kettle Hole Road, Falmouth. MA 02540 (deceased) Leighton, Joseph, Aeron Biotechnology. Inc., 1933 Davis Street #310. San Leandro. CA 44577 (deceased) Leighton. Stephen B.. National Institutes of Health, Building 13, 3WI3. Bethesda, MD 20842 Lemos, Jose Ramon, University of Massachusetts Medical Center. Worcester Foundation Campus. 222 Maple Avenue. Shrewsbury. MA 01545-2737 Lerner, Aaron B.. Yale University School of Medicine, Department of Dermatology. P.O. Box 3333, New Haven, CT 06510 Levin, Jack, Veterans Administration. Medical Center. 1 1 1 H2. 4150 Clement Street, San Francisco. CA 94121 Levine, Michael, University of California. Department MCB. 401 Barker Hall. Berkeley. CA 94720 Levine, Richard B., University of Arizona. Division of Neurobiology. Room 611. Gould Simpson Building. P.O. Box 210077. Tucson. AZ 85721-0077 Levinthal, Francoise, Columbia University, Department of Biological Sciences, Broadway and 116th Street. New York, NY 10026 Levitan, Herbert, National Science Foundation. 4201 Wilson Boulevard, Room 835, Arlington. VA 22230 Levitan. Irwin B., Brandeis University. Volen Center for Complex Systems. 415 South Street, Waltham. MA 02254 Linck. Richard VV., University of Minnesota School of Medicine. Cell Biology and Neuroanatomy Department, 4-135 Jackson Hall. 321 Church Street. Minneapolis. MN 55455 Lipicky. Raymond J., FDA/CDER/ODEI/HFD- 1 10. 5600 Fishers Lane. Rockville. MD 20857 Lisman, John E., 199 Coolidge Avenue, #902, Watertown. MA 02172- 1572 Liuzzi. Anthony, 320 Beacon Street, Boston. MA 021 16 Llinas, Rodolf'o R., New York University Medical Center. Department of Phsyiology/Biophysics. 550 First Avenue. Room 442. New York. NY 10016 Lobel, Phillip S., Boston University Marine Program. Marine Biological Laboratory, Woods Hole. MA 02543 Loew, Franklin M., Becker College. 61 Sever Street. Worcester. MA 01615-0071 Loewenstein, Birgit Rose, Marine Biological Laboratory. Woods Hole. MA 02543 Loewenstein, Werner R., Marine Biological Laboratory, Woods Hole. MA 02543 London, Irving M., Harvard-MIT. Division. E-25-551. Cambridge. MA 02 1 39 Longo, Frank J., University of Iowa. Department of Anatomy. Iowa City. IA 52442 Lorand, Laszlo, Northwestern University Medical School. CMS Biology. Searle 4-555. 303 East Chicago Avenue, Chicago. 1L 60611- 3008 Luckenbill, Louise M., Ohio University. Department of Biological Sciences, Irvine Hall, Athens, OH 45701 Macagno, Fduardo R., Columbia University. 109 Low Memorial Library, Mail Code 4306. New York, NY 10027 MacNichol Jr., Kdward F., Boston University School of Medicine. Department of Physiology, 80 East Concord Street, Boston, MA 021 IS Maglott-Dul'lield, Donna R., American Type Culture Collection, 12301 Parklawn Drive. Roik \ille. MD 20852-1776 Maienschein, Jane Ann, Ari/ona State University, Department of Philosophy. P.O. Box 872004. Tempe. AZ 85287-2004 Mainer. Robert E., The Boston Company. Inc.. One Boston Place, OBP-15-D, Boston. MA 02108 Malhon, Craig C., SUNY, University Medical Center. Pharmacology- HSC. Stony Brook, NY 11794-8651 Malchow, Robert P., University of Illinois, Department of Ophthalmology, 1855 West Taylor Street N/C 648, Chicago. IL 60612 Man. ilis, Richard S., Indiana-Purdue University, Department of Biological Science, 2101 Coliseum Boulevard East, Fort Wayne. IN 46805 Mangum, Charlotte P., College of William and Mary. Department of Biology. Williamsburg. VA 23187-8795 (deceased) Manz, Robert D., 304 Adams Street, Milton. MA 02186 Margulis, Lynn, University of Massachusetts. Department of Geosciences. Morrill Science Center, Box 35820, Amherst. MA 01003-5820 Marinucci, Andrew C., 102 Nancy Drive, Mercerville, NJ 08619 Martinez, Joe L., The University of Texas, Division of Life Sciences, 6900 North Loop 1604 West, San Antonio. TX 78249-0662 Martinez-Palomo, Adolfo, CINVESTAV-IPN, Sec. de Patologia Experimental. 07000 Mexico. D.F.A. P. 140740, MEXICO Mastroianni, Luigi, Hospital of University of Pennsylvania. 106 Dulles. 3400 Spruce Street, Philadeplna, PA 19104-4283 Mauzerall, David, Rockefeller University, 1230 York Avenue, New York, NY 10021 McC'ann, Frances V., Dartmouth Medical School, Department of Physiology. Lebanon. NH 03756 McLaughlin, Jane A., Marine Biological Laboratory. Woods Hole, MA 022543 McMahon, Robert F., University of Texas, Arlington. Department of Biology. Box 19498. Arlington, TX 76019 Meedel, Thomas, Rhode Island College. Biology Department. 600 Mount Pleasant Avenue, Providence, RI 02908 Meinertzhagen, Ian A., Dalhousie University, Department of Psychology, Halifax. NS B3H 4J 1 . CANADA Meiss, Dennis E., Immunodiagnostic Laboratories. 488 McCormick Street, San Leandro, CA 94577 Melillo, Jerry M., Marine Biological Laboratory. Ecosystems Center, Woods Hole. MA 02543 Mellon Jr., DeForest, University of Virginia. Department of Biology. Gilmer Hall, Charlottesville, VA 22903 Mellon. Richard P.. P.O. Box 187. Laughlintown, PA 15655-0187 Mendelsohn, Michael E., New England Medical Center, Molecular Cardiology Laboratory. NEMC Box 80, 750 Washington Street. Boston. MA 021 I I Merriman, Melanie Pratt, 751 1 Beach View Drive, North Bay Village. FL 33141 Meselson, Matthew, Harvard University. Fairchild Biochemistry Building, 7 Divinity Avenue. Cambridge. MA 02138 Metuzals, Janis, University of Ottawa. Department of Pathology and Laboratory Medical. 451 Smyth Road, Ottawa. Ontario K1H 8M5. CANADA Miledi. Ricardo, University of California. Irvine. Department of Psychobiology. 2205 Biology Sci. II. Irvine. CA 92697-4550 Milkman. Roger D., University of Iowa. Department of Biological Sciences, Biology Buiilding, Room 318, Iowa City, IA 52242-1324 Miller, Andrew L., Hong Kong University of Science and Technology. Department of Biology, Clearwater Bay. Kowloon, HONG KONG Mills, Robert, 10315 44th Avenue. W 12 H Street. Brandenton. FL 34210 Misevic, Gradimir, University Hospital of Basel. Department of Research. Mebelstr. 20. CH-403 1 Basel. SWITZERLAND Mitchell. Ralph. Harvard University, Division of Applied Sciences, 29 Oxford Street. Cambridge. MA 02 1 38 Members of the Corporation R75 Miyakawa, Hiroyoshi, Tokyo College of Pharmacy. Laboratory of Cellular Neurobiology. 1432-1 Horinouchi, Hachiouji, Tokyo 192-03, JAPAN Miyamoto. David M., Drew University. Department of Biology. Madison, NJ 07940 Mi/. II. Merle, Tulane University. Depanment of Cell and Molecular, Biology. New Orleans. LA 70118 Moore, John W., Duke University Medical Center. Department of Neurobiology, Box 3209. Durham, NC 27710 Moreira, Jorge E., NIH/NICHD. Department of Cell and Molecular Biol., Bethesda, MD 20852 Morin, James G., address unknown Morrell. Leyla de Toledo, Rush-Presbyterian-Street Lukes, Medical Center, 1653 West Congress Parkway, Chicago, IL 60612 Morse, M. Patricia, National Science Foundation, Room 885, Esie. Arlington, VA 22230 Morse, Stephen S., DARPA/DSO, 3701 North Fairfax Drive. Arlington, VA 22203-1714 Mote, Michael I., Temple University. Department of Biology, Philadelphia, PA 19122 Muller, Kenneth J., University of Miami School of Medicine, Department of Physiology and Biophysics. 1600 NW 10th Avenue. R-430. Miami, FL 33136 Murray, Andrew W., University of California. Department of Physiology. Box 0444, 513 Parnassus Avenue. San Francisco. CA 94143-0444 Nabrit, S. M., 686 Beckwith Street, SW, Atlanta, GA 30314 Nadelhoffer, Knute J., Marine Biological Laboratory, 7 MBL Street. Woods Hole, MA 02543 Naka, Ken-ichi, 2-9-2 Tatumi Higashi, Okazaki, 444, JAPAN Nakajima, Yasuko, University of Illinois, College of Medicine. Anatomy and Cell Biology Department. M/C 512. Chicago, IL 60612 Narahashi, Toshio, Northwestern University Medical School, Department of Pharmacology. 303 East Chicago Avenue. Chicago. IL 60611 Nasi, Enrico, Boston University School of Medical. Department of Physiology, R-406. 80 East Concord Street, Boston, MA 02118 Neill, Christopher, Marine Biological Laboratory. 7 MBL Street, Woods Hole, MA 02543 Nelson, Leonard, Medical College of Ohio. Department of Physiology. CS 10008. Toledo. OH 43699 Nelson, Margaret C., Cornell University. Section of Neurobiology and Behavior. Ithaca, NY 14850 Nicholls, John G., University of Basel, Department of Pharmacology Biocenter. Klingelbergstrasse 70. Basel, CH-4056, SWITZERLAND Nickerson, Peter A., SUNY, Buffalo, Department of Pathology, Buffalo, NY 14214 Nicosia, Santo V., University of South Florida, College of Medicine. Box 1 1. Department of Pathology, Tampa, FL 33612 Noe, Bryan D., Emory University School of Medicine. Department of Anatomy and Cell Biology, Atlanta. GA 30322 Norton, Catherine N., Marine Biological Laboratory, 7 MBL Street. Woods Hole, MA 02543 Nusbaum, Michael P., University of Pennsylvania School of Medicine, Department of Neuroscience, 215 Stemmler Hall, Philadelphia. PA 191(14-6074 O'Herron, Jonathan, Lazard Freres and Company. 30 Rockefeller Plaza. 59th Floor. New York. NY 10020-1900 Obaid, Ana Lia, University of Pennsylvania School of Medicine, Neuroscience Department, 234 Stemmler Hall, Philadelphia, PA 19104-6074 Ohki, Shinpei, SUNY at Buffalo, Department of Biophysical Sciences, 224 Cary Hall. Buffalo. NY 14214 Oldenbourg, Rudolf, Marine Biological Laboratory. 7 MBL Street. Woods Hole. MA 02543 Olds, James L., George Mason University. Krasnow Institute for Advanced Studies, Mail Stop 2A1, Fairfax, VA 22030-4444 Olins, Ada L., 45 Eastern Promenade, #7-D, Portland. ME 04101 Olins, Donald E., 45 Eastern Promenade. #7-D, Portland, ME 04101 Oschman, James L., Nature's Own Research Association, P.O. Box 5101. Dover, NH 03X20 Palazzo, Robert E., University of Kansas, Department of Physiology and Cell Biology, Lawrence, KS 66045 Palmer, John D., University of Massachusetts, Department of Zoology, 221 Morrill Science Center, Amherst, MA 01003 Pant, Harish C., NINCDS/NIH. Laboratory of Neurochemistry, Building 36, Room 4D20. Bethesda. MD 20892 Pappas, George D., University of Illinois. College of Medicine, Department of Anatomy. Chicago. IL 60612 Pardee, Arthur B., Dana-Farber Cancer Institute. D810, 44 Binney Street, Boston, MA 02 1 1 5 Pardy, Rosevelt L., University of Nebraska, School of Life Sciences, Lincoln, NE 68588 Parmentier, James L., 175 S. Great Road, Lincoln, MA 01773-41 12 Pederson, Thoru, University of Massachusetts Medical Center, Worcester Foundation Campus. 222 Maple Avenue. Shrewsbury, MA 01545 Perkins, Courtland I)., 400 Hilltop Terrace, Alexandria, VA 22301 Person, Philip, 137-87 75th Road, Flushing, NY 11367 Peterson, Bruce J., Marine Biological Laboratory, 7 MBL Street, Woods Hole. MA 02543 Pethig. Ronald, University College of North Wales. School of Electronic Engineering. Bangor. Gwynedd. LL 57 IUT, UNITED KINGDOM Pfohl, Ronald J.. Miami University, Department of Zoology, Oxford, OH 45056 Pierce, Sidney K., University of Maryland. Department of Zoology, College Park. MD 20742 Pleasure. David E., Children's Hospital. Neurology Research, 5th Floor. Ambramson Building. Philadelphia, PA 19104 Poindexter, Jeanne S., Barnard College. Columbia University, 3009 Broadway. New York, NY 10027-6598 Pollard, Harvey B., NIH/NIDDKD, Building 8, Room 401, Bethesda. MD 20892 Pollard, Thomas D., Salk Institute for Biological Studies. 10010 N. Torrey Pines Road. La Jolla. CA 92037 Porter, Beverly H., 5542 Windysun Court, Columbia, MD 21045 Porter, Mary E., University of Minnesota, Department of Cell Biology and Neuroanatomy. 4-135 Jackson Hall, 321 Church Street SE, Minneapolis, MN 55455 Potter, David D., Harvard Medical School. Department of Neurobiology, 25 Shattuck Street. Boston, MA 02115 Potts, William T., LIniversity of Lancaster. Department of Biology, Lancaster, ENGLAND Powers, Maureen K.. Vanderbilt University. Department of Psychology. 301 Arts and Science Psychology Building, Nashville. TN 37240 Prendergast, Robert A., Wilmer Institute, Johns Hopkins Hospital. 600 North Wolfe Street, Baltimore. MD 21287-9142 Price, Carl A., Rutgers University, Waksman Institute of Microbiology, P.O. Box 759. Piscataway, NJ 08855-0759 Prior, David J., Northern Arizona University. Arts and Sciences Dean's Office, Box 5621, Flagstaff, AZ 8601 1 Prusch. Robert D., Gonzaga University, Department of Life Sciences, Spokane, WA 99258 R76 Annual Report Purves, Dale, Duke University Medical Center. Department of Neurobiology. Box 3209. 101-1 Bryan Research Building, Durham. NC 27710 Quigley, James P., SUNY Health Sciences Center, Department of Pathology. BHS Tower 4. Room 140. Stony Brook. NY 1 1794-8691 Rahb, Irving VV., 1010 Memorial Drive. Cambridge. MA 02138 Rabin, Harvey. P.O. Box 4022. Wilmington. DE 19807 Rabinowitz, Michael B., Marine Biological Laboratory, 7 MBL Street. Woods Hole. MA 02543 Rafferty, Nancy S., Marine Biological Laboratory. 7 MBL Street. Woods Hole, MA 02543 Rakowski. Robert F., UHS/The Chicago Medical School, Department of Physiology and Biophysics, 3333 Greenbay Road. N. Chicago, IL 60064 Ramon, Fidel, Universidad Nacional Autonoma de Mexico. Division EStreet Posgrado E Invest.. Facultad de Medicina, 04510, D.F., MEXICO Ranzi, Silvio, Sez. Zoologia Scienze Naturali, Dip. di Biologia. Via Celoria, 26, 20133 Milano. ITALY (deceased) Rastetter, Edward B., Marine Biological Laboratory, The Ecosystems Center. Woods Hole, MA 02543 Ri'bhun. Lionel I., University of Virginia. Department of Biology, Gilmer Hall 45, Charlottesville. VA 22901 Reddan. John R., Oakland University. Department of Biological Sciences. Rochester. MI 48309-4401 Reese, Thomas S., NIH. N1NDS. Building 36. Room 2A29. Bethesda, MD 20892 Reinisch, Carol L., Marine Biological Laboratory, 7 MBL Street. Woods Hole. MA 02543 Rickles, Frederick R., 2633 Danforth Lane, Decatur, GA 30033 Rieder, {.'only L., Wadsworth Center, Division of Molecular Medicine. P.O. Box 509. Albany. NY 12201-0509 Riley, Monica, Marine Biological Laboratory, 7 MBL Street, Woods Hole. MA 02543 Ripps, Harris, University of Illinois at Chicago. Department of Ophthalmology/Visual Sciences. 1855 West Taylor Street, Chicago, IL 60612 Ritchie, J. Murdoch, Yale LIniversity School of Medicine. Department of Pharmacology, 333 Cedar Street. New Haven. CT 06510 Rome, Lawrence C., University of Pennsylvania. Department of Biology. Philadelphia. PA 19104 Rosenhluth, Jack, New York University School of Medical, Department of Physiology and Biophysics. RR 714. 400 East 34th Street, New York, NY 10016 Rosenhluth, Raja, Simon Fraser University. Institute of Molecular Biology and Biochemistry. Burnaby, BC. BC V5A IS6. CANADA Rosenh'eld, Allan, Columbia University School of Public Health. 600 West IfiSih Street. New York. NY 10032-3702 Ro.senkranz. Herbert S., 130 Desoto Street. Pittsburgh. PA 15213-2535 Roslansky, John D., 57 Buzzards Bay Avenue. Woods Hole. MA 02543 Roslansky, Priscilla F., Associates of Cape Cod. Inc., P.O. Box 224, Woods Hole, MA 02543 Ross, William N., New York Medical College. Department of Physiology. Valhalla. NY 10595 Roth, Jay S., P.O. Box 692. Woods Hole. MA 02543-0692 Rottenfusser, Rudi, Marine Biological Laboratory, 7 MBL Street. Woods Hole. MA 02543 Rowland. Lewis P., Neurological Institute. 710 West 168th Street, New York. NY 10032 Riiderman, Joan V., Harvard Medical School. Department of Cell Biology. 240 Longwood Avenue, Boston, MA 021 15 Rummel. John I)., Marine Biological Laboratory, 7 MBL Street, Woods Hole, MA 02543 Rushforth, Norman B.. Case Western Reserve University, Department of Biology. Cleveland, OH 44106 Russell-Hunter, W. D., 71 1 Howard Street, Easton. MD 21601-3934 Saffo, Mary Beth, Arizona State University West. Life Science Department, MC 2352. P.O. Box 37100. Phoenix. AZ 85069-7100 Salama, Guy, University of Pittsburgh, Department of Physiology. Pittsburgh. PA 15261 Salmon, Edward D., University of North Carolina. Department of Biology. CB 3280. Chapel Hill, NC 27514 Salvers, Abigail, University of Illinois. Department of Microbiology. 407 South Goodwin Avenue, Urbana. IL 61801 Salzberg, Brian M., University of Pennsylvania School of Medicine, Department of Neuroscience. 215 Stemmler Hall. Philadelphia. PA 19104-6074 Sanger, Jean M., University of Pennsylvania School of Medicine. Department of Anatomy. 36th and Hamilton Walk. Philadelphia, PA 19104 Sanger. Joseph W., University of Pennsylvania Medical Center, Department of Cell and Developemental Biology, 36th and Hamilton Walk. Philadelphia. PA 19104-6058 Saunders Jr., John W., Marquette University, P.O. Box 3381. Wauuoit. MA 02536 Schachman, Howard K., University of California. Molecular and Cell Biology Department. 229 Stanley Hall. #3206. Berkeley. CA 94720- 3206 Schatten, Gerald P., Oregon Health Sciences University. Oregon Regional Primate Research Center. 505 N.W. 185th Avenue. Beaverton, OR 97006 Schatten, Heide, University of Wisconsin. Department of Zoology, Madison, WI 53706 Schmeer, Arlene C., Mercenene Cancer Research Institute, 790 Prospect Street, New Haven, CT 065 1 1 Schuel. Herbert. SUNY at Buffalo, Department of Anatomy/Cell Biology, Buffalo, NY 14214 Schwartz, James H., New York State Psychiatric Institute, Research Annex, 722 West 168th Street, 7th floor, New York, NY 10032 Schwartz, Lawrence, University of Massachusetts. Department of Biology, Morrill Science Center, Amherst, MA 01003 Schweitzer. A. Nicola, Brigham and Women's Hospital. Immunology Division, Department of Pathology, 221 Longwood Avenue, LMRC 521. Boston. MA 02115 Segal, Sheldon J.. The Population Council, One Dag Hammarskjold Pla/a. New York, NY 10036 Senl't, Stephen Lamont, Neuroengineering/Neuroscience Center, P.O. Box 208205. New Haven. CT 06520-8205 Shanklin. Douglas R., University of Tennessee. Department of Pathology, Room 576, 800 Madison Avenue, Memphis, TN 381 17 Shashoua, Victor E., Harvard Medical School, Ralph Lowell Labs. McLean Hospital. I 15 Mill Street. Belmont. MA 02178 Shaver, Gaius R.. Marine Biological Laboratory. The Ecosystems Center, Woods Hole. MA 02543 Shaver, John R., Michigan State University. Department of Zoology, East Lansing, MI 48824 Sheetz, Michael P.. Duke University Medical Center. Department of Cell Biology, Bx 3709, 388 Nanalmc Duke Building. Durham. NC 27710 Slii'pro, David, Boston University. CAS Biology. 5 Cummington Street, Boston, MA 02215 Shimomura. Osamii, Marine Biological Laboratory. 7 MBL Street. Woods Hole. MA 02543 Shipley, Alan M., P.O. Box 2036. Sandwich. MA 02563 Members of the Corporation R77 Silver, Robert B., Marine Biological Laboratory. 7 MBL Street, Woods Hole, MA 0254.1 Siwicki, Kathleen K., Swarthmore College, Biology Department. 500 College Avenue. Swarthmore. PA 19081-1397 Skinner, Dorothy M., Oak Ridge National Laboratory, Biology Division. P.O. Box 2009. Oak Ridge. TN 37831 Sloboda, Roger D., Dartmouth College. Department of Biological Science. 6044 Oilman. Hanover. NH 03755-1893 Sluder, Greenfield, University of Massachusetts Medical School, Room 324. 377 Plantation Street. Worcester. MA 01605 Smith, Peter J.S., Marine Biological Laboratory. 7 MBL Street. Woods Hole. MA 02543 Smith, Stephen J., Stanford University School of Medicine, Department of Molecular and Cellular Physiology. Beckman Center. Stanford, CA 94305 Smolowitz, Roxanna S., Marine Biological Laboratory, 7 MBL Street. Woods Hole. MA 02543 Sogin, Mitchell L., Marine Biological Laboratory. 7 MBL Street. Woods Hole. MA 02543 Sorenson, Martha M., Cidade Universitana-UFRJ. Department Bioquimica Medica-ICB. 21941-590 Rio de Janerio. BRAZIL Speck. William T., Columbia-Presbyterian Medical Center. 161 Fort Washington Avenue. 14th Floor. Room 1470, New York. NY 10032- 3784 Spector, Abraham, Columbia University. Department of Ophthalmology. 630 West 168th Street. New York. NY 10032 Speksnijder. Johanna E., University of Groningen, Department of Genetics, Kerklaan 30. 9751 NN Haren, THE NETHERLANDS Spray, David C., Albert Einstein College of Medicine, Department of Neuroscience, 1300 Morris Park Avenue. Bronx, NY 10461 Spring, Kenneth R., National Institutes of Health, 10 Center Drive. MSC 1598. Building 10. Room 6N260. Bethesda. MD 20892-1603 Steele, John H., Woods Hole Oceanographic Institution, Woods Hole, MA 02543 Steinacker, Antoinette, University of Puerto Rico, Instituet of Neurobiology, 201 Boulevard Del Valle. San Ian, PR 00901 Steinberg. Malcolm, Princeton University, Department of Molecular Biology. M-18 Moffett Laboratory. Princeton, NI 08544-1014 Stemmer, Andreas C., Institut fur Robotik, ETH-Sentrum. 8092 Zurich. SWITZERLAND Stenflo, Johan, University of Lund. Department of Clinical Chemistry. Malmo General Hospital, S-205 02 Malmo. SWEDEN Stetten, Jane Lazarow, 4701 Willard Avenue. #1413. Chevy Chase. MD 20815-4627 Steadier, Paul A., Marine Biological Laboratory. The Ecosystems Center. Woods Hole, MA 02543 Stokes, Darrell R., Emory University, Department of Biology. 1510 Clifton Road NE, Atlanta. GA 30322-1100 Stommel, Elijah W., Darmouth Hitchcock Medical Center. Neurology Department. Lebanon. NH 03756 Stracher, Alfred, SUNY Health Science Center. Department of Biochemistry, 450 Clarkson Avenue. Brooklyn. NY 1 1 203 Strumwasser, Felix, P.O. Box 2278. East Falmouth. MA 02536-2278 Stuart, Ann E., University of North Carolina. Department of Physiology, Medical Research Building 206H, Chapel Hill. NC 27599-7545 Sugimori. Mutsuyuki, New York University Medical Center. Department of Physiology and Neuroscience, Room 442, 550 First Avenue, New York. NY 10016 Summers, William C., Western Washington University, Huxley College of Environmental Studies, Bellingham. WA 98225-9181 Suprenant, Kathy A., University of Kansas. Department of Physiology and Cell Biology. 4010 Haworth Hall. Lawrence. KS 66045 Swenson. Katherine I., Duke University Medical Center. Department of Molecular Cancer Biology, Box 3686, Durham. NC 27710 Sydlik, Mary Anne, Hope College, Peale Science Center. 35 East 1 2th St./P.O. Box 9000, Holland. MI 49422 Szent-Gyorgyi, Andrew G., 9 Westgate Road. Wellesley. MA 02181 Tabares, Lucia, University of Seville School of Medicine. Department of Physiology. Avda. Sanchez Pizjuan 4, Seville 41009, Spain Tamm, Sidney L., Boston University. 725 Commonwealth Avenue. Boston. MA 02215 Tanzer, Marvin L., University of Conn School of Dental Medicine. Department of Biostructure and Function. Farmmgton, CT 06030- 3705 Tasaki, Ichiji, NIMH/NIH, Laboratory of Neurobiology, Building 36. Room 2B-16. Bethesda. MD 20892 Taylor, D. Lansing, Carnegie Mellon University, Center for Flurorescence Research. 4400 Fifth Avenue. Pittsburgh, PA 15213 Taylor, Edwin W., University of Chicago, Department of Molecular Genetics, 920 E. 58th Street, Chicago, IL 60637 Teal, John M., Woods Hole Oceanographic Institute. Department of Biology. Woods Hole. MA 02543 Telfer, William H., University of Pennsylvania. Department of Biology. Philadelphia. PA 19104 Telzer, Bruce. Pomona College, Department of Biology, Thille Building, 175 West 6th Street, Claremont, CA 91711 Townsel, James G., Meharry Medical College. Department of Physiology. 1005 DB Todd Boulevard. Nashville, TN 37208 Travis, David M., 19 High Street, Woods Hole, MA 02543-1221 Treistman, Steven N., University of Massachusetts Medical Center, Department of Pharmacology. 55 Lake Avenue North, Worcester. MA 01655 Trigg, D. Thomas, One Federal Street. 9th Floor. Boston. MA 0221 1 Troll, Walter, NYU Medical Center. 550 First Avenue. New York, NY 10016 Troxler, Robert F., Boston University School of Medicine. Department of Biochemistry, 80 East Concord Street, Boston, MA 021 18 Tucker, Edward B., Baruch College. CUNY, Department of Natural Sciences, 17 Lexington Avenue. New York, NY 10010 Turner, Ruth D., Harvard University, Museum of Comparative Zoology, Mollusk Department, Cambridge, MA 02138 Tweedell, Kenyon S., University of Notre Dame, Department of Biological Sciences. Notre Dame, IN 46556-0369 Tykocinski, Mark L., Case Western Reserve University, Institute of Pathology, 2085 Adelbert Road, Cleveland. OH 44106 Tytell, Michael, Wake Forest University, Bowman Gray School of Medicine, Department of Anatomy and Neurobiology. Winston- Salem, NC 27157 Ueno, Hiroshi, Kyoto University. AGR Chemistry. Faculty of Agriculture, Sakyo. Kyoto 606-8502. JAPAN Valiela, Ivan, Boston University Marine Program. Marine Biological Laboratory. Woods Hole. MA 02543 Vallee. Richard, University of Massachusetts Medical Center. Worcester Foundation Campus, 222 Maple Avenue. Shrewsbury, MA 01545 Valois, John J., 420 Woods Hole Road, Woods Hole, MA 02543 Van Holde, Kensal E., Oregon State University, Biochemistry and Biophysics Department. Corvallis. OR 97331-7503 Van Dover, Cindy Lee, University of Alaska, P.O. Box 757220. Fairbanks. AK 99775 Vogl. Thomas P., Environmental Research Institute of Michigan. 1101 Wilson Boulevard. Arlington, VA 22209 R78 Annual Report Wainvvright, Norman R., Marine Biological Laboratory, 7 MBL Street, Woods Hole. MA 02543 Waksman, Byron H., New York University Medical Center. Department of Pathology. 550 First Avenue, New York, NY 10016 Wall, Betty, 9 George Street, Woods Hole, MA 02543 Wang, Hsien-Yu, State University of New York, University Medical Center, Physiology and Biophysics-HSC, Stony Brook. NY 1 17^4- 8633 Wangh, Lawrence J., Brandeis University, Department of Biology. 415 South Street, Waltham, MA 02254 Warner, Robert C., University of California, Irvine. Molecular Biology and Biochemistry, Irvine. CA 92717 Warren, Leonard, Wistar Institute, 36th and Spruce Streets. Philadelphia, PA 191(14 Waterbury, John B., Woods Hole Oceanographic Institution. Department of Biology, Woods Hole. MA 02543 Waxman. Stephen G., Yale Medical School. Neurology Department, 333 Cedar Street, P.O. Box 208018, New Haven, CT 06510 Webb, H. Marguerite, 184 Chestnut Street, Foxboro, MA 02035-1548 Weber, Annemarie, University of Pennsylvania School of Medicine, Department of Biochemstry and Biophysics. Philadelphia, PA 19066 Weeks, Janis C., University of Oregon, Institute of Neuroscience, Eugene, OR 97403- 1 254 Weidner, Earl, Louisiana State University, Department of Biological Sciences. 508 Life Sciences Building, Baton Rouge, LA 70803-1715 Weiss, Alice Sara, 105 University Boulevard West, Silver Spring, MD 20901 Weiss, Dieter, University of Rostock. Institute of Zoology. D- 18051 Rostock. GERMANY Weiss, Leon P., University of Pennsylvania School of Vet Medicine, Department of Animal Biology. Philadelphia, PA 19104 Weiss, Marisa C., Paoli Memorial Hospital, Department of Radiation Oncology, 255 W. Lancaster Avenue. Paoli, PA 19301 Weissmann, Gerald, New York University Medical Center, Department of Medicine/Division Rheumatology. 550 First Avenue. New York, NY 10016 Westerh'eld, Monte, University of Oregon, Institute of Neuroscience, Eugene, OR 97403 Whittaker, J. Richard, University of New Brunswick, Department of Biology, BS 4511, Frederiction, NB E3B 6E1, CANADA Wilkens, Lon A., University of Missouri, Department of Biology. 8001 Natural Bridge Road, Street Louis, MO 63121-4499 MBL Associates Wilson, Darcy B., San Diego Regional Cancer Center. 3099 Science Park Road, San Diego, CA 92 1 2 1 Wilson, T. Hastings, Harvard Medical School. Department of Physiology, 25 Shattuck Street, Boston, MA 02 1 1 5 Witkovsky, Paul, NYU Medical Center, Department of Ophthalmology, 550 First Avenue. New York, NY 10016 Wittenberg, Beatrice, Albert Einstein College of Medicine, Department of Physiology and Biophysics, Bronx, NY 10461 Wittenberg, Jonathan B., Albert Einstein College of Medicine, Department of Physiology and Biophysics, Bronx, NY 10461 Wolken, Jerome J., Carnegie Mellon University, Department of Biological Sciences, 440 Fifth Avenue. Pittsburgh, PA 15213 (deceased) Wonderlin, William F., West Virginia University, Pharmacology and Toxicology Department. Morgantown, WV 26506 Worden, Mary Kate, University of Virginia, Department of Neuroscience, McKim Hall Box 230, Charlottesville. VA 22908 Worgul, Basil V., Columbia University, Department of Ophthalmology. 630 West 16X Street. New York, NY 10032 Wu, Chau Hsiung, Northwestern University Medical School. Department of Pharmacology (S215), 303 East Chicago Avenue, Chicago, II. 6061 1-3008 Wyttenbach, Charles R., University of Kansas, Biological Sciences Department, 2045 Haworth Hall. Lawrence. KS 66045-2106 Yen, Jay Z., Northwestern University Medical School, Department of Pharmacology, Chicago. IL 6061 1 Zacks, Sumner I., 65 Saconesset Road, Falmouth. MA 02540-1851 Zigman, Seymour, University of Rochester Medical School, Ophthalmology Research, Box 314, 601 Elmwood Avenue. Rochester. NY 14640 Zigmond, Michael J., Lmiversity of Pittsburgh. S-526 Biomedical Science Tower, 3500 Terrace Street. Pittsburgh. PA 15213 Zimmerberg, Joshua J., National Institutes of Health, LCMB, NICHD, Building 10. Room 10D14, 10 Center Drive. Bethesda, MD 20892 Zottoli, Steven J., Williams College, Department of Biology, Williamstown, MA 01267 Zucker, Robert S., University of California. Neurobiology Division, Molecular and Cellular Biology Department. Berkeley. CA 94720 Zukin, R. Suzanne, Albert Einstein College of Medicine, Department of Neuroscience, 1410 Pelham Parkway South, Bronx. NY 10461 Executive Board Ruth Ann Laster, President Jack Pearce, Vice President Hanna Hastings, Treasurer Molly Cornell. Secretary Elizabeth Farnham, Membership Chair Tammy Smith Amon Duncan Aspmwall Barbara Atwood Kitty Brown Julie Child Seymour Cohen Michael Fenlon Alice Knowles Rebecca Lash Barbara Little Cornelia McMurtne Jack Moakley Joan Pearlman Virginia R. Reynolds Volker Ulbnch John Valois Kensal E. Van Holde Patrons Mr. and Mrs. David Bakalar Josephine B. Crane Foundation Dr. and Mrs. James J. Ferguson, Jr. Sustaining Associate Mr. Robert A. Jaye George Frederick Jewett Foundation Dr. and Mrs. Edward F. MacNichol. Jr. Plymouth Savings Bank Mr. and Mrs. William A. Putnam, III Supporting Associate Mrs. George H.A. Clowes Dr. and Mrs. James D. Ebert Mr. and Mrs. David Fausch Dr. and Mrs. Prosser Gifford Mr. and Mrs. Lon Hocker Mrs. Mary D. Janney Drs. Luigi and Elaine Mastroianni Dr. and Mrs. William M. McDennott Drs. Matthew & Jeanne Meselson Dr. and Mrs. Courtland D. Perkins Ms. Linda Sallop and Mr. Michael Fenlon Mrs. Anne W. Sawyer Dr. Maxine F. Singer Members of the Corporation R79 Dr. John Tochko and Mrs. Christina Myles-Tochko Mr. and Mrs. John J. Valois Drs. Walter S. Vincent and Dore J. Butler Fumilv Membership Dr. Frederick W. Ackroyd Dr. and Mrs. Edward A. Adelberg Mr. and Mrs. Douglas F. Allison Drs. Peggy and Fred Alsup Drs. James and Helene Anderson Dr. and Mrs. Samuel C. Armstrong Mr. and Mrs. Duncan P. Aspinwall Mr. and Mrs. Donald R. Aukamp Mr. and Mrs. John M. Baitsell Mr. and Mrs. William L. Banks Dr. and Mrs. Robert B. Barlow. Jr. Mr. and Mrs. John E. Barnes Dr. and Mrs. Robert M. Berne Drs. Harriet and Alan Bernheimer Mr. and Mrs. Robert O. Bigelow Dr. and Mrs. Edward G. Boettiger Mr. and Mrs. Kendall B. Bohr Dr. and Mrs. Alfred F. Borg Dr. and Mrs. Thomas A. Borgese Mr. and Mrs. Richard M. Bowen Dr. and Mrs. Francis P. Bowles Dr. and Mrs. John B. Buck Dr. and Mrs. John E. Burns Mr. and Mrs. William O. Burwell Mr. and Mrs. G. Nathan Calkins. Jr. Mr. and Mrs. D. Bret Carlson Prof, and Mrs. James F. Case Dr. and Mrs. Alfred B. Chaet Dr. and Mrs. Richard L. Chappell Dr. and Mrs. Frank M. Child, III Dr. and Mrs. Arnold M. Clark Mrs. LeRoy Clark Mr. and Mrs. James Cleary Dr. and Mrs. Laurence P. Cloud Mr. and Mrs. Lawrence H. Coburn Dr. and Mrs. Neal W. Cornell Mr. and Mrs. Norman C. Cross Mr. and Mrs. Bruce G. Daniels Mr. and Mrs. Joel P. Davis Mr. and Mrs. Richard C. Dierker Dr. and Mrs. Arthur Brooks DuBois Mr. and Mrs. John Eustis. II Mr. and Mrs. Harold Frank Mr. and Mrs. Howard G. Freeman Dr. and Mrs. Robert A. Frosch Dr. and Mrs. John J. Funkhouser Dr. and Mrs. Mordecai L. Gabriel Dr. and Mrs. David Garber Dr. and Mrs. Sydney Gellis Dr. and Mrs. James L. German, III Mr. and Mrs. Robert S. Gillette Dr. and Mrs. Murray Glusman Drs. Alfred and Joan Goldberg Mr. and Mrs. Charles Goodwin Dr. and Mrs. Philip Grant Dr. and Mrs. Thomas C. Gregg Prof, and Mrs. Lawrence Grossman Dr. and Mrs. Antoine P.O. Hadamard Mr. and Mrs. Peter A. Hall Dr. and Mrs. Harlyn O. Halvorson Capt. and Mrs. Frederick J. Hancox Drs. Alexander and Carol Hannenberg Mrs. Janet Harvey and Dr. Richard Harvey Dr. and Mrs. J. Woodland Hastings Mr. and Mrs. Gary G. Hayward Dr. and Mrs. Howard H. Hiatt Mr. and Mrs. David Hibbitt Dr. and Mrs. John E. Hobbie Drs. Francis C. G. Hoskin and Elizabeth M. Farnham Dr. and Mrs. Robert J. Huettncr Dr. and Mrs. Shinya Inoue Dr. and Mrs. Kurt J. Isselbacher Dr. and Mrs. Gary Jacobson Dr. and Mrs. Benjamin Kaminer Mr. and Mrs. Paul W. Knaplund Mr. and Mrs. A. Sidney Knowles, Jr. Dr. and Mrs. S. Andrew Kulin Dr. and Mrs. George M. Langford Dr. and Mrs. Leonard Laster Dr. and Mrs. Hans Laufer Mr. William Lawrence and Mrs. Barbara Buchanan Mr. and Mrs. Stephen R. Levy Mr. and Mrs. Robert Livingstone, Jr. Mr. and Mrs. James E. Lloyd Mr. and Mrs. Bernard Manuel Dr. and Mrs. Julian B. Marsh Mr. and Mrs. Joseph C. Martyna Mr. and Mrs. Frank J. Mather, III Mr. and Mrs. John E. Matthews Dr. and Mrs. Robert T. McCluskey Mr. Paul McGonigle Dr. and Mrs. Jerry M. Melillo Dr. Martin Mendelson Mr. and Mrs. Richard Meyers Dr. and Mrs. Daniel G. Miller Dr. and Mrs. Merle Mizell Dr. and Mrs. Charles H. Montgomery Mr. and Mrs. Charles F. Murphy Dr. and Mrs. John E. Naugle Dr. Pamela Nelson and Mr. Christopher Olmsted Mr. and Mrs. Frank L. Nickerson Dr. and Mrs. Clifford T. O'Connell Mr. and Mrs. David R. Palmer Dr. and Mrs. George D. Pappas Mr. and Mrs. Robert Parkinson Mr. and Mrs. Richard M. Paulson, Jr. Mr. and Mrs. William J. Pechilis Mr. and Mrs. John B. Peri Dr. and Mrs. Philip Person Mr. and Mrs. Frederick S. Peters Mrs. and Mr. Grace M. Peters Mr. and Mrs. George H. Plough Dr. and Mrs. Aubrey Pothier, Jr. Dr. and Mrs. Carl A. Price Mr. Allan Ray Putnam Dr. and Mrs. Lionel I. Rebhun Dr. and Mrs. George T. Reynolds Mr. and Mrs. John Ripple Dr. and Mrs. Harris Ripps Ms. Jean Roberts Drs. Priscilla and John Roslansky Dr. and Mrs. John D. Rummel Dr. and Mrs. John W. Saunders, Jr. Dr. and Mrs. R. Walter Schlesinger Mr. and Mrs. Harold H. Sears Mr. John Seder and Ms. Frances Plough Dr. and Mrs. Sheldon J. Segal Dr. and Mrs. Douglas R. Shanklin Dr. and Mrs. David Shepro Mr. and Mrs. Bertram R. Silver Mr. and Mrs. Jonathan O. Simonds Drs. Frederick and Marguerite Smith Dr. and Mrs. Hein/. Specht (Dr. Specht deceased) Drs. William and Phoebe Speck Dr. and Mrs. William K. Stephenson Mr. and Mrs. E. Kent Swift. Jr. Mr. and Mrs. Gerard L. Swope, III Mr. and Mrs. Emil D. Tietje, Jr. Mr. Norman N. Tolkan Dr. and Mrs. Walter Troll Mr. and Mrs. Volker Ulbrich Drs. Claude and Dorothy Villee Mr. and Mrs. Samuel Vincent Dr. and Mrs. Samuel Ward Mr. J. Ware and Ms. Sharon McCarthy Dr. and Mrs. Henry B. Warren Dr. and Mrs. Gerald Weissmann Dr. and Mrs. Paul S. Wheeler Dr. Martin Keister White Mr. and Mrs. Geoffrey G. Whitney, Jr. Mr. and Mrs. Leonard M. Wilson Mr. and Mrs. Leslie J. Wilson Mr. and Mrs. Dick S Yeo Dr. and Mrs. Sumner I. Zacks Individual Membership Mr. David C. Ahearn Mr. Henry Albers Mrs. Constance M. Allard Dr. Nina S. Allen Mrs. Tammy Amon Mr. Dean N. Arden Mrs. Ellen Prosser Armstrong Mrs. Kimball C. Atwood, III Dr. Serena Baccetti Mr. Everett E. Bagley Mr. C. John Berg Ms. Avis Blomberg Mrs. Elinor W. Bodian Mr. Thomas C. Bolton Mrs. Jennie P. Brown Mrs. M. Kathryn S. Brown Ms. Hennete Bull Dr. Alan H. Burghauser Mrs. Barbara Gates Burwell Mr. Bruce E. Buxton Mr. Patrick J. Calie Dr. Graciela C. Candelas Mrs. Winslow G. Carlton (deceased) Mr. Frank C. Carotenuto R80 Annual Report Dr. Robert H, Currier Mrs. Patricia A. Case Dr. Sallie Chisholm Mrs. Octavia C. Clement Mr. Allen W. Clowes Dr. Jewel Plummer Cohh Mrs. Margaret H. Cohurn Dr. Seymour S. Cohen Ms. Anne S. Concannon Mr. Robert J. Cook Prof. D. Eugene Copeland Dr. Helen M. Costello Dr. Vincent Cowling Mrs. J. Sterling Crandall Ms. Dorothy Crossley Ms. Helen M. Crossley Mrs. Villa B. Crowell Mr. Norman Dana Dr. Morton Davidson Ms. Carol Reimann De Young Dr. Mane A. DiBerardino Mrs. Shirley Dierolt Mr. David L. Donovan Mr. Stephen Doyle Ms. Suzanne Drohan Mr. Roy A. Duffus Mrs. Charles Eastman Dr. Frank Egloff Mr. Raymond Eliott Mr. William M. Ferry Mr. Robert Fitzpatrick Ms. Sylvia M. Flanagan Mr. Robert P. Flynn. Jr. Mr. John W. Folino, Jr. Mr. John H. Ford Dr. Krystyna Frenkel Mr. Paul J. Freyheit Mrs. Paul M. Fye Mr. Joseph C. Gallagher Miss Eleanor Garrield Dr. Patricia E. Garrett Mr. Charles Gilford Mrs. James R. Glazebrook Mrs. Mary L. Goldman Mr. Michael P. Goldring Mrs. Phyllis Goldstein Ms. Muriel Gould Mrs. Deborah Ann Green Dr. B. Herold Griffith Mrs. Edith T. Grosch (deceased) Mrs. Barbara Grossman Mrs. Valerie A. Hall Dr. Peter J. Hamre Ms. Mary Elizabeth Hamstrom Ms. Elizabeth E. Hathaway Dr Robert R. Haubrich Mrs Jane M. Heald Mr. Michael W. Herlihy Mrs. Nalhan Hirschfeld Mrs. Eleanor D. Hodge Mr. Roger W Huhbell Ms. Susan A. Huettner Miss Eli/ubelh B. Jackson Dr. Joseph Jacohson Mr. Raymond L. Jewett Mrs. Barbara W. Jones Mrs. Margaret H. Jones Mrs. Barbara Kanellopoulos Mrs. Joan T. Kanwisher Mrs. Sally Karush Mrs. Marcella Katz Ms. Patricia E. Keoughan Dr. Peter N. Kivy Lady Haber Kornberg Dr. Bruno P. Kremer Ms. Norma Kumin Mr. Bernard H. Labitt Mrs. Janet W. Larcom Ms. Rebecca Lash Dr. Marian E. LeFevre Dr. Mortimer Levitz Mr. Edwin M. Libbm Mr. Lennart Lindberg Mrs. Barbara C. Little Mrs. Sarah J. Loessel Mrs. Ermine W. Lovell Mr. Richard C. Lovering Mrs. Margaret M. Macleish Ms. Anne Camille Maher Mrs. Annemarie E. Mahler Mr. Patrick J. Mahoney Dr. Phillip B. Maples Mr. Daniel R. Manin Dr. G. C. Matthiessen Dr. Miriam Jacob Mauzerall Mrs. Mary Hartwell Mavor Mrs. Jane C. McCormack Ms. Suzanne McDermott Mrs. Nella W. McElroy (deceased) Dr. Susan Gerbi Mcllwain Ms. Mary W. McKoan Ms. Geraldine G. McLean Ms. Cornelia Hanna McMurtrie Mrs. Ellen L. Meigs Mr. Ted Mehllo Mrs. Grace S. Metz Mrs. Mary G. Miles Mrs. Florence E. Mixer Mr. John T. Moaklcy Mrs. Mary E. Montgomery Ms. Cynthia Moor Mr. Stephen A. Moore Mr. Alan F. Morrison Dr. M. Patricia Morse Mrs. Eleanor M. Nace Mr. William G. Neall Mrs. Anne Nelson Mrs. Catherine N. Norton Mr. Thomas J. Novitsky Mr. John J. O'Connor Dr. Renee Bennett O'Sullivan Miss Carolyn L. Parmenter Mrs. Dolores Patch-Wing Dr. John B. Pearce Ms. Joan Pearlman Dr. Judith Pederson Ms. Joyce S. Pendery Dr. Murray E. Pendleton Mr. Raymond W. Peterson Ms. Victoria A. Powell Mrs. Julia S. Rankin Mr. Fred J. Ravens, Jr. Mrs. Adell R. Rawson Dr. Robert M. Reece Ms. Anecia Kathy Regis Dr. Renato A. Ricca Dr. Mary Esther Rice Mr. John Riina Dr. Monica Riley Mrs. Alison A. Robb Mrs. Lola E. Robertson Mrs. Ruth J. Robinson Mrs. Arlene Rogers Mrs. Wendy E. Rose Ms. Hilde Rosenthal Mrs. Atholie K. Rosett Dr. Virginia F. Ross Mr. Raymond A. Sanborn Mrs. Joyce Waksman Schambacher Ms. Elaine Schott Mrs. Elsie M. Scott Sea Education Association, Inc. Dr. Cecily C. Selby Mrs. Deborah G. Senft Mrs. Charlotte Shemin Mrs. Phyllis J. Silver Mrs. Cynthia C. Smith Mrs. Perle Sonnenblick Dr. Evelyn Spiegel Dr. Guy L. Steele, Sr. Dr. Robert E. Steele Mrs. Eleanor Steinbach Mrs. Judith G. Stetson Mrs. Jane Lazarow Stetten Dr. Dorothy A. Stracher Mr. Robert Stump Dr. Maurice Sussman Mr. Albert H. Swain Mr. James K. Taylor Mrs. Alice Todd Mr. Arthur D. Trauh Ms. Natalie Trousof Ms. Ciona Ulbrich Mrs. Barbara Van Holde Dr. Kensal E. Van Holde Ms. Sylvia Vatuk Ms. Susan Veeder Mr. Lee D. Vincent Mr. Arthur D. Voorhis Mrs. Eve Warren Mr. John T. Weeks Ms. Lillian Wendorff Dr. William M. Wheeler Ms. Mabel E. Whelpley Mrs. Barbara Whitehead Mrs. A. A. Wickersham Mrs. Clare M. Wilber Mrs. Ann S. Wilke Mr. Albert Wilson Dr. T. Hastings Wilson Ms. Nancy Woitkoski Mrs. Eli/abeth S. Yntema Members (if the Corporation R81 Mrs. Donald J. Zinn Pat Hancox Arlene Rogers Hanna Hastings Lil Saunders MBL Gift Shop Volunteers Sally Karush Alice Knowles Louise Specht Cynthia Smith Marion Adelberg Donna Kornberg Peggy Smilh Barbara Atwood Evelyn Laufer Jane Stetten Caroline Banks Barbara Little Elaine Troll Harriet Bernheiiner Sally Loessel Natalie Trousof Avis Blomberg Winnie Mackey Barbara Van Holde Gloria Borgese Miriam Mauzerall Susan Veeder Kitty Brown Mary Mavor Carol Ann Wagner Elisabeth Buck Jane McCormack Mabel Whelpley Vera Clark Louise McManus Clare Wilbcr Peggy Clowes Phyllis Meyers Jewel Cobb Polly Miles Janet Daniels Florence Mixer MBL Summer Tour Guides Carol DeYoung Lorraine Mizell Fran Eastman Elizabeth Moseley Sears Crowell Alma Ebert Stacia Palmer Barbara Little Jane Foster Bertha Person Steve Oliver Becky Glazebrook Margareta Pothier Julie Rankin Muriel Gould Julie Rankm Pnscilla Roslansky Barbara Grossman Millie Rebhun Mary Ulbrich Jean Halvorson Jean Ripps John Valois Certificate of Organization Articles of Amendment Bylaws Certificate of Organization Articles of Amendment (On File in the Office of the Secretary of the Commonwealth) No. 3170 We, Alpheus Hyatt, President. William Stanford Stevens. Treasurer, and William T. Sedgwick, Edward G. Gardiner, Susan Mims and Charles Sedgwick Minot being a majority of the Trustees of the Marine Biological Laboratory in compliance with the requirements of the fourth section of chapter one hundred and fifteen of the Public Statutes do hereby certify that the following is a true copy of the agreement of association to constitute said Corporation, with the names of the subscribers thereto: We, whose names are hereto subscribed, do, by this agreement, associate ourselves with the intention to constitute a Corporation according to the provisions of the one hundred and fifteenth chapter of the Public Statutes of the Commonwealth of Mas- sachusetts, and the Acts in amendment thereof and in addition thereto. The name by which the Corporation shall be known is THE MARINE BIOLOGICAL LABORATORY. The purpose for which the Corporation is constituted is to establish and maintain a laboratory or station for scientific study and investigations, and a school for instruc- tion in biology and natural history. The place within which the Corporation is established or located is the city of Boston within said Commonwealth. The amount of its capital stock is none. In Witness Whereof, we have hereunto set our hands, this twenty seventh day of February in the year eighteen hundred and eighty-eight. Alpheus Hyatt, Samuel Mills. William T. Sedgwick, Edward G. Gardiner, Charles Sedgwick Minot, William G. Farlow. William Stanford Stevens, Anna D. Phillips, Susan Mims, B. H. Van Vteck. That the tirst meeimy nt the subscribers to said agreement was held on the thirteenth day of March in the year eighteen hundred and eighty-eight. In Witness Whereof, we have hereunto signed our names, this thirteenth day of March in the year eighteen hundred and eighty-eight, Alpheus Hyatt, President, William Stanford Stevens, Treasurer, Edward G. Gardiner. William T Sedgwick, Susan Minis, Charles Sedgwick Minot. (Approved on March 20, 1888 as follows: I hereby certify that it appears upon an examination of the within wntten certificate and the records of the corporation duly submitted to my inspection, that the require- ments of sections one. two and three of chapter one hundred and fifteen, and sections eighteen, twenty and twenty-one of chapter one hundred and six, of the Public Statutes, have been complied with and I hereby approve said certificate this twentieth day of March A,D. eighteen hundred and eighty-eight. Charles Endicoit Commissioner of Corporations) (On File in the Office of the Secretary of the Commonwealth) We, James D. Ebert. President, and David Shepro, Clerk of the Marine Biological Laboratory, located at Woods Hole, Massachusetts 02543, do hereby certify that the following amendment to the Articles of Organization of the Corporation was duly adopted at a meeting held on August 15, 1975, as adjourned to August 29. 1975, by vote of 444 members, being at leasi two-thirds of its members legally qualified to vote in the meeting of the corporation: Voted: That the Certificate of Organization of this corporation be and it hereby is amended by the addition of the following provisions: "No Officer, Trustee or Corporate Member of the corporation shall be personally liable for the payment or satisfaction of any obligation or liabilities incurred as a result of, or otherwise in connection with, any commitments, agreements, activities or affairs of the corporation. "Except as otherwise specifically provided by the Bylaws of the corporation, meet- ings of the Corporate Members of the corporation may be held anywhere in the United States. "The Trustees of the corporation may make, amend or repeal the Bylaws of the corporation in whole or in part, except with respect to any provisions thereof which shall by law, this Certificate or the bylaws of the corporation, require action by the Corporate Members." The foregoing amendment will become effective when these articles of amendment are filed in accordance with Chapter 180, Section 7 of the General Laws unless these articles specify, in accordance with the vote adopting the amendment, a later effective date not more than thirty days after such riling, in which event the amendment will become effective on such later date. In Witness whereof and Under the Penalties of Perjury, we have hereto signed our names this 2nd day of September, in the year 1975, James D. Ehert, President; David Shepro, Clerk. (Approved on October 24, 1975, as follows: I hereby approve the within articles of amendment and, the riling fee m the amount of $10 having been paid, said articles are deemed to have been filed with me this 24th day of October. 1975, Paul Guzzi Secretary' of the Commonwealth) Bylaws (Revised August 7, 1992 and December 10. 1992) ARTICLE 1— THE CORPORATION A. Name iintl Piirpiat: The name of the Corporation shall he The Marine Biolog- ical Laboratory. The Corporation's purpose shall he to establish and maintain a K82 Bylaws of the Corporation R83 laboratory or station tor scientific study and investigation and a school lor instruction in biology and natural history. B. Nondiscrimination. The Corporation shall not discriminate on the basis of age, religion, color, race, national or ethnic origin, sex or sexual preference in its policies on employment and administration or in its educational and other programs. ARTICLE II— MEMBERSHIP A. Members. The Members of the Corporation ("Members") shall consist of persons elected by the Board of Trustees (the "Board"), upon such terms and conditions and in accordance with such procedures, not inconsistent with law or these Bylaws, as may be determined by the Board. At any regular or special meeting of the Board, the Board may elect new Members. Members shall have no voting or other rights with respect to the Corporation or its activities except as specified in these Bylaws, and any Member may vote at any meeting of the Members in person only and not by proxy. Members shall serve until their death or resignation unless earlier removed with or without cause by the affirmative vote of two-thirds of the Trustees then in office. Any Member who has retired from his or her home institution may, upon written request to the Corporation, be designated a Life Member. Life Members shall not have the right to vote and shall not be assessed for dues. B. Meetings. The annual meeting of the Members shall be held on the Friday following the first Tuesday in August of each year, at the Laboratory of the Corpo- ration in Woods Hole, Massachusetts, at 9:30 a.m. The Chairperson of the Board shall preside at meetings of the Corporation. If no annual meeting is held in accordance with the foregoing provision, a special meeting may be held in lieu thereof with the same effect as the annual meeting, and in such case all references in these Bylaws, except in this Article II. B.. to the annual meeting of the Members shall be deemed to refer to such special meeting. Members shall transact business as ma\ properly come before the meeting. Special meetings of the Members may be called by the Chair- person or the Trustees, and shall be called by the Clerk, or in the case of the death, absence, incapacity or refusal by the Clerk, by any other officer, upon written application of Members representing at least ten percent of the smallest quorum of Members required for a vote upon any matter at the annual meeting of the Members, to be held at such time and place as may be designated. C. Quorum. One hundred (100) Members shall constitute a quorum at any meeting. Except as otherwise required by law or these Bylaws, the affirmative vote of a majonty of the Members voting in person at a meeting attended by a quorum shall constitute action on behalf of the Members. D. Notice of Meetings. Notice of any annual meeting or special meeting of Members, if necessary, shall be given by the Clerk by mailing notice of the time and place and purpose of such meeting at least 15 days before such meeting to each Member at his or her address as shown on the records of the Corporation. E. Wavier of Notice. Whenever notice of a meeting is required to be given a Member, under any provision of the Articles or Organization or Bylaws of the Corporation, a written waiver thereof, executed before or after the Meeting by such Member, or his or her duly authorized attorney, shall be deemed equivalent to such notice. F. Adjournments. Any meeting of the Members may be adjourned to any other time and place by the vote of a majority of those Members present at the meeting, whether or not such Members constitute a quorum, or by any officer entitled to preside at or to act as Clerk of such meeting, if no Member is present or represented. It shall not be necessary to notify any Members of any adjournment unless no Member is present or represented at the meeting which is adjourned, in which case, notice of the adjournment shall be given in accordance with Article II. D. Any business which could have been transacted at any meeting of the Members as originally called may be transacted at an adjournment thereof. ARTICLE III— ASSOCIATES OF THE CORPORATION Associates of the Corporation. The Associates of the Marine Biological Laboratory shall be an unincorporated group of persons (including associations and corporations) interested in the Laboratory and shall be organized and operated under the general supervision and authority of the Trustees. The Associates of the Marine Biological Laboratory shall have no voting rights. ARTICLE IV— BOARD OF TRUSTEES A. Powers. The Board of Trustees shall have the control and management of the affairs of the Corporation. The Trustees shall elect a Chairperson of the Board who shall serve until his or her successor is elected and qualified. They shall annually elect a President of the Corporation. They shall annually elect a Vice Chairperson of the Board who shall be Vice Chairperson of the meetings of the Corporation. They shall annually elect a Treasurer. They shall annually elect a Clerk, who shall be a resident of Massachusetts. They shall elect Trustees-at-Large as specified in this Article IV. They shall appoint a Director of the Laboratory for a term not to exceed five years, provided the term sh.ill not exceed one year if the candidate has attained the age of 65 years prior to the date of the appointment. They shall choose such other officers and agents as they shall think best. They may fix the compensation of all officers and agcnls D| the Corporation and may remove them at any time. They may fill vacancies occurring in any of the offices. The Board shall have the power to choose an Executive Committee from their own number as provided in Article V, and to delegate to such Committee such of their own powers as they may deem expedient in addition to those powers conferred by Article V. They shall, from time to time, elect Members to the Corporation upon such terms and conditions as they shall have determined, not inconsistent with law or these Bylaws. B. Composition anJ Elt'clion. ( 1 1 The Board shall include 24 Trustees elected by the Board as provided below: (a) At least six Trustees I "Corporate Trustees") shall be Members who are scientists, and the other Trustees I "Trustees-at-Large" ) shall he individuals who need not be Members or otherwise affiliated with the Corporation. (b) The 24 elected Trustees shall be divided into four classes of six Trustees each, with one class to be elected each year to serve for a term of four years, and with each such class to include at least one Corporate Trustee. Such classes of Trustees shall be designated by the year of expiration of their respective terms. (2) The Board shall also include the Chief Executive Officer, Treasurer and the Chairperson of the Science Council, who shall be ex officia voting members of the Board. (3) Although Members or Trustees may recommend individuals for nomination as Trustees, nominations for Trustee elections shall be made by the Nominating Committee in its sole discretion The Board may also elect Trustees who have not been nominated by the Nominating Committee. C. Eligibility. A Corporate Trustee or a Trustee-at-Large who has been elected to an initial four-year term or remaining portion thereof, of which he/she has served at least two years, shall be eligible for re-election to a second four-year term, but shall be ineligible for re-election to any subsequent term until one year has elapsed after he/she has last served as a Trustee. D. Removal. Any Trustee may be removed from office at any time with or without cause, by vote of a majonty of the Members entitled to vote in the election of Trustees: or for cause, by vote of two-thirds of the Trustees then in office. A Trustee may be removed for cause only if notice of such action shall have been given to all of the Trustees or Members entitled to vote, as the case may be. prior to the meeting at which such action is to be taken and if the Trustee to be so removed shall have been given reasonable notice and opportunity to be heard before the body proposing to remove him or her. E. Vacancies. Any vacancy in the Board may be filled by vote of a majority of the remaining Trustees present at a meeting of Trustees at which a quorum is present. Any vacancy in the Board resulting from the resignation or removal of a Corporate Trustee shall be tilled by a Member who is a scientist. F. Meetings. Meetings of the Board shall be held from time to time, not less frequently than twice annually, as determined by the Board. Special meetings of Trustees may be called by the Chairperson, or by any seven Trustees, to be held at such lime and place as may be designated. The Chairperson of the Board, when present, shall preside over all meetings of the Trustees. Written notice shall be sent to a Trustee's usual or last known place of residence at least two weeks before the meeting. Notice of a meeting need not be given to any Trustee if a written waiver of notice executed by such Trustee before or after the meeting is filed with the records of the meeting, or if such Trustee shall attend the meeting without protesting prior thereto or at its commencement the lack of notice given to him or her. G. Quorum and Action by Trustees. A majority of all Trustees then in office shall constitute a quorum. Any meeting of Trustees may be adjourned by vote of a majonty of Trustees present, whether or not a quorum is present, and the meeting ma\ be held as adjourned without further notice. When a quorum is present at any meeting of the Trustees, a majority of the Trustees presenl and voting (excluding abstentions) shall decide any question, including the election of officers, unless otherwise required by law. the Articles of Organization or these Bylaws. H. Transfers of Interests in Land. There shall be no transfer of title nor long-term lease of real properly held by the Corporation without prior approval of not less than two-thirds of the Trustees. Such real property transactions shall he finally acted upon at a meeting of the Board only if presented and discussed at a prior meeting of the Board. Either meeting may be a special meeting and no less than four weeks shall elapse between the two meetings. Any property acquired by the Corporation after December 1. 1989 may be sold, any mortgage or pledge of real property (regardless of when acquired) lo secure bonowings by the Corporation may he granted, and any transfer of title or interest in real property pursuant to the foreclosure or endorsement R84 Annual Report of any such mortgage or pledge of real property may be effected by any holder of a mortgage or pledge of real property of the Corporation, with the prior approval of not less ihan two-thirds of the Trustees {other than any Trustee or Trustees with a direct or indirect financial interest in the transaction being considered tor approval) who are present at a regular or special meeting of the Board at which there is a quorum. ARTICLE V— COMMITTEES A. Executive Committee. There shall be an Executive Committee of the Board of Trustees which shall consist of not more than eleven (II) Trustees, including ex officio Trustees, elected by the Board. The Chairperson uf the Board shall act as Chairperson of the Executive Committee and the Vice Chairperson as Vice Chairperson. The Executive Committee shall meet at such times and places and upon such notice and appoint such subcommittees as the Committee shall determine. The Executive Committee shall have and may exercise all the powers of the Board during the intervals between meetings of the Board except those powers specifically withheld, from lime to time, by vote of the Board or by law. The Executive Committee may also appoint such committees, including persons who are not Trust- ees, as it may, from time to time, approve to make recommendations wilh respect to matters to be acted upon by the Executive Committee or the Board. The Executive Committee shall keep appropriate minutes of its meetings, which shall be reported to the Board. Any actions taken by the Executive Committee shall also be reported to the Board. B. Nominating Committee. There shall be a Nominating Committee which shall consist of not fewer than four nor more than six Trustees appointed by the Board in a manner which shall reflect the balance between Corporate Trustees and Trustees- at-Large on the Board. The Nominating Committee shall nominate persons for election as Corporate Trustees and Trustees-at-Large. Chairperson of the Board. Vice Chairperson of (he Board, President, Treasurer, Clerk, Director of the Laboratory and such other officers, if any, as needed, in accordance with (he requirements of these Bylaws. The Nominating Committee shall also be responsible for overseeing the training of new Trustees. The Chairperson of the Board of Trustees shall appoint the Chairperson of the Nominating Committee, The Chairperson of the Science Council shall be an ex officio voting member of the Nominating Committee. C. Science Council. There shall be a Science Council (the "Council") which shall consist of Members of the Corporation elected to the Council by vote of the Members of the Corporation, and which shall advise the Board with respect to matters con- cerning the Corporation's mission, its scientific and instructional endeavors, and the appointment and promotions of persons or committees with responsibility for matters requiring scientific expertise. Unless otherwise approved by a majority of the mem- bers of the Council, the Chairperson of the Council shall be elected annually by the Council. The chief executive officer of the Corporation shall be an c.\ officio voting member of the Council D. Board of Overseers. There shall be a Board of Overseers which shall consist of not fewer than five nor more than eight scientists who have expertise concerning matters with which the Corporation is involved. Members of the Board of Overseers may or may not be Members of the Corporation and may be appointed by the Board of Trustees on the basis of recommendations submitted from scientists and scientific organizations or societies. The Board of Overseers shall be available to review and offer recommendations lo the officers. Trustees and Science Council regarding scientific activities conducted or proposed by the Corporation and shall meel from time to time, not less frequently than annually, as determined by the Board of Trustees. E. Board Committees Generallv. The Trustees may elect or appoint one or more other committees (including, but not limited to, an Investment Commiltee, a Devel- opment Committee, an Audit Committee, a Facilities and Capital Equipment Com- mittee and a Long-Range Planning Committee) and may delegate to am sin.li committee or committees any or all of their powers, except those which by law, the Arliclcs of Organization or these Bylaws the Trustees are prohibited from delegating; provided thai any committee to which the powers of the Trustees are delegated shall consist solely of Trustees. The members of any such committee shall have such tenure and duties as the Trustees shall determine. The Investment Committee, which shall oversee (he management of the Corporation's endowment funds and marketable securities sh.ill include as e.\ officio members, the Chairperson of the Board, the Treasurer and the Chairperson of the Audit Committee, together with such Trustees as may be requiixo fur not less than two-thirds of the Investment Committee to consist of Trustees. Except a> otherwise provided by these Bylaws or determined by the Trustees, any such committee may make rules lor the conduct of its business, but, unless otherwise provided by the Trustees or in such rules, its business shall he conducted as nearly as possible in the same manner as is provided by these Bylaws for the Trustees. F. Actitms Without n Meeting. Any action required or permitted to be taken at any meeting of the Executive Committee or any other committee elected by the Trustees may be taken without a meeting if all members of such committees consent to the action in writing and such written consents are filed with the records of meetings. Members of the Executive Committee or any other committee elected by the Trustees may also participate in any meeting by means of a telephone conference call, or otherwise lake action in such a manner as may, from time to time, be permitted by law. G. Manual of Procedures. The Board of Trustees, on the recommendation of the Executive Committee, shall establish guidelines and modifications thereof to be recorded in a Manual of Procedures. Guidelines shall establish procedures for: (1) Nomination and election of members of the Corporation, Board of Trustees and Executive Commiltee; (2) Election of Officers; (3) Formation and Function of Standing Committees. ARTICLE VI— OFFICERS A. Enumeration. The officers of the Corporation shall consist of a President, a Treasurer and a Clerk, and such other officers having the powers of President, Treasurer and Clerk as the Board may determine, and a Director of the Laboratory. The Corporation may have such other officers and assistanl officers as the Board may determine, including (without hmiialion) a Chairperson of the Board, Vice Chairper- son and one or more Vice Presidents. Assistant Treasurers or Assistanl Clerks. Any two or more offices may be held by the same person. The Chairperson and Vice Chairperson of the Board shall be elected by and from the Trustees, but other officers of the Corporation need not be Trustees or Members. If required by the Trustees, any officer shall give the Corporation a bond for the faithful performance of his or her duties in such amount and with such surely or sureties as shall be satisfactory to the Truslees. B. Tenure. Except as otherwise provided by law, by the Articles of Organization or by these Bylaws, the President. Treasurer, and all other officers shall hold office until the first meeting of the Board following the annual meeting of Members and thereafter, until his or her successor is chosen and qualified. C. Resignation. Any officer may resign by delivering his or her written resignation to the Corporation at its principal office or to the President or Clerk and such resignation shall be effective upon receipt unless it is specified to be effective at some other time or upon Ihe happening of some other event. D. Removal. The Board may remove any officer with or withoul cause by a vote of a majority of the entire number of Trustees then in office, at a meeting of the Board called for thai purpose and for which notice of the purpose thereof has been given, provided that an officer may be removed for cause only after having an opportunity to be heard by the Board at a meeting of the Board at which a quorum is personally present and voting. E. Vacancy. A vacancy in any office may be filled for the unexpired balance of the term by vote of a majority of the Trustees present at any meeting of Trustees at which a quorum is present or by written consent of all of Ihe Truslees, if less than a quorum of Trustees shall remain in office. F. Chairperson. The Chairperson shall have such powers and duties as may be determined by the Board and, unless otherwise determined by the Board, shall serve in thai capacity for a term coterminous with his or her term as Trustee. G. Vice Chairperson. The Vice Chairperson shall perform Ihe duties and exercise the powers of the Chairperson in Ihe absence or disability of the Chairperson, and shall perform such other duties and possess such other powers as may be determined by the Board. Unless otherwise determined by the Board, the Vice Chairperson shall serve for a one-year term. H. Director. The Director shall be the chief operating officer and, unless otherwise voted by the Trustees, the chief executive officer of the Corporation. The Director shall, subject to the direction of the Trustees, have genera! supervision of the Laboratory and control of (he business of the Corporation. Al Ihe annual meeting, the Director shall submit a report of the operations of the Corporation for such year and a statement of its affairs, and shall, from time to time, report to the Board all matters \\ ithin his or her knowledge which the inlerests of the Corporation may require to he brought to its notice I. Depin\ Director The Deputy Director, if any. or if there shall be more than one, the Deputy Directors in the order determined by the Truslees, shall, in the absence or disability of the Director, perform the duties and exercise the powers of the Director and shall perform such other duties and shall have such other powers as the Truslees may. from lime lo lime, prescribe J President- The President shall have Ihe powers and duties as may he vested in him or her by the Board. K. Treasurer and Assistant Treasurer. The Treasurer shall, subject to the direction of the Trustees, have general charge of the financial affairs of the Corporation, Bylaws of the Corporation R85 including us long-range financial planning, and shall cause to he kept accurate books of account. The Treasurer shaJl prepare a yearly report on the financial status of the Corporation to be delivered at the annual meeting. The Treasurer shall also prepare or oversee all filings required by the Commonwealth of Massachusetts, the Internal Revenue Service, or other Federal and State Agencies. The account of the Treasurer shall be audited annually by a certified public accountant. The Assistant Treasurer, if any, or if there shall be more than one, the Assistant Treasurers in the order determined by the Trustees, shall, in the absence or disability of the Treasurer, perform the duties and exercise the powers of the Treasurer, shall perform such other duties and shall have such other powers as the Trustees may, from time to time, prescribe. L. Clerk and Assistant Clerk. The Clerk shall be a resident of the Commonwealth of Massachusetts, unless the Corporation has designated a resident agent in the manner provided by law. The minutes or records of all meetings of the Trustees and Members shall be kept by the Clerk who shall record, upon the record books of the Corporation, minutes of the proceedings at such meetings. He or she shall have custody of the record books of the Corporation and shall have such other powers and shall perform such other duties as the Trustees may, from time to time, prescribe. The Assistant Clerk, if any, or if there shall be more than one, the Assistant Clerks in the order determined by the Trustees, shall, in the absence or disability of the Clerk, perform the duties and exercise the powers of the Clerk and shall perform such other duties and shall have such other powers as the Trustees may, from time to time, prescribe. In the absence of the Clerk and an Assistant Clerk from any meeting, a temporary Clerk shall be appointed at the meeting. M. Other Powers unj Dunes. Each officer shall have in addition to the duties and powers specifically set forth in these Bylaws, such duties and powers as are custom- arily incident to his or her office, and such duties and powers as the Trustees may. from time to time, designate. ARTICLE VII— AMENDMENTS These Bylaws may be amended by the affirmative vote of the Members at any meeting, provided that notice of the substance of the proposed amendment is stated in the notice of such meeting. As authori/.ed by the Articles of Organization, the Trustees, by a majority of their number then in office, may also make, amend or repeal these Bylaws, in whole or in part, except with respect to (a) the provisions of these Bylaws governing d) the removal of Trustees and (ii) the amendment of these Bylaws and (b) any provisions of these Bylaws which by law, the Articles of Organization or these Bylaws, requires action by the Members. No later than the time of giving notice of meeting of Members next following the making, amending or repealing by the Trustees of any Bylaw, notice thereof stating the substance of such change shall be given to all Members entitled to vote on amending the Bylaws. Any Bylaw adopted by the Trustees may be amended or repealed by the Members entitled to vote on amending the Bylaws. ARTICLE VIII— INDEMNITY Except as otherwise provided below, the Corporation shall, to the extent legally permissible, indemnify each person who is, or shall have been, a Trustee, director or officer of the Corporation or who is serving, or shall have served at the request of the Corporation as a Trustee, director or officer of another organization in which the Corporation directly or indirectly has any interest as a shareholder, creditor or otherwise, against all liabilities and expenses (including judgments, fines, penalties, and reasonable attorneys' fees and all amounts paid, other than to the Corporation or such other organization, in compromise or settlement) imposed upon or incurred by any such person in connection with, or arising out of, the defense or disposition of any action, suit or other proceeding, whether civil or criminal, in which he or she may be a defendant or with which he or she may be threatened or otherwise involved, directly or indirectly, by reason of his or her being or having been such a Trustee, director or officer. The Corporation shall provide no indemnification with respect to any matter as to which any such Trustee, director or officer shall be finally adjudicated in such action, suit or proceeding not to have acted in good faith in the reasonable belief that his or her action was in the best interests of the Corporation. The Corporation shall provide no indemnification with respect to any matter settled or comprised unless such matter shall have been approved as in the best interests of the Corporation, after notice that indemnification is involved, by (i) a disinterested majority of the Board of the Executive Committee, or (ii) a majority of the Members. Indemnification may include payment by the Corporation of expenses in defending a civil or criminal action or proceeding in advance of the final disposition of such action or proceeding upon receipt of an undertaking by the person indemnified to repay such payment if it is ultimately determined that such person is not entitled to indemnification under the provisions of this Article VIII, or under any applicable law. As used in the Article VIII, the terms "Trustee," "director," and "officer" include their respective heirs, executors, administrators and legal representatives, and an "interested" Trustee, director or officer is one against whom in such capacity the proceeding in question or another proceeding on the same or similar grounds is (hen pending. To assure indemnification under this Article VIII of all persons who are determined by the Corporation or otherwise to be or to have been "fiduciaries" of any employee benefits plan ol the Corporation which may exist, from time to lime, this Article VIII shall be interpreted as follows: (i) "another organization" shall be deemed to include such an employee benefit plan, including without limitation, any plan of the Corpo- ration which is governed by the Act of Congress entitled "Employee Retirement Income Security Act of 1974," as amended, from time to time, ("ERISA"); (ii) "Trustee" shall be deemed to include any person requested by the Corporation to serve as such for an employee benefit plan where the performance by such person of his or her duties to the Corporation also imposes duties on, or otherwise involves services by, such person to the plan or participants or beneficiaries of the plan; (iii) "fines" shall be deemed to include any excise tax plan pursuant to ERISA; and (iv) actions taken or omitted by a person with respect to an employee benefit plan in the performance of such person's duties for a purpose reasonably believed by such person to be in the interest of the participants and beneficiaries of the plan shall be deemed to be for a purpose which is in the best interests of the Corporation. The right of indemnification provided in this Article VIII shall not be exclusive of or affect any other rights to which any Trustee, director or officer may be entitled under any agreement, statute, vote of Members or otherwise. The Corporation's obligation to provide indemnification under this Article VIII shall be offset to the extent of any other source of indemnification of any otherwise applicable insurance coverage under a policy maintained by the Corporation or any other person. Nothing contained in the Article shall affect any rights to which employees and corporate personnel other than Trustees, directors or officers may be entitled by contract, by vote of the Board or of the Executive Committee or otherwise. ARTICLE IX— DISSOLUTION The consent of every Trustee shall be necessary to effect a dissolution of the Manne Biological Laboratory. In case of dissolution, the property shall be disposed of in such a manner and upon such terms as shall be determined by the affirmative vote of two-thirds of the Trustees then in office in accordance with the laws of the Com- monwealth of Massachusetts, ARTICLE X— MISCELLANEOUS PROVISIONS A. Fiscal Year, Except as otherwise determined by the Trustees, the fiscal year of the Corporation shall end on December 31st of each year. B. Seal. Unless otherwise determined by the Trustees, the Corporation may have a seal in such form as the Trustees may determine, from time to time. C. Execution of Instruments. All checks, deeds, leases, transfers, contracts, bonds, notes and other obligations authorized to be executed by an officer of the Corporation in its behalf shall be signed by the Director or the Treasurer except as the Trustees may generally or in particular cases otherwise determine. A certificate by the Clerk or an Assistant Clerk, or a temporary Clerk, as to any action taken by the Members, Board of Trustees or any officer or representative of the Corporation shall as to all persons who rely thereon in good faith be conclusive evidence of such action. D. Corporate Records. The original, or attested copies, of the Articles of Organi- zation, Bylaws and records of all meetings of the Members shall be kept in Massa- chusetts at the principal office of the Corporation, or at an office of the Corporation's Clerk or resident agent. Said copies and records need not all he kept in the same office. They shall be available at all reasonable times for inspection by any Member for any proper purpose, but not to secure a list of Members for a purpose other than in the interest of the applicant, as a Member, relative to the affairs of the Corporation. E. Articles of Organization. All references in these Bylaws to the Articles of Organization shall be deemed to refer to the Articles of Organization of the Corpo- ration, as amended and in effect, from time to time F. Transactions with Interested Parties. In the absence of fraud, no contract or other transaction between this Corporation and any other corporation or any firm, association, p;irtncislup or pcrsnn shall be affected or invalidated by the fact that any Trustee or officer of this Corporation is pecuniarily or otherwise interested in or is a director, member or officer of such other corporation or of such firm, association or partnership or in a party to or is pecuniarily or otherwise interested in such contract or other transaction or is in any way connected with any person or person, firm, association, partnership, or corporation pecuniarily or otherwise interested therein; provided that the fact that he or she individ- ually or as a director, member or officer of such corporation, firm, association or RS6 Annual Report partnership tn such a party or is so interested shall be disclosed to or shall have been authorizing any such contract or transaction with like force and effect as if he/she were not known by the Board ot Trustees or a majority of such Members thereof as shall be present so interested, or were not a director, member or officer of such other corporation, firm, at a meeting of the Board of Trustees at which action upon any such contract or association or partnership, provided that any vote with respect to such contract or transaction shall be taken; any Trustee may be counted in determining the existence of a transaction must be adopted by a majority of the Trustees then in office who have no quorum and may vote at any meeting of the Board of Trustees for the purpose of interest in such contract or transaction. VOLUME 197 THE NUMBER 2 BIOLOGICAL BULLETIN BIOI OGICAL BUUJH'IN CENTENNIAL ISSUE OCTOBER 1899-1999 llll BIOLOGICAL BU LI J.TIN Published by the Marine Biological Laboratory It's Wort. i_ook Brilliant: and easy. The Axioskop 2 is the microscope that brings new levels of comfort and productiv- ity to biomedical research. Renowned ICS infinity optics provide top quality images, and its operating convenience adds extra efficiency to your work. Fluorescence 4-fold. Carry out demanding fluorescence applica- tions with flexibility and optimal illumina- tion. Reflector turret for 4 fluorescence modules, 6-place excitation filter slide, plus the power of 100 watts. Motorized automation. In the Axioskop 2 MOT option, motorized functions include stage focusing and uni- versal condenser setting (including Koehler). These and other functions can be automated for high productivity and preci- sion when examining large numbers of specimens. Contact Zeiss today for the full stor , le Axioskop 2. It's smart microscopy m ear . Carl Zeiss, Inc. Microscopy & Imaging Systems One Zeiss Drive Thornwood, NY 10594 800.233.2343 Fax 91 4. 681. 7446 micro@zeiss.com www.zeiss.com ZEISS THE BIOLOGICAL BULLETIN OCTOBER 1999 Editor Associate Editors Section Editor Online Editors Editorial Board Editorial Office MICHAEL J. GREENBERG Louis E. BURNETT R. ANDREW CAMERON CHARLES D. DERBY MICHAEL LABARBERA SHINYA INOUE, Imaging ami Microscopy JAMES A. BLAKE, Keys to Marine Invertebrates of the Woods Hole Region WILLIAM D. COHEN, Marine Models Electronic Record and Compendia PETER B. ARMSTRONG ERNEST S. CHANG THOMAS H. DIETZ RICHARD B. EMLET DAVID EPEL GREGORY HINKLE MAKOTO KOBAYASHI DONAL T. MANAHAN MARGARET MCFALL-NGAI MARK W. MILLER TATSUO MOTOKAWA YOSHITAKA NAGAHAMA SHERRY D. PAINTER J. HERBERT WAITE RICHARD K. ZIMMER PAMELA CLAPP HINKLE VICTORIA R. GIBSON CAROL SCHACHINGER PATRICIA BURNS The Whitney Laboratory, University of Florida Grice Marine Biological Laboratory. College of Charleston California Institute of Technology Georgia State University University of Chicago Marine Biological Laboratory ENSR Marine & Coastal Center. Woods Hole Hunter College, City University of New York University of California, Davis Bodega Marine Lab., University of California, Davis Louisiana State University Oregon Institute of Marine Biology, Univ. of Oregon Hopkins Marine Station, Stanford University Cereon Genomics, Cambridge, Massachusetts Hiroshima University of Economics, Japan University of Southern California Kewalo Marine Laboratory, University of Hawaii Institute of Neurobiology, University of Puerto Rico Tokyo Institute of Technology, Japan National Institute for Basic Biology, Japan Marine Biomed. Inst., Univ. of Texas Medical Branch University of California, Santa Barbara University of California, Los Angeles Managing Editor Staff Editor Editorial Associate Subscription & Advertising Secretary Published by MARINE BIOLOGICAL LABORATORY WOODS HOLE, MASSACHUSETTS Cover The three-dimensional stereo anaglyph on the cover is a ventral view of a brachiolaria larva of Patiriella regularis, a starfish; the brachiolaria de- picted is about 1500 jtim in length. Serotonergic neurons in the larva were stained with a rabbit antiserum and appear, in confocal fluorescent mi- croscopy, as bright dots lining the ciliated bands and brachiolar arms. The image (which should be viewed through the stereo glasses provided with this issue) is composed of 145 optical sections and was reconstructed as described in the article by Francis Chee and Maria Byrne (p. 123). Immunoreactive serotonergic cells are already visible in the gastrulae of echinoderms; but they increase in number and form an increasingly com- plex neural system as development proceeds. Be- cause the immunoreactivity is associated with the ciliary bands of free-swimming, planktotrophic lar- val forms — as well as with their sensory structures and buccal cavity — the serotonergic system has been thought to coordinate the locomotory and feeding behaviors of these larvae. In Iheir paper. Chee and Byrne focus on the larval stages of Patiriella regularis, which are all plank- totrophic; thus the development of the serotonergic system can be monitored throughout development, from the gastrula. through the brachiolaria (the last larval stage), and on to metamorphosis. The authors have used confocal fluorescence microscopy to re- construct the development of the serotonergic ner- vous sytsem in three dimensions and have related the increase in complexity to morphogenetic changes in the larvae. They have demonstrated a complex network of cells with varicose processes that connect the preoral and postoral ciliated bands, supporting the hypothesis that this network is reg- ulating larval feeding and swimming. In a related article in this issue (see p. 115), Michael Dailey and his colleagues use the mamma- lian brain as a model to show how multidimensional confocal fluorescence microscopy can enhance studies of biological structure and function. The images in this article are fine examples of the tech- niques described, and readers should use the stereo glasses to examine them. This is the third in a series of papers on Concepts in Imaging and Microscopy; the series is supported by the Optical Imaging Association, which has also provided the stereo glasses. Finally, this issue marks the end of The Biolog- ical Bulletin's first century of publication and the beginning of its second. The four small images on the cover, below the anaglyph, show how the face of the journal changed as the decades passed, biol- ogy expanded, the world shrank, and scientific pub- lishing entered its greatest revolution since the in- vention of movable type. A metamorphosis is certainly at hand, but the nature of the imago re- mains unresolved. CONTENTS VOLUME 197, No. 2: OCTOBER 1999 EDITORIAL IMMUNOLOGY Greenberg, Michael J. A century of science: The Biological Bulletin looks back — and forward . 113 IMAGING AND MICROSCOPY Dailey, Michael, Glen Marrs, Jakob Satz, and Marc Waite Concepts in Imaging and Microscopy: Exploring biolog- ical structure and function with confocal micros- copy 115 NEUROBIOLOGY AND BEHAVIOR Chee, Francis, and Maria Byrne Development of the larval serotonergic nervous sys- tem in the sea star Patiriella regu/aris as revealed by confocal imaging 123 Hartline, O.K., E.J. Buskey, and P.H. Lenz Rapid jumps and bioluminescence elicited by con- trolled hydrodynamic stimuli in a mesopelagic cope- pod, Pleuromamma xiphica 132 Harrison, Paul J.H., and David C. Sandeman Morphology of the nervous system of the barnacle cypris larva (Balanus amplutnte Darwin) revealed by light and electron microscopy 144 PHYSIOLOGY Gainey, Louis F., Jr., Kelly J. Vining, Karen E. Doble, Jennifer M. Waldo, Aurora Candelario-Martinez, and Michael J. Greenberg An endogenous SCP-related peptide modulates cili- ary beating in the gills of a venerid clam, Mercenaria mercenaria 159 DEVELOPMENT AND REPRODUCTION Saigusa, Masayuki, and Hiroshi Iwasaki Ovigerous-hair stripping substance (OHSS) in an es- tuarine crab: purification, preliminary characteriza- tion, and appearance of the activity in the developing embrvos 174 Shirae, Maki, Euichi Hirose, and Yasunori Saito Behavior of hemocytes in the allorejection reaction in two compound ascidians, Bottyllus scalaris and Sywz- plegma replant 188 ECOLOGY AND EVOLUTION Skorokhod, Alexander, Vera Gamulin, Dietmar Gun- dacker, Vadini Kavsan, Isabel M. Muller, and Werner E.G. Muller Origin of insulin receptor-like tyrosine kinases in marine sponges 198 Grain, Jennifer A. Functional morphology of prey ingestion by Placetron wosnessenskii Schalfeew Zoeae (Crustacea: Anomura: Lithodidae): 207 SHORT REPORTS FROM THE 1999 GENERAL SCIENTIFIC MEETINGS OF THE MARINE BIOLOGICAL LABORATORY FEATURED ARTICLE Rome, Lawrence C. Introduction. Bringing the script to life: the role of muscle in behavior 225 Rome, Lawrence C., Andrei A. Klimov, and Iain S. Young A new approach for measuring real-time calcium pumping and SR function in muscle fibers 227 PHYSIOLOGY Malchow, Robert Paul, and David J. Ramsey Responses of retinal Muller cells to neurotransmitter candidates: a comparative study 229 Clay, John R., and Alan M. Kuzirian Fluorescence localization of K+ channels in the membrane of squid giant axons 231 Ruta, Vanessa J., Frederick A. Dodge, and Robert B. Barlow Evaluation of circadian rhvthms in the Limnlus eve. . . 233 CONTENTS: VOLUME Novales Flamarique, Iriigo, and Ferenc I. Harosi Photoreceptor pigments of the blueback herring (Aloia aestevalis, Clupeidae) and the Atlantic silver- side (Mfnitiiii mi'iii/lin, Atherinidae) 235 Hanley, Janice S., Nadav Shashar, Roxanna Smolowitz, William Mebane, and Roger T. Hanlon Soft-sided tanks improve long-term health of cul- tured cuttlefish 237 King, Alison J., Shelley A. Adamo, and Roger T. Hanlon Contact with squid eggs increases agonistic behavior in male squid (Loligo f>ealei) 256 CELL MOTILITY PISCINE NEVROBIOLOGY A.\L> BEHAVIOR Zottoli, S.J., F.R. Akanki, N.A. Hiza, D.A. Ho-Sang, Jr., M. Motta, X. Tan, K.M. Watts, and E.-A. Seyfarth Physiological characterization of supramedullary/ dor- sal neurons of the cunner, Tuutogolfilmis adspersus. . . . Fay, R.R., and P.L. Edds-Walton Sharpening of directional auditory input in the descend- ing octaval nucleus of the toadfish, Opasnus tau ....... Kaatz, Ingrid M., and Phillip S. Lobel Acoustic behavior and reproduction in five species of Corycoras catfishes (Callichthvidae) .............. Lobel, Phillip S., and Lisa M. Ken- Courtship sounds of the Pacific damselfish, Abudefduf sordidus (Pomacentridae) ..................... Oliver, Steven J., and Elise Watson Threat-sensitive nest defense in domino damselfish 239 240 241 Price, Nichole N., and Allen F. Mensinger Predator-prey interactions of juvenile toadfish, Opsa- iiu\ Ian .................................... Tang, Kathleen Q., Nichole N. Price, Maureen D. O'Neill, Allen F. Mensinger, and Roger T. Hanlon Temperature effects on first-year growth of cultured oyster toadfish, Opsanus tau .................... 24(i Bearer, E.L., M.L. Schlief, X.O. Breakefield, D.E. Schu- back, T.S. Reese, and J.H. LaVail Squid axoplasm supports the retrograde axonal transport of herpes simplex virus 257 Gould, Robert, Concetta Freund, Frank Palmer, Pam- ela E. Knapp, Jeff Huang, Hilary Morrison, and Doug- las L. Feinstein Messenger RNAs for kinesins and a dvnein are lo- cated in neural processes 259 Fukui, Yoshio, Taro Q.P. Uyeda, Chikako Kitayama. and Shinya Inoue Migration forces in Dictyostelium measured by centri- fuge DIG microscopy 260 Tran, P.T., P. Maddox, F. Chang, and S. Inoue Dynamic confocal imaging of interphase and mitotic microtubnles in the fission yeast, S. pombe 262 Maddox, Paul, Arshad Desai, E.D. Salmon, T.J. Mitchi- son, Karen Oogema, Tarun Kapoor, Brian Matsumoto, and Shinya Inoue Dynamic confocal imaging of mitochondria in swim- ming Tftrahymena and of microtubule poleward flux in Xenopus extract spindles 263 Wollert, Torsten, Ana S. DePina, and George M. Lang- ford Effects of vanadate on actin-dependent vesicle motil- ity in extracts of clam oocytes 265 CHEMORECEPTION AND BEHAVIOR Mjos, Katrin, Frank Grasso, and JeUe Atema Antennule use by the American lobster, Homann americanus, during chemo-orientation in three turbu- lent odor plumes 249 Hanna, John P., Frank W. Grasso, and Jelle Atema Temporal correlation between sensor pairs in differ- ent plume positions: A study of concentration infor- mation available to the American lobster, Humartis inni'rn(tnti\, during chemotaxis 250 Zetder, Erik, and Jelle Atema Chemoreceptor cells as concentration slope detec- tors: preliminary evidence from the lobster nose . . . 252 Berkey, Ci istin, and Jelle Atema Individual recognition and memory in HII//KIIIH amem/i//ii\ > >l<--female interactions 253 McLaughlin, L she C., Jennifer Walters, Jelle Atema, and Norman V aimvrighl Urinary protein coiuenlration in connection with agonistic interactions m Haimini*, nmmcanus 254 CELL AND DEVELOPMENTAL BIOLOGY Billack, Blase, Jeffrey D. Laskin, Michael A. Gallo, and Diane E. Heck Effects of a-bungarotoxin on development of the sea urchin Arbacia puncttdatu 267 Silver, Robert B., and Nicole M. Deming Leukotriene B4 as calcium agonist for nuclear enve- lope breakdown: an enzymological sur\'ey of endo- membranes of mitotic cells 268 Weidner, Earl, and Ann Findley Extracellular survival <>1 an intracellular parasite (Spraffiii'ii l/>/ih/i, Microsporea) 270 Kaltenbach, Jane C., William J. Kuhns, Tracy L. Simp- son, and Max M. Burger Intense concanavalin A staining and apoptosis of peripheral flagellated cells in larvae of the marine sponge Microfionti prulifrnt: significance in relation to morphogenesis 271 CONTENTS: VOLUME COMPARATIVE BIOCHEMISTRY Harrington, John M., and Peter B. Armstrong A cuticular secretion of the horseshoe crab, Limulus polyphemus: a potential anti-fouling agent Asokan, Rengasamy, and Peter B. Armstrong Cellular mechanisms of hemolysis by the protein limu- lin, a sialic-acid-specific lectin from the plasma of the American horseshoe crab. Limiting polyphermis Biswas, Chhanda, and Peter B. Armstrong Identification of a hemolvtic activity in the plasma of the gastropod Sustain canaliculatum Kiihns, William J., Max M. Burger, and Eva Turley Hyaluronic acid: a component of the aggregation factor secreted by the marine sponge, Microciona pro- lifera Popescu, Octavian, Key Interior, Gradimir Misevic, Max M. Burger, and William J. Kuhns Biosynthesis of tyrosine O-sulfate by cell proteoglycan from the marine sponge, Microciona frrolifrra Vasse, Aimee, Alice Child, and Norman Wainwright Prophenoloxidase is not activated by microbial sig- nals in Limulus poliiplirnnis Ogunseitan, O.A., S.L. Yang, and E. Scheinbach The 8-aminolevulinate dehydratase of marine Vibrio alginolyticus is resistant to lead (Pb) Hoskin, Francis C.G., Diane M. Steeves, and John E. Walker Substituted cyclodextrin as a model for a squid en- zyme that hydrolyzes the nerve gas soman Zigman, Seymour, Nancy S. Rafferty, Keen A. Rafferty, and Nathaniel Lewis Effects of green tea polyphenols on lens photooxida- tive stress ECOLOGY AND EVOLUTION Mondrup, Thomas Salinity effects on nutrient dynamics in estuarine sediment investigated by a plug-flux method 275 276 283 285 Pease, Katherine M., L. Claessens, C. Hopkinson, E. Rastetter, J. Vallino, and N. Kilham Ipswich River nutrient dynamics: preliminary assess- ment of a simple nitrogen-processing model Wolfe, Felisa L., Kevin D. Kroeger, and Ivan Valiela Increased labiliiv of estuarine dissolved organic ni- trogen from urbanized watersheds Evgenidou, A., A. Konkle, A. D'Ambrosio, A. Corcoran, J. Bowen, E. Brown, D. Corcoran, C. Dearholt, S. Fern, A. Lamb, J. Michalowsky, I. Ruegg, and J. Cebrian Effects of increased nitrogen loading on the abun- dance of diatoms and dinoflagellates in estuarine phytoplanktonic communities Cubbage, Andrea, David Lawrence, Gabrielle Tomasky, and Ivan Valiela Relationship of reproductive output in Acartia tonsa, chlorophyll concentration, and land-derived nitrogen loads in estuaries in Waquoit Bay, Massachusetts Canfield, Susannah, Luc Claessens, Charles Hopkinson Jr., Edward Rastetter, and Joseph Vallino Long-term effect of municipal water use on the water budget of the Ipswich River Basin Young, Talia, Sharon Komarow, Linda Deegan, and Robert Garritt Population size and summer home range of the green crab, Carriniu nu'ti»ii.\, in salt marsh tidal creeks Komarow, Sharon, Talia Young, Linda Deegan, and Robert Garritt Influence of marsh flooding on the abundance and growth of Fundulus hettrvclitus in salt marsh creeks . . . Widener, Justin W., and Robert B. Barlow Decline of a horseshoe crab population on Cape Cod Kerr, Lisa M., Phillip S. Lobel, and J. Mark Ingoglia Evaluation of a reporter gene system biomarker for detecting contamination in tropical marine sedi- ments. . 289 290 294 ORAL PRESENTATIONS 287 Pl'BLISHED BY TlTLE ONLY 297 299 300 303 307 THE BIOLOGICAL BULLETIN THE BIOLOGICAL BULLETIN is published six times a year by the Marine Biological Laboratory. 7 MBL Street. Woods Hole, Massachusetts 02543. Subscriptions and similar matter should be addressed to Subscription Manager. THE BIOLOGICAL BULLETIN. Marine Biological Laboratory, 7 MBL Street, Woods Hole, Massachusetts 02543. Subscription per year (six issues, two volumes): $205 for libraries; $95 for individuals. Subscription per volume (three issues): $102.50 for libraries; $47.50 for individuals. Back and single issues (subject to availability): $40 for libraries; $20 for individuals. Communications relative to manuscripts should be sent to Michael J. 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Other than these charges for authors' alterations, The Biological Bulletin does not have page charges. Reference: Biol. Bull. 197: 113-114. (October 1999) A Century of Science: The Biological Bulletin Looks Back — and Forward The first number of The Biological Bulletin was pub- lished in October 1899; so with the current issue we cele- brate the 100th anniversary of this journal. The founder was Prof. C. O. Whitman, director of the Marine Biological Laboratory at Woods Hole, Massachusetts (MBL), who aimed to publish short articles with simple illustrations as rapidly as possible. Progress was halting at first: a prede- cessor, the Zoological Bulletin (with C. O. Whitman and W. M. Wheeler as editors, but unassociated with the MBL). failed in 1898 after two volumes: and The Biological Bul- letin itself ceased publication for a time, also after two volumes.1 But guided by general policies set out in a pro- spectus written in June 1902,2 the Bulletin began to function smoothly under the editorship of Frank R. Lillie. Those policies, paraphrased below, have informed the operation of the journal ever since: • The Bulletin will be published under the auspices of the Marine Biological Laboratory. • Its scope will include "Zoology, General Biology, and Physiology": it is a general interest journal. • It will contain original articles, occasional reviews, re- ports of work and lectures at the MBL: and preliminary statements of important results will be special feature. • It will meet the need for rapid publication of results. • It will be open to contributions from any source. A general journal. The Biological Bulletin was meant to be, in some measure, an institutional journal; and indeed, the Annual Report of the MBL Corporation has been pub- lished by the Bulletin since 1908. Moreover, the scientific agenda of the MBL has always been very broad, so the close association of the Bulletin with the Laboratory has ensured that the scope of the journal would also be so. Probably every editor has considered this characteristic of the journal. Indeed, Alfred C. Redrield ( 1941 ) called it "one of the most perplexing problems of policy with which the ... editor 1 The history of The Biological Bulletin has been described thoroughly twice before: by Alfred C. Redfield ( 1941 ). who was Managing Editor of this journal from 1930 to 1942; and (on the occasion of the 100th anni- versary of the founding of the MBL) by Pamela L. Clapp ( 1988), who was Editorial Assistant to Charles B. Metz. the Editor from 1980-1989. 2 The prospectus is published in the Tenth Report for the Years 1903- 1906 including Financial Report from 1900-1906, Section XI Publica- tions, pp. 42-44. (1907) Marine Biological Laboratory. Woods Holl. Massachusetts. must deal." and he concluded that "in this day of special- ization [in 1941!} one journal at least should present a rather broad cross-section of biology as a whole." This is not a necessary conclusion, but it has been accepted by the seven editors that succeeded Lillie. Today we expect — beyond technical competence — that a publishable submission will contain data resulting from the experimental testing of some hypothesis, and that it will advance its own area signifi- cantly. Moreover, we suppose that if such an investigation is not too narrowly focused, it is likely to interest a generous fraction of our diverse readership. Of course, the actual scope of The Biological Bulletin has always been determined by those who contribute manu- scripts: and the composition of that pool of potential au- thors— which is unconstrained by policy — has changed markedly through the years. In the decade 1930-1940, roughly 700 papers were published in the Bulletin; of these, 29% originated from the MBL, and 40% were written by members of the Corporation. These values began to decline during the late '60s, as investigators at the MBL (as else- where) began to send their work to specialty journals. The pace of specialization increased markedly, so that in the most recent decade (1989-1999), only about 10% of arti- cles were authored by members of the Corporation. More- over, the rate of acceptance by foreign authors, which began to increase in the '60s. reached 30% of papers published in the last decade. So today more than ever before, the con- tributors to the Bulletin are widely distributed throughout a shrinking world. We are able, in a way, to ask these contributors whether The Biological Bulletin is actually a general journal, be- cause authors select the headings under which their papers are published. The profile varies from issue to issue, but over 50% of recent articles have appeared under the rubric of either Physiology or Development & Reproduction; 20% are listed under Ecology & Evolution; and a quarter are distributed between Cell Biology and Neurobiology & Be- havior. These data suggest that, although the pool of authors is different than it was a century ago. the scope of published material is similar to that in 1903 or 1941. On the other hand, of the animals reported on in the past two years, all but 6 were invertebrates from 13 different phyla, mostly marine, and mostly molluscs, crustaceans, cnidarians, and echinoderms. Insects are virtually absent, only one paper is about nematodes, and of 37 molluscan articles, only one is 114 about Aplysia: current marine biomedical models appear largely in specialty journals. The subject matter in Bulletin articles is diverse and thus difficult to characterize in brief. But it is predominantly experimental and functional, and the functions tend to be complex, sometimes appearing in un- usual, primitive, or extremeophilic animals, and often at the intersection of fields, e. g.. the development of symbiosis; or neural or pheromonal regulation of development, activity, or metamorphosis. In summary. The Biological Bulletin, like all general journals, has its special focus. We might predict that so long as its publication is under the auspices of the MBL, the scope of the Bulletin will remain general; but its focus might well shift with time or new leadership. Enhancing the contents. Although The Biological Bul- letin contains primarily research reports, successive editors have leavened the diet with reviews, some based on lec- tures. The series of lectures on Concepts in Imaging and Microscopy, one of which appears in this issue, is exem- plary. During the past decade, the Bulletin has also pub- lished symposia and workshops, about one per year, on a variety of topics. These proceedings have also served to broaden the scope of the journal. As part of its association with the MBL, The Biological Bulletin has, since 1936, published the abstracts of the General Scientific Meetings held at the Laboratory each summer. Investigators, postdoctoral fellows, and students present their work at these meetings, so the abstracts pro- vide a snapshot of the research taking place at the MBL. The abstracts have been enhanced since 1991: they are longer and contain a figure or table; moreover, they are reviewed and are thus more credible and valuable than the old ab- stracts were. In the past nine years, about 420 of these short reports have been published, which has also served to widen the focus of the Bulletin. Response to electronic publishing. Printing, design, graphic techniques, and the quality of paper have improved slowly over the past 100 years, brightening and enhancing the data published in The Biological Bulletin, as in other journals. This improvement in appearance is symbolized by the images on the cover of this issue. But these technical advances are trivial compared with the sea change that electronic commu- nication has brought in the last decade. This revolution is not close to peaking, but it has already fundamentally altered the way that scientists do business. To date. The Biological Bulletin has responded to the potential for electronic publishing with three online prod- ucts: the Compendia, the Marine Models Electronic Record (MMER), and the Keys to the Invertebrates of the Woods Hole Region (The Keys). These products, or their contents, are composed of relatively independent units of data or methods. I he) are therefore well adapted to navigation and viewing on-screen and are amenable to the advantages of online editing a,"d publication, especially continual updat- ing. The Ct>iupeii:lia consist of tabulated data: e.g., compo- sition of physiological solutions, breeding seasons and ga- mete characteristics, and invertebrate anesthetics and relaxants (in review). The MMER is a completely electronic journal devoted to the collection, culture, and preparation of marine animals for experimentation. The Keys, first pub- lished in 1954, were particularly useful to researchers who are not systematists; unfortunately, this guide is now out- of-date, but it is undergoing revision online. These three products are accessible on the home page of Biological Bulletin Publications at: www.mbl.edu/BiologicalBulletin/. Biological Bulletin Publications also manages the classical print journal. Tables of contents and the abstracts of articles published in each issue of the Bulletin are published elec- tronically as soon as they have all been accepted and proof- read. Moreover, videos and data supplemental to published articles are also maintained online. The full text of the papers in The Biological Bulletin are still not available electronically; but this state of affairs cannot continue forever. Our readers do not want to store paper journals. They want to store a collection of articles, selected from a variety of journals, and tailored to their specific needs. Overwhelmingly, now, these papers are ob- tained, not as reprints, but by photocopying or, where avail- able, by downloading from the internet. More important, our authors expect that, when the paper is accepted and the editorial process is complete, their paper will be distributed as rapidly and widely as possible. If this expectation is to be met, then The Biological Bulletin should be published elec- tronically, and since fees limit distribution, access should be free of cost to readers. The present Biological Bulletin Publications could pro- duce an online journal, but two major, well-ventilated ques- tions remain: First, who should pay for this significant service to authors? Probably authors — at least in part. At present, however. The Biological Bulletin has no page charges, and revenue comes almost entirely from libraries. Second, although paper documents last for hundreds of years, electronic storage technology turns over in about five; so if we only publish electronically, how do we solve the problem of archiving? Clearly paper archival copies must be produced. But who will pay for them? Probably the librar- ies— at least in part. In any event, scientific publication will be revolutionized in the next decade, and The Biological Bulletin — if it survives its inevitable transmogrification — will bear about as much physical resemblance to its earlier life as a butterfly does to a caterpillar. — MICHAEL J. GREENBERG, Editor-in-Chief References Clapp, P. L. 1988. The history of The Biological Bulletin. Bwl. Bull. 174: 1-3. Kcdtield, A. C. 1941. Annual report of the Marine Biological Laboratory for the year 1940. Report of the Managing Editor. Biol. Bull. 81: 12-17. Reference: Bio/. Bull- 197: 115-122. (October 1999) Concepts in Imaging and Microscopy Exploring Biological Structure and Function with Confocal Microscopy MICHAEL DAILEY. GLEN MARRS1, JAKOB SATZ1, AND MARC WAITE Department of Biological Sciences and l Program in Neitroscience. The University of lomi. Iowa Citv, Iowa 52242 Abstract. Confocal microscopy is providing new and ex- citing opportunities for imaging cell structure and physiol- ogy in thick biological specimens, in three dimensions, and in time. The utility of confocal microscopy relies on its fundamental capacity to reject out-of-focus light, thus pro- viding sharp, high-contrast images of cells and subcellular structures within thick samples. Computer controlled focus- ing and image-capturing features allow for the collection of through-focus series of optical sections that may be used to reconstruct a volume of tissue, yielding information on the 3-D structure and relationships of cells. Tissues and cells may also be imaged in two or three spatial dimensions over time. The resultant digital data, which encode the image, are highly amenable to processing, manipulation and quantita- tive analyses. In conjunction with a growing variety of vital fluorescent probes, confocal microscopy is yielding new information about the spatiotemporal dynamics of cell mor- phology and physiology in living tissues and organisms. Here we use mammalian brain tissue to illustrate some of the ways in which multidimensional confocal fluorescence imaging can enhance studies of biological structure and function. Received 26 March 1999: accepted 21 July 1999. To whom correspondence should be addressed: Dr. Michael Dailey. Dept. of Biological Sciences. 335 Biology Building, University of Iowa. Iowa City, IA 52242. E-mail: michael-e-dailey@uiowa.edu This is the third in a series of articles entitled "Concepts in Imaging and Microscopy." This series is supported by the Optical Imaging Association (OPIA) and was introduced with an editorial in the April 1998 issue of this journal (Bio/. Bull. 194: 99). Other articles in the series are listed on Tin- Biological Bulletin'f, website at . ll namics of glial cells responding to neural tissue injury (U. ms et a!.. 1996; Dailey and Waite. 1999). In each case, i ! -.mimic features of cell structure and movement could be \ < i-d in a near-native tissue environment. One of i and i -utilized features of confocal microscopy is the ability to image dynamic cell and tissue structures in four dimensions (4-D); that is. in three spatial dimensions over time (e.g., Kriete and Wagner. 1993; Konijn et ai. 1996; Errington et ul.. 1997; Zimmermann and Siegert. 1998). This can be accomplished by collecting stacks of confocal images at set time intervals The resultant time series of confocal image stacks can be used to reconstruct 3-D views of dynamic cell and tissue development. Mark Cooper's group has elegantly applied this approach to early CONFOCAL MICROSCOPY 119 Figure 3. Time-lapse sequence shows the dynamics of axon growth and contact with a dendrite in a developing rat hippocampal slice. Neurons were labeled with a fluorescent membrane dye, Dil. To image growth of neuronal processes in three dimensions, stacks of 16 optical sections spanning 30 ^xm in the axial dimension (2-^im ;-steps) were collected at time intervals of 6 min. Images in the top sequence represent a simple axial projection of the 16 images in the through-focus stack. The bottom series of images are red-green stereo images of the same data to provide depth information (viewing requires red-green or red-blue stereo glasses). A thin axon (arrow) extends parallel to a dendrite (arrowhead). Note the long thin filopodia at the leading edge of the growth cone (0 min), which advances (18 min) and bifurcates (arrows, 36 min). The left branch of the growth cone contacts the adjacent dendrite, and the axon growth is subsequently reoriented in that direction (54 min). A time-lapse movie of the axon growth is available for viewing on The Biological Bulletin Website at . zebrafish development (Cooper, 1999), demonstrating the power of time-resolved, 4-D confocal imaging in a fully intact, experimental vertebrate preparation. In Figure 3 we illustrate the use of 4-D confocal imaging to capture the dynamic behavior of an axonal growth cone extending and contacting a dendrite within a rat hippocampal brain slice. Imaging Cell and Tissue Physiology With increasing frequency, it is becoming necessary — and feasible — to gather information about both the structure and physiology of the biological specimen. This is espe- cially essential for studies on neural tissue, where spatial and temporal patterns of electrical and chemical signals play critical roles in brain function. Optical imaging of the phys- iology of individual cells within the context of a 3-D tissue can provide a powerful means of exploring tissue organiza- tion and function. Within a single field of view, the activity of many tens or hundreds of cells may be observed simul- taneously. This can help elucidate physiological features of populations of cells, reveal distinct functional properties and relationships of different cell types, and define func- tional domains within a tissue. In conjunction with the various fluorescent probes used in cell physiology, confocal imaging can provide information on absolute values of, as well as transient changes in, membrane potential, pH, intracellular calcium, and several other ions and physiological factors. For example, fluores- cent calcium indicator dyes (such as fluo-3) have been used often to investigate the dynamics of intracellular calcium fluctuation in a variety of cell and tissue preparations. Such studies have helped define the spatiotemporal aspects of intra- and inter-cellular calcium signals (Cornell-Bell et al., 1990; Cleemann et al.. 1998; Wier et al.. 1997). Confocal physiological imaging also has been feasible for studies in thick brain tissue slices (Dani e t al., 1993; van den Pol et al., 1992; Dailey and Smith, 1994; Komuro and Rakic, 1996; Guerineau et al.. 1998) and in other complex neural prep- arations, such as the intact zebrafish (Cox and Fetcho, 1996) and the neuromuscular junctions of frog (Reist and Smith. 1992) and fly (Karunanithi et al., 1997). Figure 4 illustrates the use of confocal imaging to examine, in cultured brain tissue, the spatiotemporal patterns of intra- and inter-cellu- lar activity in neuroglial cells in response to a physiological perturbation. Many calcium imaging experiments that use laser confo- cal microscopy have employed nonratiometric calcium in- dicator dyes (e.g., fluo-3, calcium green), primarily because the most popular ratiometric dyes (fura-2 and indo-1) re- M. DAILEY ET AL .ICTOsec 200 sec 200um Figure 4. Physiological time-lapse imaging reveals changing spatiotemporal patterns of intracellular cal- cium (Ca2+) activity in brain tissue in response to potassium (K.f) depolarization. The slice was loaded with fluo-4 AM (Molecular Probes, Eugene, OR), a membrane-permeant fluorescent indicator of intracellular calcium, and mounted in an open chamber for imaging. Single confocal scans were collected at 7-s intervals to detect changes in fluorescence intensity, which reflect changes in intracellular calcium levels. Each panel (left, center, right) is a composite of three images acquired at three slightly different time-points (7 s apart) and encoded red, green, or blue. Thus, the colors represent points in time when cells are active. Inactive cells appear black, and cells with sustained high calcium levels appear white. The left panel, corresponding to a time-point prior to Kf depolarization, shows a low level of spontaneous calcium activity (few colored cells). The center panel, taken just after addition of medium containing high (9 mM) K ' . shows a much higher level of calcium activity in cells. Note that the small colored patches (corresponding to individual, active cells; arrows) are dispersed across the field of view. In the right panel, taken about 100 s later, the isolated cell activity has diminished, and a new pattern of activity emerges corresponding to groups of 5-15 synchronously active cells within patches that are 100-200 fiin in diameter. The active cells are probably astrocytes, and the emergence of synchronously active groups of neighboring cells probably represents electrical (gap junction) coupling among astrocytes (Charles, 1998; Harris-White et ni, 1998). A lime-lapse movie is available for viewing on The Biological Bulletin Website at . quire excitation wavelengths in the ultraviolet (UV) range. Such short-wavelength lasers are expensive and thus less widely available; moreover, chromatic aberration problems associated with UV excitation make confocal microscope design very challenging (Blinton and Lechleiter. 1995). However, several studies have shown that ratiometric phys- iological data can be obtained by visible wavelength con- focal imaging. These studies utilize two calcium-sensitive dyes (fluo-3 and fura red) simultaneously (Lipp and Niggli. 1993, 1994; Schild et a!., 1994), or a calcium sensitive (fluo-3) and a calcium-insensitive (rhodamine) dye in com- bination (Strieker. 1996). Since many physiological events occur on a very fast time-scale, an imaging system must sample at a sufficiently high rate to resolve such events. Many of the early confocal systems were severely limited by the rate at which they "ere able to collect and store images. This limitation is inc; , singly being overcome in two ways. First, with stan- dard r scanning confocal microscopes, the sampling rate can hi jd by reducing the size of the field over which image d;n . t'lected. In the extreme case, the "field size" is reduced to a vigle line that can be repeatedly scanned at high rates ( Mi Hz), a so-called line-scanning mode. This yields limited spatial information, but provides the ex- tremely high time-resolution necessary for resolving fast physiological events such as neural synaptic activity (Schild etal.. 1994; Korkotian and Segal, 1998; Yuste etui.. 1999). Second, several video rate or "real-time" confocal sys- tems have been developed, some of which are capable of collecting over one hundred .v-v (2-D) images per second. These systems have been utilized to study preparations as diverse as individual mesenchymal cells (Vesely and Boyde. 1996), perfused whole rat heart (Hama ft 100) and larvae were mounted on welled slides in a drop of Fluoro- guard antifade reagent (Bio-Rad). and the coverslips were sealed with nail polish. Slides were viewed immediately or stored at 4°C in the dark. Slides refrigerated for several months showed no sign of fading when examined. All times quoted are postfertilization. The specimens were examined with a confocal laser scanner coupled to an epifluorescence microscope (a Bio Rad MRC600 scanner and a Zeiss Axiophot microscope or an MRC1024 and an Olympus BX 60). The 488-nm line of the krypton/argon laser was used with a 520DF32-nm filter block. Various numbers of optical sections were collected at different depth intervals. The depth of collection was deter- mined by the thickness of the specimen and the degree of immunolabeling. The number of immunoreactive cell bod- ies was determined by optically sectioning the various lar- vae. All images are displayed in ventral or lateral view, anterior at the top of the page. Image projections (extended focal length) were created using Confocal Assistant (Soft- ware version 4.02), and three-dimensional (3D) stereo ana- glyphs were produced using Laser Sharp (Bio-Rad Labora- tories) and Confocal Assistant. Computer animations were produced using a Silicon Graphics XS24 4000 with Voxel View Ultra software. Patiriella regularis larvae were prepared for scanning electron microscopy according to Byrne and Barker ( 1991 ) and examined with a Philips XL30 at 10 Kv. Results Giistmlii The first cells exhibiting specific 5-HT-like immunoreac- tivity occurred in mid gastrulae (Fig. 1A), about 24 h postfertilization. As the gastrulae began to elongate, these cells formed a partial dome-like array across the animal region and included monopolar. bipolar, and tripolar cells (Fig. IB). Varicosities were occasionally observed on these processes (Fig. 1C). Both the soma and the neurites of these cells were immunopositive (Fig. 1C). In cross section the cells spanned the epithelium (Fig. ID). With 3D computer reconstructions or extended focus projections, these cells were shown to be pyramidal. Control gastrulae (H = 15), were nonfluorescent. 5-HT NEUROGENESIS IN A LARVAL SEA STAR 125 Figure 1. Confocal images showing bodies and processes of cells with 5-HT-like immunoreactivity in early and advanced gastrula. (A) A projection from 16 optical sections taken at 4.5-/j,m intervals of a mid gastrula shows cells with 5-HT-like immunoreactivity (arrowheads) scattered in the epithelium. C, cilia. Bar. 63 jam. (B) Advanced gastrula. projection created from 14 images at 4.5-ju.m depth intervals showing the concentration of immunoreactive cells (arrowheads) in the animal half. Bar. 95 /am. (C) 5-HT-like immunoreactivity in a tripolar nerve cell in a 34-h gastrula. ax, axonal-like processes; v, varicosities; neb. nerve cell body. Bar. 16 (j,m. (D) Advanced gastrula/early bipinnaria epithelium (e) showing nerve cell bodies (neb) and axonal-like processes (ax) traveling along the basal lamina in a single confocal section. Bar. 20 ju.m. Bipinnaria Prior to the opening of the mouth, early bipinnariae had a distinct stomodaeal invagination while the blastopore was still located at the vegetal pole (Fig. 2A). The larvae could now be orientated according to their dorsoventral axis (Fig. 2 A). A 144-/xm-thick projection reconstructed from 32 op- tical sections showed that cells with 5-HT-like immunore- activity were abundant on both sides of the larva (Fig. 2A). On the ventral surface, the cells around the stomodaeum, about 10 in number, were monopolar and marked the posi- tion at which the adoral ciliated band will form (Fig. 2 A). A collection of bipolar ovoid immunoreactive cells on the dorsal surface was positioned roughly opposite the stomo- daeal invagination (Fig. 2A). At about 48 h postfertilization, the mouth opened. The larvae were further elongated and the anus opened ven- trally. With completion of the gut, the larvae were able to feed. As seen above, 5-HT-like immunoreactivity was conspicuous in the cells surrounding the mouth, which marked the position of the developing adoral ciliated band (Fig. 2B). A few immunoreactive cell bodies were also observed on the upper right region of the buccal cavity (Fig. 2B). Immunoreactive cells and processes on the mid-dorsal surface formed an incomplete ring that wrapped partially around the larva but did not extend to the ventral surface (Fig. 2C). Axonal-like processes from these cell bodies extended towards the posterior end of the larva (Fig. 2C). Although the fate of the immunore- active cells in this ring could not be followed, their mid-body position indicates that they were subsequently 126 F. CHEE AND M. BYRNE Figure 2. Confocal optical projections of early hipinnariae. (A) Early bipinnaria reconstructed from 32 optical sections. Cells with 5-HT-like immunoreactivity can be clearly seen around the stomodaeal invagination (si) and the dorsal surface (arrowheads) of the larva. (B) Projection of 5 confocal sections from the ventral side of a 48-h bipinnaria. 5-HT-like immunoreactivity is present in cell bodies (arrowheads) in the adoral ciliated band (adcb), and a few immunoreactive cells are also present on the upper left-hand side of the mouth (m). (C) Projection of 5 optical sections from the dorsal side of the larva in panel B. Immunoreactive cell bodies (neb) and axonal-like processes (ax) form a band partially wrapping around the larva. Bars. 95 /j.m. incorporated into the serotonergic tracts associated with the preoral and postoral ciliated bands. As the preoral, postoral and adoral ciliated bands devel- oped, the oral hood was also beginning to form (Fig. 3 A, B). Serotonin-like immunoreactivity was observed along the ciliated bands in the form of monopolar cell bodies with axonal-like tracts following the path of these bands (Fig. 3 A). By this stage, a ganglion was evident at the anterior end of the larva. This anterior ganglion ( AG) consisted of the immunopositive cells and processes innervating the preoral and postoral ciliated bands and processes intercon- necting these bands (Fig. 3A). Advanced bipinnaria (about 18 days old) underwent a distinct shape change with the formation of an extension at the anterior end of the larva (Fig. 4A, B). The three ciliated bands were well developed in these larvae (Fig. 4A, B). Internally, the larva had a well-developed gut. and the right and left enterocoels had formed. The distribution of immu- noreactive cells in these bands, discussed below, was con- sistent in all larvae examined (n = 100). Adoral cilititeil huiul The adoral ciliated band was located along the posterior margin of the mouth and was characteristically paraboloidal (Fig. 5A). Along this band were densely packed cells with 5-HT NEUROGENESIS IN A LARVAL SEA STAR 127 Figure 3. Three-dimensional red/green anaglyph and a scanning electron micrograph (false colored) of early bipinnariae. (A) A 3-D lateral view of an early bipinnaria showing immunoreactive cell bodies and axonal-like tracts following the ciliated bands. The anterior ganglion (ag) has formed and connects the preoral (procb) and postoral (pocb) ciliated bands, adocb. adoral ciliated band; o. esophagus; s, stomach; i, intestine. Bar, 95 /j.m. (B) Ventral view of an early bipinnaria at the same stage as in panel A. The ciliated bands are developing, but the anterior extension has not yet formed. Black arrowheads, preoral ciliated band; white arrowheads, postoral ciliated band; white arrow, adoral ciliated band; in. mouth; a. anus. Bar, 100 /u,m. 5-HT-like immunoreactivity; the apical ends of these cells extended to the edge of the ciliated epithelium. These cells were pyramidal and connected basally \-ui a thick immu- nopositive tract (Fig. 6A). Compared with the other ciliated bands, the adoral ciliated band had the highest concentration Figure 4. Scanning electron micrographs of a hipinnaria showing fully developed ciliated bands (arrowheads and arrows): (A) Ventral view of a bipinnaria with a flexed oral hood and mouth (m) open showing the position of the adoral ciliated band (adcb). (B) Lateral view of a bipinnaria showing the anterior extension of the oral hood, top right-hand side. Double-ended arrow indicates the anterior region where the anterior gan- glion links the preoral and postoral ciliated bands. Arrowheads, preoral ciliated band; arrows, postoral ciliated band: Bar, 200 /urn. of immunoreactive cells and processes, forming the adoral nerve plexus (ADNP). The ADNP innervated the epithe- lium of the adoral ciliated band. Confocal optical sectioning revealed that the apical region of these cells protruded to the exterior of the ciliated band epithelium. Computer anima- tions (data not illustrated) and a 3D anaglyph (Fig. 5A| showed that this plexus was also connected by serotonergic processes with the nerve plexus in the preoral transverse band via two thin (approximately 2.5 pirn) lateral immuno- reactive tracts. Preoral ciliated band The preoral ciliated band was located on the ventral surface of the larva and outlined the oral hood (Fig. 4A). Where this band traversed the larva above the mouth (pre- oral transverse region), a large number of flask-shaped cell bodies with 5-HT-like immunoreactivity (x = 21, SE = 0. 1 n = 10 larvae) were found in the epithelium (Fig. 4B). From these cell bodies, confocal sectioning into the larva from the ventral surface revealed axonal-like processes from the basal portion of the cells extending inwards toward the buccal cavity (Fig. 4B). In its lateral region, the preoral ciliated band contained a few immunopositive cells scat- tered along its path. Occasionally, a collection of cell bodies forming a pair of lateral ganglia were seen in the lateral region of the postoral ciliated band. These structures were not seen in all larvae and appear to be ephemeral. In the late bipinnaria, a ganglion developed at the anterior end of the larva. This ganglion consisted of immunoreactive cells 128 F. CHEE AND M. BYRNE Figure 5. Bipinnaria: 3-D anaglyph and a high-magnification confocal image projection of the anterior ganglion. Images were constructed from a series of optical sections covering a distance of 132 /urn. (A) 3-D anaglyph of a bipinnaria detailing the serotonergic nervous system following the pathway of the ciliated bands. White arrowheads, serotonergic connection between preoral and adoral ciliated bands; s, stomach; m, mouth; o, esophagus; arrows, immunoreactive coelomic cells. Bar, 200 JJITI. ( B ) A projection of the anterior ganglion in a late bipinnaria. Parallel axonal-like tracts on the opposing sides of the preoral ciliated band (prcb) and the postoral ciliated bands (pocb) interconnecting in a fine network of processes with 5-HT-like immunoreactivity. axt, axonal-like processes; neb, nerve cell bodies. Bar, 50 /im. associated with the preoral and postoral ciliated bands and a network of varicose processes spanning the two bands. This structure innervated the two bands and is a prominent neu- roanatomical feature in the bipinnaria of Patiriella rei>iultiiix with a similar shape. Three-dimensional recon- structions of the tripolar cells revealed that they were pyramidoid. a structure not previously reported. This ob- servation would, however, be dependent on the imaging 5-HT NELIROGENESIS IN A LARVAL SEA STAR 129 Figure 6. Confocal images detailing immunnreactive cells in ciliated bands. (Al Image from 15 optical sections (total thickness 139 /im) showing nerve cell bodies (neb) with 5-HT-like immunoreactivity and an axonal-like tract (axt and arrowsl in the adoral ciliated band. The entire band is immunoreactive. Note that the apical region of the neuron-like cells extend to the edge of the epithelium of the ciliated band. Arrowheads, cilia projecting into the buccal cavity. Bar. 20 /nm. (B) The preoral and postoral ciliated bands of a fully developed bipinnaria. Note the greater number of cells with 5-HT-like immunoreactivity (neb) present in the preoral ciliated band (procb) compared with the postoral ciliated band (pocb). The preoral ciliated band has immunoreactive processes (ip and lateral arrowheads) extending towards the buccal cavity, c and arrowheads, cilia. Bar, 63 f±m. (C) Nerve cell bodies (ncbl and arrowheads) in the right lateral postoral ciliated band. Bar, 50 |um. technique employed. Monopolar cells were the most com- mon type of immunoreactive cell in the preoral and postoral ciliated hands, whereas multipolar pyramidal cells were the most common cell type in the adoral nerve plexus (ADNP) of the adoral ciliated band. The presence of the apical projection arising from the cell bodies in the ADNP suggests that this plexus may have a sensory role. This interpretation is similar to that of Ko- matsu et til. (1991), who defined sensory neurons in the bipinnaria of Luidia senegalensis as neurons whose apical surface contacts the external environment. Strathmann ( 1975) demonstrated that the cilia of the adoral ciliated band in bipinnariae are involved in carrying food particles into the esophagus. It is possible that the adoral ciliated band of P. regularis plays a gustatorial function under the influence of serotonergic activity in the ADNP. In the bipinnaria of P. regularis. serotonin-like immuno- reactivity was conspicuous in the adoral ciliated band, in the preoral and postoral ciliated bands, and in the anterior ganglion. The adoral ciliated band was strongly fluorescent and connected to the preoral ciliated band by an immuno- reactive tract. Detection of this connection was possible through generation of 3D anaglyphs from confocal optical sections, which allowed visualization, and tracing of the complex immunostained network with respect to larval anatomy. On-screen animations (Chee and Byrne, 1997) were also employed to view immunolabeled larvae to de- termine the structure and direction of the immunolabeled 130 F. CHEE AND M. BYRNE Figure 7. Confocal image showing immunoreactive cells (arrow- heads) in the intestinal wall (il and surrounding the anus (a): Bar. 50 jum. processes. Although serotonergic immunoreactivity of the adoral ciliated band has been described in several studies (Nakajima, 1988; Moss et ai, 1994). the connections be- tween the adoral and preoral ciliated bands have not been seen before. The conventional epi fluorescence microscopy used in these earlier studies would not, however, have allowed resolution of this fine structure. Our observations demonstrate the presence of an extensive serotonergic com- munication network that connects all the ciliated bands and may govern reactions to stimuli and generate the behavioral patterns associated with feeding and swimming. Optical sections through the oral region revealed that the immunoreactive cells in the preoral ciliated band gave rise to basal immunoreactive processes that project dorsally along the roof of the buccal cavity. The high density of immunoreactive cells in the region of the preoral ciliated band along the buccal opening suggests that these cells may play a sensory role in feeding. Selection and rejection of particles during feeding is thought to be associated with sensory cells in the buccal cavity (Strathmann, 1975). The cell processes in the roof of the buccal cavity in the larvae of P. regularis may connect with receptor sites that lie within the buccal cavity and are involved in particle selec- tion in feeding. The 5-HT immunopositive tract connecting the adoral and preoral ciliated bands indicates a serotonergic link between the adoral ciliated band and the preoral ciliated band; this link could be important in feeding. The anterior ganglion (AG) is first seen in early bipinna- ria prior to formation of the anterior extension. As this extension develops, the ganglion becomes more intricate, forming a highly complex network. In advanced bipinnaria the AG consisted of prominent, strongly fluorescent tracts traversing the anterior region of the preoral and postoral ciliated bands. The anterior ganglion was the only seroto- nergic connection between these ciliated bands. In contrast to that suggested for sea stars (Lacalli, 1994), the AG of P. regularis does not split to form a pair of lateral ganglia. On the contrary, the pair of lateral ganglia occa- sionally seen in P. regularis (Chee and Byrne, 1997) are distinct from the AG. This bilateral collection of nerve cells may be homologous to those seen in other sea star larvae (Nakajima, 1988; Moss et al, 1994), and it also corresponds to the position of peptidergic GFNSALMFamide (SI ) gan- glia seen in P. regularis (Byrne et ai, 1999). At the brachiolaria stage, the AG is incorporated into the attachment complex, which contains many serotonergic neuronal-like cells suggested to be involved in the settle- ment process (Chee and Byrne, 1999). What appears to be a serotonergic AG has been observed in other echinoderms. Immunocytochemical labeling with anti-serotonin in the auricularia larvae of a holothuroid produced a structure described as an apical ganglion (Burke et al., 1986). Unlike the AG of P. regularis, this structure was not composed of many immunoreactive tracts. Serotonergic AG that differ structurally from that of sea stars but are still anterior in position have been extensively described for sea urchin plutei (Bisgrove and Burke, 1987; Bisgrove and Raff, 1989; Nakajima et al., 1993). The AG in the bipinnaria of P. regularis is similar to the anterior concentration of serotonergic neurons, variously called apical organs or apical ganglia, characteristic of many invertebrate larvae (Lacalli, 1994). These appear to be highly conserved structures in marine invertebrate larvae and are thought to have a sensory function (Lacalli, 1994; Marois and Carew, 1997). The function of the AG and the significance of the connection between the preoral and postoral ciliated bands of P. regularis are not known. The position of the AG, considered together with the bipinna- ria's anterior direction of swimming, suggests that it may have a sensory role in directional swimming; a similar function has been suggested for the apical ganglion of other invertebrate larvae (Marois and Carew, 1997). Moreover, in an ultrastructural study of the bipinnaria of Luiilia seuega- lensis, sensory cells were found in the preoral and postoral ciliated bands ( Komatsu et ai, 1991 ) in the region where the AG is located in P. regularis. Immuno-electron microscopic examination of thin sections from the anterior region of P. regularis would be needed to determine whether similar cells are present in this species. The anterior ganglion in P. regularis is also similar to non-serotonergic neuronal structures in other asteroids. Similar catecholaminergic anterior structures in the bipin- naria of Archaster typicits were described as a "fluorescent anastomosis" (Chen ct ai, 1995). Nakajima (1987) de- scribed a similar catecholaminergic structure as a "fibrous network" in the bipimiariae of Asterias ainurensis. We 5-HT NEUROGENESIS IN A LARVAL SEA STAR 131 believe that confocal imaging would reveal that these struc- tures are similar to the AG in P. regultiris. This study presents the most detailed immunocytochem- ical description of the development of the serotonergic system in a larval sea star. The organization of the seroto- nergic nervous system in the bipinnaria of Patiriella regu- luris reflects the bilateral symmetry of the larva. A striking bilateral symmetry is also seen in the SI -like peptidergic system in P. ref>ularis (Byrne et ai, 1999). For a complete picture of the expression of serotonin in nerve-like cells during development, we will continue this study in the brachiolaria of P. regultiris through metamorphosis. Inter- estingly, serotonin has never been localized immunocyto- chemically in the nervous system of adult sea stars. It appears that complex serotonergic innervation is a feature common to the swimming and feeding larval form across a range of marine invertebrate phyla. Changes in expression of serotonin in the lecithotrophic larvae of the other Patiri- ella species are being examined to document the evolution of neurogenesis in these asteroids. Acknowledgments We thank Paulina Selvakumaraswamy, Anna Cerra and Paula Cisternas, and Gillian Anderson for their comments and help with the manuscript. Ray Ritchie kindly supplied the algal cultures. Tony Romeo at the Electron Microscope Unit at the University of Sydney also provided assistance. This work was supported by an Australian Research Coun- cil grant. 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LENZ1 1 Bekesy Laboratory of Neumbiology, Pacific Biomedical Research Center, Universitv of Hawaii at Manoa, 1993 East-West Rd., Honolulu, Hawaii 96822; and 2 Marine Science Institute, University of Texas at Austin. 750 Channelview Drive, Port Aransas, Texas 78373 Abstract. Actively vertically migrating mesopelagic copepods are preyed upon by a wide variety of fishes and invertebrates. Their responses to predatory attacks in- clude vigorous escape jumps and discharge of biolumi- nescent material. Escape jumps and bioluminescent dis- charges in the calanoid copepod Pleuromamma xiphias were elicited by quantified hydrodynamic disturbances. Brief weak stimuli (peak water velocity 64 ± 21 /im s~') elicited weak (peak force 6.5 dynes) propulsive responses ("jumps") and no bioluminescence. Moderate stimuli (1580 ± 780 ju,m s~') produced strong propulsive re- sponses consisting of long trains of coordinated power strokes by the four pairs of swimming legs ("kicks"). Peak forces averaged 42 dynes. Strong stimuli (5520 ± 3420 ju,m s~') were required to elicit both a jump and a bioluminescent discharge. In several cases, multiple stimuli were needed to evoke bioluminescence, given the limits on stimulus magnitude imposed by the apparatus. Repeated bioluminescent discharges could be evoked, but this responsiveness waned rapidly. Latencies for the jump response (14 ± 4 ms) were shorter than for the accompanying bioluminescent discharge (49 ± 26 ms). The higher threshold for eliciting bioluminescent dis- charge compared to escape jumps suggests that the cope- pods save this defense mechanism for what is perceived to be a stronger threat. Received 13 May 1999; accepted 6 August 1999. * Author to whom correspondence should be addressed. E-mail: danh(<"phrc lunv.ui.edu Introduction Planktonic copepods are preyed upon by a wide variety of fishes and invertebrates (Hopkins and Baird, 1985; Hopkins ct a/.. 1996). Thus, predator evasion strategies are key to the survival of these animals in pelagic communities. Plank- tonic copepods respond to perceived attacks with rapid and powerful escape "jumps" (Singarajah, 1969, 1975; Strick- ler, 1975). The Augaptiloidea (Calanoida), which typically inhabit the mesopelagic region, possess the ability to dis- charge bioluminescent material (Clarke et «/., 1962; Her- ring, 1988). These discharges are thought to either startle a potential predator away or misdirect a possible attack (David and Conover, 1961; Morin, 1983; Young, 1983). Although we have a qualitative understanding that biolumi- nescent discharges in these calanoids are used as a defense mechanism, we know less about how these discharges are triggered in the natural environment. In the laboratory, electrical stimulation and mechanical agitation are routinely used to elicit bioluminescent discharges (e.g., Latz et al., 1987. 1990; Widder, 1992). However, we know little about the magnitude of stimuli required to elicit this behavior. Neither do we understand the relationship between the escape jump and the bioluminescence. We addressed some of these questions in a laboratory study, working with tethered Plettromamma xiphias. This calanoid is a metridi- nid (Augaptiloidea) and belongs to a widespread and abun- dant genus in this group. Here we report on the minimum hydrodynamic stimuli necessary to elicit a jump response, and how this compares to the minimum stimulus that trig- gers bioluminescence. By concurrently monitoring jump behavior with a force transducer and bioluminescence with 132 COPEPOD JUMPS AND BIOLUMINESCENCE 133 a photomultiplier tube, we are able to describe the temporal sequences for the two behaviors following a quantitative stimulus. Materials and Methods Collection Animals were collected at night (2000 to 2200 h). about 1 mile offshore from Keauhou Bay, Kona, Island of Hawaii, at a depth of 70 to 100 m. A plankton net (0.5-m diam, 333-/j.m mesh) was towed from a small boat at idle speeds (<2 knots) for 15 to 20 min. Within 2 h of collection the animals were sorted into jars with clean seawater, cooled to 6°C, and flown, in coolers, to Oahu. Once the animals were brought into the laboratory (within 16 h of collection), they were kept in the dark at 6° to 8°C. Every 2 to 3 days the copepods were fed under dim red light with a mixture of Artemia nauplii and Isochrysis galbana cells. Tethering Copepods were affixed to aluminum wire tethers with cyanoacrylate glue (Borden or Loctite) under red light in an otherwise darkened room. They were corralled in a droplet of seawater, which was then drawn down until a portion of the dorsal prosome was briefly exposed to air. The wire, with some glue on its tip, was applied and held in place while the animal was reimmersed. During this procedure the animals typically bioluminesced in response to the tactile stimulation. Once a copepod was glued and transferred to the experimental setup, 3 h were allowed to elapse before it was tested for mechanical sensitivity. Good experimental animals had high mechanical sensitivity, maintained their swimming appendages in the promoted position (tucked under the body, anteriorly directed), and were biolumines- cently competent. In the experiments presented, the animals maintained their mechanical sensitivity for at least 2 days, although the force produced during the jump typically de- clined. Toward the end of the experiments we observed either a loss in sensitivity or a failure to maintain the swimming appendages in the promoted position. All ani- mals were still bioluminescently competent at the end of the experiments and responded to direct tactile stimulation with a discharge. While on the tether, copepods were fed Iso- chrysis galbana. Health To test the bioluminescent competence of P. xiphias, five specimens were tested for total mechanically stimulable luminescence (TMSL) using methods described in Buskey and Swift ( 1990). A single P. xiphias was placed in each of five liquid scintillation vials containing 10 ml of filtered seawater. After allowing the copepods to recover for about 2 h from the disturbance of being transferred, we placed a vial inside an integrating sphere (Labsphere, Polane coated) and stimulated bioluminescence by stirring the vial with a battery-powered test-tube stirrer until no additional biolu- minescence was detected. Bioluminescence was quantified using a photomultiplier tube (PMT; Hamamatsu R464) and a photon-counting photometer (Hamamatsu C1230). Values for TMSL of P. xiphias ranged from 5.3 X 1010 to 5.5 X 10" photons, with a mean of 2.4 X 10" photons. These results are similar to previously measured values of TMSL for P. .xiphias (Buskey and Swift, 1990; Latz el al, 1990) and indicate that our experimental animals were capable of full bioluminescence and were in good physiological con- dition. Experimental protocol The experimental setup is diagrammed in Figure 1 and is described in detail in Lenz and Hartline (1999). After the tethered copepod was positioned in the apparatus, red back- ground lights were turned off, and illumination was switched to infrared light from four Optek OP-293A LEDs emitting 875 ± 20 nm and positioned about 1 cm behind the animal, outside of the field of view of the video camera. Hydrodynamic stimuli were generated using a piezoelectric pusher to control movement of a plastic sphere of either 3- or 5-mm diameter positioned about 3 mm in front of the animal. At maximum amplitude, the experimental sphere was displaced vertically by 40 jum. A behavioral response was elicited at threshold by vertical movements of the larger sphere of less than 0.5 /AID. Water displacement at the rostrum, approximately parallel to the long axes of the first antennae, was calculated based on the dipole attenuation expected of near-field laminar water flow (Kalmijn, 1988; Gassie et al., 1993). Although there are some errors and approximations inherent in this indirect approach to deter- mining stimulus magnitude (see Gassie et al., 1993, and Lenz and Hartline, 1999, for detailed discussion), it is widely used in behavioral and physiological studies on hydrodynamic reception in aquatic organisms (e.g., Coombs etal., 1989: Bleckmann, 1994; Coombs, 1994) and provides a reasonable measure given uncertainties in such factors as the location of receptors. Computer-controlled stimuli in- cluded short and long sinusoidal movements ranging in frequency from 50 to more than 1000 Hz. Force measurement During a rapid swim the copepod exerted a force on the tether. The displacement this produced along a horizontal axis, roughly parallel to the copepod' s body axis, was measured with a fiberoptic displacement sensor (Philtec 88N) positioned opposite to a small reflective disk mounted on the tether (Fig. 1). The force was calibrated by pushing 134 A From Computer D. K. HARTLINE ET AL To Computer B Photometer To Computer To VCR Microscope Figure 1. Diagram of the experimental setup. (A) Side view showing the positions of the dipole (sphere) and the glued copepod. The sphere used in the stimulus was either 3 or 5 mm in diameter. The distance between the center of the sphere and the rostrum of the animal ranged from 3 to 5 mm. (B) View from the top. Location of the photometer, dissecting microscope, and camera are shown. The experimental dish was made out of microscope slides and designed to allow positioning of the equipment at right angles to the glass. against the tether with a wire, the deflection of which had been calibrated using weights. Force-transducer responses were monitored with an oscilloscope, digitized at 42 kHz per channel, and stored on computer. Resonance frequency of the transducer ( 1.5-2 kHz) was kept as high as possible while maintaining sufficient sensitivity for measurements. The transducer was underdamped, with an overshoot of around 20% to abruptly applied (0.5 ms rise) forces; it had a dampn time-constant of 4 ms. Force signals were filtered at 2 kHz wilt an 8-pole Bessel filter. Further details of the recording system are given in Gassie et til. ( 1993) and Lenz and Hartline (1999). Mounted perpendicular to the view presented in Figure 1 A were a photometer, a dissecting microscope, and a video camera (Fig. IB). Each of these instruments faced one of the five sides of the experimental chamber. Light from the I-R LEDs was blocked from the photometer with an interfer- ence filter (center wavelength 480 nm), and background recordings were very low. The spatial and temporal patterns of bioluminescent emission of P. xiphitis were recorded on videotape using a Cohu monochrome CCD (charge-cou- pled-device) camera (30 fps) fitted with a 55-mm Micro- N1KKOR macro lens, coupled to a Videoscope Interna- tional KS-1381 microchannel plate image intensifier. The video output signal was recorded on a Mitsubishi HU-770 videocassette recorder. The stimulus-trigger from the com- puter also triggered a 30-ms-long flash in an I-R LED, producing a single video frame with an elevated light level. This was used to correlate video with force and PMT records, which thus had an uncertainty of 30 ms. Charac- teristics of the bioluminescence monitored by the PMT could frequently be used to estimate the relative timing with higher temporal resolution. Lii;ht measurement The bioluminescent emissions of P. xipliicis were mea- sured in two ways: with a photomultiplier photometer and with an image intensifier. In early experiments, photometer measurements were made using a Hamamatsu C1230 pho- ton counter and a Hamamatsu R464 PMT. This system was convenient for measuring the total integrated biolumines- cence emitted by P. xiphias. but it did not provide the temporal resolution necessary to accurately measure flash kinetics since it integrates counts over 0.1 -s intervals. It was replaced with a Pacific Instruments model 126 wide-range photometer using an EMI QL-30 PMT. Amplified voltage from the PMT was sent directly to the computer and digi- tized along with other components of the data stream. Be- fore and after being shipped to Hawaii, both photometer systems were calibrated using cultures of bioluminescent bacteria (Photobacterium sp.) and a calibrated Quantalum 2000 luminescence photometer with a highly stable silicon photodiode sensor. A secondary standard ( UC emission standard made from Sylvania Type 132 blue phosphor, peak wavelength 455 nm) was also calibrated. The secondary standard was measured frequently to allow for calibration of readings of bioluminescence. Results Sudden hydrodynamic disturbances were capable of elic- iting behavioral responses in Plciironuiinmn xiphias; we interpret these responses as "rapid swims." or "jumps." In COPEPOD JUMPS AND B1OLUMINESCENCE 135 tethered animals, a complex temporal pattern of force de- velopment followed closely on the presentation of such a stimulus. Figure 2A shows a typical response to a brief (2-ms) water movement of peak-to-peak amplitude com- puted at 3.83 jum at the copepod's rostrum. Following a short latency ("L"), there was an abrupt rise ("R") in for- ward propulsive force. Then a relatively rapid return past zero force to a smaller reverse force ("Rv") led to the development of a second forward component. As in a pre- vious study on the epipelagic copepod Umlinula vulgaris (Lenz and Hartline, 1999), we interpret these propulsive units to be kicks generated by the combined power strokes of the four pairs of swimming legs (pereiopods). The fea- tures of strong locomotor responses in P. xiphias were 20 30 Time (ms) 50 100 150 Time (ms) 200 Figure 2. Force record of a fast swim response of a Pleiiminnniiiui xiphias adult female to a suprathreshold hydrodynamic stimulus. (A) Expanded temporal scale showing the first four kicks of the response. (B) Record showing the complete response to the stimulus. Stimulus: 700 H/,, 1.5 cycles, maximum water velocity of 8400 /mi s~' at the rostrum. Piezoelectric transducer: PZL-060; vertical movement of sphere: 40 ;um; sphere diameter: 5. 1 mm; distance from center of ball to rostrum: 4.4 mm (BPL97-8.D04, second trace). similar in most respects to those of U. vnlgaris (Lenz and Hartline, 1999). They were characterized by short latencies, measured from the onset of the stimulus to the onset of the forward propulsion, typically around 10 ms (minimum: 6 ms). A weak brief backward propulsion, or "preparatory movement." was observed in some animals immediately preceding the forward propulsion (e.g.. Fig. 3A, "Pr"). Following the peak of forward propulsion, there was often an irregular pattern of peaks and valleys for the remainder of the short stroke duration (mean: 8.7 ms. Table I). As in U. vulgaris (Lenz and Hartline, 1999) and Calanux helgo- landicus (Svetlichnyy, 1987), the major peaks can be as- signed to the individual strokes of pereiopod pairs. Minor peaks caused by resonance in the underdamped force-trans- ducer system were also often apparent (Fig. 2A "res"). The distinct reverse propulsion following the termination of the forward phase was a feature found consistently in P. xiphius but not in previous studies on U. vulgaris. A pattern of multiple kicks in quick succession characterized a strong response to a stimulus. This is illustrated in Figure 2B, which shows the same response as Figure 2A on a com- pressed time scale. In P. xiphius, a train of kicks was typical, producing a cohesive propulsive response we term a "jump." Within the train, kicks occurred at repetition rates of 80 Hz (Table I; range 59 to 98 Hz). Response depended on stimulus magnitude With the experimental setup described, we were able to monitor jumps and bioluminescence simultaneously. As with other copepods we have tested, P. xiphias is very sensitive to water movement. Figure 3 shows records from the PMT and the force transducer at three stimulus intensi- ties. We observed several degrees of response, graded with the intensity of the stimulus (Table II). Figure 3A shows a "weak" response given to the lowest intensity of a 1.5-cycle stimulus that elicited a measurable response in this animal. Peak water velocity produced by this stimulus at the rostrum was calculated to be 50 jum s ' (BPL97-10: Table II). Neither the PMT nor the image intensifier recorded any sign of bioluminescence. The force trace shows first one small 12-dyne kick followed by a 100-ms delay and then three additional kicks. The cumulative force impulse generated by these kicks (the integral of force over time; related to total distance moved in a linear viscous medium) reaches only 0.2 dyne-second. In general, a weak response consisted of a brief force transient, which often barely registered on the force transducer (e.g., mean of 6.5 dynes. Table 1). These weak responses consisted of a small number of pro- pulsive events (e.g., 1-3) with moderate latencies (15-20 ms). We term them "weak kicks," but determining what is involved in their production awaits high-resolution cinema- tography. As in Figure 3A, a weak kick was sometimes 136 D. K HARTLINE ET AL. I- CO CO 2. io9 o 0 cf 40 1g 20 I 0 8-20 o LL 50 100 150 ^* CO I109 -^ 0 I- 1 40 'c? 20 I 0 8-20 o LL 200 Time (ms) 50 100 1 50 200 w 50 100 200 Time (ms) 50 100 150 200 Figure 3. Behavioral responses of a Pleuromamma xiphias adult female to three stimulus intensities. Hydrodynamic stimulus was produced hy a piezoelectric transducer (PZL-060) with a 5.1 -mm sphere, the center of which was located 4.3 mm from the animal's rostrum. (A) Photomultiplier tube (PMT) and force records showing response to a small stimulus: 700 H/., 1.5 cycles, maximum water velocity of 50 iim s ' at the rostrum (vertical peak-to-peak movement of sphere: (1.22 /xm). (B) PMT and force records showing response to a moderate stimulus: 700 Hz, 1.5 cycles, maximum water velocity of IdOO ,um s ' at the rostrum (vertical peak-to-peak movement of sphere: 7.1 /j,m). (C) PMT and force records showing response to a large stimulus: 700 Hz, 8 cycles, maximum water velocity of XWO iim s"1 at rostrum (vertical peak-to-peak movement ol sphere: 40 jtxm). Response is to fourth stimulus in a series of five presented at l-s intervals (the animal also luminesced to the fifth presentation). Estimated times of video frames shown in Figure 5 indicated with marks along the time axis. (Dl Integral of force over time for the force records shown in A, B. and C. Arrows indicate stimulus presentation Bar in C indicates the length of time the stimulus was on ( 1 1.5 ms). Stimulus length in A and B: 2 ms (BPL97-IO.D02, D04, D0f». followed ( :' SO-200 ms period of quiescence and then a cluster oi elayed, sometimes stronger, kicks. As stimulus intensity was progressively increased above the threshold level, a point was passed at which the intensity of the response increased abruptly (Tables I, II). Figure 3B shows force and PMT records for a stimulus intensity that is COPEPOD JUMPS AND BIOLUMINESCENCE Table I Characteristics of escape response elicited bv a hydrodynamic stimulus 137 Experiment Sex Weak kick force (dynes) Max kick force (dynes) Latency (ms) Kick duration (ms) Kick frequency (Hz) BPL97-3 M 7.5 ± 1.7 60.0 ± 6.4 12.9 ± 9.1 8.1 ± 1.2 89 ± 3 (5) (5) (6) (4) (5) BPL97-6 M 4.7 24.8 ± 6.8 11.5 ± 1.0 6.9 ± 0.6 98 ± 5 (1) (5) (6) (5) (6) BPL97-8 F 4.3 55.9 ± 3.5 7.4 ± 0.6 8.5 ± 0.7 89 ± 5 (1) (6) (6) (6) (6) BPL97-10 F 10.4 ± 3.2 36.8 ± 6.8 1 1 . 1 ± 3.3 11.3 ± 0.8 75 ± 4 (8) (19) (16) (8) (8) BPL97-II F 5.5 ± 1.5 34.2 ± 6.8 15.6 ± 1.7 8.8 ± 2.4 56 ± 5 (3) (4) (4) (6) (7) Weak kick forces were measured from escape responses to near-threshold stimuli. Maximum kick force, latency, kick duration, and kick frequency were all measured from responses to suprathreshold stimuli. Maximum kick force refers to the largest force produced in a train of kicks. Latency and kick duration were measured as shown in Figure 2 (L. D). Kick frequency was calculated by averaging the number of kicks over time either for the complete jump or over the data record (200 ms) in the cases where the jumps extended beyond the sampling window. Means and standard deviations are given; sample size (in parentheses) indicates the number of measurements used for the mean and SD. 30 times higher than that shown in Figure 3A. The PMT record shows no sign of bioluminescence. However, many characteristics of the force record are substantially aug- mented (Fig. 3B). The response typically involved multiple strong kicks with maximum forces produced by individual kicks registering nearly 40 dynes (Fig. 3B). The force impulse produced in the example shown in Figure 3B over a 75-ms interval approached 1 dyne-second (Fig. 3D). In general, such "strong" responses were elicited at stimulus strengths 15 to 30 times above threshold for the weak kicks (Table II). Peak amplitudes (mean = 42 dynes) were greater by a factor of 5 or more than for the weak kicks (Table I). Duration of individual kicks averaged 8.7 ms (Table I). The overall envelope of peak forces during a jump was "spindle" shaped (Fig. 2B). The first few kicks increased progres- sively in amplitude, then continued with several (sometimes 35 or more) kicks, and finally tapered off somewhat before ending. Figure 4 shows an example of the initial phase of one of these very long spindle-shaped jumps. In a multiple- stimulus protocol, the first or second stimulus of a train of five at 1.5-s intervals usually evoked the longest spindle- shaped jump. Further increase in stimulus intensity would in some cases result in a bioluminescent discharge. Figure 3C shows Table II Calculated water velocities that elicited behavioral responses: weak kicks, strong kicks, and strong kicks and bioluminescent discharges Weak kick response Strong kick response Jump + biolum response Expt Sex Stim Velocity (ju.m s ') Stim Velocity ( yum s ' ) Stim Velocity (u.m s~') BPL97-3 M S700 70 S700 2220 S700 2220 BPL97-6 M S700 66 S700 1170 S700 6590 BPL97-7 F ND ND ND ND S700 8420 BPL97-8 F S700 84 S700 840 ND ND BPL97-9 F S700 28 S700 890 ND ND BPL97-10 F S700 50 S700 1570 F700 8860 BPL97-11 F S700 89 S700 2770 F700 8860 BPL97-1 F F700 58 ND ND F700 1830 BPL96-1 M ND ND ND ND F700 1830 Mean 64 1580 5520 SD 21 780 3420 Water velocities at the copepod were calculated using dipole equations. Sinusoidal vertical movements of sphere at 700 Hz were either short (S700. 1.5 cycles) or long (F700. 8 cycles). ND = not determined: threshold could not be established. 138 D. K. HARTLINE ET AL. 50 100 150 Time (ms) 200 B Q. E 0 50 100 Time (ms) 150 200 Figure 4. (A) Force record of a long series of multiple kicks in response to a large stimulus in Pteuromamma .\iphias. adult female. (B) Integral of force over time for the force record shown in A. Stimulus: 700 Hz. 1.5 cycles, maximum water velocity of 8900 jim s~ ' at rostrum. Piezoelectric transducer: PZL-060; sphere diameter: 5.1 mm: distance from center of ball to rostrum: 4.4 mm: vertical peak-to-peak movement of sphere: 40 /uni. the response of the same animal as in Figures 3A and 3B to a stimulus with an amplitude 180 times greater (and of longer duration) than threshold for eliciting the weak jump. Both a jump and a bioluminescent discharge were produced. The jump response was initiated well before (18 ms) the bioluminescence (Fig. 3C, top panel). The bioluminescent discharge started at the end of the second kick in a train of five and lasted for about 200 ms. The integrated force for the train of kicks was about 0.8 dyne-second (Fig. 3D). Biolu- minescence was usually accompanied by strong spindle- shaped jumps. Near threshold for bioluminescence. dis- charges were likely to be given in response to one of the later stimuli in a sequence of five, and were thus not clearly associated with the strongest (= longest) jump. We were not able to elicit bioluminescent discharges to hydrodynamic stimuli in all cases. This was not due to a lack of bioluminescent competence, as electrical stimuli or more vigorous mechanical disturbance would invariably elicit bioluminescence even if our strongest hydrodynamic stimulus would not. In five experiments, we obtained thresholds for both jump and bioluminescence. and the mean and standard deviations for the stimulus intensities are shown in Table II. The mean threshold of computed peak water velocity for a jump response was 64 /urn s~ ', whereas that for eliciting bioluminescence was 5520 jam s~'. The variability of the threshold for bioluminescence was greater than that for the jump. On average the stimulus magnitude had to be 90 times greater to elicit bioluminescent discharge than to produce a weak jump, but this ratio ranged from 30 to 180 in the five experiments. Once we established a threshold for bioluminescence for an experimental animal, we usually were able to elicit bioluminescence multiple times at that stimulus level, sometimes within half an hour from the previous discharge. Water velocity was not the only stimulus characteristic that affected the likelihood of a bioluminescent discharge, as shown in Table II. Stimulus length was important: the multi-cycle sinusoidal stimulus (F700) was more effective than the 1.5-cycle one (S700; Table II). Furthermore, re- peated presentation of stimuli in quick succession was even more effective. In these cases, the animals would respond with only a jump to the first and second stimulus presenta- tions, but would bioluminesce as well as jump to the sub- sequent one or two stimuli. Characteristics of evoked bioluminescence In our tethered animals, bioluminescence typically (though not always) outlasted the jump. The PMT record in Figure 3C shows that bioluminescence was initiated at about 30 ms post-stimulus, corresponding to the second kick. It lasted throughout the recording period, although by 200 ms post-stimulus it was well along an exponential decay. Excerpts from the corresponding video record are shown in Figure 5. Taken at 30 frames per second (fps), with the frame following stimulus delivery tagged by a light flash, the first frame shows no bioluminescence and the onset of the major kick transients occur in this interval. Bioluminescence begins to appear from the region of the abdomen in the next frame, and reaches a peak in the third. Its near-absence from the last two frames is partly a result of decay and partly that much of the material has left the field of view. Thirty-five minutes later a second trial for the same animal as in Figures 3 and 5 elicited an escape as well as a bioluminescent discharge from both head and abdomen (Fig. 6). The animal bioluminesced in response to the sec- COPEPOD JUMPS AND BIOLUMINESCENCE 139 Figure 5. Video frames showing the bioluminescent discharge associated with the records in Figure 3C. The pre-stimulus frame is a composite of the 10 frames preceding stimulus presentation. The next five video stills are from frames 3-7, counting the first post-stimulus frame as 1 (30 fpsl. Ventral-posterior aspect of animal faces camera. Discharge is primarily from abdominal glands. Broken outlines up to frame 3 indicate position of body prior to stimulation. ond stimulus of a train of five. It was somewhat more delayed (50-ms latency) and shorter (100-ms duration) than the earlier response (peak amplitude could not be measured owing to saturation of the PMT), but the jump was twice the length (10 kicks versus 5). Records of jumps and bioluminescent discharges from a male Pleuromamma xiphias are shown in Figure 7. In this case the animal completed its jump before the biolumines- cence. This example was chosen to illustrate a double re- action. The animal responded with two sets of kicks and matching bioluminescent discharges. The discharges were small and short in duration. The animals routinely push the bolus of bioluminescence away from them by flicking their urosomes. This can be seen in Figure 7 as the streaks of bioluminescence move across the screen. The force gener- ated by this behavior is very small compared to the pereio- pod power strokes and does not register on the force record. Comparing this record with the data from the female of Figures 3. 5. and 6 shows the differences that occur when the pereiopods beat during emission of bioluminescent ma- terial. The combined kicking and bioluminescence produce the explosion of bioluminescence seen in the video frames. This is in contrast to the male (Fig. 7). in which the lumi- nescent material clung to the urosome, presenting a streaky appearance. Temporal relations bet\\'een jump and bioluminescence The rapid swim was always initiated before the biolumi- nescence, as illustrated in Figure 8. a scatter plot of jump latencies versus bioluminescence latencies. All points are above the line with a slope of one. Rapid swims were initiated within 7 to 20 ms (mean ± SD = 14 ± 4 ms), whereas bioluminescence latencies ranged from 20 to 50 ms (with one very delayed response that started at 110 ms; mean ± SD = 49 ± 26 ms). In general, the longer the rapid swim latency the greater the delay for the bioluminescence, although the correlation coefficient was not significant (/• = 0.508, n = 8). Bioluminescent discharges in response to the hydrodynamic stimulus were typically short, lasting from 50 to 350 ms. Luminescence often (e.g.. Figs. 3C; 7), but not always (Fig. 6), extended well after the termination of the jump. Discussion Escape jumps Like all pelagic calanoids, mesopelagic Pleuromamma xiphias has an impressive escape jump at its disposal. When sensitivity to water perturbations and jump kinematics mea- sured in tethered animals are compared to similar data for neritic Undinula vulgaris (Lenz and Hartline, 1999), a pat- 140 D K HARTLINE ET AL 50 100 150 Time (ms) 200 Figure 6. (A) Force record of a second response from the animal in Figures 3 and 5. Comparison shows variability in propulsion and bioluminescence. Note the greater duration of the jump and the shorter duration of the bioluminescent discharge. Stimulus: second in a train of live identical to that for Figures 3 and 5, delivered 35 min following. (B) Video frames 3-7 and 10 following stimulus, showing bioluminescent discharge associated with records of A (30 fps). Broken outline indicates position of body prior to stimulation. Note luminescent discharge from cephalic gland. tern of characteristics emerges that is similar in broad scope but distinctive in detail. P. xiphias sensitivities (—60 /xm s~') are similar to. though perhaps somewhat lower than, those in U. vulgaris (—40 /xm s~'). Minimum latencies for P. xiphias (—6 ms) were distinctly longer than for U. vulgaris ( -2 ms). This difference in reaction times is in part explained by the lack of myelination of nerve fibers in the Augaptiloidea (Davis et al., 1999). Peak forces of kicks from U. vulgaris showed a small gradation in magnitude as a function of the strength of the triggering stimulus and over the course of an escape jump. In contrast, those of P. xiphias exhibited a much wider range, with a 5- to 10-fold differ- ence between the weak kicks produced to near-threshold stimuli and the strongest kicks in the middle of a spindle- shaped jump. The strongest kicks registered in our apparatus by U. vulgaris (100 dynes) were almost twice the peak forces measured from P. xiphias (Table I). In U. vulgaris, the initial one or two kicks were the strongest, whereas in P. xiphias, the strength of kick built up over several cycles, and then waned, giving rise to the spindle-shaped enve- lope. Although both species produced multiple kicks in response to threshold and well supra-threshold stimuli. U. vulgaris consistently produced fewer (2-3 typical; up to 9) than did P. xiphias (5-10 typical; up to 35). For comparably sized animals, this should result in longer jump distances in the latter species. This expectation is in agreement with casual observations made while attempt- ing to catch P. xiphitis in an open vessel: jumps of tens of centimeters are not atypical, while those of U. vulgaris are shorter (3 to 5 cm). Bioluminescent discharges can he evoked hv hydmdynaniii • stimuli Pleuromamma xiphias will produce a bioluminescent dis- charge to a brief water disturbance; tactile stimulation is not COPEPOD JUMPS AND BIOLUMINESCENCE 141 B o o .c Q. O 3< 0.5 0 31 41 61 71 81 1 00 200 Time (ms) 300 Figure 7. Response of Pleuromamma xiphuis adult male to a large hydrodynamic stimulus. (A) PMT and force records. (B) Video frames of bioluminescent discharge. The pre-stimulus frame is a composite ot 10 frames preceding stimulus presentation. The next five video stills are post-stimulus frames 3-8. Approximate times of frames are indicated with marks along time axis of A. Note the occurrence of two jumps and two separate bioluminescent discharges, spaced 150 ms apart. Stimulus: 700 Hz, 1.5 cycles, maximum water velocity of 6600 p,m s~ ' at rostrum. Piezoelectric transducer: PZL-060; sphere diameter: 5. 1 mm; distance from center of ball to rostrum: 4.8 mm, vertical peak-to-peak movement of sphere: 40 ^m. Broken outlines indicate position of body prior to stimulation and a portion of the stimulating sphere in lower left corner; posterior view of animal with dorsal toward lower right corner of frames. Glowing material appears associated with abdominal glands (BPL97-6.D03). required. The magnitude of the stimulus required varied among experimental animals, but in general was signifi- cantly greater than that sufficient to trigger strong escape jumps (velocities of 2000 to 9000 /urn s ~ ' ). When presented with a threat, P. xiphias preferentially responds with an escape jump. However, if the threat is prolonged or persists as in the case of repetitive strong stimulation, then the jump is more likely to be accompanied by a bioluminescent discharge. Widder ( 1992) found a similar pattern for Gaus- sia princeps. During a train of electrical stimulation (3 s~') G. princeps would respond with an escape alone until the fifth stimulus, when it finally produced a bioluminescent discharge as well. Bioluminescence is delayed compared to the jump Bioluminescence was always initiated after the onset of a jump sequence. Although the numbers of animals tested were insufficient for complete reliability, in two animals of our study (both males), jumps were completed before the bioluminescence began. In four others (all females), the bioluminescent discharge commenced during the train of kicks. This resulted in a qualitative difference in the visual effect of the bioluminescence. the luminescent bolus being swept along by water propelled posteriorly by the power strokes. An animal that bioluminesces after it has stopped swimming would seem more likely to become a victim of a predatory attack if the luminescent bolus attracts a predator. 142 D. K. HARTLINE ET AL. C/) O c Q) CO 120 80 E 40 _ o CO 0 • Fern X Male I I I 1 0 10 20 Jump Latency (ms) Figure 8. Scatter plot of fast swim latencies (x axis) versus biolumi- nescence latencies (v axis). Squares: adult females (H = 7); crosses: adult male (n = 1 1. All points lie above the line, which has a slope of one. The possibility that there might be a sexual difference in the response patterns is intriguing. Characteristics of biolitminescence and its relation to other cases reported in the literature The kinetics and spatial patterns of bioluminescence re- leased by copepods have been studied for copepods stimu- lated with electrical pulses (Latz et al. 1987; Bowlby and Case. 1991) and for copepods stimulated by mechanical disturbance of undefined frequency and intensity (Latz et ai, 1990). For the large mesopelagic copepod Gaussiu princeps, Bowlby and Case ( 1991 ) identified three types of flash in response to single electrical stimuli: a fast flash of about 2-s duration, a long flash of 7-s duration, and a slow flash of 17-s duration. Latz et al. (1987) found two compo- nents to flashes in P. xiphias stimulated with a single electrical pulse: a fast component that reached maximum intensity in < 100 ms and a slow component that reached peak intensity in > 600 ms. Double flashes with fast and slow characteristics were also observed. Using an intensi- fied video system, he observed that the fast component originated from thoracic and abdominal glands, without obvious discharge of bioluminescent material away from the body; the slow component of flashes was caused by the discharge of luminescent fluid from the abdominal organ. Flashes with similar kinetics were observed for P. xiphiax exposed to mechanical stimulation from a stirring paddle with three tines rotated at 2000 rpm for < 1 s. Since the spatial relationship between the copepod and the rotating tines is unknown during the stimulation period, neither the frequency nor the intensity of mechanical stimulation is known. In addition to strong hydrodynamic stimulation caused by the velocity of the water and the shear created by the spinning tines, mechanical stimulation is possible through direct contact of the copepods with the tines or by contact with the walls of the scintillation vial following an escape jump. In our observations of bioluminescence evoked by hydrodynamic stimuli of known intensity, only fast flashes were observed. In contrast to the observations of Latz et al. (1990). we observed bioluminescence having fast flash kinetics originating from abdominal glands, and with obvious discharge of bioluminescent material away from the body. We have noted that a copepod' s ability to produce a second bioluminescent discharge shortly after a previous one is not necessarily precluded. Thus recovery times mea- sured in TMSL protocols (8-24 h) are probably overly long for most natural situations. Ecological significance Vertically migrating copepods such as Pleuromamma xiphias are important components of mesopelagic food webs, and Pleuromamma spp. are often preferred prey of mesopelagic fish (Hopkins and Baird. 1985; Hopkins et ai. 1996). To help them avoid predation, these copepods have evolved several defensive behaviors, including vertical mi- gration (Bennett and Hopkins, 1989), strong escape jumps (Buskey et al., 1987; present study), and bioluminescence (Clarke et al.. 1962). In contrast to the diversity of strategies possessed by P. xiphias. neritic Undinula vtilgaris appears to have relied on enhancing the speed and strength of the escape response itself as a survival mechanism (Davis et al., 1999; Lenz and Hartline, 1999). The production of light in an otherwise dark environment may at first seem counter- intuitive as a defense mechanism against visual predators; discharge of bioluminescence while the predator is still remote might help the predator locate its prey. However, the higher stimulus threshold for eliciting bioluminescence compared to escape jumps suggests that copepods save this defense for what are perceived to be the strongest threats by predators in close proximity. Mesopelagic predators have sensitive eyes adapted to low light levels, and the discharge of bioluminescence when the predator is nearby may serve to temporarily blind and confuse the predator (Buck, 1978; Morin, 1983). Since copepods initiate escape jumps prior to release of bioluminescence, and leave behind distinct drop- lets or clouds of bioluminescent material (Widder, 1992), the bioluminescent discharge may also serve as a decoy to confuse visual predators (Morin, 1983). Acknowledgments We thank P. Couvillon, B. Kodama. and S. Lum for major alterations to the experiment room: C. Kosaki tor administrative assistance; H. Akaka and A. Davis for tech- COPEPOD JUMPS AND BIOLUMINESCENCE 143 nical assistance; C. Unabia for providing the algal cultures; and P. Cunningham for making the copepod collections possible. The Natural Energy Laboratory of Hawaii pro- vided access to the laboratory facilities at Keahole Point, Hawaii. This is University of Texas Marine Science Insti- tute Contribution number 1118. The research was supported by NSF grant OCE 95-21375. Literature Cited Bennett, J. L., and T. L. Hopkins. 1989. Aspects of the ecology of the calanoid copepod genus Pleuromamma in the eastern Gulf of Mexico. Conlrih. Mar. Sci. 31: 119-136. Bleckmann, H. 1994. Reception of Hydrodynamic Stimuli in Aquatic and Semiaquatic Animals. G. Fischer, New York, 1 15 pp. (Progress in Zoology vol. 41). Bowlby, M. R., and J. F. Case. 1991. Flash kinetics and spatial patterns of bioluminescence in the copepod Gaussia princeps. Mar. Biol. 110: 329-336. Buck, J. B. 1978. Functions and evolutions of bioluminescence. Pp. 419-460 in Bioluminescence in Action. P. J. Herring, ed. Academic Press, New York. Buskey, E. J., and E. Swift. 1990. An encounter model to predict natural bioluminescence. Limnol. Oceanogr. 35: 1469-1485. Buskey, E. J., C. G. Mann, and E. Swift. 1987. Photophobic responses of calanoid copepods: possible adaptive value. J. Plankton Res. 9: 857-870. Clarke, G. L., R. J. Conover, C. N. David, and J. A. C. Nicol. 1962. Comparative studies of luminescence in copepods and other pelagic marine animals. J. Mar. Biol. Assoc. UK 42: 541-564. Coombs, S. 1994. Neartield detection of dipole sources by the goldfish (Carassius auratus) and the mottled sculpin (Coitus bairdi). J. Exp. Biol. 190: 109-129. Coombs, S., R. R. Fay, and J. Janssen. 1989. Hot-film anemometry for measuring lateral line stimuli. J. Acoust. Soc. Am. 85: 2185-2193. David, C. N., and R. J. Conover. 1961. Preliminary investigation on the physiology and ecology of luminescence in the copepod Metridia lucens. Biol. Bull. 121: 92-107. Davis, A. D., T. M. Weatherby, D. K. Hartline, and P. H. Lenz. 1999. Myelin-like sheaths in copepod axons. Nature 398: 571. Gassie, D. V.. P. H. Lenz, J. Yen, and D. K. Hartline. 1993. Mech- anoreception in zooplankton first antennae: electrophysiological tech- niques. Bull. Mar. Sci. 53: 96-105. Herring, P. J. 1988. Copepod luminescence. Hydrobiologia 167/168: 183-195. Hopkins, T. L., and R. C. Baird. 1985. Aspects of the trophic ecology of the mesopelagic lish Lampan\ctus a/alits (Family Myctophidae) in the eastern Gulf of Mexico. Biol. Oceanogr. 3: 285-313. Hopkins, T. L., T. T. Sutton, and T. M. Lancroft. 1996. The trophic structure and predation impact of a low latitude midwater fish assem- blage. Prog. Oceanogr. 38: 205-239. Kalmijn, A. J. 1988. Hydrodynamic and acoustic field detection. Pp. 83-130 in Sensory Bio/ogv of Aquatic Animals, J. Atema, R. R. Fay, A. N. Popper, and W. N. Tavolga, eds. Springer Verlag, New York. Latz, M. R., T. M. Frank, M. R. Bowlby, E. A. Widder, and J. F. Case. 1987. Variability in Hash characteristics of a bioluminescent cope- pod. Biol. Bull- 173: 489-503. Latz, M. R., M. R. Bowlby, and J. F. Case. 1990. Recovery and stimulation of copepod bioluminescence. J. Exp. Mar. Biol. Ecol. 136: 1-22. Lenz, P. H., and D. K. Hartline. 1999. Reaction times and force production during escape behavior of a calanoid copepod. Undinula vulgaris. Mar. Biol. 133: 249-258. Morin, J. G. 1983. Coastal bioluminescence: patterns and functions. Bull. Mar. Sci. 33: 787-817. Singarajah, K. V. 1969. Escape reactions of zooplankton: avoidance of a pursuing siphon tube. J. Exp. Mar. Biol. Ecol. 3: 171-178. Singarajah, K. V. 1975. Escape reactions of zooplankton: effects of light and turbulence. J. Mar. Biol Assoc. UK 55: 627-639. Strickler, J. R. 1975. Swimming of planktonic Cyclops species (Cope- poda, Crustacea): pattern, movements and their control. Pp. 599-613 in Swimming and Flying in Nature, Vol. 2, T. Y.-T. Wu, C. J. Brokaw, and C. Brennan. eds. Plenum Press, New York. Svetlichnyy, L. S. 1987. Speed, force and energy expenditure in the movement of copepods. Oceanology 27: 497-502. Widder, E. A. 1992. Mixed light imaging system for recording biolu- minescence behaviours. J. Mar. Biol. Assoc. UK 72: 131-138. Young, R. E. 1983. Oceanic bioluminescence: an overview of general functions. Bull. Mar. Sci. 33: 829-845. Reference: Biol. Bull. 197: 144-158. (October 1999) Morphology of the Nervous System of the Barnacle Cypris Larva (Balanus amphitrite Darwin) Revealed by Light and Electron Microscopy PAUL J. H. HARRISON* AND DAVID C. SANDEMAN School of Biological Science, University of New South Wales, Sydney, Australia 2052 Abstract. The central nervous system of the cypris larva of Balanus amphitrite consists of a brain and posterior ganglion. The neuropil of the brain includes protocerebral and deutocerebral divisions, with nerve roots from the pro- tocerebrum extending to the eyes and frontal filaments, and nerve roots from the deutocerebrum extending to the first antennae (antennules) and cement glands. The neuropil of the posterior ganglion includes subesophageal and thoracic divisions, with nerve roots from subesophageal divisions extending to the gut, and nerve roots from each of the six thoracic divisions extending to their corresponding thoracic appendage. The antennular nerve is the major peripheral extension of the nervous system and is composed in pail by afferent fibers that innervate setae on the antennules. The cyprid nervous system is small, containing fewer than 2000 neurons, but is well organized for coordinating a response to settlement cues. Introduction The cyprid (cypris larva) is the final larval stage of the barnacle. Cyprids are specialized for settlement (Anderson, 1994), a behavioral process in which a site is selected for permanent attachment and metamorphosis (Anderson, 1994; Walker, 1995). Cyprid settlement is known to be mediated by specific environmental cues (Clare. 1995; Walker. 1995), but little is known about the mechanisms of cue detection and, in particular, how the detection of cues results in the centrally coordinated motor patterns of settle- ment behavior. Cyprids are highly mobile and bear numerous sense or- Received 22 March 1999; accepted 27 July 1999. * Current address: Department of Biology, Georgia State University. PO Box 4010. Atlanta, Georgia 30302. gans (Walley, 1969). The nauplius eye (= median eye) is present during the cyprid stage and is remodeled into the adult ocelli during metamorphosis (Takenaka et ai, 1993). A pair of compound eyes are also present, which are unique to the cyprid. These develop during the final naupliar stage and are lost during metamorphosis (Walley, 1969; Hallberg and Elofsson, 1983). The compound eyes are closely asso- ciated with a pair of frontal filaments (Walker. 1974), and many setae are located on the antennules (Nott and Foster, 1969; Nott, 1969; Clare and Nott, 1994; Glenner and H0eg, 1995), thoracic appendages (Glenner and H0eg, 1995), cau- dal rami (Walker and Lee, 1976; Glenner and H0eg, 1995), and carapace valves (Walker and Lee, 1976; Jensen et ai. 1994; Glenner and H0eg, 1995). Many of these setae are thought to function as mechano- and chemoreceptors. Pu- tative sensory structures located on the carapace include dermal pits, wheel organs (Elfimov, 1995), and lattice or- gans (Jensen et nl.. 1994; H0eg et ai. 1998). Recently, cilia-type dendritic segments were shown to innervate the lattice organs, suggesting a chemosensory function (H0eg et ul.. 1998). To date, morphological studies of the cyprid have fo- cused primarily on external structures (Elfimov, 1995), and particularly on the antennules because of the role played by these appendages during settlement (Nott and Foster, 1969; Nott. 1969; Moyse et al.. 1995). Fewer details are available on the internal organization of the cyprid. Walley (1969) described the larval development of Semibalanus bal- anoides (previously Balanus balanoides) and outlined the nervous system and major sense organs of both the cyprid and nauplius. Other studies have shown that the antennules (Nott and Foster, 1969), frontal filaments (Kauri, 1961; Walker, 1974), dermal pits (Walker and Lee, 1976), lattice organs (H0eg et ai. 1998), and cement glands (Walker, 1971; Okano et ai, 1996) are innervated, but the nerves 144 CYPRID NEUROANATOMY 145 associated with each of these structures have not been traced back to the central nervous system. The cyprid is well equipped to detect settlement cues, but little is known about the underlying role of the nervous system. Recent studies have suggested that cyprid settle- ment behavior is affected by exposing cyprids to certain neuroactive substances (Clare et ai, 1995; Kon et ai, 1995; Yamamoto et ai. 1995, 1996: Okano et ai. 1996, 1998). Studies aimed at investigating the underlying mechanisms of settlement would benefit from a detailed account of the cyprid nervous system. We report here the results of an anatomical study of the central nervous system and the major sense organs of the cypris larva of B. amphitrite. gained from microdissection, semithin serial sections, and electron microscopy. We find that the central nervous sys- tem is made up of about 2000 neurons and that it contains regionalized neuropils, many of which are linked to periph- eral sense organs. Although the cyprid nervous system is small, it is well organized, which is consistent with the cyprids' need to detect and respond to multiple cues for settlement. Materials and Methods Cyprids used in this study were obtained from a labora- tory culture of Balanus amphitrite (see DeNys et ai, 1995). The selected individuals were between 1 and 3 days old (nauplius-cyprid molt = day 0), were active, and had clear (i.e., non-milky) carapaces, obvious cement glands, and compound eyes. Dissection of the animals provided a useful overview of their structure, including the placement of the antennules and limbs within the bivalved carapace and the gross organization of internal organs. Specimens were placed on a stereomicroscope and dissected using tungsten microscalpels and pins (Conrad et ai, 1993). Individuals were placed in a calcium-free saline (in mmol • 1~', 485 NaCl, 13 KC1, 10 MgCU 10 HEPES, pH 7.4) to reduce movement and secured (ventral surface upward) to a sili- con-coated microscope slide using either tungsten pins or a nontoxic, rapid-setting silicon adhesive (Kwik-Sil, World Precision Instruments). A cut along the ventral midline allowed separation of the carapace valves to expose the central nervous system and internal organs of the cephalon (see Figs. 1, 2). The carapace valves, antennules, and tho- racic appendages were then secured with fine (<10 ju,m diameter) tungsten pins. The secured preparation was trans- ferred to a fixed-stage Olympus BH-2 microscope and viewed and photographed using water immersion objec- tives. Fixation and embedding. Larvae were placed in equal volumes of 0.4 M MgCU and 0.22 jam-filtered seawater (FSW) and gently agitated for up to 3 h. which had the effect of relaxing the carapace adductor muscles and expos- ing the antennules. Specimens were cooled to 4°C for 30 thoracic appendages 100u.m Figure 1. The cypris larva of Balumis amphitrite. (A) Light micro- graph of a live cyprid with antennules and thoracic appendages extending from the bivalved carapace. Internal structures visible include a compound eye (CE), translucent oil cells (OC), and a cement gland (CG). Frontal filaments (FFl extend posterior to each antennule. (B) Longitudinal section of the cyprid showing the central nervous system, which consists of a brain and posterior ganglion. The brain connects via paired circumesophageal connectives (not visible in this section I to the posterior ganglion. A single antennular nerve (AnN) and its associated antennular soma cluster (ASC) are visible. The ASC contains the somata of bipolar sensory neurons. Also apparent in this section are densely stained oil cells, the oral cone, esoph- agus, and gut. The compound eyes and cement glands are located lateral to this plane and are not seen (0.5 /urn section; stained with toluidine blue and osmium tetroxide). min, transferred to chilled (4°C) fixative consisting of 2.5% glutaraldehyde and 2.0% formalin in FSW (pH 8.2; 950 mosmol). The formalin used was prepared fresh from para- formaldehyde (37% w/v paraformaldehyde in H2O). Micro- wave treatment was used to facilitate the penetration of fixative. For microwave fixation, specimens were placed in 20-ml glass vials filled with chilled fixative; the vials were secured in a beaker filled with chilled water, which in turn was placed in a beaker of crushed ice. Microwave treatment continued until the water in the beaker reached a tempera- ture of 37°C (typically 50 s). Specimens were then removed from the oven and allowed to cool to ambient temperature; fixation continued overnight. The following day, specimens were rinsed in FSW for 1 h (three changes of 20 min each), post-fixed in 2% osmium tetroxide (in H-.O) for 30 min, 2% uranyl acetate (in H2O) for 20 min, dehydrated through an ethanol series, cleared in propylene oxide, exposed to 146 J. H HARRISON AND D. C. SANDEMAN thorax PMC PMC posterior Ventral Figure 2. Schematic drawings of the cypnd nervous system and major organs in longitudinal and horizontal planes. (A-B) The body of the cyprid is organized in two main compartments, the cephalon and the thorax. The bivalve carapace encloses anterior (AMC) and posterior (PMC) mantle cavities about the cephalon and thorax respectively. The brain, compound eyes (CE), median eye (ME), and cement glands (CG) are contained within the cephalon. The antennules extend from the cephalon and bear the adhesive discs and putative chemoreceptive and mechanoreceptive sensilla. The brain connects with the posterior ganglion via circumesophageal connectives (CC). The posterior ganglion and gut are contained within the thorax. Six pairs of thoracic appendages (TA) and a pair of caudal rami (CR) extend from the thorax. (C-D) The orientation of neural structures in the cyprid depends on the relative position of the antennules and thoracic appendages, both of which extend beyond the carapace when the c\pnd either swims or contacts the substratum (C), or can be withdrawn for protection (D). Planes are identified on the basis of the orientation ot the nervous system in (C). Other abbreviations: ASC. antennular soma cluster; OC. oil cell; FF, frontal hlamein. increasing concentrations of Araldite epoxy resin, and Hat- embedded on microscope slides. Flat embedding allowed the orientation of the specimen to be determined using a light microscope. The Araldite was then removed from the slides by cold shock (using liquid nitrogen) and specimens cut from the blocks and remounted on Araldite stubs for sectioning. Lii>!ii microscopy. Twelve animals were serially sec- tioned at cither 0.5 or 1.0 /j,m (six in sagittal plane, three frontal, and three horizontal — see Fig. 2 for orientation) with a Reichen -Jung ultramicrotome and diamond histology knife. Sections were transferred to microscope slides and stained with toluidine blue (17r in 6<7r borax, 0.6<7r boric acid, pH 8.3)ormethyleneblue (1% in 0.1% borax, pH 8.0); reconstructions were made from camera lucida drawings and photographs and with the aid of PC-based. Adobe Illustrator software. Electron microscopy. Specimens for transmission elec- tron microscopy were prepared as described above, sec- tioned at 60-70 nm on a Reichert-Jung ultramicrotome using a diamond knife, and viewed and photographed using a Hitachi H-7000 transmission electron microscope. For scanning electron microscopy, anesthetized and fixed ani- mals were washed for 10 min in H^O (three changes of 3 min each) with sonication during the first two steps, dehy- drated, and transferred to acetone for critical-point drying. Dried specimens were mounted on microscope stubs with double-sided carbon tape, then gold coated and photo- graphed on a Leica/Cambridge S-360 scanning electron microscope. Results General anah>in\ Live cyprids of B. amphitrite typically measure 500-550 /am in length from the rostral to the caudal end of the carapace (Fig. 1A) (Glenner and H0eg, 1995), but minor size variations occurred in our cultured animals. When fixed, the average dimensions for 1 2 individuals were 480 /LUTI in length, 220 /xm in height, and 1 70 /xm across the broadest part of the carapace. The body of the B. amphitrite cyprid, like that of 5. balanoides (Walley, 1969). is arranged as two separate compartments, the cephalon and thorax (Fig. 2). A bivalved carapace encloses anterior and posterior mantle cavities around the cephalon and thorax respectively (Fig. 2). The cephalon houses the brain, eyes, and cement glands, to- gether with many large, densely staining oil cells (Figs. 1, 2), which are thought to supply energy for the lecithotrophic larva and its subsequent metamorphosis (Walley, 1969). First antennae (antennules) project anteriorly from the cephalon and can be extended well beyond the carapace during temporary attachment (Fig. 2C), or completely re- tracted within the anterior mantle cavity (Fig. 2D). Frontal filaments extend from the ventral surface of the cephalon, posterior to the antennules (Figs. 1A, 2A, 6C). The thorax houses the posterior ganglion and the undifferentiated gut (Figs. 1, 2). Six pairs of thoracic appendages and the caudal rami project from the ventral surface of the thorax, which may extend beyond the ventral edge of the carapace or be completely withdrawn within the posterior mantle cavity (Fig. 2C-D). The central nervous system (CNS) can be seen in near- sagittal section (Fig. IB) and is drawn schematically in Figure 2. The CNS consists of a cerebral ganglion, or "brain." linked by paired circumesophageal connectives to a CYPRID NF.UROANATOMY 147 posterior ganglion. Nerve roots extend from central ganglia toward peripheral organs (Figs. 1,2). The major peripheral nerves include the antennular nerve and thoracic nerve roots (Fig. 2A). The relative position of neural structures depends on the degree of contraction of the appendages. When the appendages are fully extended (e.g., Fig. 2C), the central nervous system lies essentially flat along the ventral surface, and this orientation is used to identify planes throughout this study. When the appendages are withdrawn (e.g., Fig. 2D), the central nervous system can bend to an angle of 60° relative to the longitudinal axis, and the antennular nerve bends accordingly to accommodate flexion of the antennule. The bruin and associated structures The brain (Figs. 3, 4) is composed of centrally positioned neuropil surrounded by the somata of central cells (Figs. 3, 4). The neuropil is composed of fine fibers and nerve endings and on close inspection contains membrane-bound vesicles and densely staining clefts typical of invertebrate synapses (Fig. 4C). Central somata are relatively uniform in size, measuring 4-8 /u,m in diameter, and form an outer layer of between one and five cells thick (Figs. 3, 4). The somata of these cells typically contain lightly stained gran- ular nuclei and have a relatively thin layer of cytoplasm between the nucleus and cell membrane (Fig. 3B-C). Dis- tinct clusters project neurites together in bundles to the central neuropil (Fig. 3B-C); based on a calculation of soma volume, we estimate the total number of neuronal cells in the cyprid brain to be approximately 750. The brain is enclosed in a thin sheath, but the broad perineural glial layer present beneath the sheath in decapod crustacean ganglia (Sandeman, 1982) is absent. Small, densely stained, spindle-shaped glial cells lie between the somata and central neuropil and delineate neuropil regions (Fig. 3C). Two broad areas of neuropil can be discerned in the cyprid brain, and the lateral lobes of each division link via transverse fiber tracts (Fig. 4). The anterior division is connected to the eyes and frontal filaments via the optic tracts, and the posterior division is connected to the anten- nules via the antennular nerves (Fig. 4A). These divisions appear similar to the protocerebral and deutocerebral divi- sions of the decapod brain. We find no evidence of a tritocerebrum in the cyprid, which is consistent with the absence of antenna II during this stage (Sandeman el ul., 1992). The protocerebntm. The protocerebrum can be subdi- vided into three regions, based on connections with periph- eral sense organs and delineation by spindle-shaped glia (Fig. 5). We refer to these regions as the dorsofrontal neuropil. optic lobe neuropil, and median protocerebral neuropil. The dorsofrontal neuropil is dorsal to the proto- cerebral commissure (Fig. 5) and receives input from the median eye. The optic lobe neuropils are located within the anterolateral extensions of the brain and are linked to more posterior regions of the protocerebrum via the optic tract (Figs. 4, 5). Each optic lobe neuropil receives input from the adjacent frontal filament and compound eye (Figs. 4. 5). The median protocerebral neuropils elongate along the antero- posterior axis of the brain (Fig. 5) and are not directly linked with peripheral sense organs. Neurites from surrounding somata project into the median protocerebral neuropils (Figs. 3B. C) and longitudinal fibers that extend from the posterior regions of these neuropils contribute to the cir- cumesophageal connectives (Fig. 5). Lateral lobes of the median protocerebral neuropil connect via the protocerebral commissure (Figs. 4. 5). The nauplins eve (= median eye) is located on the an- terodorsal margin of the brain. The nauplius eye has been studied in B. amphitrite hawaiiensis (Takenaka el ai, 1993) and in 5. balanoides and B. crenutiis (Kauri, 1961 ). In the B. amphitrite cyprid, the nauplius eye is composed of three pigment "cups" (two lateral and one ventral), with each cup containing four retinular cells. Axons from each of the three pigment cups were traced to the dorsofrontal neuropil. In one of three preparations in which these axons were traced, however, some axons appeared to bypass the dorsofrontal neuropil and contribute directly to the protocerebral com- missure. A frontal filament is attached to the anteromedial margin of each compound eye. The fine structure of the frontal filament in the nauplius of S. balanoides has been described previously (Walker, 1974). In the relaxed cyprid, the fila- ments extend beyond the carapace margin (Figs. 1A. 6C) and each contains large internal vesicles in its basal region (Fig. 6B). A frontal filament tract connects each frontal filament to its adjacent optic lobe neuropil (Figs. 5. 6B). The structure of the compound eye in B. amphitrite is consistent with that described for S. balanoides (Walley, 1969: Hallberg and Elofsson, 1983). Each eye is located within a lateral "pocket" of the cephalon and composed of radially arranged ommatidia, each with a spherical lens and underlying retinular cells (Figs. 2, 5, 6A, B). Retinular cell axons converge to form a short optic nerve (Figs. 6A. B). which emerges from the medial surface of each compound eye and projects anteriorly to the optic lobe neuropil (Figs. 5. 6A, B). The deutocerebrum. The deutocerebrum can be subdi- vided into two distinct regions, which we call the circular deutocerebral neuropils and median deutocerebral neuropils (Figs. 3B, 4A, 5). All peripheral nerves associated with the deutocerebrum travel within the antennular nerves. The circular deutocerebral neuropils are located lateral and slightly posterior to the brain-antennular nerve junction (see Fig. 3B). These neuropils are clearly delineated by glial cells and, based on their position and shape, are possible candidates for olfactory lobes. However, glomeruli that characterize the olfactory lobes in many animals (Hallberg 148 P. J. H. HARRISON AND D. C. SANDEMAN Figure 3. (A) Horizontal section through the cyprid brain. The brain is composed mainly of centrally positioned neuropil (NP) and surrounding cell somata. The anterior portion of the brain extends laterally to form the optic lobes. Also apparent are the compound eyes (CE), cement glands (CG). and cross-sections of the cement ducts (*), which extend to the adhesive discs of the antennules (see also Figure 7). OC, oil cell. (B) Longitudinal section of the cyprid brain showing clusters of somata with primary neurites (arrows) that project to the neuropil (NP). The circular deutocerebral neuropil (see also Fig. 5) is outlined. (C) Electron micrograph of the brain in near sagittal section highlighting projections of neurons (single arrow) into the central neuropil (NP) and the delineation of the neuropil by spindle-shaped glia (double arrows). ME, median eye. t'/ <;/., 1992; Hildebrand and Shepherd, 1997) were not seen in this region. Lateral lobes of the median deutocerebral neuropil are linked by the deutocerebral commissure and receive primary neurites from surrounding cell somata, par- ticularly those located ventrolaterally to this neuropil. Pos- terior projections from the median protocerebral neuropils contribute to the circumesophageal connectives and travel in bundles distinct from those associated with the median protocerebral neuropil (Fig. 5). The antennules and associated cement glands are inner- vated by the antennular nerves, which link to the deutoce- rebral neuropil (Fig. 4 A, B). Detailed morphological de- scriptions of the cyprid antennule are available for both B. ainphitrite (Clare and Nott. 1994; Glenner and H0eg, 1995) CYPR1D NEUROANATOMY 149 1 .0 urn j| Figure 4. Two distinct neuropil dmsions in the cyprid brain as seen in (A) longitudinal and (B) horizontal planes. (A) The anterior, or protocerehrul. neuropil (PNP) is associated with the optic tract (OpT); the posterior, or deutocerebral. neuropil (DNP) is associated with the antennular nerve (AnN). (B) Protocerebral and deutocerebral commissures (PC. DC respectively) are the transverse fiber tracts that link lateral lobes of these two major neuropil divisions. (C) Electron micrograph of the neuropil showing many dark clefts (e.g.. single arrow) and membrane-bound vesicles (e.g.. double arrow) indicative of synapses. and S. bukmoides (Nott and Foster, 1969). We provide a brief description here to account for the neural innervation of this appendage. The antennule. represented schematically in Figure 7, consists of four articulating segments. Segment I projects ventrally from the cephalon and attaches to the slightly longer and slender segment II. Segment III func- tions as an adhesive disc and is used for attachment to the substratum (Nott and Foster. 1969; Nott. 1969; Walker, 1971). Segment IV is the terminal segment and extends laterally from the disc. Cuticular setae project from the antennular segments, particularly from the disc (Nott and Foster, 1969; Moyse el al, 1995) and from segment IV (Nott and Foster, 1969; Gibson and Nott. 1971; Clare and Nott. 1994; Glenner and H0eg. 1995). Two large cement glands are associated with the antennules (Fig. 7). These are located within the cephalon, posterior to the compound eyes (Figs. 1, 2, 7; Walker, 1971), and ducts from these glands extend the length of the antennule to open through the adhesive disc. A muscular sac surrounds each duct ( Walley, 1969) near the base of the antennule. In addition to the cement glands, antennulary glands are present in segment II of the antennule. which are thought to mediate the con- trolled release of adhesive used for temporary attachment (Nott and Foster. 1969; Walker. 1971). The antennular ncn-e extends from the ventrolateral mar- gin of the brain to the distal region of the antennule (Fig. 7). Distally, the antennular nerve is composed almost exclu- sively of neural processes associated with the distal setae (Figs. 8, 9). The external morphology of setae on segment IV has been described previously (Clare and Nott, 1994; Glenner and H0eg, 1995). There are nine setae on the fourth segment, winch are arranged in terminal and subterminal rows (Fig. 8); their associated neural processes can be seen in cross-section of segment IV (Fig. 9A). Most neural processes in this segment are between 0.5 and 1.0 jum in diameter and contain between one and three mitochondria 150 P. J. H. HARRISON AND D. C. SANDEMAN OpN circuruesophageal \ connective Posterior Figure 5. Schematic drawing of the neuropils and fiber tracts in the cyprid brain, showing further possible subdivision. The protocerebrum includes the paired optic lobe neuropils (OLN), median protocerebral neuropils (MPN), and an unpaired dorsofrontal neuropil (DFN). Optic lobe neuropils receive input from compound eyes (CE) via the optic nerve (OpN). and from frontal filaments (FF) vi'i; the frontal filament tract (FFT). The small dorsofrontal neuropil (DFN) lies anterior to the PC and receives input via the median eye (ME). Median protocerebral neuropils are not directly linked to peripheral sense organs, but the lateral lobes are con- nected by the protocerebral commissure (PC). The deutocerebrum includes paired circular and median deutocerebral neuropils (CDN, MDN, respec- tively). Peripheral nerves from the deutocerebrum form the antennular nerve (AnN), and lateral lobes of the median deutocerebral neuropil are connected by the deutocerebral commissure (DC). Longitudinal fiber tracts extend from the posterior of both the MPN and MDN and contribute to the circumesophageal connectives. (Fig. 9B). Narrow "cilia-type" dendritic profiles (0.1-0.2 ju,m in diameter), which can be identified by a 9 X 2 + 2 microtubule arrangement, are also present (Fig. 9C). The larger processes can be traced as far as the distal portion of this segment, whereas the outer dendritic segments of smaller cilia-type processes extend into each of the four short subterminal setae (Fig. 9C). Approximately 50 /um from the brain, the antennular nerve is associated with a cluster of neurons, which we refer to as the antennular soma cluster (Fig. 10). This group of cells was referred to as the antennular "ganglion" by Walley ( 1969), who proposed that the somatu were those of motor- neurons that had migrated out from the brain. From light micrographs, the cells in this cluster appear to be bipolar, with processes extending both proximally to the brain and distallv along the antennular nerve. The somata, which measure 6-8 /nm in length, have large nuclei and a rela- tively thin layer of cytoplasm (Figs. IOB, 3C). From elec- tron micrographs, we found no evidence of branching or of synapses within the antennular soma cluster, which leads us to conclude that these cells do not form a ganglion in the usual sense. The morphology of these cells and their asso- ciation with the antennular nerve suggest that they are receptor cells and are, therefore, possible candidates for chemoreceptors or mechanoreceptors whose dendrites ex- tend to the distal setae. The antennular nerve splits midway between the anten- nular soma cluster and the brain (Fig. 7). sending a fine branch toward the cement duct. This fine branch splits again before reaching the duct, and minor branches project toward both the cement gland and the muscular sac. These branches of the antennular nerve travel adjacent to the cement duct and are difficult to trace in serial section. They are more obvious, however, during dissection of this region and will typically separate from the collecting duct following slight enzymatic treatment (0.01 mg • ml~ ' trypsin for 5 min). We were unable to trace projections of these fine branches beyond the muscular sac. The posterior ganglion and associated structures The posterior ganglion is composed largely of centrally positioned neuropil and fiber tracts surrounded by neuronal somata (Fig. 1 1 A). The somata in this region, like those in the brain, measure 4-8 ^im in diameter, contain lightly stained granular nuclei, and are gathered into clusters with neurites that project together to the central neuropil. Densely stained glial cells are present and delineate the neuropil (Fig. 1 1 A, B). The posterior ganglion is composed of several fused divisions. Longitudinal fiber tracts extend through the length of this ganglion, and individual divisions can be discerned by the presence of transverse commissures (Fig. 1 1 A). We identified six divisions in the posterior portion of this ganglion as thoracic divisions on the basis that paired nerve roots extend ventrally from each division toward the corresponding thoracic appendage (Figs. 2, 11A-C). We were unable to determine whether a seventh thoracic divi- sion, which might be expected in cirripedes (see Grygier, 1987), was present in the cyprid. Individual divisions are more difficult to distinguish in the anterior portion of the ganglion, which elongates laterally and is compressed lon- gitudinally (Fig. I IA). However, we identified three divi- sions in this region (from two of the three preparations sectioned in the horizontal plane), which might reflect the presence of the three pairs of gnathopods that can be seen with either scanning electron microscopy or light micros- copy (Fig. 12B). Thoracic appendages and the candid mini. Six pairs of thoracic appendages (thoracopods) and the paired caudal rami extend from the ventral surface of the thorax. The extrinsic muscles of thoracic appendages and the caudal rami attach dorsally in the thorax and can be seen in both TYPRID NEUROANATOMY B 151 Figure 6. Compound eyes and frontal filaments. (A) The optic nerve (OpN) connects the compound eye (CE) to the optic lobe neuropil (OLN). (B) Frontal filaments are closely associated with compound eyes, and a transverse section through the base of a frontal filament (FF) is shown. The frontal filament tract (FFT) connects the frontal filament with the optic lobe neuropil proximal to the point of entry of the optic nerve (OpN) (see Fig. 5). (C) Scanning electron micrograph of the anterior ventral surface of a cyprid in which the cephalon is extended from the bivalve carapace. Frontal filaments (FF) extend from the ventral surface of the cephalon. posterior to the antennules. horizontal and longitudinal sections (Fig. 11 A, B). Paired nerve roots to the thoracic appendages extend from each thoracic division (Fig. 1 IB, C). Paired nerve roots extend to the caudal rami, but unlike those to thoracic appendages, are not associated with an obvious ganglionic division. Instead, these nerve roots appear to extend from a terminal loop of the longitudinal fibers (Fig. 11 A, D). Oral cone. The gnathopods of the cyprid form an oral cone, which opens to the ventral surface of the cephalon (Figs. 1, 2, 12A). The cyprid does not eat. and gnathopods are rudimentary during this stage (Walley. 1969). There are no obvious nerves connecting the gnathopods to the central nervous system in B. aiuphi trite. However, the posterior ganglion extends laterally in the region adjacent to the oral cone. It is likely that the three ganglionic divisions located adjacent to the oral cone reflect the presence of three pairs of gnathopods and are, therefore, referred to as subesopha- geal divisions. Esophagus and digestive system. The digestive system of the cyprid is not fully developed (Walley, 1969). The esoph- agus has an oral opening (Fig. 12A), but we found no evidence of a rostral opening of the digestive system. In some sections the esophagus appears to be closed in the region where it passes between the cephalon and thorax (Fig. 12A, B), but it remains possible that this represents a sectioning artifact. Fine nerves can be traced from the dorsal surface of the subesophageal ganglionic divisions to the esophagus and midgut (Fig. 12B). These nerves are most obvious when they converge to pass between the cephalon and thorax, but they disperse among the cells surrounding the midgut (Fig. 12C). Discussion Our results show that the cypris larva of B. amphitrite has a well-developed nervous system that, in spite of being relatively small, contains the full complement of neural elements necessary for mediating complex interactions with 152 P. J. H. HARRISON AND D. C. SANDEMAN muscular sac sub-terminal sensilla antennular soma cluster (ASC) cement gland terminal sensilla Figure 7. (A) Schematic drawing of the amennule to show the extension of the antennular nerve and its association with the collecting duct of the cement gland. The antennule has tour segments (I-IV). The antennular nerve consists of neural processes associated with the distal setae (innervation of setae not drawn here, but see Figs. 8, 9). The antennular nerve is also expected to contain the motor neurons to the antennular musculature. The antennular soma cluster (ASC), located near the base of the antennule, contains bipolar cells, which are candidates for receptor cell somata whose dendrites innervate the distal setae (discussed in text). Midway between the ASC and the brain, a small branch of the antennular nerve extends to the muscular sac, a node of muscle surrounding the collecting duct of the cement gland, and a separate branch extends toward a cement gland. the environment. The presence of regionalized neuropils. some of which clearly receive input from peripheral sensory structures, suggests a level of neural integration that goes beyond simple reflexive responses. Observations of complex behavior displayed by cyprids during settlement support the claim that the nervous system has the capacity for more than simple reflex responses. For example, cyprids of S. haltinoiiles settle gregariously in response to a proteinaceous cue associated with the cuticle of conspecifics (Crisp and Meadows, 1962; Gabbott and Larman, 1987). Upon encountering this cue, however, the cyprids' response is not indiscriminate. For example, in "favored" areas (those containing conspecifics), individual cyprids will still conduct a meticulous inspection phase and reject the substratum if the immediate barnacle density is too high or if the surface topography is inadequate (Crisp, 1961). The cyprids' need to settle is reflected by the fact that the barnacle nervous system is most "complete" during the cyprid stage. The cyprid has a well-developed brain and a large investment in cephalic sense organs, whereas the brain is greatly reduced in the naupliar stages and almost com- pletely absent in the adult barnacle (Walley. 1969). The "upgrade" from the nauplius to the cyprid nervous system is CYPRID NEUROANATOMY 153 Figure 8. (A) The setae on the fourth antennular segment (IV) are arranged in terminal and subterminal rows. (A) The four subterminal setae (sts) do not articulate at the base. These are short and taper toward the tip with a general morphology similar to that of the aesthetasc (olfactory) sensillu described in other crustaceans. The terminal row consists of two plumose setae (a. h). a short, sickle-shaped seta (c). a longer "sculptured" seta with many cuticular ridges along its length (d), and a shorter unsculp- tured seta (e). Terminology following Clare and Nott (1994) and Glenner and Hoeg ( 1995). (B) Higher power micrograph highlighting the articulat- ing bases of two of the terminal setae (b. d). (C) High power of the distal tips of setae a, b. and d. The setules of a and b are folded back on themselves, and seta d appears open-ended. 9A. 9C, 9D in (A) refer to cross-sectional planes shown in Figure 9. consistent with the cyprids' need to detect and actively respond to settlement cues. The restructuring to the adult nervous system (Walley, 1969). in which most of the ante- rior neuropil regions and cephalic sensory structures that we describe here degenerate, is presumably an adaptation to sedentary life. The nen'ous svstem and associated structure* Neural input from cephalic sense organs is structurally organized in discrete neuropils within the cyprid brain (see Fig. 5). The cephalic sensory input in the cyprid can be summarized as follows: primary nerves from the median eye project to the dorsofrontal neuropil; primary nerves from each compound eye form an optic tract and project to the optic lobe; primary nerves from each frontal filament form a frontal filament tract and project also to the optic lobe: and primary nerves that innervate setae on the anten- nule project to the deutocerebrum. Eyes and frontal filaments. Optic nerves connect the compound eyes with their adjacent optic lobe neuropils (Figs. 5. 6A, B). Under the light microscope, this neuro- pil appears to be unstructured, lacking the geometrically ordered segments seen in the optic lobes of decapods (Dahl, 1965). Nevertheless, compound eyes are morpho- logically well developed in the cyprid (Hallberg and Elofsson, 1983). and the fact that these eyes are present only during the cyprid stage is suggestive of a significant role in settlement. The exact function of these eyes is not yet known. Crisp (1955) argued, on the basis of the simple structure of these eyes, that image formation was unlikely, but suggested a role in mediating responses to fine-scale topographic features such as cracks and grooves. It is likely that compound eyes enable the de- tection of reflected light levels (Yule and Walker. 1984), thereby mediating light-guidance behavior (Barnes et ai, 1951 ). However, this function might equally be attributed to the median eye. The frontal filament tract connects each of the frontal filaments with the adjacent optic lobe (Fig. 6B). proximal to the point of entry of the optic nerve (Figs. 5. 6A-B). The optic lobe of decapods includes neuropil divisions of the lamina ganglionaris, external and internal medullae. terminal medulla, and hemiellipsoid body (Dahl, 1965). We were unable to identify these divisions in the cyprid and have therefore chosen to use the general term of optic lobe. The exact nature of the frontal filaments and their function remains a contentious issue. Frontal organs are found in many Crustacea but, to date, frontal filaments of barnacle larvae have been considered only as pressure sensors on the basis of their suspected homology with the SPX organs (or organ of Bellonci) of Pericarida (Kauri. 1964; Walker, 1974). Antennules. The antennules play a role as attachment organs during exploration and settlement (Nott and Foster. 1969) and have been implicated in the detection of chemical and physical cues (Nott and Foster, 1969; Walker, 1971; Clare et ai, 1994; Clare and Nott. 1994; Walker, 1995). The antennular nerve extends through the length of the anten- nule (Fig. IB) and during dissection can be dissected into smaller individual bundles. Recently, electrical activity has been recorded from this nerve in response to chemical and mechanical stimulation of the distal segments of the anten- nule (Harrison, 1998). The setae on the fourth antennular segment, and the nerves that innervate them, vary considerably in their morphology (Figs. 7-9). The functional properties of individual setae on this segment are not known. How- ever, the innervation of segment IV suggests that these are sensilla. and some appear to be morphologically similar to chemoreceptors and mechanoreceptors of other Crustacea (Nott and Foster, 1969; Bush and Laverack. 1982; Heimann, 1984; Schmidt, 1989; Clare and Nott, 1994). The external morphology of the four subterminal setae, for example, is similar to that of the olfactory 154 P. J. H. HARRISON AND D. C. SANDEMAN A Figure 9. Innervation of antennular segment IV (see Fig. SA for orientation). (A) The dendritic profiles in segment IV vary in diameter. Most profiles are between 0.5 and 1.0 /urn in diameter and contain between one and three mitochondria (single arrow). Smaller-diameter processes (typically 0.1-0.3 /im in diameter) are also present (double arrows), and in some cases a 9 X 2 + 2 pattern of microtubules is apparent (*). (B) Enlargement of part of (A) to highlight the variation in the number of mitochondria in neural profiles. (C) Transverse section through a single subterminal seta showing the outer dendritic segments (ods) that extend into each of the subterminal setae. At this level, the dendrites are contained within an electron-dense tube (arrow), which is encircled by two glial cells. (D) A transverse section through seta e (see Fig. 8a) reveals densely stained peripheral structures (arrow) that possibly support this seta. Neural processes were not observed to extend into this seta, nor into anv of the other terminal setae. aesthetascs of Decapoda (Hallberg et al.. 1992; Clare and Nott, 1994). Furthermore, our results show that up to six outer dendritic segments (each 0.1-0.2 ;um in diameter) project into the lumen of each subterminal seta (Fig. 9C). The outer dendritic segments are contained within a central cavity bordered by electron-dense material and a pair of ensheathing cells. This arrangement is similar to that reported for olfactory aesthetascs of crayfish (Tier- ney et til., 1986). It is generally accepted that the setae on the antennule include both chemoreceptors and mechanoreceptors (Clare and Nott, 1994; Clare, 1995). but the location of the somata of receptor cells has not been shown. In an effort to locate receptor cell somata. we traced serial sections of the antennule and were led to the bundle of cells that form the antennular soma cluster (Fig. 10). The cells in this bundle are 6-8 /u,m in length. 4-6 jum in width, and are located about 150 jum from the fourth segment of the antennule. Interestingly, the size and shape of these soma is again consistent with olfactory receptor neurons of many decapods (Laverack and Ardill. 1965; Snow, 1973; Tierney ct til.. 1986; Hallberg et al., 1992). These cells, however, are located at the base of the antennule in the cyprid and not. as in decapods, at the base of the sensilla. The antennules are used for temporary attachment dur- ing surface exploration, which enables the cyprid to "walk" across the substratum (Nott and Foster. 1969; Walker. 1971). This involves the controlled release of cement from the adhesive disc via the antennulary glands (Nott and Foster. 1969; Walker, 1971; Okano et til., 1996) and the coordination of motor activity. Motor neurons to the antennular musculature are expected to travel in the main branch of the antennular nerve. The CYPRID NEUROANATOMY 155 Figure 10. Longitudinal sections of the untennular soma cluster (ASC). (A) Light micrograph of the ASC shows its position relative to the brain, and association with the antennular nerve (AnN). The bipolar cells in this cluster are likely candidates for receptor cells that project to distal setae. (B) Electron micrographs of the ASC do not reveal synapses in this region, as would be expected for motor neurons, suggesting that these cells do not form a ganglion of antennular motorneurons as previously suggested (discussed in text). location of efferent cell soma within the central nervous system is not known, but cells located ventrolateral to the median protocerebral neuropil that project anteriorly in the deutocerebrum are possible candidates (see Fig. 3B). The location of cells that control the release from the antennular glands for temporary attachment and the ex- plosive release from cement glands for permanent attach- ment remains to be shown. Thoracic appendages and caudal mini. The nerves that project to the thoracic appendages are, together witli the antennular nerve, the most obvious peripheral extensions from the central nervous system. Thoracic appendages are used for swimming and bear many setae (Glenner and H0eg, 1995). These appendages, however, serve a natatory func- tion, and it is not known whether the setae play a sensory role. Setae are also present on the caudal rami (Walker and Lee, 1976; Glenner and H0eg, 1995), and behavioral obser- vations suggest that caudal rami might play a sensory role (Crisp and Barnes, 1954). The nervous system and settlement Cyprids settle in response to a range of environmental cues. It follows that the cyprid nervous system must sort and process input from various sense organs, and coor- dinate an appropriate behavioral response. We have traced neural connections between the central nervous system and many of the peripheral sense organs, but connections to the lattice organs (Jensen et al.. 1994b; H0eg et al.. 1998) and other sensory structures on the carapace remain to be shown. The small size of the cyprid raises questions about the behavioral capacity of this 156 P. J. H, HARRISON AND D. C. SANDEMAN 20um Figure II. Light micrographs of the posterior ganglion. (A) Horizontal section showing regions of neuropil (NP), parallel fiber tracts (FT) and transverse fiber tracts (*). The transverse fibers distinguish individual divisions of this ganglion. The parallel fiber tracts end in a terminal loop (arrow). Extrinsic muscles of the thoracic appendages are seen in cross-section (double arrows). (B) A longitudinal section through the posterior ganglion. Paired nerve roots (*) extend from the ventral portion of each ganglionic division to the corresponding thoracic appendage. Extrinsic muscles of the thoracic appendages are seen (arrows). (C) Higher power of a thoracic nerve root (*) extending venlrally from the posterior ganglion. (Dl Paired nerve roots (*) extend from the terminal loop of the transverse fiber tract to the caudal rami. organism (Rittschof et «/., 1998). However, the large investment in sensory structures, each of which links to a discrete neuropil within the brain, suggests that the cyp- rid nervous system has the capacity for a relatively so- phisticated level of neural processing. Acknowledgments We thank Renate Sandeman for discussion and advice during the course of this work and particularly for advising on many of the techniques used. We also thank Holly Gate CYPRID NEUROANATOMY 157 20|am Figure 12. Light micrographs showing possible innervation of the esophagus and gut. (A-B) The esophagus of the cyprid opens on the ventral surface through the oral cone (consisting of vestigial mouthpart appendages). In these sections the esophagus appears to be closed in the region where it passes between the cephalon and thorax. Nerve roots arising from the anterior ganglionic divisions can be traced along the length of the esophagus and extend toward the gut (arrows). Innervation of mouthpart appendages was not observed. (C) The neurites of ventrally located cells project to the neuropil of anterior ganglionic divisions, but few nerves could be traced out of this region. and two anonymous reviewers, whose comprehensive feed- back was used to significantly improve the manuscript. Literature Cited Anderson, D. T. 1994. Barnacles. Structure. Function. Development and Evolution. Chapman & Hall. London. 357 pages. Barnes, H., D. J. Crisp, and H. T. Powell. 1951. Observations on the orientation of some species of barnacles. J. Aiiim. Ecol. 20: 227-241. Bush, B. M. H., and M. S. Laverack. 1982. Mechanoreception. Pp. 399 — 168 in The Biologv of Crustacea. Vol. 3. Neurobiology, Structure and Function. 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GREENBERG2 1 Department of Biological Sciences, University of Southern Maine, Portland, Maine 04104; and - the Whitney Laboratory of the University of Florida, 9505 Ocean Shore Blvd., St. Augustine, Florida 32086-8623 Abstract. The activities of both the lateral and frontal cilia of Mercenaria mercenaria were unaffected, either by the two endogenous SCP-related peptides AMSFYFPRM- amide and YFAFPRQamide, or by FMRFamide (all at 10~h M). Dopamine (DA) inhibited the lateral cilia; the mean ECSO was 2 X 1(T6 M. The peptide YFAFPRQamide— but neither AMSFYFPRMamide nor FMRFamide — antago- nized the inhibition induced by DA; this effect was depen- dent on both time and dose. At a DA concentration of 5 X 1(T7 M, the effect of YFAFPRQamide appeared within 20 min and became maximal within 40-60 min; the mean EC50 at these times was 4.7 X 10" " M. If the concentration of DA was increased to 10~6 M, the maximal effect of the peptide was delayed to 50 min, and the mean EC50 in- creased to 1.1 X 10~7 M. Particle transport by the frontal cilia was inhibited by 5-hydroxytryptamine (5HT); the mean EC,,, was 5.7 X 10~7 M. Again, only YFAFPRQ- amide had an antagonistic effect on the 5HT-induced inhi- bition. At a 5HT concentration of 10~6 M, the effects of YFAFPRQamide did not appear until 45 min; the mean EC50 was 10~6 M. When radioimmunoassayed with an SCP antiserum, the elution profile of a gill extract overlapped those of the SCP-related peptides that had previously been identified in extracts of whole animals. These data suggest that all three SCP analogs occur in the gill. Immunohisto- chemistry of the gill, carried out with a monoclonal anti- body raised to SCPB, stained many varicose neuronal fibers. Received 7 April 1999; accepted 22 July 1999. * To whom correspondence should be addressed. Department of Bio- logical Sciences, University of Southern Maine. P.O. Box 9300, Portland. ME 04104-9300. E-mail: gainey@usm.maine.edu Most of these were associated with the gill musculature, but a sparse innervation of the filaments underlying the cilia was also observed. Some fluorescent nerve cell bodies were also seen in the gill tissue. Our results are consistent with the hypothesis that YFAFPRQamide modulates branchial activities — muscular as well as ciliary — that are associated with feeding. Introduction Molluscs are ciliary organisms; they are among the larg- est animals, yet cilia perform mechanical functions that, in many other taxa, are carried out primarily by muscles. For example, cilia are responsible for locomotion in gastropods as large as the lightning whelk Busycon contrarium and the helmet conch Cassis tiiberosa (see Gainey, 1976; and Miller. 1974. respectively). Cilia are particularly well known for generating the currents that provide for respira- tion and feeding in all bivalves, except the Septibranchia. These currents are substantial; clearance rates generated by cilia in the American oyster Crassostrea virginica are as high as 24 to 27 l/li (Loosanoff and Nomeijko, 1946; Col- lier, 1959 [both cited in Foster-Smith, 1975]). The ctenidial water currents are created by the lateral cilia (Purchon, 1968; Morton, 1983), although the abfrontal cilia may contribute between 30% and 40% of the flow in Myti- lus edulis (Jones and Richards, 1993). The control of lateral ciliary activity, especially in Mytilus, has been studied for nearly a century (early work reviewed in Aiello, 1960; Paparo, 1972, 1985). In brief, the ciliated cells of bivalve gills are electrically coupled (Motokawa and Satir. 1975; Murakami and Machemer, 1982; Saimi et al, 1983b; Stom- 159 160 L. F, GAINEY, JR. ET AL, me], I984a), and branches of the branchial nerve run be- neath the lateral and frontal ciliated cells (Aiello and Guideri, 1965; Paparo, 1972; Owen, 1974; Aiello, 1979). Apparently not all of the ciliated cells are innervated, but those that do receive neural input are reported to act as pacemakers (Paparo. 1972). Both 5-hydroxytryptamine (5HT) and dopamine (DA) have been localized in the branchial nerves of M\tilu.\ (Paparo and Finch. 1972; Stefano and Aiello, 1975). More- over, electrical stimulation of either the cerebrovisceral connective or the branchial nerve at a stimulus frequency of 1 0 Hz increased the rate of beat of the lateral cilia, whereas stimulation at a frequency of 20 Hz decreased the rate. These excitatory and inhibitory effects of electrical stimu- lation were blocked, respectively, by serotonergic and do- paminergic antagonists (Catapane et ai, 1978, 1979; Cata- pane, 1983). Applied exogenously to isolated gills. 5HT stimulates the lateral cilia of all bivalves studied to date, including those of Mercenaria mercenaria (see Aiello, 1962, 1970, 1990; Paparo. 1972; Motokawa and Satir. 1975: Catapane. 1983). In contrast to the effect of 5HT, the response of lateral cilia to DA is variable. For example, the lateral cilia ofMytilus edulis, Crassostrea virginica (Paparo and Aiello, 1970; Catapane, 1983; Paparo, 1985), Ostrea edulis, Mercenaria mercenaria, and Modiolus modiolus (Gainey and Shumway, 1991 ) are inhibited by DA; but the lateral cilia of Geukensia (= Modiolus) demissa (Catapane, 1983). Argopecten irradians, and Mya arenaria (Gainey and Shumway. 1991 ) are unaffected. In summary, both 5HT and DA are present in the gills of at least some bivalves, and they appear to serve as endogenous transmitters regulating, in part, the activity of the lateral cilia. Dose-response curves for 5HT and DA (see Catapane. 1983) show that the lateral cilia of Mytilns have a maximal beat frequency of about 25 beats/s, and that synchronous beating is lost below about 10 beats/s. Between these nar- row limits (i.e.. 25 and 10 beats/s), the cilia respond in a graded manner, both to stimulation by 5HT and to inhibition by DA. At the lower limit (10 beats/s), these compounds seem to be activating a simple on-off switch. That such a switch controls pumping in intact animals has yet to be demonstrated unequivocally (Stefano et ai. 1977; Jorgen- sen. 1989; Jones and Richards. 1993). In contrast to the lateral cilia, which transport water, the frontal cilia receive material that has been retained by the branchial filter and transport it to the food grooves; there it is packaged in mucus and carried to the labial palps (Pur- chon. 1968; Morton. 1983; Murakami. 1989). The frontal cilia are therefore intimately involved in feeding, and their activity is correlated with the rate of mucus secretion (Ai- ello, 1979). Beyond that generality, the pharmacology and control of the frontal cilia iv poorly understood (reviewed by Aiello, 1990). The inconsistency between the raime of clearance rates in intact bivalves and the pharmacology of isolated gill cilia, as well as the distinct functions of cilia in different tracts, suggests that ciliary activity is probably not controlled by motoneurons that release only dopamine or serotonin. Stud- ies of a pair of neurons in the pedal ganglia of the nudi- branch Tritonia diomedea clearly show that, in this mollusc, peptides are also involved. These neurons innervate the locomotory cilia on the foot of Tritonia, augment the fre- quency of ciliary beat when stimulated, and synthesize and store a family of three pedal peptides (Peps). Moreover, the action of these peptides mimics neuronal stimulation by increasing the frequency of ciliary beating (reviewed by Willows et ui, 1997). The beat frequency of vertebrate cilia, particularly those of airway epithelia, are also regulated by neuropeptides. including Substance P (Lindberg and Mercke, 1986; Lindberg et til., 1986; Lindberg and Dolata, 1993; Aiello et ai, 1991 ); vasoactive intestinal polypeptide (VIP) (Lindberg et ul, 1988); neuropeptide Y (NPY) (Cervin et ul.. 1991; Wong et ul.. 1998); endothelin (Tamaoki et ul., 1991); and vasopressin (Tamaoki et til.. 1998). Among bivalved molluscs, three members of the SCP- related family of peptides have been isolated from the quahog Mercenaria mercenaria: lAMSFYFPRMamide. AMSFYFPRMamide, and YFAFPRQamide; the second peptide is likely a degradation product of the first. Further- more, high levels of these peptides occur in the gills, and SCP-related immunoreactivity has been localized to neural fibers in the gill. But though these peptides affect gut motility in the clam (Candelario-Martinez et ai, 1993), their effects upon ciliary activity in the gill have not yet been tested. We have, therefore, examined two of these SCPs — YFAFPRQamide and AMSFYFPRMamide— as well as DA, 5HT, and another neuropeptide. FMRFamide, for their actions upon both the lateral and frontal gill cilia of Mer- cenaria. The results indicate that one of the peptides, YFAFPRQamide, modulates the effects of the amines. Pre- liminary results of this study were presented to the Society for Integrative and Comparative Biology (Gainey et ai. 1997). Materials and Methods Animals Quahogs (Mercenaria mercenaria L.) were obtained from Poquoson and Wachapreague, Virginia. The animals were held at I()"C in natural seawater (309M on a 12 h light/dark cycle. Individuals were held a minimum of 3 days prior to use. The preparation Gills were dissected away from the body wall distal to the visceral ganglia and were then separated into demibranchs. The dissection caused the beatint: of the lateral cilia to cease MODULATION OF CILIARY ACTIVITY 161 for an hour or more. But once the beating had resumed, the frequency remained unchanged for up to 24 h. Therefore, the gills were excised between 4 and 15 h before an exper- iment. Dorsoventral strips about 1 cm wide were cut from the isolated demibranchs and pinned to strips of rubber band that had been glued with rubber cement to the bottom of petri dishes (4.7 cm diameter). The dishes were filled with 5 ml of artificial seawater (recipe in Welsh et «/.. 1968). Drugs Peptides were synthesized at the Protein Chemistry Core Facility of the Interdisciplinary Center for Biotechnology Research at the University of Florida, Gainesville. Dopa- mine (DA) and 5-hydroxytryptamine (5HT) were purchased from Sigma Chemical, St. Louis, Missouri. Responses of the lateral cilia The activity of the lateral cilia was measured as follows: Isolated, pinned-out strips of gill were placed on the stage of a compound microscope and observed at a magnification of 100X. The substage illuminator on the microscope was replaced with a mirror, and the rate of beating of the lateral cilia was determined by their synchrony with a Pasco Sf- 921 1 strobe light. Details of the measurement procedure are described in Gainey and Shumway (1991). At the outset of each experiment, we would locate an area of the gill with well-defined metachronal waves and with frequencies between 12 and 25 beats/s. Once the initial rate was measured, the gill was not moved, and the same patch of cilia was used for all subsequent measurements. In all but the initial set of experiments on the effects of the peptides alone, two pieces of gill were used on separate microscopes, with one of these pieces serving as a control. Larger quahogs (7 to 9 cm long) had lateral cilia that were consistently less sensitive to DA than those of smaller quahogs (5 to 7 cm). Moreover, the sensitivity of the lateral cilia of the smaller quahogs followed a seasonal pattern; they were less sensitive to DA from April to June. There- fore, all of the experiments reported here were done with gills from smaller animals and were carried out from June to April. Effects of peptides: ( 1 ) Stimulation. Freshly dissected gill strips showing no lateral ciliary activity were exposed to one of the peptides at 10~6 M; controls were untreated. 5HT at 10~6 M was used as a positive control because it excites quiescent lateral cilia of Mercenaria mercenariu (Aiello, 1970). The rate of beat of the lateral cilia on the treated and control strips was measured hourly for 3 h. In a separate observation, we examined 10 areas on each strip of gill for the presence or absence of metachronal waves; the percent- age of areas with metachronal waves (percent activity) was taken as an estimate of the ciliary activity of the strip. The data on rate and on percent activity were analyzed with ANCOVA with time as a covariate; the analysis was per- formed with the general linear models (GLM) procedure in SAS, version 6. Effects of peptides: (2) Inhibition. Isolated strips of gill with active lateral cilia were exposed to one of the peptides at 10~6 M: controls were untreated. Measurements were made every 2 min for the first 10 min and then at 20. 40, and 60 min. The effects of the peptides on the rate of ciliary beating were evaluated with a two-way ANOVA with treat- ment, time, and treatment*time as factors; the analysis was performed with the GLM procedure in SAS. Effects of peptides and DA. Previous studies have shown that 10' 4 M DA will, within several minutes, completely arrest the lateral cilia of Mercenaria (Gainey and Shumway, 1991). This effect is temporary because DA slowly oxi- dizes, and the cilia eventually return to their initial rate of beating. To assess the effects of the peptides on this DA arrest, we exposed isolated demibranch strips to concentra- tions of 10~6 M of each peptide; 10 min later, the same demibranch was exposed to 10~4 M DA. Controls were exposed only to 10~4 M DA. The activity of the lateral cilia was recorded every 2 min until it returned to the initial rate. In some instances, the ciliary beating on one of the gill strips did not return to its initial rate before the end of the exper- iment; data of this type were designated censored. The results were analyzed with the Wilcoxon test with the lifetest procedure in SAS; this program adjusts for censored data. Dose-dependent effects. In these experiments, two strips taken from the same demibranch were pinned out and observed with separate microscopes. The initial rate of ciliary beating of both strips was then determined. There- after, the measurement of ciliary activity at any time was expressed as a fraction of the initial rate; i.e.. the fractional rate of beat. After a drug of interest was applied, the fractional rate of the treated strip was corrected by subtrac- tion of the fractional rate of the control strip. This fractional difference was taken as the measure of peptide effect and was used as the ordinate on dose-response curves. ( 1 ) Dopamine. In these experiments, oxidation of DA was retarded with an ascorbic acid buffer as described by Malanga (1975a). DA was added to the treated strip; the control strip was untreated; and the rate of beat of both strips was determined every 10 min for 1 h. Each pair of gill strips was used to measure only one dose of DA. Because DA is inhibitory, the value of the fractional difference becomes larger and more negative with dose. Therefore, to make the DA dose-response plots more comprehensible, the effect was expressed as the adjusted fractional difference: 1 1 + (fractional ratetreated - fractional rateconm,,)]. (2) YFAFPRQamide. After the measurement of initial rate, a dose of peptide was added to the treatment strip. Ten minutes later, DA (either 5 X 10~7 A/ or 10~6M) was added to both the treatment and control gills. Thereafter, rates 162 L. F. GAINEY, JR. ET AL. were measured every 10 min for 1 h. The fractional differ- ence was used as the measure of effect in the dose-response curves; notice that when the response of peptide is maximal, no inhibition by DA is observable, so the fractional rate of the control strip approximates zero. Responses of the frontal cilia The activity of the frontal cilia was measured as follows: Isolated, pinned-out strips of gill were observed at a mag- nification of 100X with a compound microscope, and the activity of the frontal cilia was determined by the rate of transport of polystyrene microspheres (diameter, 0.85-1.0 jiun; Polysciences, Inc., Warrington, Pennsylvania). The time (in seconds) required for these particles to travel 0.5 mm was measured with a stopwatch and an ocular micro- meter. Particle transport rates (mm/s) were expressed as a fraction of the initial rate. During the experiments on pep- tides, five readings were taken on each gill strip at each time. But during the experiments on the effects of the peptides and 5HT, three readings were made at each time. Once the initial rate was measured, the gill strips were not moved, and the same gill filaments were used for all sub- sequent measurements. Effects of peptides. Isolated strips of gill were exposed to one of the peptides at 10~6 M; controls were untreated. Particle transport rates were measured every 5 min for 25 min. Initial analysis of these data indicated a positive cor- relation between the standard deviation and the mean of the fractional initial rate. Therefore, the data were transformed with natural logarithms, which removed this correlation. The effects of the peptides on the frontal cilia, as well as the effects of the peptides plus 5HT, were evaluated using repeated measures ANOVA; the analyses were performed using the GLM procedure in SAS. Effects of peptides with 5-hydroxytryptamine. We found that 5HT inhibits the rate of particle transport by the frontal cilia. To assess the effects of the SCPs on this inhibition, we first exposed isolated strips of gill to the SCPs at 10~h M: 10 min later, the same gill strip was exposed to 10~A M 5HT. Control strips were exposed only to 10~6 M 5HT. Particle transport rates were measured every 15 min for I h. Dose-dependent effects. We followed almost the same protocols and analyses that were used to examine the effects of DA and of YFAFPRQamide plus DA on the lateral cilia. The exceptions were that (1) readings were taken every 15 min for I h; and (2) three replicate readings on each gill strip at each time were averaged, and the average rates were expressed as a u lion of the average initial rate of each strip. In the experiments on the effects of YFAFPRQamide plus 5HT. the concen ration of 5HT was 10 '' M. Regression cnuilvses, significance levels All dose-response curves, and the concentrations of ago- nists giving half-maximal responses (ECM1), were estimated, at each time, from a logistic model (response =: !/(! + e<(3<>+j3i' g dose)^ wjt|1 a nonijnear regression procedure (Nlin) in SAS. E tests were used to compare the regression lines using a general linear test approach (Bates and Watts, 1988; Neter el ai, 1990). In most instances, means are reported with their standard errors and sample sizes. All test statistics, including ANCOVA and ANOVAs. were consid- ered significant at probabilities less than 0.05. Detection of SCPs in chini i>ill Radioimmunoassay of a fractionated extract. In 1993, Candelario-Martinez et al. tabulated the distribution of SCP-related immunoreactivity among the tissues of M. nter- cenaria (see their table I). In this paper, we present the unpublished immunoreactive profile of the SCPs in gill, which were obtained as follows. Gills from 20 animals were extracted in acetone. The extract was evaporated, and the aqueous portion was loaded onto a Prep- 10 Octyl column ( 10 X 100 mm. 4 ml/min) and eluted with a gradient of acetonitrile ( 16%- 40% over 30 min) in water with 0.1% trifluoroacetic acid. Fractions were collected every half minute and analyzed by radioimmuno- assay; elution patterns were plotted from these data. Details of the fractionation and the assay are set out in Candelario- Martinez et ul. (1993). Immunohistochemistry. Small, rectangular pieces of tis- sue were cut from the outer demibranchs of several clams; the samples usually included the ventral edge of the gill and were 2-3 mm wide and 3-5 mm high. A few minutes after dissection, the tissues were fixed in a solution of paraform- aldehyde, prepared freshly as follows. A solution of para- formaldehyde (4 g in 45 ml distilled water) was heated at 60"C for 10 min, clarified by the addition of I N NaOH. brought to a final volume of 50 ml, and cooled on ice for about 20 min. Thereafter. 50 ml of 0.2 M sodium potassium phosphate buffer (SPB) was added, together with 15 g of sucrose to approximate the osmolality of seawater. The tissues were left in this fixative overnight at 4UC. After fixation, the tissues were rinsed twice (5 min each) with Tris buffered saline (TBS; pH 7.4), and then placed in 30% sucrose/PBS and left overnight at 4°C. The tissues were then embedded in Tissue Tek O.C.T., frozen, and sectioned (10 /u,m). The sections were collected onto gela- tin-coated slides, and stored at -80°C for at least 24 h prior to staining. The sections were preincubated for 30 min at 37°C in TBS containing 0.1% Triton X-100 and 2% normal goat serum. The preincubation medium was then poured off and replaced with the primary antibody — a monoclonal raised to SCPB (Masinovsky et ul.. 1988)— which was diluted 1:100 MODULATION OF CILIARY ACTIVITY 163 in the preincubation medium. After 4 h in the primary antibody at room temperature, the sections were rinsed three times (5 min each) in TBS. and secondary antibody — fluoresceine isothiocyanate-conjugated goat anti-mouse IgG (FITC-GAM IgG) — was then applied to the sections; incu- bation continued for 2 h, at room temperature, in the dark. The sections were then washed once for 5 min in TBS containing 10 ju,g/ml of 4',6-diamidino-2-phenylindole (DAPI), an ultraviolet-excitable, nucleic acid-binding dye. The sections were washed twice more (5 min each) in TBS, and coverslips were applied; the mounting medium was 60% glycerol/TBS containing p-phenylenediamine (PPD). Controls were treated as described above, except that the primary antibody, before being applied to the sections, was incubated overnight, at 4°C, on a rotating shaker, with either YFAFPRQamide or AMSFYFPRMamide (10~3 M). Micrographs were generated with a Leica/Leitz DMRB microscope equipped with filters that allow the mutually exclusive visualization of fluorochrome and DAPI staining. Digital images were gathered with a Humumatsu color chilled 3CCD camera (C5810) and were prepared for print- ing with Adobe Photoshop. Results Lateral cilia Peptides. Analysis of preliminary experiments on gill strips exposed to either AMSFYFPRMamide, YFAFPRQ- amide, or FMRFamide (all at 10~6 M) revealed that none of the peptides had any significant stimulatory or inhibitory effect upon the activity of the lateral cilia: stimulation (ciliary rate), F(3 liy, = 0.85, P = 0.47; stimulation (percent activity), FOJ9> = 0.37, P = 0.78 (Table I); inhibition (ciliary rate), F(3^s) = 0-26> P = 0.85. In the experiment on the inhibition of spontaneous ciliary activity, the mean rate of beat of the control cilia, as well as those treated with any Table I Responses of quiescent lateral cilia exposed to peptides and 5HT at 1Q-" M Treatment Frequency (beats/s) SE % activity SE Control 11 1. 36 5.5 6 AMSFYFPRMa 12 1. 31 5.5 6 YFAFPRQa 10 [. 30 5.5 6 FMRFamide 12 1 30 5.5 6 5HT* 12 1. 57** 5.8 6 Controls were untreated; n = number of gill-strip preparations. Re- sponses are frequency (beats/s); or as the percent occurrence of metachro- nal waves in 10 separate areas of each gill (% activity). The data are all expressed as least square means. * Excluded from the ANCOVA in text. ** Significantly greater (P < 0.05) than the control and the peptides. Table II Comparison t>f int'iui tinie\ to recover}' (—SE) for lateral cilia exposed siimiltaneousl\ to peptides (10"'' M) and DA (10 J Mj. and for lateral cilia exposed onlv lo DA I/O J M) Recovery time Treatment Treated (mm) Control (min) P** AMSFYFPRMu 62 ± 9.26 60 ± 9.44 5 0.60 YFAFPRQa 43 ± 9.04 64 ± 8.19 6 0.02 FMRFamide 69 ± 9.16 62 ± 5.36 6 0.93 n = number of gill strip preparations. ** P values were generated with a Wilcoxan test. of the three peptides, was 25 beats/s (;; = 9 gills for each treatment). Peptides and dopaminc. To determine whether the pep- tides might alter the activity of lateral cilia inhibited by DA, we exposed isolated gill strips to individual peptides at 10 M, and 10 min later to l(r4 M DA. Control strips were exposed only to DA. An ascorbic acid buffer was omitted in these experiments, so DA oxidized and the ciliary beat recovered. The recovery times of lateral cilia exposed, at first, to either AMSFYFPRMamide or FMRFamide, and then to DA, were not significantly different from those of the controls (DA only. Table II). In contrast, the lateral cilia of gill strips exposed to YFAFPRQamide and then to DA returned to their initial rates within 42 ± 9.0 min, whereas the DA controls required 64 ± 8.2 min to return to their initial rates; these times are significantly different (Table II). Dose-dependent effects. The adjusted fractional differ- ences, measured at several times, were plotted against the log of the DA concentration, and the family of calculated regression lines is set out in Figure 1A. This graph shows that the effects of DA appear within 10 min of the treatment and remain constant for 1 h; there is no statistical difference among the regression lines plotted in Figure 1A (F(1(U2()) = 0.22, P = 0.99). The mean ECSO, as estimated from the regression parameters, is 2.0 X 10~6 M (±6.9 X 10"s M). But the dose-response curves are very steep; the change from 90% to 10% maximal activity is effected by an in- crease of only half a log unit in the concentration of DA (3-4 JJ.M). Moreover, Figure IB shows that the response is essentially biphasic; i.e., the cilia are either beating or not at a DA concentration of about 3 y.M. The dose-dependent effects of YFAFPRQamide on DA- treated cilia were studied on gills exposed to 5 X 10~7 M DA. This concentration of the amine was chosen because it was predicted (from the dose-response regression equation) to inhibit the cilia by 17% of their initial rate; thus the peptide could, in theory, either potentiate or inhibit the effects of DA. The actual response of the control gills to DA was quite variable, ranging from 10% to 100% inhibition of the original rate, but YFAFPRQamide always had an 164 L. F. GAINEY. JR. ET AL. antagonistic effect on the action of DA. That is, gill strips treated with varying concentrations of the peptide and 5 • 1CF7 M DA were inhibited less than gill strips exposed to DA alone. The threshold for the effect of YFAFPRQamide was about 5 X 10 ~ '" M. The maximal response (i.e., complete block of inhibition) was produced by about 1CTS M. Moreover, the antagonistic effects of the peptide were time dependent. A set of dose-response regression lines produced at 10-min intervals shows that the effects of the peptide began to appear within 20 min after the addition of DA. The regression lines from 40 to 60 min are not statis- tically different (F(4.44) = 0.71. P > 0.05; Fig. 2A). U 1.0 O 0.4 0.2 0.0 I I I I I I -9 -8 -7 -6 log [DA (M)] -5 -4 B = 0.45, P = 0.64); the mean EC50 for these times is 1.1 X 10~7 M (±2.6 X 10~s M). Finally, a Mann-Whitney U test revealed that the mean EC5(I of gills exposed to YFAFPRQamide and 10~ft M DA was significantly greater than that of gills exposed to YFAF- PRQamide and 5 X 10 7 M DA (P = 0.04). Thus, the latency of the peptide response cannot be due entirely to permeability. Beat frequencies. The lateral cilia used in dose-response studies, including controls, beat in metachronal waves at frequencies from 7 to 27 beats/s, or they did not beat at all. That is, no metachronal waves appeared at frequencies lower than 7 beats/s (n = 888 on 148 pieces of gill). Frontal cilia Peptides. Preliminary experiments on gill strips exposed to the three peptides, all at 10^ft A/, revealed that none of the peptides had a significant effect on the activity of the frontal cilia: AMSFYFPRMamide. F(I781 == 0.05, P == 0.81; YFAFPRQamide, FIK7X) = 1.66, P = 0.20; FMRFamide, ^(i 48i = 3.18, P = 0.08; the mean rate of particle transport in all cases was 0.29 mm/s. SCPs and 5-hydroxytryptamine, To determine whether the two SCPs might alter the activity of frontal cilia inhib- ited by 10~6 M 5HT. we first exposed isolated gill strips to either AMSFYFPRMamide or YFAFPRQamide (10~6 M) and then. 10 min later, to 10~6 M 5HT. Controls were exposed only to 5HT. Neither peptide had a significant effect upon the 5HT-induced inhibition: AMSFYFPRM- amide. F(U61 = 1.22, P = 0.28; YFAFPRQamide, F(1 8) = 3.93. P = 0.08. Dose-dependent effects. In Figure 4A, the regression lines of the adjusted fractional difference are plotted against the log of the 5HT concentration and time. The graph shows that the inhibitory effects of 5HT on the frontal cilia appear within 15 min and remain constant for 1 h; there is no statistical difference between these regression lines (F(6 ,4) = 0.32, P = 0.92). The mean ECW is 5.7 X 10"7 M (±3.5 X 10~7 M). 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Pavlova, and N. E. Phillips. 1997. Modulation of ciliary beat frequency by neuropeptides from identified molluscan neurons. J. Exp. Biol. 200: 1433-1439. Wong, L. B., I. F. Miller, and D. B. Yeates. 1991. Pathways of substance P stimulation of canine tracheal ciliary beat frequency. J. Appl. Physiol. 70: 267-273. Wong, L. B., C. L. Park, and D. B. Yeates. 1998. Neuropeptide Y inhibits ciliary beat frequency in human ciliated cells via nPKC, inde- pendently of PKA. Am. J. Ph\siol. 275: 440-448. Reference: Bio/. Bull. 197: 174-187. (October I4W) Ovigerous-Hair Stripping Substance (OHSS) in an Estuarine Crab: Purification, Preliminary Characterization, and Appearance of the Activity in the Developing Embryos MASAYUKI SA1GUSA AND HIROSHI IWASAKI Department of Biology, Faculty of Science, Okuyama University, Tsushima 2-1-1 (General Education Buildings), Oka\amu 700-8530, Japan Abstract. Ovigerous-hair stripping substance (OHSS) is an active factor in crab hatch water (i.e., filtered medium into which zoea larvae have been released). This factor participates in stripping off the egg attachment structures (i.e., egg case, funiculus, and the coat investing ovigerous hairs) that remain attached to the female's ovigerous hairs after larval release. Thus this activity prepares the hairs for the next clutch of embryos. OHSS activity of an estuarine crab. Sesarma haematoc/ieir, eluted as a single peak on molecular-sieve chromatography, but this peak still showed two protein bands at 32 kDa and 30 kDa on SDS-PAGE. The two protein bands stained with a polyclonal antiserum raised to the active fractions from molecular-sieve chroma- tography. Moreover, antibodies purified from this poly- clonal OHSS antiserum also recognized both the 32-kDu and 30-kDa bands. OHSS immunoreactivity and biological activity were associated with the attachment structures that remained connected to the ovigerous hairs after hatching. In developing embryos, both protein bands could be stained immunochemically at least 10 days before hatching. But OHSS biological activity appeared only 3 days before hatching. The immunoreactive protein bands were not ob- served in the zoea, but OHSS bioreactivity was present, though greatly reduced. The 32-kDa protein, at least, is probably an active OHSS, and the 30-kDa protein band may also be OHSS-related. The OHSS appears to be produced and stored by the developing embryo. Upon hatching, most of the material may be trapped by the remnant structures, and the remainder is released into the ambient water. Received 16 January 1997; accepted 16 June 1999. Introduction After egg-laying, the embryos of intertidal and estuarine crabs — and indeed most decapod crustaceans — are encased in a thick, protective capsule composed of two or three layers; these capsules are then attached to the female's ovigerous hairs through the funiculus and investment coat (Yonge, 1937, 1946; Cheung, 1966; Goudeau and Lachaise, 1983). The capsule breaks open during or after embryonic development, and hatching occurs (Davis, 1968, 1981; Saigusa, 1997). The funiculus and investment coat, as well as the broken egg capsule, remain attached to the hairs after hatching (for further details, see Saigusa, 1994). But at the time of hatch- ing, an active factor we call ovigerous-hair stripping sub- stance (OHSS) is released from the embryo and causes these remnant structures to slip off the hairs (Saigusa, 1994). The stripping of these remnant structures is very important be- cause it leaves the hairs clean, unbroken, and thus prepared to incubate the next clutch of embryos (Saigusa, 1995). The funiculus. the coat that wraps the ovigerous hairs, and the outermost layer of the egg capsule are all composed of the same material (Saigusa ct <;/.. unpub. data), and OHSS may be an enzyme that acts on this material. In many other animals, a hatching enzyme is released from the embryos upon hatching and digests the layers of the egg case (e.g., Yamagami. 1988; Lepage and Cache. 1989; Roe and Lennarz, 1990; Helvik et ai, 1991). However, there is no evidence, even after electron microscopy studies, that the layers of the egg capsule encasing crab embryos are di- gested by OHSS (Saigusa et , 50 X o 01 23456 78910 Fraction number Conlro1 Fraction number 234567 89 B 55.6 39.2 26.6 14.3 kDa Figure 2. Purification of OHSS activity by molecular-sieve chroma- tography with protein analysis on SDS-PAGE. (A) The pass-through fractions (60 ml) from anion-exchange chromatography were concentrated by ultrafiltration, and an aliquot (500 /il) was applied to molecular-sieve chromatography; fractions are I ml. Open circles (O): protein concentra- tion in each fraction (280 nm absorption). Solid triangles (A): the OHSS activity of each fraction; bioassays carried out for 1.5 h with one (or two) ovigerous setal segments per fraction; error bars: standard deviation. Con- trol assay (A bottom right). Downward-pointing arrows indicate the mo- lecular masses of marker proteins: glutamate dehydrogenase (55.6 kDa). aldolase (39.2 kDa). and trypsin inhibitor (20.1 kDa). (B) Analysis, by SDS-PAGE. of the proteins in each fraction (1-9) eluted from molecular- sieve chromatography. The polyacrylamide gel was transblotted to a PVDF membrane which was stained with Coomassie brilliant blue. The marker proteins were glutamate dehydrogenase (55.6 kDa), aldolase (39.2 kDa), triosephosphate isomerase (26.6 kDa), and lysozyme (14.3 kDa). The two bands that appear in fractions 6 and 7 (arrows to the right) have molecular masses of 32 kDa and 30 kDa. 180 M. SAIGUSA AND H IWASAKI B 55.6 39.2 —I 26.6 14.3 kDa 40 23 -«- 12 kDa 100 r 80 - 40 20 1.5 h abed 100 r 80 - 60 - 40 20 2.0 h I abed Figure 3. Distribution of OHSS activity in a polyacrylamide gel divided into four equal segments after SDS-PAGE. (A) The gel. Numbers at the left of the small arrows indicate the molecular masses of the standards (see Fig. 2B). The molecular mass limits of each gel segment are indicated by the large arrows. (B) Bioassay with two or three ovigerous setal segments. Open bars indicate OHSS activity of each gel segment. Incubation periods for the bioassay: 1.5 h and 2.0 h. Stippled bars: controls; the ovigerous setal segments were incubated in DW for 1.5 h and 2.0 h. Error bars: standard deviation. nificant difference between the two experiments. Solutions maintained for longer times (100 and 120 h) decreased in activity in both experiments. Figure 4C shows the pH dependency of OHSS activity. After an assay incubation of 1 h, the pH optimum was quite broad, about 7.0-11.0. Specificity of antibodies and affinity purification An antiserum raised from molecular-sieve chromatogra- phy (fractions 6 and 7) detected only two strong protein bands on SDS-PAGE, and they appeared in fractions 6-8 (Fig. 5). These immunostained protein bands clearly corre- sponded to the peak of OHSS activity in Figure 2A. The antibodies that had stained the 32-kDa and 30-kDa protein bands were purified by immunochemical affinity to determine the specificity of binding. As shown in Figure 6A, the antibodies eluted from the 32-kDa protein band on SDS-PAGE stained the 30-kDa protein as well; and the antibodies eluted from the 30-kDa band similarly stained both the 30-kDa and 32-kDa proteins. B 100- 80- S 60^ w w O 40- 20- 0 — i — 20 — i — 40 — i — 60 — i — 80 100 Temperature ( C ) 100- 80- 60- 40 -i 20- — i — 20 — i — 40 — i — 80 60 80 100 Time of incubation ( h ) 120 100-i 80- 60- 40- 20- 0 f i * i 0 24 6 81012 pH Figure 4. Characteristics of OHSS activity. (A) Heat stability. Active solutions were incubated for 15 mm at each temperature, and OHSS activity was bioassayed for 1 h. (B) Effects of prolonged incubation (up to 5 days). For each time, active solutions were maintained at either 4°C (O) or 23°C (A), and the activity was bioassayed for 1 h. (C) Dependence of activity on pH. Buffers: 100 mM Na-acetate (•): 100 mM Tns-HCl (A); 100 mM Gly-NaOH (O). The activity was bioassayed for 1 h. Broken line in (C) indicates the least-square regression curve fitted to the data. Error bars: standard deviation of three experiments. ACTIVE SUBSTANCE IN CRAB HATCH WATER 181 Fraction number 23456789 B Fraction number 556 392 266 — 143- kDa 2 34567 8 9 * Figure 5. Specificity of the polyclonal antiserum raised against OHSS. (A) SDS-PAGE of fractions eluted from molecular-sieve chromatography. The polyacrylamide gel was transblotted onto a PVDF membrane and stained with Coomassie brilliant blue. (B) Immunostaining of the PVDF membrane with a polyclonal antiserum raised to OHSS. Numbers to the left show the molecular masses of the standards shown in Figure 2B. The intensely immunostained bands in fractions 6-8 have masses of about 32 kDa and 30 kDa (arrows to the right). In this experiment (Fig. 6A), however, the 32-kDa band was at most 2 mm apart from the 30 kDa band on the gel. In addition, the PVDF membrane was not stained with CBB, except the narrow strip that had been cut on both sides (5 mm in width), because the antibodies were not bound to CBB-stained proteins. So these protein bands could have been incompletely separated. In Figure 6B, concentrated active fractions from molec- ular sieve chromatography (6 and 7 in Fig. 2A) were elec- trophoresed for a longer period (9 h), which produced a gap of 5-6 mm between the upper and lower bands. The whole membrane was stained by Ponceau S, and each protein band (2 mm in width) was subjected to affinity purification. Again, the antibody raised from the 32-kDa band bound to the 30-kDa band as well; and the antibody produced from the 30-kDa band also recognized the 32-kDa band (Fig. 6B). 55.6- 39.2- 26.6 14.3- kDa B 55.6 39.2 26.6 14.3 kDa i a Figure 6. Immunostaining with antibodies affinity purified from SDS-PAGE bands that had OHSS activity. SDS-PAGE of the proteins from molecular-sieve fractions 6 and 7 (see Fig. 2A) was carried out for either 3.5 h (A) or 9h (B), the longer electrophoresis producing the greater separation between the active bands at 32 kDa and 30 kDa. The bands were cut out of the PVDF membranes, incubated with a polyclonal antiserum raised to OHSS, and the bound antibodies were eluted (see Methods). These antibodies, obtained from the short and long electrophoresis, were applied to two new SDS-PAGE runs, both of 3.5 h; these final runs are shown in this figure. Immunostaining was effected by antibodies bound (a) to protein in the upper band (32 kDa), and (b) to the protein in the lower band (30 kDa). The strips to the left of the two panels were stained with Coomassie brilliant blue (A) and Ponceau S (B); the numbers (with arrow) are the molecular masses of the standard molecules (identified in Fig. 2B). 182 M. SAIGUSA AND H. IWASAKI OHSS activity in the post-hatching remnants of the embryo attachment system After hatching, the broken egg cases, funiculus, and in- vestment coat remain attached to the ovigerous hairs. The female picks these remnant attachment structures off the ovigerous hairs, but this must occur after the OHSS released with the hatch water has been greatly diluted in the estuary. We therefore examined the possibility that at least some OHSS is present in the remnant attachment structures them- selves. The remnants were collected and stained with FITC- conjugated, OHSS antiserum (Fig. 7). OHSS was clearly detected all over the remnants, including the prezoeal cuti- cles. In contrast, the egg capsule, funiculus, and investment coat did not react to the FITC-conjugated OHSS antiserum when the embryos were squeezed in the egg cases (not shown). When samples for electrophoresis and immunoblotting were prepared from remnants that had been crushed and denatured, only the 32-kDa band was detected (Fig. 7, right). Remnants stored at — 20°C were thawed and crushed, and suspensions of this material (not centrifuged) were bioas- sayed for 1.5 h and 2.0 h. As shown in Table I, strong OHSS was detected in this solution. In another experiment, the remnants were thawed and crushed, and then treated with 5%, 10%, and 20% Triton-X. After centrifugation for 20 min ( 15,000 rpm), the supernatant was bioassayed for 1.5 h and 2.2 h and showed strong OHSS activity, particularly at the longer incubation (Table II). Appearance of OHSS in the developing embryos Because OHSS immunoreactivity and biological activity occur in the remnants after hatching, we determined when. during development, these activities would appear. Immu- nochemical and biological observations were made. Immunochemistry. Embryo clusters (one-third of an ovigerous seta) were taken at successive times from a single female and crushed; the suspensions were denatured with the lysis buffer before centrifugation. As shown in Figure 8, the 32- and 30-kDa proteins became noticeable at least 2 weeks before hatching. The 30-kDa band was the stronger of the two bands at 10 and 6 days before hatching; but its intensity declined and became very faint in embryos 4 h before hatching; and it disappeared completely in post- hatched zoeas. In contrast, the 32-kDa band appeared later than the 30-kDa band, but it was still quite clear in embryos 4 h before hatching, and again was not detected in the post-hatched zoeas. In addition to the two bands of OHSS- related protein, an immunoreactive band appeared at about 55 kDa, from 14 to about 2 days before hatching (Fig. 8). In another female, the supernatant and the sediment were denatured separately by the lysis buffer after the embryo clusters had been crushed and centrifuged. In the superna- tant (Fig. 9 A). OHSS first appeared as a weak 32-kDa band 14 days before hatching. The 30-kDa band was noticeable 10 days before hatching, and the amount increased abruptly in the embryos collected 6 days before. This protein de- creased markedly just before hatching, but was still visible 4 h before hatching. On the other hand, the sediment contained broken egg cases, funiculi, the investment coat, ovigerous hairs, prezoeal cuticles, and probably only a portion of the em- bryos. When this material was centrifuged and the superna- tant was examined on the same female (Fig. 9B), the 30- kDa band appeared weakly in the lanes derived from embryos collected 10, 6, and 4 days before hatching; but it was absent at other times. In contrast, the 32-kDa band 55.6 39.2 26.6 32 kDa Figure 7. Immunochemica] staining of the structures remaining attached to a female's ovigerous hairs after hatching. Left: the iminunoblot of an extract of the remnants subjected to SDS-PAGE. Arrowhead: 32-kDa protein band. Numbers to the left show the molecular masses of the standard molecules (as in Fig. 2B). Right: the remnants stained with polyclonal FITC-conjugated OHSS antiserum. ci1: broken egg case:/: funiculus; pc: prezoeal cuticle; oh: female ovigerous hair (see fig. 2 in Saigusa. 1994). Scale: 100 /nl. ACTIVE SUBSTANCE IN CRAB HATCH WATER 183 Table I iatissiiv iff suspensions of crushed attachment slniciu ificr hatching Incubation* 1.5 h 2.0 h Control t Experiment 1.2 ± 2.4%(5) 81.1 ± 7.4%(6) 3.8 ± 6.3%(6) 93.5 ± 4.3%(4) * Values are the mean percentage of unbroken, stripped ovigerous hairs on each whorl; number of whorls on the subdivided segments of the ovigerous seta is given in parentheses. t Incubation of embryo clusters in distilled water. became very distinct 6 days before hatching, and was vir- tually the only band that appeared from 2 days before, until hatching. A 55-kDa band was also detected in the sediment, but was less clear than in the supernatant (Fig. 9A). Bioassay. Embryo clusters (one-third of an ovigerous seta) were detached from a single female, and were frozen at — 20°C. The embryo clusters were thawed and crushed in distilled water, centrifuged, and the OHSS activity in the supernatant was bioassayed for 1.5 h and 2.2 h (Fig. 10A). The OHSS activity began to appear 3 days before hatching, became very strong 6 h before hatching, and was markedly decreased in the zoeas. The sediment of the embryo clusters was also examined (Fig. 10B). The OHSS activity was again detectable 3 days before hatching; the activity increased near hatching and, like the supernatant, was very strong 6 h before hatching. The zoeas, again, had weak OHSS activity. Discussion Ovigerous-hair stripping substance (OHSS) from an es- tuarine terrestrial crab was purified through three steps of chromatography (Fig. 2A). The activity eluted as a single peak on molecular-sieve chromatography, but still showed two protein bands at 32 kDa and 30 kDa on SDS-PAGE (Fig. 2B). Affinity purified antibodies raised to the active fractions (6 and 7 in Fig. 2A) also bound to two bands at 32 kDa and 30 kDa, corresponding to the bands shown in Figure 2B. Furthermore, immunochemical staining indi- cated that OHSS-related 32-kDa and 30-kDa bands appear in developing embryos at least 10 days before hatching. This time course, however, did not correspond to the ap- pearance of OHSS activity as bioassayed with ovigerous setal segments. These features invite the following four issues for discussion. Stability of OHSS As shown in Figure 4B, purified OHSS retains its activity for at least 80 h, even at room temperature. When crude or concentrated hatch water was frozen to temperatures lower than -20°C, OHSS was stable. But instability appeared after thawing. For example, active fractions that were eluted from the hydrophobic column lost most of their activity within 12 h (unpub. data). Crude hatch water contains multiple proteases, and these proteases digest casein (Katsube el al, 1999; see also Saigusa, 1996), and they are not excluded by the hydropho- bic chromatography shown in Figure 1. So the disappear- ance of OHSS activity could be due to its digestion by the caseinolytic proteases that still remain in active solutions. These caseinolytic proteases do, however, bind to an anion exchange column (MONO-Q) and are therefore ex- cluded with this chromatography. Thus, caseinolytic pro- teases are not present in the materials applied to the column for molecular-sieve chromatography (Fig. 2 A). Conse- quently, purified OHSS retains its activity for a long period even at room temperature (Fig. 4B). Evidence that the 32-kDa hand is active OHSS As shown in Figure 2A, OHSS activity always appeared in fractions 6-8 on molecular-sieve chromatography. On SDS-PAGE, 32-kDa and 30-kDa protein bands appeared very clearly in these fractions (Fig. 2B). The second seg- ment (b) of the gel strip containing these two bands cer- tainly showed OHSS activity (Fig. 3). An additional band (22 kDa) appeared in fractions 5 and 6 (Figs. 2B and 5 A), but did not correspond to the OHSS activity bioassayed with ovigerous setal segments (compare Fig. 2 A with Fig. 2B). Thus, we might speculate that OHSS consists of either or both of the two protein bands at 32 kDa and 30 kDa. Antibodies raised from fractions 6 and 7 stained the 32-kDa and 30-kDa protein bands (Fig. 5B). These bands also appeared in fraction 8, indicating a good correspon- dence with the bioassayed OHSS activity (Fig. 2 A). OHSS is clearly detected all over the remnants that remain on the ovigerous hairs after hatching (Fig. 7). However, only the Table II Bioassay with the extract from ihc remnants with the detergent (Triton-X) Incubation* 1.5 h 2.2 h Control t 5f/r Triton-X 10% Triton-X 20% Triton-X 3.6 ± 8.7%(7) 27.5 ± 9.3%(4) 72.5 ± I5.19H3) 88.3 ± 7.3%(3) 5.8 ± 7.99H5) 92.0 ± 5.79H3) 91.0 ± 2.8%(3) 98.5 ± 2.6%(4) * Values are the mean percentage of unbroken, stripped ovigerous hairs on each whorl; number of whorls on the subdivided segments of ovigerous seta is given in parentheses. t Incubation of embryo clusters in distilled water. 184 M. SAIGLISA AND H. IWASAKI Days before hatching 14d 10d 6d 4d 2d 1d 4h Z REM 55.6 39.2 26.6 — 32 kDa 14.3 -> kDa Figure 8. The appearance of immunoreactive OHSS in developing crab embryos. Embryo clusters (one- third of an ovigerous seta) were detached from a single female, crushed, and then denatured with the lysis buffer. The extracts (supernatant) were subjected to SDS-PAGE, and blots were stained immunochemically with the polyclonal OHSS antiserum. d (or /?): days (or hours) before hatching. Z: post-hatched embryos (zoeas) released from the same female. REM: remnant structures remaining attached to the female's ovigerous hairs after hatching. Numbers to the left are the molecular masses of the same markers shown in Figure 2B Note that only the 32-kDa band is delected in the remnant structures (see Fig. 7). 32-kDa protein band was detected in these remnants (Fig. 8). Furthermore, a strong OHSS activity was also detected in these remnants (Table I), as well as in the supernatant after treatment with detergent (Table II). Hence, we specu- late that the 32-kDa band is an active OHSS. On the other hand, we cannot yet hypothesize that the 30-kDa protein band also has OHSS activity. Since purified antibodies recognized both the 32-kDa and 30-kDa bands (Figs. 6A and 6B). we suppose that these two proteins have very similar sequences; i.e., the bands detected by immu- nochemical staining are probably both OHSS-related pro- teins. The OHSS antiserum detected not only 32-kDa and 30- kDa proteins, but also a band at 55 kDa (Figs. 8 and 9). When the active fractions 6 and 7 of molecular-sieve chro- matography were studied (Fig. 3A). this band was not detected at all. In contrast, when the crushed embryos (Figs. 8 and 9) were examined, the 55-kDa band often appeared clearly; but it did not appear in the remnant (Fig. 7). The 55-kDa band also appeared on molecular-sieve chromatog- raphy (Fig. 2B), but it did not have OHSS activity (Fig. 2 A). This band also did not appear in any fraction (1-8) of immunostained PVDF membranes (Fig. 5B). So the 55-kDa band in Figure 2B might be different from that appearing in Figures 8 and 9. As shown in Figure 8. the 55-kDa protein appeared clearly between 14 days and 4 days before hatching; and it became very faint after 2 days before hatching. Figure 9A 556 39 2 266 A Days before hatching 14d 10d 6d 4d 2d 1d 4h Z -»»*»- A 01 DM Days before hatching 14d 10d 6d 4d 2d 1d 4h Z Figure 9. Appearance of immunoreactive OHSS in developing crab embryos. Embryo clusters (one-third of '• wigerous seta) were detached from a single female, crushed, and then centrifuged. The supernatant and n ni svere separately denatured with lysis buffer after centrifugation. (A) The supernatant subjected to PAGE, and the blots immunostained. (B) Extracts of precipitate of the crushed embryos run on SDS-PAGE ami niiiiunoMained. n: is secreted from the embryos as a 57-kDa form on SDS-PAGE, but it is converted to a 33-kDa form during purification. This conversion was speculated to be due either to autolysis or to proteolysis by a different protease contained in the sample solution. Acknowledgments We thank Dr. Masatsugu Hatakeyama, Kobe Univer- sity, for help in raising the polyclonal antiserum. Thanks are also due to Dr. Yasushi Yamamoto, Biochemistry Laboratory, and the students of the Molecular Biology Laboratory, for their kind help and many suggestions about SDS-PAGE and immunochemical techniques. Dr. Tadashi Akiyama helped M. S. in the collection of hatch water in 1994. Supported by Grant-in-Aid for Scientific Research (C) (2) from the Ministry of Education, Science, Sports, and Culture, Japan, to M. S. (Nos. 06839017 and 08833009. Marine Biology; No. 10836014, Natural History). Also supported by Narishige Zoological Science Award to M. S. in 1997. Literature Cited Cheung, T. S. 1966. The development of egg-membranes and egg at- tachment in the shore crab. Carcinus nuienas, and some related deca- pods. J. Mar. Biol. Assoc. UK 46: 373-400. Davis, C. C. 1968. Mechanisms of hatching in aquatic invertebrate eggs. Oceanogr. Mar. Biol. Aniui. Rev. 6: 325-376. Davis, C. C. 1981. Mechanisms of hatching in aquatic invertebrate eggs. II. Oi-twiogr. Mur. Biol. Awui. Rev. 19: 45-123. Goudeau, M., and F. Lachaise. 1983. Structure of the egg funiculus and deposition of embryonic envelopes in a crab. Tissue Cell 15: 47-62. Helvik, J. V., D. O. Oppen-Bernsten, and B. T. Walther. 1991. The hatching mechanism in Atlantic halibut (Hippoglossus hippoglossus). Int. J. r>ev. Biol. 35: 9-16. Kalsube, Y., M. Terajima. and M. Saigusa. 1999. Caseinolytic pro- tease in crab hatch water: preliminary studies of purification, charac- terization, and its putative role. Proc. Arthropod. Embryol. Soc. Jpn. 34: 1-9. I :u iiiinli. U. K. 1970. Cleavage of structural proteins during the assem- bly of the head of bacteriophage T4. Nature 227: 680-685. Lepage, T., and C. Cache. 1989. Purification and characterization of the sea urchin embryos hatching enzyme. J. Biol. Cliem. 264: 4787 — 1793. Roe, J. L., and W. J. Lennarz. 199(1. Biosynthesis and secretion of the hatching enzyme during sea urchin embryogenesis. J. Biol. Chem. 265: 8704-871 I. Saigusa. M. 1982. Larval release rhythm coinciding with solar day and tidal cycles in the terrestrial crab Sesarina. Biol. Bull. 162: 371-386. Saigusa. M. 1992. Control of hatching in an estuarine terrestrial crab. I. Hatching of embryos detached from the female and emergence of mature larvae. Biol. Bull. 183: 401-408. Saigusa, M. 1993. Control of hatching in an estuarine terrestrial crab. II. Exchange of a cluster of embryos between two females. Biol. Bull 184: 186-202. Saigusa, M. 1994. A substance inducing the loss of premature embryos from ovigerous crabs. Biol. Bull. 186: 81-89. Saigusa. M. 1995. Bioassay and preliminary characterization of oviger- ous-hair stripping substance (OHSS) in hatch water of crab larvae. Biol. Bull. 189: 175-184. Saigusa. M. 1996. Two kinds of active factor in crab hatch water: ovigerous-hair stripping substance (OHSS) and protease. Biol. 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Two constituent proteases of a teleostean hatching enzyme: concurrent syntheses and packaging in the same secretory granules in discrete arrangement. De\: Biol. 149: 349-356. Yonge, C. M. 1937. The nature and significance of the membranes surrounding the developing eggs of Homarus vu/garis and other Decapoda. Proc. Zool. Sue. Land. (Ser. A). 107: 499-517. Yonge, C. M. 1946. Permeability and properties of the membranes surrounding the developing egg of Homarus vit/garis. J. Mar. Biol. Assoc. UK 26: 432-43S. Reference: Biol. Bull. 197: 188-197. (October 1999) Behavior of Hemocytes in the Allorejection Reaction in Two Compound Ascidians, Botryllus scalaris and Symplegma reptam MAKI SHIRAE1*, EUICHI HIROSE:, AND YASUNORI SAITO1 1 Shimoda Marine Research Center, University ofTsukiiba. Shimoda 5-10-1. Shi-noka 415-0025. Japan: uiitl Department of Chemistry, Biology and Marine Science, Faculty of Science, University of the Ryiikyns, Nishihara, Okinawa 903-0213. Japan Abstract. In botryllid ascidians, the type of allorejection reaction differs among species. Comparative studies of these different reactions contribute to our understanding of the allorecognition and nonself-rejection system. We stud- ied the morphology of hemocyte behavior during allorejec- tion reactions in two species. Botryllus scalaris and Svm- p/egma reptans, which stand at important points in botryllid phylogeny. In B. scalaris, phagocytes mediated hemocyte aggregation, resulting in interruption of blood flow just after vascular fusion of incompatible colonies. Although previ- ous studies indicate that morula cells (MCs) play a central role in the rejection reaction, the MCs of B. scalaris did not participate in the rejection reaction. Colonies of S. reptans showed two types of allorejection reaction that started at different points in the process of vascular fusion between two colonies. In both types of rejection reaction, the MCs played a central role and behaved similarly to those of all botryllids except B. scalaris and a botryllid from Israel. These observations suggest that the differences in hemocyte behavior and allorecognition site observed in this study reflect the variation in allorejection reactions among botryl- lids. Introduction Colony specificity is a type of self-nonself recognition and rejection reaction against conspeeifics that occurs in many colonial forms of animals; allogeneic colonies fuse to form a single mass or reject each other when the colonies come into contact at their growing edges, whereas synge- Received 30 September 1998; accepted 26 June 1999. Abbreviations: MCs, moriih cell-. * To whom correspondence should he addressed. Email: shirae@ kurofune.shimoda.tsukuba.ac.jp neic colonies always fuse with each other. The phenomenon has been studied in various species in different phyla (e.g., Porifera. Cnidaria, Bryoz.oa, and Urochordata), and the sig- nificance of the fusion and the rejection to survival has been discussed. However, it is difficult to reach a reasonable conclusion by comparing the features of colony specificity among a broad range of species because they differ in body plan and physiological condition. In botryllid ascidians, the morphological process of fu- sion and rejection reactions in colony specificity has been described in detail for five species: Botrvllus primigenus (Oka and Watanabe, 1957, 1960;Tanaka, 1973; Tanaka and Watanabe, 1973; Taneda and Watanabe. 1982a, 1982b); Botryllus schlosseri (Sabbadin, 1962; Boyd et ai, 1990); Botrylloides simodensis (Mukai and Watanabe, 1974; Hi- rose et ai, 1988. 1997); Botrylloides fuscus (Hirose et ai, 1994, 1997); and Botrylloides violaceus (Hirose et ai, 1988). In these botryllid ascidians, the processes of fusion are essentially the same. When rejection reactions occur, they interrupt the progress of fusion, and the beginning stages of rejection differ among species. That is, the varia- tion in allorejection types is thought to be caused by differ- ences in the allogeneic recognition stage in the colony fusion process (Taneda et ai, 1985; Saito et ai. 1994). Hemocytes play important roles in the allorejection reac- tions of botryllid ascidians (Taneda et ai, 1985; Saito et ai, 1994). An earlier comparative study on the ultrastructure of hemocytes in some botryllids, including Botryllus scalaris and Syniplc^ma reptans, showed that hemocytes could be commonly classified into five morphological types: hemo- blasts, morula cells (MCs), granular leukocytes, vacuolated cells, and phagocytes (Shirae. unpub. data). Transient cells in differentiation were also observed. We hypothesize that I 88 HEMOCYTE BEHAVIOR IN ASCIDIAN ALLOREJECTION 189 Figure 1. Allorejection reaction in Botryllus scalaris under a stereomi- croscope. Two incompatible colonies are in contact at their growing edges. Their tunics and ampullae are fused, and hemocyte aggregation in the fused ampullae induces interruption of blood flow (arrowheads), am. ampullae; tv, tunic vessels; z. zooids. Bar = 0.5 mm. differences in these hemocyte characteristics reflect the va- riety in allorejection reactions among botryllids. Botryllus scalaris is thought to be the most primitive botryllid species known to date, based on its manner of sexual reproduction (Mukai, 1977; Mukai el al., 1987; Saito et ai, 198 la, 1981b; Saito and Watanabe, 1985). Recently, the molecular phylogeny of botryllids, based on 18S rRNA sequencing, indicated that B. scalaris branched off from the lineage earlier than the other botryllids studied thus far (Cohen et al.. 1998). In B. scalaris, the allorejection reac- tion begins at the latest stage of the fusion process, that is, soon after fusion of blood vessels and the beginning of blood exchange between two incompatible colonies (Saito and Watanabe, 1982). Therefore, the rejection reaction in B. scalaris may be the most primitive type of rejection among botryllid ascidians (Saito et al., 1994). However, there are few descriptions of hemocyte behavior in the rejection reaction to compare with hemocyte behavior of other bot- ryllids. Symplegimi reptans, of the family Styelidae, might be the nearest phylogenetic relative of botryllids for the following reasons. Styelidae is a sister family of Botryllidae (Berrill, 1936). and some researchers (cf. Kott, 1969) argue that botryllid ascidians should be classified in a subfamily Bot- ryllinae, belonging to an enlarged family Styelidae. Among compound styelids, S. reptans is the species most similar to botryllids morphologically. In botryllids and S. reptans, all zooids in a colony are interconnected via a common vascu- lar system, and vascular ampullae occur at the growing edge of a colony. Furthermore, the occurrence of colony speci- ficity in S. reptans was reported, on the basis of stereomi- croscopic observation, by Mukai and Watanabe (1974). They showed that the manner of colony specificity of S. reptans resembles that of botryllids. In this study, we used light and electron microscopy to investigate the processes of allorejection reactions in B. scalaris and 5. reptans, paying special attention to the behavior of hemocytes. In B. scalaris, we found that the hemocytes with a central role in the rejection reaction are not morula cells (MCs) but phagocytes. In 5. reptans, we describe two types of allorejection reaction — the particular combination of allogeneic colonies determines which type occurs. Finally, we discuss the variation in the allorejection reaction in botryllids based on the behavior of hemocytes. Materials and Methods Colonies of Botryllus scalaris and Symplegma reptans were collected in the vicinity of Shimoda (Shizuoka Prefec- ture), Japan. They were attached to glass slides and reared in culture boxes immersed in Nabeta Bay near the Shimoda B Figure 2. Reaction reaction in Botryllus scalaris. (A) Vascular fusion has just occurred and blood of the two incompatible colonies is exchanged. Arrow indicates the fusion point of two ampullae (am) between the incompatible colonies. (B) Hemocytes gradually aggregate in the fused vessels. Arrowheads indicate clusters of aggregating hemocytes. (C) Blood flow is interrupted by the mass of aggregating hemocytes in a few minutes. A, B, and C are the same magnification. Bar = 100 /urn. 190 M. SHIRAE ET AL. Marine Research Center. University of Tsukuba. Colonies that grew well were used for the experiments. Fusion experiments to test fusibility (compatibility) be- tween two colonies were routinely carried out as follows. A small piece was cut from the periphery of each colony with a razor blade. Two allogeneic or syngeneic colony pieces of the same size were brought into contact at their growing edges on a glass slide and were allowed to adhere to the slide in a moisture chamber for 30 min. These colony pieces were subsequently reared in a running seawater aquarium and were observed periodically under a binocular stereomi- croscope. Specimens undergoing fusion or rejection were fixed for 2 h on ice in a 2.57c glutaraldehyde solution containing 0.45 M sucrose buffered with 0. 1 M sodium cacodylate at pH 7.4. The fixed specimens were rinsed in the same buffer and were then postfixed with 1% osmium tetroxide in the same buffer without sucrose for 1.5 h. After dehydration through an ethanol series, the specimens were cleared with //-butyl glycidyl ether and embedded in low-viscosity epoxy resins. Thick sections were stained with 1.0% toluidine blue and examined under a light microscope. Thin sections were double-stained with uranyl acetate and lead citrate and then examined with a Hitachi HS-9 transmission electron micro- scope at 75 kV. Results Allorejection reaction in Botryllus scalaris In B. scalaris, as in other botryllids. autogeneic or syn- geneic colony pairs fuse and form a single mass. The fusion process was the same as that of other botryllids (Katow and Watanabe. 1980; Saito and Watanabe. 1982). First, the tunics of two colonies fused. Second, the ampullae (termini of blood vessels) penetrated into the opposite colony and the ampullar tips came into contact with the sides of opposite ampullae (tip-to-side contact); at these contact points the colonies fused with each other. Blood started to flow be- tween the two colonies, and finally those colonies formed a single mass. The rejection reaction between allogeneic col- onies began after the fusion of opposing ampullae (Fig. 1 ). Soon after blood exchange through the fused vessels of the two colonies (Fig. 2A), hemocytes aggregated in the vas- cular lumen of the fused ampullae (Fig. 2B, arrowheads). These aggregates formed a cluster that plugged the opening of the fused ampullae (Fig. 2C) and interrupted the blood flow within a few minutes. Histological observations of the rejection area in the same stage are shown in Figure 3A. As the aggregation gradually proceeded, the volume of hemo- cyte clusters in the blood vessels increased (Fig. 3. B and C). Subsequently, the vessels collapsed in the rejection area, the surrounding tunic disintegrated, and the two colonies became separate. The behavior of the hemocytes, as observed by electron microscopy, is shown in Figures 4 and 5. Immediately after ve jife. • « n : «?* 5 B ve Figure 3. Histological sections in rejection reaction area in B. xcalaris. (A) Immediately after interruption of blood exchange. Arrow indicates fusion point of vascular epithelia. (B) About 10 h after interruption of blood exchange. Arrow indicates fusion point of vascular epithelia. (C) Two days after interruption of blood exchange. Three clusters encapsulated by phagocytes are shown, t. tunic; ve, vascular epithelia; arrow indicates fusion point of vascular epithelia; arrowheads indicate periphery of hemo- cyte clusters. A. B. and C are the same magnification. Bar = 50 fj.m. the stoppage of blood flow, the cell density of the phago- cytes (including hyaline amebocytes, macrophage-like cells, and signet ring cells, as described by Ballarin et ai, 1994) increased significantly in the rejection reaction area. The phagocytes had variable shapes, contained many round granules of 0.5 /urn in diameter, and often engulfed other hemocytes. Moreover, the phagocytes adhered to other he- mocytes and began to aggregate with one another inside the fused ampullae (Fig. 4A). After about half an hour, hemo- cytes in the aggregates were packed more tightly in the fused ampullae (Fig. 4B), and some of them disintegrated. The aggregates in the ampullae gradually enlarged by HEMOCYTE BEHAVIOR IN ASCIDIAN ALLOREJECTION 191 Figure 4. Hemocytes in allorejection reaction in Botiyllus scalaris. (A) Several phagocytes adhere to a hemocyte. (B) Aggregation. (C) Phagocytosis. Arrowheads indicate outer edges of a phagocyte that surrounded and engulfed a hemocyte cluster. (D) Encapsulation. Arrowheads indicate the periphery of a phagocyte that encapsulated a hemocyte cluster, m, encapsulated and disintegrated morula cell: p. phagocyte. Bars = 2 urn. B Figure 5. Morula cells in Borryllus scalaris: (A) inside the vascular lumen in an allorejection reaction; (B) in an intact colony, m, morula cell. Bars = 2 joim. 192 M SHIRAE ET AL. am am . Figure 6. The rejection type of Sympli ^inn IV/>MJ» that is initiated before fusion of ampullae. (A) View of allorejection under stereomicroscope. Ampullae of two incompatible colonies make contact at their growing edges, and infiltrated hemocytes (arrowheads) are observed at the tips of ampullae. (B) Contact area of two incompatible colonies. Arrows indicate the contact point of these two colonies. Many hemocytes infiltrate the tunic and disintegrate, and the surrounding tunic is broken, am. ampulla; I. tunic. Bars: A = 200 /urn: B = 50 /j,m. recruiting cells from the circulating blood. Furthermore. 3 to 4 h after the stoppage of blood flow, phagocytes engulfed other hemocytes or hemocyte clusters in the rejection areas of blood vessels (Fig. 4C). The hemocyte clusters were encapsulated by phagocytes (Fig. 4D), and the encapsulated clusters sometimes attained a diameter of about 50 juin. All types of hemocytes, including MCs. were engulfed or en- capsulated by phagocytes. In the allorejection reaction of other botryllids. MCs always infiltrate the tunic from the blood vessels and disintegrate, but in the allorejection reac- tion of B. scalarix, MCs showed neither infiltration nor disintegration; they also did not show any morphological change inside the blood vessels (Fig. 5). Allorejection reactions in Symplegma reptans In botryllid ascidians the ampullae exhibit tip-to-side fusion after extension into the facing colony (Katow and Watanabe, 1980). In S. reptans, the fusion process in auto- geneic and syngeneic colony pairs has been reported (Mukai and Watanabe, 1974) as tip-to-tip fusion: ampullae of the two colonies did not extend into the facing colony and the ampullae of both colonies came into contact with each other at their tips. But as far as we observed, tip-to-side fusion of ampullae always occurred in the fusion reaction of S. reptans, as in botryllid ascidians. Unlike botryllids, S. reptans showed two types of al- lorejection reaction. The first type was the same as that HEMOCYTE BEHAVIOR IN ASCIDIAN ALLOREJECTION 193 B Figure 7. The rejection type of Symplcximi re/nans that is initiated after fusion of ampullae. (A) View of rejection area under stereomicroscope. Arrowheads indicate fused ampullae of two incompatible colonies and aggregation of hemocytes in their ampullae. (B) Inside of fused blood vessels during rejection reaction. Single arrows indicate fused points of vascular epithelia in the two incompatible colonies. Arrowheads indicate phagocytes engulfing other hemocytes. Double arrowheads indicate disintegrating cells, tv, tunic vessels; z, zooid. Bars: A = 500 pm: B = 10 /urn. described by Mukai and Watanabe (1974). In this type of rejection reaction, when two allogeneic colonies came into contact with each other, their tunics fused, but their ampul- lae neither extended into the opposite colony nor fused with each other. Then, within about 12 h of the contact hemo- cytes infiltrated the tunic from the ampullar tips at the contact area (Fig. 6A). Then, at the contact area, the tunic around the infiltrating hemocytes disintegrated (Fig. 6B). In the second type of rejection reaction, which was dis- covered in this work, fusion of blood vessels and exchange of blood occurred between two incompatible colonies as it does in fusion between compatible colonies. Twelve to fourteen hours after the fusion, hemocytes began to aggre- gate in blood vessels, and cell aggregation progressed inside the fused ampullae. Thereafter, the vessels were filled with aggregated hemocytes until finally the blood exchange was interrupted completely, within about 24 to 48 h after vas- cular fusion (Fig. 7A). Histological observations showed phagocytosis and disintegration of cells in this area (Fig. 7B). In addition, hemocytes infiltrated the tunic mainly from the fused ampullae, and these hemocytes and the surround- ing tunic disintegrated. With respect to hemocyte behavior, the two types of rejection reaction were similar. In both types, most of the hemocytes infiltrating the tunic were MCs. During the re- jection reaction. MCs disintegrated inside and outside the blood vessels (Figs. 8 and 9), as in some botryllids (such as Botryllus priniigeHits and Bntiylliis schlosseri). Disintegra- 194 M. SHIRAE ET AL. B Figure 8. Hemocytes and vascular epithelium in the rejection type initiated before fusion of ampullae in Symplexma reptans. (A) Blood vessel and outside of blood vessel. (B) Disintegration of morula cell inside blood vessel. (C) Disintegration of infiltrating morula cell in tunic. Arrowheads indicate electron-dense liber in the tunic, m. infiltrating morula cell; t. tunic: ve. vascular epidermal cell. Bars = 2 p.m. tion of MCs in the tunic promoted disintegration of the tunic matrix. Phagocytosis occurred in both types of rejection reaction, but encapsulated hemocyte clusters were not found. In the first type of rejection reaction (rejection before vascular fusion), tunic disintegration was limited to the regions surrounding the tips of ampullae in the contact area, and it was more intensive than in the second type (rejection after vascular fusion). However, MC disintegration and phagocytosis inside blood vessels was more common in the second type of rejection reaction than in the first. In the second type of rejection, electron-dense material that might have been discharged from MCs was often found in the vascular lumen (Fig. 9, arrowheads). This material was most abundant in the fused ampullae. The second type of rejec- tion reaction in 5. reptans progressed more slowly than the rejection ^action of B. scalahs. In both types of rejection reaction in v reptans, cellular junctions between the epi- thelial cells ui unpullar tips became loose, and some gaps appeared (arrow in Fig. 8A). Some 5. reptans colonies could exhibit both types of rejection reaction, but others showed only the first type (Fig. 10). The fusion experiments with the same pair of colonies were repeated several times and always showed the same results. Discussion In five botryllids, Botr\llus primigenus, Botryllus schlos- seri, Botrylloides simodensis, Botrylloides fuscus, and Bot- rylloides violaceiis, the rejection reaction between incom- patible colonies starts before the fusion of ampullae, although the beginning stages differ among species. These reactions involve activation of a few types of hemocytes, especially MCs, which are ubiquitous hemocytes in ascid- ians (Wright, 1^81 ). In contrast to these allorejections ac- companied by MC activation, the first stage of the rejection reaction in B. scalaris was hemocyte aggregation in fused ampullae mediated by phagocytes, especially hyaline ame- bocytes. The hemocyte clusters clearly caused the interrup- tion of blood exchange between incompatible colonies, as HEMOCYTE BEHAVIOR IN ASCIDIAN ALLOREJECTION 195 Figure 9. Hemocytes and vascular epithelium in the rejection type initiated after fusion of ampullae in Svmplegma reptans. (Al Hemocytes in blood vessel where the hemocytes have not aggregated yet. Phagocytosis often occurred. (B) Aggregation of hemocytes mediated by highly electron-dense material. (C) Disintegration of morula cell inside blood vessel. (D) Disintegration of infiltrating morula cell in tunic. Arrowheads in B, C, and D indicate highly electron-dense material inside blood vessel. Arrows in D indicate disintegrated tunic around morula cells, m, infiltrating morula cell; p, phagocyte; t. tunic; ve, vascular epidermal cell. Bars: A and B = 5 fim; C and D = 2 /im. shown in Figure 2. We labeled this phagocyte-mediated rejection reaction as P-type rejection, to distinguish it from the M-type rejection reaction primarily mediated by MCs in the other botryllids studied thus far. In S. re/nans, the two types of allorejection reaction shown here could be regarded as M-type rejection, although the second type did not in- volve remarkable MC infiltration. Morula cell infiltration, which occurs in allorejection in most botryllid species, might be facilitated by the change of permeability in the ampullar epithelium (Taneda and Watanabe, 1982a). This rejection reaction is always accompanied by disintegration of MCs, and their disintegration occurs not only outside but also inside of the ampullae in some botryllids (Rinkevich et al., 1994. 1998: Shirae. unpub. data). Therefore, we regard the standard character of M-type rejection as disintegration of MCs and discharging of their vacuolar contents. Both MCs and phagocytes are known to have important functions in the defense system of ascidians (Wright, 1981; Raftos, 1990; Cammarata et al.. 1997). In the M-type re- jection in botryllids and in S. reptans, the accumulation of the electron-dense material at the allorejection area is dis- tinctive. Moreover, in the second type of rejection in S. reptans, that material seems to mediate hemocyte aggrega- tion, resulting in interruption of blood exchange. Since phenoloxidase activity was demonstrated in MCs (Ballarin ct ul., 1995), the electron-dense material might be a mela- 196 M. SHIRAE ET AL. A B C D E F G \ f 1 1 1 1 1 A \ 1 1 1 1 1 B \ 1 1 1 1 C \ f 2 2 D \ 2 2 E \ f F \ G c) F=G ihe first type of rejection 2. — — : the second type of rejection f, n : the fusion reaction Figure 10. A pairwise allorejection assay among seven strains of Symplegma reptans (a) and relationships among S. reptans colonies concerning allorejection/fusion type (h). nin-like substance. A recent cytochemical study by Ballarin <7 i/l. ( 1998) suggested that phenoloxidase activity of MCs caused cytotoxicity in the allorejection reaction of Botryllus schlosseri. However, in the P-type rejection reaction of B. scalaris, such a defense system might not act. In B. scalaris, phagocytes might involve some functions in mediation of hemocyte aggregation. Consequently, the diversity of he- mocyte characteristics among botryllid ascidians may lead to the observed variation in allorejection. Comparative stud- ies of hemocyte histochemistry might further highlight the variation in allorejection systems of botryllids. A "fusion-rejection" reaction resembling the allorejection reaction in B. scalaris was described in an Israeli Botryl- loides (Rinkevich et <> itions. Orientation: anterior is top left, posterior is bottom right. Figure 3. Lateral view of the oral region of first-stage Placetron wosnessenskii zoea, showing locations and orientations of the mouthparts. Orientation: anterior is to the right, posterior to the left; ventral is at the top of the figure, and dorsal at the bottom. ingestion, but were difficult to observe because of their position deep in the preoral chamber. Posterior to the paragnathal lobes is a large, immobile projection of the sternite (Figs. 2, 3), which completes the posterior boundary of the preoral chamber. The sides of the chamber are bordered, anteriorly to posteriorly, by the highly mobile mandibles, maxillules, and maxillae. Mandibles. The cutting edges of the mandibles are di- vided into distinct incisor and molar processes and are asymmetrical with respect to the armament of these pro- cesses. The incisor process of the left mandible (Fig. 4b) is slightly larger than that of the right (Fig. 4a), consisting of one very large tooth and three or four smaller denticulate teeth. The left molar process is a large, rounded section of the mandible bearing two to four sen-ate or denticulate ridges culminating in a group of jagged denticles at the extreme (dorsal) edge. The right mandible (Fig. 4a) also bears a large, denticulate incisor process that includes one very large tooth, but in contrast to the left bears approxi- mately 6 to 10 smaller denticulate teeth. The molar region of the right mandible has a complex jagged margin and several serrate ridges. The cutting edges of the mandibles are aligned in a ventrodorsal plane, perpendicular to the sagittal plane of the animal. The incisor processes are positioned farthest ventrally, adjacent to the oral opening. The mandibles have rolling ventral-dorsal (down-up), mesial-lateral (inside-outside). and anterior-posterior (front- back) components to their movement. Each mandibular motion begins with a lateral-to-mesial movement that brings the cutting edges together and ends with a mesial-to-lateral movement that draws them apart. The ventral-dorsal rolling motions serve to tear and subsequently grind the prey by bringing the two mandibles together in an arcing slice, with contact made first at the extreme (dorsal) ends of the molar regions. Simultaneously, the mandibles make short anterior- posterior motions that grind the ridges of the molar regions against each other. The point of contact between the man- dibles is then smoothly shifted from the molars to the incisors over the duration of the arcing movement. Al- though the actions of the mandibles bring the grinding INGESTION BY LITHODID ZOEAE 211 . 25 mm Figure 4. Illustrations of the preoral chamber features and mouthparts of first-stage Plact'tnm wosnessenskii zoeae: (a) labrum; (b) paragnaths; (c) right mandible; (d) left mandible: (e) maxillule; (f) maxilla. molar regions together first, prey entering the oral region initially contact the tearing incisor processes. As the incisors are drawn across each other, the rolling motion of the mandibles results in a mouthward push of torn material from the incisors to the molar regions, to be crushed by the next mandibular roll. In this way. food is broken into sequentially smaller fragments by the mandibles before entering the oral cavity. Maxillules. Each maxillule (Fig. 4c) has a three- segmented endopodite located distolaterally; a large, toothed, mesially directed basal endite; and a smaller setose coxal endite that is also mesially directed. The setae of the three endopodal segments are (progressing distally): one simple, one pappose, and three plumodenticulate or pappose setae. The basal endite is armed with three large cuspidate teeth, each tooth with two to five denticles, and often with one very small, naked tooth developing between the others. The basal endite also bears two submarginal serrate or serrulate setae. The coxal endite bears six to eight marginal setae that are plumodenticulate, serrate, simple, or a com- bination of these types and one submarginal serrate seta. The maxillules position the food prior to ingestion. An overall mesial-lateral motion is most common, with the entire maxillule moving as a lever that uses the basal at- tachment as the fulcrum, and the tips of the endites describ- ing a large arc. Anterior-posterior motions of the maxillule are also observed, with the base of the appendage again serving as the fulcrum. The endopodite of each maxillule is muscularized and seems to be capable of some independent motion both in the anterior-posterior and mesial-lateral di- rections. The radically different morphologies of the endopodite and coxal and basal endites facilitate the plasticity of func- tions that this appendage is capable of performing. The denticulate teeth of the basal endite are used primarily for grasping and holding a captured metanauplius. The row of denticles on each tooth presumably aids in maintaining a grip on a struggling metanauplius. and although many of the metanauplii captured struggled energetically, very few of them were able to escape from the maxillules. The two submarginal setae on the basal endite may also aid in this grasping and holding. The three-segmented endopodite was seen to act as an outer guide for captured metanauplii, with the distal setae considerably extending the effective reach of the appendage in the anterior direction. The mobility of this endopodite adds a certain amount of fine control to the positioning of large prey. The location of the coxal endite was often difficult to distinguish in the videotapes, but was clearly at the posterior end of the preoral chamber during the final phase of ingestion. This endite was sometimes used to sweep small fragments of loose food back into the preoral chamber during phases 2 and 3 of ingestion. The setae of the coxal endites of the two maxillules were also seen to over- lap each other along the midline of the zoea when the 212 J. A. GRAIN maxillules were not in motion, thus forming a stationary border to the preoral chamber when at rest. Maxillae. Each maxilla (Fig. 4d) of the first zoeal stage consists of a large scaphognathite (exopodite fused with epipodite) on the lateral portion of the appendage, a weakly bilobed endopodite and two bilobed endites on the mesial portion. The scaphognathite bears four to six plumose setae on the outer margin; the proximal lobe is completely fused with the protopod. There are two or occasionally three small groupings (one to five setae per grouping) of setae on the distal portion of the endopodite, in combinations of serrate, plumodenticulate. simple, and pappose setae. The coxal endite is divided into two distinct lobes, with three plumo- denticulate or serrate marginal setae and one plumodenticu- late or pappose submarginal seta on the distal lobe. The proximal lobe of the coxal endite bears two plumodenticu- late marginal setae, a ridge with four or five plumodenticu- late setae, and one plumodenticulate submarginal seta. A similar division into two distinct lobes is characteristic of the basal endite. with the distal lobe bearing three plumo- denticulate or pappose marginal setae and one plumoden- ticulate submarginal seta. There are three or four plumo- denticulate or pappose marginal setae and one plumoden- ticulate or pappose submarginal seta on the proximal lobe of the basal endite. The maxillae beat continuously during ingestion, with the entire appendages moving water over the thoracic area. The scaphognathites, with their fringe of plumose setae, create a large functional "paddle" that increases the amount of current flow over the developing gill buds at the bases of the maxillipeds and adjacent area. This beating pattern is apparently respiratory, as it is in adults, and is undisturbed by the motions of adjacent append- ages. Involvement of maxillae in prey ingestion was not observed. Behavioral observations: ingestion of Artemia metanauplii Not all of the videotaped zoeae were observed to feed. Many individuals failed to capture or attempt to capture metanauplii. Those that successfully captured and ingested Anemia metanauplii nearly always exhibited an initial pe- riod of several minutes during which no metanauplii were captured. This interval prior to capture and ingestion of the first metanauplius tended to be longer than the pauses be- tween subsequent captures. Initial capture preceded a rapid series of capture and ingestion events, after which the zoea was apparently satiated and ceased feeding. At this point, the appendages even failed to respond to direct contact with the moulhparts by an Artemia metanauplius. In two separate sequences, the zoea spent several minutes merely holding, twisting, rotating, and finally rejecting a metanauplius cap- tured during the preceding feeding event. Zoeae captured and ingested metanauplii both while ac- tively swimming and while stationary. Prey manipulation and ingestion were apparently unaffected by movements of the exopodites and endopodites of the maxillipeds. which were used only for locomotion. Similarly, positioning of the abdomen and telson had no apparent effect on ingestion mechanisms. Because of complications in the use of untethered larvae, compounded by limitations of both field of view and of field of focus, only one sequence was recorded in which the ingestion of an Artemia metanauplius was traceable in its entirety (Fig. 5). Partial sequences in which only portions of the ingestion process were recorded were more numerous. Consequently, ingestion events have been separated into three consecutive phases to analyze appendage motions throughout the ingestion process. Each phase is easily dis- tinguished and possesses a distinct beginning and end. Phase 1. During the initial phase of Artemia ingestion, only the maxillules are in direct contact with the prey item. As noted above, the principal movements of the maxillules involve the rotating of the entire appendage from the base. In this way, they push the prey around or catch it between opposing endites. and the whole maxillule acts as a large paddle. The maxillules coordinate simultaneous and alter- nating motions as necessary to manipulate prey into position for ingestion. The first motion of ingestion is a rapid out- ward fling of the paired appendages, followed by an inward squeezing action, bringing the endites of the maxillules into contact with the metanauplius as it enters the preoral cham- ber. Metanauplii are held between the basal endites of the opposing maxillules, with the endopodites acting as outer guides and presumably aiding in stabilization of the strug- gling prey. Symmetrical motions of the opposing maxillules control the anterior-posterior positioning of the metanau- plius, while asymmetrical motions are involved with lateral repositioning of the metanauplius or with rotating it about its long axis. In two sequences, the metanauplius was turned end over end by the maxillules employing both symmetrical and asymmetrical motions. Metanauplii can be grasped by the maxillules in any position, but are usually manipulated into either a head-first or tail-first position (with the long axis of the metanauplius perpendicular to the long axis of the zoea) prior to ingestion. In some cases, ingestion se- quences are initiated with the metanauplius in a sideways position (with the long axis parallel to the long axis of the zoea), but are completed with the metanauplius in a head- first or tail-first position. Variations of this basic sequence were seen with two or more metanauplii involved. In four of the five multiple ingestion sequences analyzed, a newly captured metanau- plius was used to push a partially ingested metanauplius into the mandibles. In two separate sequences, two metanauplii were ingested simultaneously, although sequential inges- tions were far more common. In two of the analyzed INGESTION BY LITHODID ZOEAE 213 Figure 5. Selected frames from a sequence of ingestion of an Arieniiii metanauplius by a first-stage Placetron wosnessenskii zoea at 0, 7. 13. 26. 27. 37 (phase 1 ), 45.0 (phase 2). 45.8 and 46.3 (phase 3) seconds elapsed time: (a) photographs from images captured from a videotaped sequence; (h) illustration drawn from photographs in (a) to enhance clarity. 214 J. A. GRAIN maxillule Figure 6. Coordination of maxillule and mandible movements during phase 2 of ingestion of an Arteinui metanauplius by a first-stage PUiceiron wosnessenskii zoea. Approximate duration: 0.1 to 0.6 s. sequences, phase 1 of ingestion was shortened to merely the fling and capture steps, which were followed immediately by the maceration associated with phase 2. Recorded phase 1 duration ranged from 0.2 to 5.9 s in sequences involving only a single metanauplius. and from 6.1 to 233.0 s in sequences involving two or more meta- nauplii. Mean durations were 3.9 (;i = 14; SD = 5.8) and 56.8 (n = 5; SD = 93.2) s respectively. Phase 2. This phase of ingestion involves the maxillules, mandibles, and labrum. Phase 2 begins with the first contact of the metanauplius with the mandibles and ends with the loss of direct contact with the prey item by the basal endites of the maxillules. The maxillules, after manipulating the metanauplius into position in phase 1, begin pushing the prey toward the mouth, bringing it into contact with the mandibles. In one sequence, phase 2 was initiated by re- peated nudging of the metanauplius into and out of range of the mandibles by the maxillules. The maxillules and labrum press the metanauplius against the mandibles as the latter tear and grind the prey on its way into the mouth. Two separate ratcheting mechanisms were observed, each of which maintains a steady net movement of the metanauplius into the mouth, while enabling the appendages to renew points of contact for leverage. First, the maxillules and mandibles alternate motions, maintaining a firm grip on the metanauplius as it is masticated (Fig. 6). The maxillules press the prey against the mandibles as they shred bits of tissue with their rolling motions. The mandi- bles then hold the metanauplius as the maxillules regrasp it once every two or three mandibular rotations, or as seen in two sequences, with each mandibular roll. Second, the man- dibles and the labrum work in conjunction to prevent food from escaping from the mouth when the mandibles are on the recovery stroke of their motion (Fig. 7). The labrum moves downward, pinning the metanauplius in the preoral chamber with its spiny processes as the mandibles return to their initial (lateral) position in preparation for the next grinding roll, with a 1:1 ratio of alternating movements. The recorded total durations of phase 2 range from 6.5 to 150 s, with a mean duration of 49 s (n = 1 1: SD = 45.7). Phase 3. This phase of ingestion begins when the basal endites of the maxillules are no longer in contact with the prey, and continues until the entire metanauplius has passed through the mandibular region into the oral cavity. The mandibles and labrum continue to work together as in phase 2 until the entire metanauplius has been ingested. Although the maxillules are apparently no longer in direct contact with the prey, they often continue to make the sweeping mesial-lateral motions associated with phase 2, and direct small, easily lost fragments of masticated food back into the preoral chamber. A variation of phase 3 was seen in four sequences, when the maxillules pushed one metanauplius through the final stage of ingestion with a second metanau- plius. The range of durations recorded for this phase was from 4 to 84 s, with a mean duration of 23.2 s (/; = 9; SD = 31.6). mandible Figure 7. Coordination of mandible and lahrum movements during phases 2 and 3 of ingestion ot an Aitt'inin metanauplius by a tirst-slage Plact'lron wmnesscnskii zoea. Approximate duration: 0.1 to 0.3 s. INGESTION BY LITHODID ZOEAE 215 Behavioral observations: capture of algal cells Although several instances of algal capture by zoeae were observed, only one, in which a Pronicentniin niicuns cell was caught and ingested, was recorded on videotape. This capture took place very quickly, with the maxillules and mandibles drawing laterally, then held open momentarily before closing over the algal cell, which had apparently been drawn into the mouth by the suction created in the oral region. In preliminary gut fluorescence experiments. P. wosnessenskii ingested four species of unicellular algae: Cryptomonas sp. (approximately 12 X 7 jum), Prorocen- triini micans Ehrenberg (approximately 39 X 30 /urn), Gv- rodinium sp. (approximately 40 X 35 /im), and Isochrysis galbana Parke (approximately 4X3 /urn). These experi- ments did not contain sufficient numbers of replicates to determine clearance rates, but did indicate that the zoeae were able to ingest a range of particle sizes. Discussion Comparison of morphological descriptions A comparison of the present description of the mouth- parts of first-stage P. wosnessenskii zoeae with that of Haynes ( 1984) reveals few differences. Haynes commented on asymmetry between the left and right mandibles and on the jagged ridge of denticles located at the extreme end of the molar process of each, as noted here. The descriptions of the maxillules and maxillae differed only slightly in termi- nology and degree of detail. Mechanical functions of setae Function can sometimes be inferred by the types and placement of setae on an appendage. Detailed descriptions of setal types and locations, using both light and scanning electron microscopy, have been instrumental in revealing the function of the corresponding appendages or body areas (e.g.. Roberts. 1968; Farmer. 1974; Factor, 1978; Ajmal- Khan and Natarajan, 1981; Ohtsuka and Onbe, 1991: La- valli and Factor, 1992). In some cases specific setal types have been linked to specific functions, especially in feeding and grooming (e.g.. Barker and Gibson. 1977; Kunze and Anderson, 1979; Schembri, 1982; Fryer. 1983; Pohle. 1989). Crustacean setal types have been described in detail by many authors, as reviewed in Jacques (1989). Lavalli and Factor ( 1992), in their work on the lobster Homarus ameri- caiuis. used light microscopy to produce detailed descrip- tions of the range and locations of setal types found on the mouthparts of larval and juvenile lobsters. They grouped the setae into 13 categories based on external features, espe- cially the form and position of setules. Each category in- cluded variations in form, and several of the setal types found on the mouthparts of first-stage P. wosnessenskii zoeae fit into Lavalli and Factor's (1992) groupings. The setae found on the tips and edges of the feeding appendages of P. wosnessenskii were often more setulose than those on the inner surfaces, a condition that is also reported for crayfish by Thomas ( 1970), lobsters by Factor (1978) and Lavalli and Factor (1992), and leucosids by Schembri ( 1982). Fanner ( 1974) reported smoother setae on the inner surfaces of the mouthparts of Nephrops norvegicus (Linnaeus) than on the fringes, attributing this pattern of distribution to the different functions of the two locations. Farmer ascribed primarily a gripping function to the setae located on the insides of the mouthparts, a role for which setulose setae would be less suited because of the recurring need for removal of bits of trapped food. Following this line of reasoning, the submarginal serrulate setae on the inner surface of the maxillules of P. wosnessenskii zoeae are well suited for gripping: their minute scales enhance the ability to clutch food without retaining or becoming clogged by small particles. Similarly, the serrate setae on the endites of the maxillules and maxillae may perform gripping functions along with grooming of other setose appendages, as sug- gested by several authors, e.g.. Roberts (1968). Farmer (1974), and Schembri (1982). A variety of functions have been reported for setae armed with long setules. such as the plumose, pappose, and plu- modenticulate setae found on the maxillules and maxillae of P. wosnessenskii zoeae. Plumose setae like those found in this study on the scaphognathites of the maxillae aid in generating water currents for respiration (Thomas, 1970; Farmer, 1974; Factor, 1978; Ajmal-Khan and Natarajan, 1981; Schembri, 1982). filter feeding (Rubenstein and Koehl. 1977: Cheer and Koehl. 1987). and locomotion (Fryer, 1983). If the setules are closely spaced, the boundary layer surrounding each will overlap that of adjacent setules, increasing the effective area of the appendage. As the thick- ness of these boundary layers changes with velocity, a given group of setae can effectively function either as large solid areas or as sieves (Cheer and Koehl. 1987). In this respect, although the planar configuration of the setules on plumose setae may cause them to function as flat paddles, the seem- ingly haphazard arrangement of the setules on the shafts of pappose setae such as those found on the maxillary endites of P. wosnessenskii may in turn cause the setae to function more like cylinders. Plumodenticulate setae on the maxillules and maxillary endites of P. wosnessenskii may act as a combination of pappose and denticulate setae. Schembri (1982) observed that the tendency toward smaller denticles on the distal portions of plumodenticulate setae served to direct stray particles toward the tips of the setal shafts. The large num- ber of these setae on the endites of the maxillae and max- illules of first-stage P. wosnessenskii zoeae, coupled with the diversity of secondary processes, suggests that they may 216 J. A. GRAIN be quite versatile, performing grasping, brushing, cleaning, and current-controlling functions. The chemosensory and tactile roles of setae have been studied in detail by a number of authors, including Shelton and Laverack (1970). Derby (1982). Atema (1985), and Lavalli and Factor ( 1992). Regrettably, the electron micros- copy and neurological experimentation necessary to at- tribute chemosensory or tactile function to specific setal types was beyond the scope of this study. Behavioral and morphological analysis The wide ranges of time for each phase of ingestion were due in large part to the variability of levels and types of activity in which the zoeae were engaged. Lapses in inges- tion activity were frequent, often corresponding with in- creased swimming or capture of a second metanauplius, but in some cases not attributable to any obvious external cause. The shortest phase durations were recorded for zoeae that were actively engaged in sequential capture and ingestion events, and were often followed by long periods of reduced interest in feeding. Although not usually included in most morphological descriptions, the functional significance of the puragnathal lobes warrants mention. It has been observed that in at least some crustaceans the paragnaths are mobile and are me- chanically coupled with the mandibles (Wales, 1982). The paragnaths of P. wosnessenskii zoeae were not observed to move in this study, which is consistent with the findings of Alexander (1988), who noted that although the paragnaths of anomurans do not move by themselves, they can pas- sively shift position as the mandibles open. Alexander also noted that the many large setae on the inner margins of unoimiran paragnaths seem to aid in food retention. This appears to be true in P. wosnessenskii. The labrum is another feature of crustacean anatomy often omitted from morphological descriptions. The func- tional significance of this structure and its complement of spines and setae in prey ingestion by zoeal P. wosnessenskii cannot be overlooked. In all of the observed sequences, the labrum was instrumental in maintaining a steady movement of food into the mouth. Its location at the opening to the oral chamber places it in position to give food items the final push into the loregut as well as to grip large prey items while the mandibles reposition themselves between mo- tions. Similar accounts of labrum and mandible coordina- tion in other crustaceans can be found in Manton ( 1977) and Schembri (1982). The mandibles are the masticating appendages, responsi- ble for all tearing, shredding, and grinding of the prey prior to entry into the mouth. Although the rotational motion of the mandibles brings the molar processes into contact with each other before the rest of the cutting edges, the prey item actually contacts the ventrally located incisor processes first. The incisor portion of the cutting edge tears off large pieces of the prey, the rotational motion of the mandible then aids in pushing those pieces farther toward the mouth to be further shredded and ground by the molar processes. The asymmetry of the molar processes of the left and right mandibles and the complexity of these regions provides a variety of masticating surfaces. As these surfaces are mov- ing past each other, the prey is torn, shredded, and ground into tiny pieces in the final preparation for ingestion. The maxillules manipulate prey during the initial phase of ingestion. The radically different morphologies of the en- dopodite and coxal and basal endites facilitate a wide range of functions. The denticulate teeth of the basal endite were used primarily for grasping and holding a captured meta- nauplius. The row of denticles on each tooth presumably aids in maintaining a firm grip on a struggling metanauplius. and although many of the metanauplii captured struggled energetically, very few of them escaped from the grip of the maxillules. The two submarginal setae on the basal endite may also aid in this grasping and holding process. The three-segmented endopodite acts as an outer guide for the manipulation of captured metanauplii, with the distal setae extending its effective reach in the anterior direction. En- dopodite mobility enhances fine control over the positioning of a large prey item. The position of the coxal endite was often difficult to distinguish in the videotapes, but it clearly was at the posterior end of the preoral chamber during the final phase of ingestion. The range of motion demonstrated by manipulation of preserved specimens shows that the coxal endite is capable of at least some movement indepen- dent of the rest of the maxillule. Small fragments of floating food were often swept back into the preoral chamber by the coxal endite during phases 2 and 3 of ingestion. The setae of the coxal endites of the two maxillules overlapped each other along the midline of the zoea when the maxillules were not in motion, thus forming a stationary border to the preoral chamber when at rest. Fryer ( 1983) observed that the naupliar maxillules of the anostracan Branchinecta ferox (Milne-Edwards) retained food particles in a similar manner. The lack of observed feeding function of the endites of the maxillae off. wosnessenskii zoeae is probably related to their more posterior position in the zoeal stages compared to the adults, in which they are presumed to be involved in ingestion. In adult crabs, all of the feeding appendages, including the three pairs of maxillipeds, overlap one another obliquely and are limited in their posterior extension by the sternites of the pereopods. In this arrangement, the maxil- lary endites are more directly in the path of the food parti- cles than they are in the larvae. Greenwood ( 1972) observed that the movements of the endites in adult hermit crabs were coupled with those of the scaphognathites. resulting in a continuous beating, "threshing the sides of the food" in the preoral chamber. It is also quite possible that the setae of the INGESTION BY LITHODID ZOEAE 217 maxillary endites of both zoeae and adult crabs aid in the ingestion of smaller food items either through physical contact with the food or by the creation of a strong feeding current. Other possible food sources for zoeae: ecological implications For some decades, the focus of studies on zoeal diet has been its effect on growth and survival under laboratory and natural conditions (e.g., Kurata, 1959; Sulkin, 1975, 1978; Paul ct ul.. 1979; McConaugha, 1985; Harms and Seeger. 1989; Paul et ul., 1989; Epifanio el ul.. 1991). There is increasing agreement that under natural conditions, zoeae are unlikely to encounter zooplankton prey in the concen- trations routinely used in laboratory rearing experiments, and therefore they can probably utilize a variety of food sources (Incze and Paul, 1983; Harms and Seeger, 1989; Paul et ul.. 1989; Epifanio et ul.. 1991). Sulkin found that the zoeae of Cullinectes sapidus Rathbun, a brachyuran crab that cannot successfully complete development to metamor- phosis on purely algal diets, nonetheless ingested unicellu- lar organisms (Sulkin. 1975). The lithodid crab Parulifh- odes cumtschuticu (Tilesius) can be reared in the laboratory on polychaete larvae or Artemia nauplii, but not on a diet made up solely of diatoms (Kurata, 1959). However, Paul et ul. (1989) found that when first-stage P. camtschatica zoeae ingested phytoplankton soon after hatching, they molted to the second zoeal stage at higher rates than those that did not. The algal diet did not sustain the zoeae through metamor- phosis, and an increasing dependence on carnivory through the zoeal stages was hypothesized for this species (Paul et ul., 1989). First-feeding P. wosnessenskii zoeae are able to take advantage of a wide range of prey items and probably rely on a variety of planktonic food sources throughout their development. Factor and Dexter (1993) found that the larvae of the brachyuran crab Carcinus muenus (Linnaeus) could capture suspended algal cells, and hypothesized that the setose mouthparts were involved in suspension feeding. In prelim- inary gut fluorescence experiments, P. wosnessenskii in- gested unicellular algae covering a range of sizes. How these zoeae capture small algal cells is not known, but we assume that the mechanism is similar to that seen for in- gestion of a Prorocentnun micuns cell. In the one sequence of particle capture recorded on videotape, a P. micuns cell was captured and ingested using a "fling and clap" method similar to that described for copepods by Koehl and Strick- ler (1981). The mouthparts were flung outward, enlarging the space between them, thus drawing the cell into the mouth. The mouthparts were then closed over the cell, squeezing water out through the spaces between the setae and endites. as seen in algal capture by copepods (Koehl and Stockier, 1981). This study demonstrates that P. wosnessenskii zoeae can utilize prey items ranging from unicellular algae (Crypto- monus sp., Pmroccntnim micuns. Gyrodinium sp.. and Iso- cluysis gulbiinu) to relatively large, active zooplankton (Ar- temiu sp. metanauplii). McConaugha ( 1985) identified three criteria for suitability of prey items as food sources for larval crustaceans: ( 1 ) appropriate size for capture and con- sumption. (2) adequate concentration, and (3) essential di- etary nutrients to meet the larvae's needs for survival, growth, and metamorphosis. Natural plankton assemblages are varied in composition both spatially and temporally. The ability to capture and ingest a variety of sizes and shapes, expanding the diversity of prey species that meet Mc- Conaugha's first criterion, increases the probability that the zoea will be able to fulfill its nutritional requirements for successful development. Acknowledgments This project was funded by a grant from the PADI Foun- dation. 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Ecol. 34: 29-41. Thomas, W. J. 1970. The setae of Austropotomobius pallipes (Crusta- cea: Astacidae). J. Zoo/. Lund. 160: 91-142. Wales, W. 1982. Control of mouthparts and gut. Pp. 165-191 in The Biologv of Crustacea, Volume 4. Academic Press, New York. Reports of Papers Presented at THE GENERAL SCIENTIFIC MEETINGS OF THE MARINE BIOLOGICAL LABORATORY, Woods Hole, Massachusetts 16 to 18 August 1999 Program Chairs: BARBARA BOYER, Union College WILLIAM ECKBERG, Howard University CHARLES HOPKINSON, Ecosystems Center, MBL ROBERT PAUL MALCHOW, University of Illinois at Chicago Each of these reports was reviewed by two members of a special editorial board drawn from the research community of Woods Hole, Massachusetts. Reviewers included scientists from THE MARINE BIOLOGICAL LABORATORY, THE WOODS HOLE OCEANOGRAPHIC INSTITUTION, AND THE NATIONAL MARINE FISHERIES SERVICE. SHORT REPORTS FROM THE 1999 GENERAL SCIENTIFIC MEETINGS OF THE MARINE BIOLOGICAL LABORATORY FEATURED ARTICLE Rome, Lawrence C. Introduction. Bringing the script to life: the role of muscle in behavior 225 Rome, Lawrence C., Andrei A. Klimov, and Iain S. Young A new approach for measuring real-time calcium pumping and SR function in muscle fibers 227 Oliver, Steven J., and Elise Watson Threat-sensitive nest defense in domino damselfish (Dascyllm iilhiwllii) 244 Price, Nichole N., and Allen F. Mensinger Predator-prey interactions of juvenile toadfish, Opsa- n us tau 246 Tang, Kathleen Q., Nichole N. Price, Maureen D. O'Neill, Allen F. Mensinger, and Roger T. Hanlon Temperature effects on first-year growth of cultured oyster toadfish, Op\//ini.\ l/iu 247 PHYSIOLOGY Malchow, Robert Paul, and David J. Ramsey Responses of retinal Miiller cells to neurotransmitter candidates: a comparative study 229 Clay, John R., and Alan M. Kuzirian Fluorescence localization of K.' channels in the membrane of squid giant axons 231 Km. i. Vanessa J., Frederick A. Dodge, and Robert B. Barlow Evaluation of circadian rhythms in the I.imitlu.s eye. . . 233 Novales Flamarique, Iriigo, and Ferenc I. Harosi Photoreceptor pigments of the blueback herring (Alosa aestevalis, Clupeidae) and the Atlantic silver- side (Menidia menidia. Atherinidae) 235 Hanley, Janice S., Nadav Shashar, Roxanna Smolowitz, William Mebane, and Roger T. Hanlon Soft-sided tanks improve long-term health of cul- tured cuttlefish . 237 PISCINE NEUROBIOLOCY AND BEHAVIOR Zottoli, S.J., F.R. Akanki, N.A. Hiza, D.A. Ho-Sang, Jr., M. Motta, X. Tan, K.M. Watts, and E.-A. Seyfarth Physiological characterization of supramedullary/ dor- sal neurons of the dinner, Tautogolalmu athpersus. . . . 239 Fay, R.R., and P.L. Edds-Walton Sharpening of clirectic mal auditor)' input in die descend- ing octaval nucleus of the toadfish, (>/>//.\i/n.\ Itni 240 Kaatz, Ingrid M., and Phillip S. Lobel Acoustic behavior and reproduction in five species of Cmycoroi catfishes (Callichthyidae) 241 Lobel, Phillip S., and Lisa M. Ken- Courtship sounds of the Pacific damselfish, Abudefduf .wrdidus (Pomacentridae) 242 CHEMORECEPTION AND BEHAVIOR Mjos, Katrin, Frank Grasso, and Jelle Atema Antennule use by the American lobster, Homanis americanus, during chemo-orientation in three turbu- lent odor plumes 249 Hanna, John P., Frank W. Grasso, and Jelle Atema Temporal correlation between sensor pairs in differ- ent plume positions: A study of concentration infor- mation available to the American lobster, Homams americanus, during chemotaxis 250 Zetder, Erik, and Jelle Atema Chemoreceptor cells as concentration slope detec- tors: preliminary evidence from the lobster nose . . . 252 Berkey, Cristin, and Jelle Atema Individual recognition and memory in Homaru\ americanus male-female interactions 253 McLaughlin, Leslie C., Jennifer Walters, Jelle Atema, and Norman Wainwright Urinary protein concentration in connection with agonistic interactions in Homarus americanus 254 King, Alison J., Shelley A. Adamo, and Roger T. Hanlon Contact with squid eggs increases agonistic behavior in male squid (Lnligo pealef) 256 CELL MOTILITY Bearer, E.L., M.L. Schlief, X.O. Breakefield, D.E. Schu- back, T.S. Reese, and J.H. LaVail Squid axoplasm supports the retrograde axonal transport of herpes simplex virus 257 221 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS Gould, Robert, Concetta Freund, Frank Palmer, Pam- ela E. Knapp, Jeff Huang, Hilary Morrison, and Doug- las L. Feinstein Messenger RNAs for kinesins and a dynein are lo- cated in neural processes 259 Fukui, Yoshio, Taro Q.P. Uyeda, Chikako Kitayama, and Shinya Inoue Migration forces in Dictyostelium measured by centri- fuge DIC microscopy 260 Tran, P.T.. P. Maddox, F. Chang, and S. Inoue Dynamic confocal imaging of interphase and mitotic microtubules in the fission yeast, ,S. pombr 262 Maddox, Paul, Arshad Desai, E.D. Salmon, T.J. Mitchi- son, Karen Oogema, Tarun Kapoor, Brian Matsumoto, and Shinya Inoue Dynamic confocal imaging of mitochondria in swim- ming Tetrahymena and of microtubtile poleward flux in Xenopus extract spindles 263 Wollert. Torsten, Ana S. DePina, and George M. Lang- ford Effects of vanadate on actin-dependent vesicle motil- ity in extracts of clam oocytes 265 CELL AND DEVELOPMENTAL BIOLOGY Biswas, Chhanda, and Peter B. Armstrong Identification of a hemolytic activity in the plasma of the gastropod Busyron canalirulatum 276 Knlins. William J., Max M. Burger, and Eva Turley Hvaluronic acid: a component of the aggregation factor secreted by the marine sponge, Microdona pro- lifera 277 Popescu, Octavian, Rey Interior, Gradimir Misevic, Max M. Burger, and William J. Kuhns Biosynthesis of tyrosine O-sulfate by cell proteoglycan from the marine sponge, Microdona prolifern 279 Vasse, Aimee, Alice Child, and Norman Wainwright Prophenoloxidase is not activated by microbial sig- nals in Limulm polyphemus 281 Ogunseitan, O.A., S.L. Yang, and E. Scheinbach The 6-aminolevulinate dehydratase of marine Vibrio alginolytirus is resistant to lead (Pb) 283 Hoskin, Francis C.G., Diane M. Steeves, and John E. Walker Substituted cyclodextrin as a model for a squid en- zyme that hydrolyzes the nerve gas soman 284 Zigman, Seymour, Nancy S. Rafferty, Keen A. Rafferty, and Nathaniel Lewis Effects of green tea polyphenols on lens photooxida- tive stress 285 Billack, Blase, Jeffrey D. Laskin, Michael A. Gallo, and Diane E. Heck Effects of a-bungarotoxin on development of the sea urchin Arbada punttulala 267 Silver, Robert B., and Nicole M. Deming Leukotriene B4 as calcium agonist for nuclear enve- lope breakdown: an enzymological survey of endo- membranes of mitotic cells 268 Weidner, Earl, and Ann Findley Extracellular survival of an intracellular parasite (Spragitea lophii, Microsporea) 270 Kaltenbach, Jane C., William J. Kuhns, Tracy L. Simp- son, and Max M. Burger Intense concanavalin A staining and apoptosis of peripheral flagellated cells in larvae of the marine sponge Microdona prnlifrra: significance in relation to morphogenesis 271 COMPARATIVE BIOCHEMISTRY Harrington, John M., and Peter B. Armstrong A cuticular secretion of the horseshoe crab, Linndii\ polyphemus: a potential anti-fouling agent 274 Asokan, Rengasamy, and Peter B. Armstrong ( Cellular mechanisms of hemolysis by the protein limu- lin, a sialic-acid-specific lectin from tlie plasma of the American horseshoe crab, Limulm pol\plu>mus 275 ECOLOGY AND EVOLUTION Mondrup, Thomas Salinity' effects on nutrient dynamics in estuarine sediment investigated by a plug-flux method 287 Pease, Katherine M., L. Claessens, C. Hopkinson, E. Rastetter, J. Vallino, and N. Kilham Ipswich River nutrient dynamics: preliminary assess- ment of a simple nitrogen-processing model 289 Wolfe, Felisa L., Kevin D. Kroeger, and Ivan Valiela Increased lability of estuarine dissolved organic ni- trogen from urbanized watersheds 290 Evgenidou, A., A. Konkle, A. D'Ambrosio, A. Corcoran, J. Bowen, E. Brown, D. Corcoran, C. Dearholt, S. Fern, A. Lamb, J. Michalowsky, I. Ruegg, andj. Cebrian Effects of increased nitrogen loading on the abun- dance of diatoms and dinoflagellates in estuarine phytoplanktonic communities 292 Cubbage, Andrea, David Lawrence, Gabrielle Tomasky, and Ivan Valiela Relationship of reproductive output in Acartia tonsa, chlorophyll concentration, and land-derived nitrogen loads in estuaries in Waquoit Bay, Massachusetts 294 Canfield, Susannah, Luc Claessens, Charles Hopkinson Jr., Edward Rastetter, and Joseph Vallino Long-term effect of municipal water use on the water budget of the Ipswich River Basin 295 LIST OF MBL REPORTS 223 Young, Talia, Sharon Komarow, Linda Deegan, and Kerr, Lisa M., Phillip S. Lobel, and J. Mark Ingoglia Robert Garritt Evaluation of a reporter gene system biomarker for Population size and summer home range of the green detecting contamination in tropical marine sedi- crab, Carcimis mmnus, in salt marsh tidal creeks 297 ments 303 Komarow, Sharon, Talia Young, Linda Deegan, and Robert Garritt Influence of marsh flooding on the abundance and growth of Fundulus htteroclitus in salt marsh creeks . . . 299 ^^ PRTSENT \TIONS Widener, Justin W., and Robert B. Barlow Decline of a horseshoe crab population on Cape 300 PUBLISHED BY TITLE ONLY 307 Reference: Biot. Bull. 197: 225-226. (October Introduction to the Featured Article Bringing the Script to Life: the Role of Muscle in Behavior A goal of Neurosc ience 's soon-to-be-completed "Decade of the Brain" is to integrate nervous function from molecule to behavior. But behavior does not begin and end in the nervous system. Muscles actuate the movements that constitute most behaviors — and if the properties of the muscle are not "tuned" to the overall biomechanics of the body, then the behavior will not occur as planned in the brain. Thus, the properties of the muscles and the overall biomechanics of the body are also determinants of behavior, sometimes equal in importance to that of the nervous system. Two central concepts dictate that experimental approaches to the properties of muscles must also be integrative. First, one cannot understand how a muscle is designed without knowing exactly what it does during normal behavior ( 1 ). and second, the adaptation of muscle for different motor activities takes place at the molecular level. Our goal over the past few years has therefore been to understand, from the level of molecular biophysics to whole-animal biomechanics, how muscles are designed to power different activities. This ambitious goal has only become feasible because of a revolution that has taken place in the field of integrative muscle physiology and biomechanics over the past decade. This revolution has been largely fueled by technological improvements in three areas: biophysics, whole-animal measurements, and computer modeling. The development of new biophysics technologies (e.g., Ca2+-sensitive dyes and caged compounds) enables us, for the first time, to measure the kinetics of the pertinent molecular processes of muscle contraction. Indeed, we will soon begin to develop a model of muscle contraction that is based on principles of chemical kinetics. At the organismal end of the scale, development and miniaturization of transducers (e.g.. sonomicrometry, strain gauges) and telemetry now facilitate measuring the performance of muscles in the animal during normal behavior. Further, recent development of musculoskeletal modeling systems and 3-D laser scanners permits us to construct virtual animals with anatomical features that are exactly like those of living animals. Because the movements of these virtual animals are dictated by biomechanics rather than an animator's imagination, simulation of a set of muscle contractions will allow us to test how whole-animal motor performance would be altered if the kinetics of a molecular process were changed in a specific manner. This approach will show how changes in single molecular components might affect whole-animal performance in real life, and thus how simple genetic changes might have affected the evolution of behavior. Finally, this current revolution in muscle physiology and biomechanics would not have been possible without the development of exceptional experimental models. Fish have led the way in this regard. In contrast to the mixing of different fiber types that occurs in the muscles of mammals and most other vertebrates, the different muscle fiber types in fish are organized into large, homogenous, and anatomically separated regions that are visible to the naked eye. In fish, as opposed to mammals, we can monitor which motor activities are powered by each fiber type because we can implant electromyography electrodes in these anatomically separate regions. In addition, one can dissect bundles of fibers that are all of the same type, so that the mechanical, biochemical, and ultrastructural properties of each muscle fiber type can be determined. The fish that best exemplifies the diversity of muscular function and design is the toadfish (Opsamis tan) (Fig. 1. left). These animals rest on the bottom and wait for a fish or crustacean to pass; then they grab the prey with their immense jaws. The swimming muscles of the toadfish reflect this sluggish behavior. Their slow-twitch muscles, which in most fish are used for steady swimming, are almost nonexistent; the few that do occur are among the slowest ever measured — they work at about 1 Hz. Toadfish "fast"-twitch white muscle, used in a short burst of activity to capture prey, is also unimpressive — it has about the same speed as ,s7mr-twitch muscle in other fish. But when it comes to calling for a mate, the male toadfish is an athlete of Olympic stature. The muscles that surround his heart-shaped swimbladder (Fig. 1, center) alternately contract down on the organ and relax at up to 200 Hz to produce the "boat-whistle" mating call. In fact, the swimbladder muscle is the fastest vertebrate muscle known. The diversity of muscles in toadfish strikingly illustrates how muscle can be so exquisitely tuned for one 225 226 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS Figure 1. Left: The toadfish Opsanus tau (Photo by A. Kuzirian). Center: Toadfish swimbladder; the red swimbladder muscle runs circiimferentntlh- around the white swimbladder (Photo by L. Nelson). Right: Molecular processes of muscle activation and relaxation (Drawing: T. Clark). behavior that it can't be used for another. Neither the red nor the white swimming muscle can physically produce high-frequency sounds because it can't relax fast enough. By contrast, at the low frequencies of steady swimming, the swimbladder muscle's extremely low mechanical power output and extremely high energetic cost make it unable to power locomotion (2). The challenge of studying the adaptations of the fastest vertebrate muscle is irresistible. My colleagues and 1 (3, 4) had already determined that the immense speed of the swimbladder is principally due to its extremely fast relaxation rate, which in turn is due to three specific adaptations: the swimbladder removes Ca2 + from the cytoplasm 50-fold faster than the red muscle does (Fig. 1, right: Step 4). swimbladder troponin C has a far faster Ca2 + off-rate than red muscle (Step 5), and swimbladder crossbridges have a 50-fold faster detachment rate constant than red muscle (Step 6). In our attempt to further dissect the molecular mechanisms responsible for the swimbladder's rapid clearing of Ca~ + , we ran into a snag in distinguishing the three competing mechanisms. One of these mechanisms is fast Ca2 + pumping by the sarcoplasmic reticulum (SR); but two other mechanisms — binding of Ca2 + to the soluble protein pan/albumin and binding to the SR-Ca2 + pumps themselves — may also make a very large contribution. Consider that when toadfish call with the swimbladder muscle, it is not a single twitch but a series of many contractions that is produced. When the muscle relaxes after a stimulus, the interval before the next stimulus (Vino s) is not long enough for all the Ca2+ to be pumped back into the SR. So Ca2+ is left attached to the parvalbumin and the Ca2+ pumps, and thus less Ca2+ is pumped back into the SR than was originally released. During calling, therefore, the SR may ultimately run out of Ca2+ to release. Perhaps reflecting this limitation, toadfish call in short bursts rather than continuously. Therefore, if we could determine the relative importance of these three mechanisms we could understand, not only how the system is designed to be so quick, but why these animals behave as they do. Unfortunately, these processes can't be differentiated in an intact cell. We turned, therefore, to permeabilized ("skinned") muscle cells because the parvalbumin can be washed out through the leaky cell membrane while the SR membrane remains intact. The following paper describes the development of a new technique for measuring Ca2+ uptake by the SR in skinned muscle fibers. This technique is a small but necessary step towards solving the puz/.le of how these biological machines have evolved to perform so many functions. It will also help us better understand the biological basis of behavior. — Lawrence C. Rome August 1999 Literature Cited I Rome, L. C., R. P. Funke, R. M. Alexander, G. Lutz, H. D. .1. N. Alclridge, F. Scott, and M. Freadman. 1988. Nature 355: 824-X27 2. Rome, L. C., and S. L. Lindstedt. 1998. News Plmiol. Sci. 13: 261-268. 3. Rome, L. C., D. A. Syme, S. Hollinj;Horth, S. L. l.indstedt, and S. M. Baylor. 1996. Pmc. Null. Acud. Si-i. USA 93: K(W5-SIO(). 4 Rome, L. C., C. Cook, D. A. Syme, M. A. Connaughton, M. Ashley- Ross, A. A. Klimov, B. A. Tikunov, and Y. E. Goldman. 1999. Pr,,c. Null. Acud. Sci. USA 96: SS3I. FEATURED ARTICLE 227 Reference: Bio/. Bull. 197: 227-228. (October A New Approach for Measuring Real-time Calcium Pumping and SR Function in Muscle Fibers Lawrence C. Rome{, Andrei A. Kliniov, and lain S. Yoiiii}f (Biology Department, University of Pennsylvania, Philadelphia, Pennsylvania 19104) Different muscles are designed to perform a wide variety of motor activities that extend over a large range of frequencies. To increase our understanding of the molecular basis for these differ- ent designs, we are attempting to model the activation-relaxation cycle of different muscles by obtaining kinetic information about each of the constituent processes (1, 2). An important component in muscle relaxation is Ca2+ sequestration by sarcoplasmic retic- ulum (SR)-Ca2 + pumps, but the pump turnover rate in different fiber types is not known precisely. Previous measurements of SR function have generally in- volved one of two approaches. One approach involves homog- enizing muscle, making SR preparations and measuring the rate of Ca2+ accumulation in the presence of oxalate (e.g., 3). It is unclear, however, whether homogenization alters the pumping rate compared to that of intact SR in muscle fibers. Further, the presence of oxalate probably increases the rate of accumulation above that in normal fibers by reducing the free Ca2 + concen- tration inside the SR. An alternate approach involves physio- logical measurements of intracellular free [Ca2 + ] using calcium sensitive dyes (e.g., 1, 4). However, this approach provides direct information only about the small amount of Ca2 + that is actually free in the myoplasm. The total movements of Ca2 + can only be estimated by modelling the large quantities of cither Ca2 + buffers in the cell (e.g., parvalbumin and troponin; 4). but the amounts and the kinetics of these calcium buffers are not always known in detail. We have developed a technique and an instrument that permit direct measurements of Ca2+ sequestration by intact SR of muscle fibers as a function of [Ca2+free]. Single muscle fibers are exposed to 50 /ig/ml saponin (20 min), which permeabli/.es the cell membrane, but leaves the SR membrane intact (5). The "skinned" muscle fibers are then bathed in solutions that contain various concentrations of Ca2 + frce; and calcium sequestration (i.e., Ca2+ leaving the bath) is monitored by using a Ca2+ sensitive dye, FURA-2. This approach solves two important problems. First, during the "skinning" procedure, most of the intracellular Ca2+-buffer (the soluble parvalbumin) leaks out of the cell membrane and is washed away. Second, the amounts of remaining intracellular buffers (e.g.. troponin), become very small compared to the total buffer in the bath, which is well defined in terms of amounts and kinetics, and can be accurately calibrated. Hence Ca2 + sequestration by the SR can be accu- rately measured as a removal of Ca2+ from the bathing solution. ' To whom correspondence should be addressed. This approach, unfortunately, involves a high bath volume to fiber volume ratio. The fiber diameter (and hence fiber volume) must be kept small because of potential diffusion limitations. Further, the necessity of stirring the bathing solution rapidly in the long and thin chamber fitted to the morphology of muscle fibers prevents reduction of the bath volume below a critical value. In this study we used a bath volume of 5 /xl and typical fiber volumes of -5 nl. giving a bath to fiber volume ratio of about 1000:1. We overcame the large bath volume to fiber volume ratio by using three approaches to optimize the detection of the fluores- cence change associated with the uptake of Ca2+. First, by using FURA-2 as the main Ca2+ buffer (in addition to its use as Ca2+ indicator), we maximized the absolute fluorescence change for a given uptake of Ca2 ' . By avoiding using addi- tional buffers (e.g.. EGTA), at low [Ca2 + free], we achieved a nearly stoichiometric fluorescent change in one FURA molecule for every Ca2+ molecule taken up by the SR. Second, we also improved detection by employing only a low concentration (50 fj.M) of the FURA Ca2 ' buffer. Note that at high [Ca2 + lrec|. the ATP in the solution also bound Ca2 + , thus leading to a decline in sensitivity. Third, as illustrated in the optics diagram (Fig. IB), the FURA was excited at 400-420 nm. With this excita- tion, only unbound FURA absorbs light (6). Hence the emission measured at 480-610 nm with a solid state photodetector is proportional to the concentration of unbound FURA. Thus, those solutions containing high [Ca2 + ,ree] have low fluores- cence. This is important because, under conditions where Ca2 + sequestration was measured (i.e., high [Ca2 + free]). Ca2+ se- questration was observed as an increase in fluorescence over a low initial fluorescence (Fig. 1C), thereby further improving detection. In addition to the optics for measuring the fluorescence change, the calcium sequestration device contained a cuvette in which the muscle fiber is positioned and in which solutions containing the appropriate FURA and Ca2 + concentrations can be very rapidly exchanged ( — 200 ms. Fig. 1A). Because the cuvette is well stirred (with air jets — Fig. I A), the FURA fluorescence can be sampled anywhere within it, except in the small volume occupied by the fiber itself. For development of this technique, our preparation consisted of one or two toudfish swimbladder muscle fibers with a max- imum diffusion distance of about 20 /nm. This fiber type is thought to have the highest density of Ca2 ' pumps (7) and the fastest calcium transient of any vertebrate muscle ( 1 ). Our base solution *^ W 400 800 1200 1600 c lonomycin L 7 ATP Adn Ach Dop Glu / Figure 1. Changes in inlrucellular calcium levels in isolated retinal cells upon superfusion of a variety of neurotransmitter agents. In- creases in internal calcium were indicated by increases in the ratio of Fura-2 fluorescence induced by stimulation with 334 and 380 nm light. (A) Responses from mi isolated tiger salamander Miiller cell, showing that of the agents tested, only ATP was able to produce an increase in the ratio of 334/380 fluorescence. This result was typical of the majority of the cells tested. (B) Responses from another tiger salamander Miiller cell. In this cell, as in 47% of the others tested, both acetylchotine and ATP induced an increase in tin1 ratio of 334/380 fluorescence. The increase induced h\ acetylcholine was always smaller and more tran- sient than that produced by ATP. (CJ Responses from t\vo skate Miiller cells. None of the neurotransmitter candidates induced changes in intracellular calcium levels in these cells. However, ionomycin, a calcium ionophore. induced a significant increase in the 334/380 ratio, indicating that the svstem could indeed detect increases in intracellular calcium. (D) Responses from an isolated external horizontal cell from the skate. As expected, the neurotransmitters GABA and glutamate, and the glutamate analoi; kainate. all produced significant increases in intracellular calcium /crc/v. All drugs were bath superfused ill u concentration of I mM. except lonomycin. In the latter case, 2 y.1 of a 5 mM solution was added to the central we/I of the dish, resulting in a final concentration of 50 p,M. Superfusion was turned off during the application of ionomycin. Abbreviations: Adn. adenosine; GABA. gainma-atniut) butyric acid: Ach, acetylcholine; Dop, doparnine; glu, g/iitamalc: \TP, adenn.\inc Iripliospliale. 230 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS Table I Cells responding with an increase in intracelliiliir calcium after addition of neiirotransmitter agents ATP Acetylcholine Glulamate GABA Dopamine Adenosine Tiger 64(65) 16(34) 5 (42) 2 (40) 1 (15) 5 (34) Salamander Miiller Skate 1 (24) — (21) I (22) -(12) — (17) — (17) Muller The number of cells tested in each condition is shown in parentheses. The number of cells responding with an increase in calcium (defined as producing a 334/380 ratio greater than 25% of the initial baseline) is shown to the left of the parentheses. A dash ( — ) indicates that none of the cells tested responded with an increase. All chemicals were applied at 1 mM concentrations via bath superfusion. alternately with 334 and 380 nm light every 2 seconds, and the resultant fluorescence, emitted through a 520 nm filter, was quan- tified with a Zeiss Attofluor calcium imaging system. Drugs were applied onto the cells, in random order at about 1-2.5 ml/min via a gravity superfusion system. Cells were rinsed for at least two minutes with Ringer in between applications of drugs. A response was considered to have been induced when the ratio of 334/380 fluorescence exceeded, by 25%, the baseline level recorded prior to the application of a drug. A transient increase in calcium was frequently observed when the flow of Ringer was started; typically, drugs were applied 2 min after this initial transient had declined to a steady baseline level. We examined the responses of these cells to applications of 1 mM gamma-aminobutyric acid (GABA), glutamate, acetylcholine, adenosine, adenosine triphosphate (ATP) and dopamine. As first reported by Keirstead and Miller (12). extracellularly-applied ATP elicited a marked increase in calcium in the tiger salamander Muller cells (Fig. 1A). Of 65 cells examined, ATP induced an increase in calcium in 64 cells. Acetylcholine was less consistent in raising internal calcium, producing smaller and more transient calcium increases in 47% of the cells tested (Fig. IB; see also Table I). These data are of interest in light of work by Wakakura i' i at. (13), demonstrating that one-half of all rabbit Muller cells examined responded to acetyicholine with increases in calcium. We were, however, unable to evoke an increase in calcium with 1 mM glutamate. despite previous evidence supporting the presence of metabotropic glutamate receptors on the Muller cells of the tiger salamander (12). This result was unexpected, but we note that glutamate has also been reported to be ineffective in inducing calcium waves in Muller cells in the intact retina of the tiger salamander (7). None of the other neurotransmitters caused an increase in calcium in the majority of cells tested. The same array of neuroactive agents were ineffective in in- creasing calcium in 24 skate Muller cells. Positive controls to ensure that the imaging system was working properly included demonstrations: (a) that increases in calcium were observed in skate Muller and horizontal cells upon application of the calcium ionophore ionomycin (Fig. 1C); and (b) that glutamate and GABA both increased calcium levels in isolated skate retinal horizontal cells (Tig. ID), as had been described previously (14). Electrophysiological data also support significant differences in the responses of the Muller cells of the two species to neurotrans- mitter agents. For example, skate Muller cells respond to GABA with a brisk electrical current mediated by GABAa receptors (9), while no such similar current has yet been reported in tiger salamander Muller cells. While we cannot rule out the possibility that the enzyme used to produce isolated cells might have altered the properties of some of the membrane proteins present in the cells, our results suggest that Muller cells of disparate species can differ widely in their responses to neurotransmitter agents. Con- sequently, the conditions under which Muller cells might alter the function of neighboring neurons is likely to differ from species to species. The authors would like to thank Kasia Hammar and Rudi Rottenfusser for guidance and assistance in using the Zeiss Attofluor system, Naomi Rosenkranz for help with cell culture. Richard Sanger for computer and equipment assistance, and Peter J. S. Smith and Barbara Innocenti for comments on the manuscript. This research was supported by grant EY0941 1 from the National Eye Institute, grant DBI-9605155 from the National Science Foun- dation, and grants NCCR P41 RR01395 and R21 RR12718 from the National Center for Research Resources. Literature Cited l. 7. 8. Carter-Dawson, L., F. Shen, R. S. Harwerth, E. L. Smith 3rd, M. L. Crawford, and A. Chuang. 1998. Exp. Eye Res. 66: 537-45. 2. Newman, E. A. 1985. Trends Neurosci. 8: 156-159. 3. Newman, E. A. 1996. J. Neurosci. 16: 159-168. 4. Sarthy. P. V. 1982. J. Neurosci. Methods 5: 77-82. 5. Wilbold, E., and P. G. Layer. 1998. Histol. Histopathol. 13: 531- 552. 6. Schwartz, E. A. 1993. Neuron 10: 1 141-1 144. Newman, E. A., and K. A. Zahs. 1997. Science 275: 844-847. Newman, E. A., and K. A. Zahs. 1998. J. Neurosci. 18: 4022- 4028. 9. Malcnow, R. P., H. H. Qian, and H. Ripps. 1989. Proc. Nat/. Acad. Sci. U.S.A. 86: 4226-4230. 10. Newman, E. A. 1985. / Neurosci. 5: 2225-2239. 1 1 . Linser, P. J., K. Smith, and K. Angelides. 1985. J. Comp. Neural. 237: 264-272. 12 Keirstead, S. A., and R. F. Miller. 1997. Glin 144-203. 13. Wakakura, M., I. Utsunomiya-Kawasaki, and S. Ishikawa. 1998. Graefe's Arch. Clin. Exp. Ophrhalmol. 236: 934-939. 14 Haugh-Scheidt, L., R. P. Malchow, and H. Ripps. 1995. J Physiol. 488: 565-576. PHYSIOLOGY 231 Reference: Biol. Bull 197: 231-232. (October Fluorescence Localization of K+ Channels in the Membrane of Squid Giant Axons John R. Clay and Alan M. Ku-irian (Marine Biological Laboratory, Woods Hole, Massachusetts 02543) Modern neuroscience can be said to have begun with the sem- inal work of Hodgkin and Huxley (1) who demonstrated the existence of Na+ and K+ specific conductances in the membrane of squid giant axons. These conductances underlie the action potential that propagates along the nerve and then triggers con- traction of the mantle, i.e., the jet-propelled escape response (2). In the last few years, Na+ and K+ channel proteins from this prep- aration have been cloned and sequenced (3. 4). Moreover, a polyclonal antiserum has been raised against a portion of the NH-,-terminal amino acid sequence of the SqKvlA K* channel (4) — an intracellular epitope. We have used this antiserum to localize with fluorescence immunocytochemistry K* channels in the axonal membrane. Because the target for the antibody we have used is intracel- lular. we necessarily had to deliver it to the interior of the axon. We were unable to accomplish this by external application of the antibody, even with Triton-X-100, because of barriers out- side the axolemma — particularly the basement membrane and the glial cell layer (5). We circumvented this problem by using intracellular perfusion. This is a novel approach for histologists, but not for electrophysiologists, who have for decades used intracellular perfusion together with the axial wire voltage- clamp technique to investigate the effects of intracellular fac- tors on ionic currents in squid giant axons (6. 7). The method used is illustrated in Figure 1. The axoplasm was extruded with a roller (Fig. 1A). A small piece or "plug" of axoplasm was left near one end of the axon. The preparation was then placed in a Lucite chamber and tied onto a small hook (Fig. IB). A small hole was cut in the axon. and a glass cannula was guided into the axon through the remaining plug of axoplasm. The axon was ligated onto the cannula, and perfusion with a I -ml syringe was initiated. The syringe was attached to the apparatus which held the cannula in place (not shown). Throughout the experiment, the exterior surface of the central portion of the axon ( 1 cm length) was superperfused with filtered seawater (fsw). The standard intracellular perfusion buffer consisted of 100 mM K glutamate and 500 mM sucrose (pH = 7.2; referred to below as "standard buffer"). Even though we were able to introduce antibodies inside the axon with this preparation, no immunostaining was observed un- less we used procedures to circumvent the inner cortical layer of axoplasm that remains after extrusion (5). This layer prevents antibodies from reaching the inner membrane surface. Specifically, we used either potassium iodide (KI) — a chaotropic agent — to completely wash away the cortical layer, or Triton-X-100 to per- 1 Laboratory of Neurophysiology, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892. meabilize the layer. In the former experiments, we perfused for 10 min with 300 mM K] and 500 mM sucrose, and then for 1 h with standard buffer containing primary K* channel antibody (1:500 dilution). After a wash with standard buffer for 30 min, we perfused with secondary antibody (goat anti-rabbit/Oregon Green. Molecular Probes Inc.. Eugene, Oregon) at the recommended dilution of 1:200. followed by a wash with standard buffer for 30 min. In the experiments with Triton-X-100, we perfused with standard buffer containing 4% paratbrmaldehyde for 1 h, followed by perfusion with 0.2% Triton-X-100 in standard buffer for 1 h. Antibodies were applied as above. The axon was subsequently tied closed at both ends, removed from the chamber, and mounted on a slide with Aqua Poly/Mount (Polysciences, Warrington, Penn- sylvania). The preparation was viewed either with a confocal laser microscope or conventional epifluorescence. Control preparations were treated in the same way as the test axons. but with the K + channel antibody replaced by an antibody that does not cross-react with squid. Observed by conventional epifluorescence microscopy, the test axons were intensely immunofluorescent in discrete areas or patches throughout the membrane, with a spacing of —25 ju,m between patches (Fig. 2A). A pattern in the immunostaining was not clearly apparent, although the punctate patches of fluorescence appeared to lie along linear elements longitudi- nally oriented along the axon. The result in Figure 2A was obtained with the KI pretreatment described above. A control preparation (different axon) for these conditions is shown in Figure 2B in which the SqKvlA antibody was replaced with a K+ channel antibody that does not crossreact with squid. All other procedures were the same as for the test result. A clear fluorescence signal was not observed, which demonstrates lack of nonspecific binding by the secondary antibody (Fig. 2B). Results similar to those in Figure 2A were obtained when fixation and permeabilization of the cortical axoplasm layer was carried out with Triton-X-100, as described above. Using con- focal laser microscopy, we observed in test preparations a roller axon A 1 «-Q axon 1 j fsw 4 cannula ^ T) l 1 Figure 1. Sclu'innrii' diagram illustrating the method for intracellular perfusion of antibodies in the w/mlasinic con: the unsupported a\«nal membrane folded and collapsed as it \\-as mounted mi the slide Hop panel). The bottom pan of the image lin focus) illustrates paiches «/ immunqfluorescence from Mm membrane surface's. I'hc 1,1/1 purl «/ the image loin of locus) .showed .sunning similar to thai in the hottoni part of the inia,gi- when it mis brought into focus. B. Epiflno- rescenl linage of an a\on idi/tcrcnl than the one described in A) perfused \\ilh a K channel antihoilv that does not < rossrcact with st/niil. All other experimental procedures \verc the same as the lest a\on. as described in the >c\l. The bar below this image represents /fill ij.ni (same lor A ami K). dittuse signal throughout the membrane — not present in the control axons — in addition to the patches of intense immuno- fluorescence shown in Figure 2A. Preliminary results with immunogold suggest that the immunofluoreseence is attribut- able to K+ channels in the a.xonal membrane rather than to K + channel containing vesicles which have not been removed dur- ing extrusion of the axoplasm. Our results provide further support to the argument that SqKvlA mediates the classic delayed rectifier potassium ion current in the squid giant axon (4). Punctate ion-channel immunofluoreseence has been observed in a number of preparations. The results most relevant to this study are those of Johnson et ul. (8). They demonstrated, in cultured Aplysin axons. clusters of Na+ channels that were separated by regions where channels were not present, i.e.. similar to nodes of Ranvier in myelinated axons (9). Our results are consistent with the punctate nature of immunofluoreseence observed in Aplysin axons, but the patches of fluorescence in squid axons appear to be uniform throughout. We speculate that the membrane patches of ion channels suggested by the results in Figure 2A are targets for K+ channel-containing vesicles, which we have isolated from the axoplasm and which may be involved in turnover of ion channels in the membrane (J. R. Clay and A. M. Kuzirian. unpublished). The punctate distribu- tion of immunostaining is very similar to clustering of Kvl channels by PDZ proteins in heterologous expression systems ( 10). We note that the squid SqKvlA K+ channel does have a consensus sequence for PDZ protein binding at its C term- inus (4. 10). However, it is premature to speculate on the role of PDZ proteins in the clustering of K1 channels in squid axons. We gratefully acknowledge William Oilly and Zora Lebaric for generous gifts of their K4 channel antibodies. Literature Cited 1. Hodgkin, A. L., and A. F. Huxley. 1952. J. Physiol. iLond.l. 116: 449-472. 2. Otis, T. S., and W. F. Gilly. 1990. Proe. Nat/. Aead. Sci. U.S.A. 87: 291 1 -29 1 5. 3. Rosenthal, J. J. C., and W. F. Gilly. 1993. Pmc. Nail. Acad. Set. U.S.A. 90: 10026-1(1030. 4. Rosenthal. J. J. C.. R. G. Vickery. and W. F. Gilly. 1996. J. Gen. Physiol. 108: 207-219. 5. Brown, A., and R. J. Lasek. 1990. Pp. 235-302 in St/itid as Exper- imental Animals. D. L. Gilbert, W. J. Adelman, Jr., and J. M. Arnold, eds. Plenum Press, New York. 6 Baker. P. F., A. L. Hodgkin, and T. I. Shaw. 1961. Nature 190: 885-887. 7. Adelman. W. J., Jr., and D. L. Gilbert. 1964. J. Cell. Comp. Physiol. 64: 423-428. 8. Johnson, W. L., J. R. Dyer, V. F. Castellutd, and R. J. Dunn. 1996. J. Neiirosei. 16: 1730-1739. 9 Dugandzija-Novakovic, S., A. G. Koszowski, S. R. Levinson, and P. Shrager. 1995. / Neurosei. 15: 492-503. 10. Sheng, M., and M. Wyszynski. 1997. Bioesxayx 19: 847-853. PHYSIOLOGY 233 Reference: Biul. Bull. 197: 233-234. (October IW9) Evaluation of Circadian Rhythms in the Limulus Eye Vanessa J. Riita, Frederick A. Dodge, and Robert B. Barlow (Marine Biological Laboratory, Woods Hole, Massachusetts 02543) The visual system of the horseshoe crab, Limulus polyphemus, shows a remarkable circadian rhythm in sensitivity. About the time of sunset, a circadian oscillator in the brain of the animal generates efferent optic nerve signals to the lateral eye modulating both its structure and function. The overall increase in sensitivity nearly compensates for the reduction in ambient light at night (1,2). High nighttime visual sensitivity appears to be important for mating, a visually guided behavior that the animals can accomplish as well at night as in the day (3). The mechanisms underlying the elegant adaptation of the Limulus eye to dim nighttime illumination in- clude anatomical changes in the retina that increase photon catch at the expense of spatial resolution, and physiological changes that enhance the summation of photon events at the expense of tem- poral resolution. Here we evaluate further the effect of the circadian clock on two properties that shape the dynamics and strength of the eye's response. We first asked whether the elevated nighttime response resulted from a clock-induced reduction in the strength of lateral inhibitory interactions between ommatidia. We then asked whether the loss of temporal resolution at night resulted from an increase in the dispersion of latencies of optic nerve responses. We report here that the circadian clock does not change the latencies of the optic nerve responses but decreases the strength of lateral inhibition at night. To evaluate the possible clock influence on the strength of lateral inhibitory interactions, we compared the steady state optic nerve discharge of a single dark-adapted ommatidium over a wide range of light intensities during the day and at night. To maximize the inhibitory influences exerted on the recorded ommatidium. we uniformly illuminated the entire eye by placing a Teflon diffusing screen in contact with the cornea and illuminating it with a large (5 mm) light pipe (4). We recorded the intensity response function of the single optic nerve fiber during the day. At dusk efferent optic nerve fiber activity begins to transform the retina to its highly sensitive nighttime state, and we tracked the transformation by measur- ing the shift in threshold of the dark-adapted ommatidium. We define threshold as the intensity of brief (35 ms) test flashes to which the optic nerve responds 50% of the time; test flashes are triggered at 20 s intervals allowing complete decay of lateral inhibition. We remeasure the intensity-response function about 2100 h when the eye has attained its nighttime state. Figure 1A shows the intensity response functions for a single dark-adapted ommatidium during the day and at night. Plotted are the steady-state optic nerve responses as a function of the log of the light intensity. To compare the level of lateral inhibitory interactions in the two states of the retina, we subtracted the mean rate of spontaneous activity from all steady-state responses. We then shifted the nighttime intensity-response function to the right to account for the increase in sensitivity as measured by the threshold shift (5). Barlow and Fraioli (4) reported that inhibitory effects are not detectable at low firing rates ( — 1-2 impulses/s). Note that in Figure 1A the "Night" and "Day" functions separate above —5 impulses/s. The elevated nighttime responses indicate a weakening of lateral inhibition. At firing rates greater than 10 impulses/s another circadian mechanism comes into play — one that increases response gain. The increased gain at night is thought to result from a reduction in the efficacy of a voltage-dependent light adapting mechanism which acts to reduce the eye's response to light during the day (5). The contribution of increases in response gain to the separation of the two functions limits our ability to evaluate the clock's role in lateral inhibition strength at higher light intensities. For a more quantitative measure of the changes in lateral inhibition at night, independent of the changes in gain, we designed a second experiment in which lateral inhibition was induced by antidromic stimulation of the optic nerve trunk. We recorded the response of a single, optically-isolated ommatid- ium to a steady 30-s light stimulus and every two seconds applied a brief ( 100 ms) train of four current pulses to the optic nerve trunk. To avoid transients at the onset of illumination, we discarded the response to the first antidromic stimulus, averaged the following 14 responses, and plotted the two-second cycle averages in Figure IB. Brief periods of inhibition occur imme- diately following the antidromic stimuli (0.9-1.0 s). In addition to exerting inhibition, antidromic stimulation also activates the efferent fibers, simulating the circadian clock and driving the retina into the higher sensitivity nighttime state ( 1 ). We reduced the light intensity by over 300 times to compensate for the increased sensitivity of the eye and maintain roughly the same firing rate as in the daytime state. Note that the depth and duration of inhibition in the nighttime state (thick black line) are less than in the daytime state (thin black line). As measured by the change in response rate, inhibition is approximately 60% less in the nighttime state. Other experiments exhibited equiv- alent or smaller decreases in inhibition. After cessation of efferent input, inhibition (broken line) recovered to that re- corded during the daytime state. To monitor response latencies of the dark-adapted retina, we measured the delay of the optic nerve response to a brief (35 ms) test flash. As in the experiment shown in Figure 1A, we adjusted the intensity of the test flash to produce a threshold response of 50%. Figure 1C shows a histogram of the latencies of response for both the day and nighttime state of the retina. During the day, the average latency of response at threshold was 140 ± 21 ms. After converting the eye into the nighttime state by delivering current pulses to the optic nerve, the threshold intensity decreased tenfold, but the average response latency remained unchanged (145 ± 22 ms). There is no significant change in either the length of the 234 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS 30T latency of response or in the distribution of latencies as a conse- quence of circadian input. We conclude that the clock increases retinal sensitivity with a concomitant reduction in lateral inhibition but without a significant change in the latency between the absorption of a photon and the discharge of an optic nerve impulse. Literature Cited 1 Barlow . R. B., S. J. Bolanowski, and M. L. Brachman. 1977. Sci- ence 197: 86-89. 2. Barlow, R. B., S. C. Chamberlain, and J. Z. Levinson. 1980. Sci- ence 210: 1037-1039. 3 Barlow, R. B., R. B. Ireland, and L. Kass. 1982. Nature 296: 65-66. 4. Barlow, R.B., and A. J.Fraioli. 1978. J. Gen. Physiol. 71: 699-718. 5. Barlow, R. B., E. Kaplan, G. H. Renninger, and T. Saito. 1987. / Gen. Phvsiol. 89: 353-378. 30T B Seconds 150 T (ft 3) B § 100- Q. W • Night 0) o: uDay **— 0 50- 6 0- t I •fj 0.1 0.2 Latency (s) Figure I. Input from the circadian cluck in the nighttime eye increases sensitivity by weakening lateral inhibition hut does not affect the dispersion of response latencies. (A) Intensity functions of the steady-state response of a single dark-adapted ointnatidiittn in a Limulus eve during the da\ and at night. The steady-state response is defined as the last 2 s of a 5-s light flash, although at low intensities the flash duration and the count interval were increased to compensate for variability in the spike discharge. The whole eye was uniformly illuminated by placing a Teflon diffusion screen over the eve. At log 1 = 0 the light incident on a single ommatiilium was ~10 photons/s. {B) Inhibition of the response of a single ommatidium by antidromic stimulation of the optic nerve in the daytime state (thin black line), in the simulated nighttime state (thick black line), and after recovery back to the daytime state (broken line}. (C) The dispersion of optic nerve response latencies is not affected bv efferent input. The histogram for the daytime state includes the latencies of 275 responses and that for the nighttime state includes the latencies of 280 responses. PHYSIOLOGY 235 Reference: «/«/. Bull. 197: 235-236. (October 1999) Photoreceptor Pigments of the Blueback Herring (Alosa aestevalis, Clupeidae) and the Atlantic Silverside (Menidia menidia, Atherinidae) Inigo Noviiles Flamarique and Ferenc I. Harosi (Marine Biological Laboratory, Woods Hole, Massachusetts 02543) Since the discovery of an ultraviolet (UV) visual pigment in the retina of the Japanese dace ( 1 ). other freshwater fishes have been found to possess UV pigments (1). Among marine species, UV pigments have been reported for the adults of only one tropical species (3). Here we examine the pigments in the retinal cones from two marine coastal species, the blueback herring and the Atlantic silverside. The blueback herring is strictly a saltwater species (stenohaline), whereas the Atlantic silverside is euryhaline; i.e., it can live in a wide range of salinities. Both the Atlantic silverside and the juvenile blueback herring inhabit coastal surface waters (<10 m depth) where light from 320-800 nm is available to stimulate all potential photoreceptor types (UV, blue, green, and red) present in fish retinas. Adult blueback herring also dwell in deeper waters (50-100 m) where UV light is absent and the spectrum narrower and centered around 560 nm (4). We used the technique of microspectrophotometry (MSP) to measure photoreceptor pigments in situ. After enucleation of the eye from a recently euthanized dark-adapted animal, pieces of retina were extracted and teased apart in Ringer solution. These pieces were placed between coverslips. and individual photorecep- tors lying on their sides were examined with the dichroic mi- crospectrophotometer (5). Sample measurements were carried out by placing a beam of light (0.6 X 2 /urn cross section) on the structure of interest and taking an average of eight consecutive transmission scans from 270-648 nm. Similarly, reference mea- surements were recorded from adjacent areas devoid of tissue, and sample absorbance was computed in 2 nm increments (5). The blueback herring showed single cones, double cones, and rods. The single cones were sensitive to blue or short (S) wave- lengths with maximum absorbance (Amax) at 447 nm. The double cones were composed of a green or middle (M) wavelength- sensitive cone (Amux = 517 nm) and a red or long (L) wavelength- sensitive cone (Amax = 566 nm). The Am.ix of the rod was 510 nm (Figs. 1A, B). The average spectral bandwidth at half maxima for the cones and the rod was about 4000 cm '. This measurement suggests that the primary visual pigment chromophore in this retina is vitamin A, (6). The retina of the Atlantic silverside also showed rods, as well as single and double cones; but the distal parts of the cones' inner segments and the pigment spectra were different from those in the blueback herring. Double cones in the Atlantic silverside were of two kinds, M/L and L/L pairs. The average Amax of the M pigment was 472 nm. while that of the L pigment was 580 nm (Fig. ID). M/L pairs exhibited pinkish globular structures at the distal end of their inner segments, termed ellipsosomes (7). These organelles were first documented in the double cones of killifishes and gup- pies and are known to be of mitochondria! origin (7). The M ellipsosome had the highest absorbance. with a spectral profile indicative of cytochrome-c (Fig. IE). Ellipsoid absorbance by the L member was always lower than that of the M member (Fig. IF). L/L double cones showed an ellipsosome in one member with absorbance similar or higher than that of the L member in M/L pairs, and minute traces of cytochrome-c in the other member. There were two types of single cones: large ones (S cones) with inner segment structures that looked exactly like ellipsosomes. but did not contain any cytochrome-c (pseudoellipsosomes. Fig. IF), and smaller ones with normal ellipsoids. The average Amax of the S cones was 410 nm (Fig. 1C). Although we were unable to secure good average spectra from the smaller cones, our records none- theless show that they contain a UV pigment (Amax around 365 nm). The average spectrum of the rod peaked at 5 1 1 nm (Fig. 1C), and its half bandwidth was —4010 cm" '. The latter is indicative of a visual system predominantly based on vitamin A,. The pigment complement found in the retina of the blueback herring resembles that of other stenohaline fishes and may be the basis for maximal quantal catch over an extensive depth range, > 0-100 m (8). Fishes that live closer to the water surface, where UV and blue wavelengths are more prevalent, may instead benefit from a pigment complement shifted toward shorter wavelengths, as with the Atlantic silverside. If the presence of UV cones in the Atlantic silverside were confirmed, the visual pigment complement of this fish would then be similar to those of the killifishes, with Amax at 360. 410. 470 and 570 nm for the cones, and at 510 nm for the rods (2). Beyond the visual pigments, these euryhaline species also share the presence of ellipsosomes in double cones, which were previously hypothesized to act as optical filters for the reduction of /3-band absorption by M cones (7). A similar mech- anism could operate here to improve discrimination and contrast detection at short wavelengths. Since the Atlantic silverside is often found in the same habitat as the killifishes, the similarities in their visual systems may be due to convergent evolution. Literature Cited 1. Harosi, F. I., and V. Hashimoto. 1983. Science 222: 1021-1023. 2. Harosi, F. I. 1991. Vision Res. 34: 1359-1367. 3 McFarland, W. N., and E. W. Loew. 1994. Vision Kes. 34: 1393- 1396. 4. Novales Flamarique, I., and C. W. Hawryshyn. 1993. Can. J. Fish. Aquat. Sci. 50: 1706-1716. 5. Harosi, F. I. 1987. ./. Gen. Physiol. 89: 717-743. 6. Bridges, C. I). B. 1967. Vision Res. 1: 349-369. 7. MacNicholl, E. F. Jr., Y. W. Kunz, J. S. Levine, F. I. Harosi, and B. A. Collins. 1978. Science 200: 549-552. 8. Harosi, F. I. '.996. Biol. Bull. 190: 203-212. 236 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS B 0.04 0.03 0.02 (0 .g o to -0.01 M 0) u 0.04 0.03 0.02 0.01 0 350 400 450 500 550 600 Wavelength (nm) 650 -0.01 350 400 450 500 550 600 650 Wavelength (nm) 0.04 S I 8 0.04 0.03 0.02 0.01 -0.01 -0.01 M 350 400 450 500 550 600 650 Wavelength (nm) 350 400 450 500 550 600 650 Wavelength (nm) 0.35 0.35 -0.05 -0.05 Ellipsosome (L) Pseudoellipsosome (S) ^^^H^VTN^^ 400 450 500 550 600 Wavelength (nm) 650 400 450 500 550 600 Wavelength (nm) 650 1. Photoreceptor pigments »t ' the hint-hack herring ami the Atlantic silverside. (A. Bt Blnchack herring: average spectral absorbances of the S (n = .(). M (n = 13) anil L (n = //) cones, ami the rod (R. n = 6). (C D) Atlantic silverside: average spectral ab.wrbances of the S (n = 9), M (n = //) an,/ • (n = 15) cones, and ilic nut (R, n = 8). (E. F) Atlantic xilversule: t\/>ical ellipsosomes of the M and L members of double cones, ami l>M'ii,l<:clnr , ,./ the S cone. A value of II I? has been added to the L ellipsusome trace to separate it from that oftheSpseudoelttpsosome. Ellipsosomes exhibited the typi • \tnchrome-c spectrum with a prominent Sorcl ly) peak at 414 nm, and secunthin- (3 and a peaks nt 5 IK inn and 54K nm. respectively. PHYSIOLOGY 237 Reference: Bml. Bull. 197: 237-238. (October 1999} Soft-sided Tanks Improve Long-term Health of Cultured Cuttlefish Janice S. Hanle\, Nadav Shashar1, Roxanna Smolowit-, William Mebane, and Roger T. Hanlon (Marine Resources Center, Marine Biological Laboratory, Woods Hole, Massachusetts 02543). The common European cuttlefish. Sepia officinalis Linnaeus. 1758. is being cultured in captivity to provide experimental ani- mals for biological and biomedical research, and for testing meth- ods applicable to larger-scale mariculture. This species adapts quite well to captivity, and several generations of cuttlefish have been grown in facilities in Europe and the United States (e.g., 1-5). However, while jetting away from other animals during social interactions, or from human observers who have inadvertently startled them, cuttlefish occasionally collide into the walls of holding tanks (3). Moreover, recent work by Boal et al. (7) shows that social interactions (many of them deleterious) increase sub- stantially when sub-adult and adult cuttlefish are maintained or cultured in higher densities, which they usually are. The accumu- lated damage to the skin caused by the collisions (see also 6) often contributes substantially to mortalities in the course of the one- year life cycle. Repetitive collision causes trauma, particularly to the posterior mantle tip. because the cuttlefish are most often jetting backwards when they hit the wall. The mantle tip is par- ticularly vulnerable because the layer of muscle and skin overlying the posterior tip of the cuttlebone is quite thin. As a result, focally extensive, deep ulcerative dermatitis and cellulitis develop. We term this posterior mantle tip dermatitis (PMTD). Severe cases result in hemolymph loss, bacteremia. and death. Subacute. chronic cases result in fibrosing cellulitis and abnormal cuttlebone formation at the posterior apex; occasionally, the apex of the cuttlebone fractures. Because culturing a cuttlefish through its life cycle is expensive, methods that will increase survival to adult- hood need to be developed. We counted collisions for a total of 7 h over 3 days; the cuttlefish were males of about 1 kg. and females of about 0.5 kg. Collisions with tank walls can occur quite frequently: we counted rates of 0.75-1 .25 events per animal per hour in a variety of tanks (n = 4 tanks), even when the animals were relatively calm (e.g., when they were not sexually active, when the tanks contained only females or immature animals, and when human distraction was minimized). These rates increased to over 4.0 wall collisions per animal per hour when the animals were sexually active (e.g., when two males were placed in a tank with mature females, or when human observers startled the cuttlefish). Hulet et al. (8) suggested that a "bumper system" be installed along the walls of squid holding tanks to reduce skin damage from collisions with the hard surfaces. Modifying their concept, we have developed a simple system in which black plastic cushioning curtains were hung parallel to, and 10 cm away from, the inner surfaces of the tank walls (Fig. 1 ). The resulting holding arena was smaller — 1.65 X 1.3 m (surface area = 2.145 m2) — than the 1 Hebrew University, H. Steinitz Intel-university Institute, P.O. Box 469, Eilat 8800, Israel. control tank. Water depth was held at 0.28 m. providing an overall volume of 0.6 m\ An identical control tank ( 1.85 X 1.5 X 0.28 m, surface area = 2.775 m2. volume = 0.777 m3) was set adjacent to the experimen- tal tank. Animals in both tanks experienced the same water input, outside disturbances, light conditions, cleaning schedule, etc. Since no space was taken by cushioning curtains, the cuttlefish could roam through the entire volume of the control tank. Hulet et al. (8) used cushioning walls that created a slope between the bottom of the tank and its hard walls. Although this design greatly reduced the impact when the side surfaces were hit, it restricted water movement behind this angled curtain, so the medium became increasingly anoxic, and hydrogen sulfide built up. The new tank design circumvents the water quality problem. First, we are using a flow-through seawater system and no sub- strate. We improved water circulation by placing the cushioning curtains parallel to the tank walls and by pumping fresh seawater into the area between the curtains and the walls. Two airlift pumps were used, each working at 9.45 1/min (2.5 gal/minute); the pumps were placed near the curtains at opposite sides of the tank and moved water from behind the curtains to the central area. The volume of water behind the curtains was 176.4 liters, and with both pumps operating that entire volume could be exchanged approxi- mately every 9.3 minutes. No significant difference in pH or in oxygen concentration was found between the water in the control tank and that in the center arena of the experimental tank, or in the area between the cushioning curtains and the walls of the experi- mental tank. Two sequential trials were conducted. In each trial. 4 sexually mature cuttlefish (2 males and 2 females, all free of any body Water input 1.5m Water out 9 ......... > \Airlift 1.3m indicates water flow 1.85m 1 .65 m Figure 1. Setup for testing the use of cushioning curtains in cuttlefish /milling tanks. Left, a control tank. Right, the experimental tank with the cushioning curtain (hold lines) set along its sides. Two airlifts moved water from behind the curtain to the center of the tank, thus maintaining high- i/U(ilir\' water on hoth sitlc,\ of the curtain. 238 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS injury) were placed in each tank. Hence, the density in the exper- imental tank was slightly higher than in the control tank (0.54 vs. 0.69 nr per cuttlefish, respectively). The health of these adult animals was monitored throughout the trials. Trial 1 lasted for 40 days. On day 1 1 , one of the cuttlefish in the control tank developed PMTD. On day 17, a second cuttlefish in the control tank developed PMTD. None of the animals in the experimental tank showed any signs of PMTD. although two animals developed unrelated illnesses. Trial 2 was conducted with four new healthy cuttlefish in each tank (2 males, 2 females). In the control tank. PMTD first occurred at day 7, and this animal died on day 1 1 ; thus animal density in this tank was lower during the remainder of the trial. By day 26, the three remaining animals in this tank showed PMTD; one died on day 42, one on day 49, and one is still alive at this writing (day 105). Once again, in contrast, none of the animals in the experi- mental tank developed PMTD. More importantly, the second trial is still in progress, and all four cuttlefish in the experimental tank are completely free of PMTD at day 105. Overall, for both trials, none of the 8 cuttlefish in the tank equipped with the cushioning sides exhibited PMTD. as compared with 6 out of 8 (75%) of the animals in the control tank (\2, P < 0.014). The new tank design increases the maintenance effort in rearing the cuttlefish. Eight additional surfaces must be scrubbed as com- pared with traditional hard-sided holding tanks. Furthermore, the soft sides of the tank are more difficult to scrub than hard surfaces. Nevertheless, the efforts are worthwhile because cuttlefish nur- tured through the life cycle in captivity have a relatively high value. Although our tank design did not reduce the number of social interactions among cuttlefish, which occur even at low densities (7). it reduced the resulting skin damage experienced by the animals, and thus increased their longevity. Acknowledgments We thank Geoff Till for help in culturing the animals, and Aaron Hoffman for animal observations. This study was partly supported by NSF Grant IBN 9729598. Literature Cited 1. Schroeder, W. 1966. Sber. Ges. nalitif. Frenmle (N.F.) 6: 101-107. 2. Pascual, E. 1978. Invest. fV.v) mule in courtship eoloration. fc) jemulc. both about l> cm total length. <5.4%) and siieeessful (34.6%) strike* (n = 7H) is plotted n^iiinsl ilisliinee Inn) from prey til strike initiation. PISCINE NEUROBIOLOGY AND BEHAVIOR 247 occurred at 1.5 to 2.0 cm (Fig. 1 ). Strike success was similar when compared to prey number remaining, indicating that earlier preda- tion events did not influence predation success. Juvenile toadfish are effective short-range ambush predators. At strike distances between 0 and 1.5 cm they strike successfully 76. 2% of the time. But between 1.5 and 2.0 cm. strike success is mixed, and it continues to decline with distance. Future experi- ments will examine the efficacy of prestrike orientation on preda- tion success. We thank L. C. Rome, S. M. Highstein, W. Mebane, and J. Hanley for their contributions to this study. We also show our appreciation to M. Tytell and C. Browne, directors of the under- graduate Marine Models in Biological Research program. The experiment was funded by the Lawrence Scholarship of Connect- icut College and the NSF grant NSFDBI-9605155. Literature Cited 1. Schwartz, F. J., and B. W. Dutcher. 1963. Trans. Am. Fish. Soc. 92: 170-173. 2. Bisker, R., M. Gibbons, and M. Castagna. 1989. J. Shellfish Res. 8: 25-31. 3. Edds-Walton, P. I,., and R. R. Fay. 1997. Hear, Res. Ill: 1-21 4. Rome, L. C., and S. L. Lindstedt. 1998. News Pliysiol. Sci. 13: 261-268. 5. Mensinger, A. F., and S. M. Highstein. 1999. J. Com/). Nenrol. 410: 653-676. 6. Tang, K. Q., N. N. Price. M. D. O'Neill, A. F. Mensinger, and R. T. Hanlon. 1999. Bi,,l. Bull. 197: 247-248. 7. Gosline, W. A. 1996. Em-iron. Biol. Fishes. 47: 399-405. 8. Walton, W. E., S. S. Easter, C. Malinoski, and N. G. Hairston. 1994. Can. J. Fish. Aquat. Sci. 51: 2017-2026. Reference: Biol. Bull. 197: 247-248. (October 1999) Temperature Effects on First- Year Growth of Cultured Oyster Toadfish, Opsanus tan Kathleen Q. Tang (Marine Biological Laboratory, Woods Hole, Massachusetts ){, Nichole N. Price2, Maureen D. O 'Neill, Allen F. Mensinger . and Roger T. Hanlon The toadfish, Opsanus tan. has been the focus of scientific- research for more than a century. Since its development was first outlined by Ryder ( 1 1. it has continued to be an important marine model for muscle (2). auditory (3), and vestibular physiology (4), as well as for nerve regeneration (5). Recent studies (6) have established baseline blood chemistries to help understand biolog- ical requirements of the toadfish in captivity. Despite the many studies of toadfish physiology, little is known about the basic life history of the animal, such as its growth rate and nutritional requirements. Recently, the availability of wild-caught toadfish has declined, making it difficult for the Marine Biological Laboratory (MBL) to provide the necessary numbers and size classes for research. In addition, captive populations of adults have been affected by diseases such as bacterial pericarditis, Flexibacter sp. dermatitis, and parasitic infestations (7). To provide researchers with healthy toadfish and reduce pressure on wild populations, a mariculture program has been initiated to examine husbandry techniques needed to propagate captive populations. To our knowledge, Op- sanus tau has never been cultured. This paper summarizes aspects of growth and survival during the first year posthatching. Two toadfish nests (with guardian males) were transported to the Marine Resources Center of the MBL from Waquoit Bay in early July 1998. Physical detachment of the larval fish from the nest was completed by late July. One hundred (100) juvenile toadfish from an original pool of about 200 fish were randomly selected in mid-October for this study and separated into 90-1 fiberglass tanks ( 130 x 70 x 10 cm). Each tank was provided with rock and sandy substrate and artificial habitats (PVC pipe). Half of 1 Washington University. St. Louis, Missouri 631 10. " Connecticut College. New London, Connecticut. 3 Washington University School of Medicine, St. Louis, Missouri 631 10. the toadfish were maintained at a constant temperature (Temper- ature A) of 19.5° ± 1°C. The other toadfish were maintained at predominantly cooler temperatures (Temperature B) using ambient (October-November 1998, 10°-15°C) or recirculating seawater (December 1998 -May 1999 13° ± 2°C). These fish were subse- quently switched to ambient in June 1999 and experienced tem- peratures ranging from 1 5°C to 23°C by the end of July 1999. Each experimental group was further subdivided into two groups con- sisting of 10 (1 1.3 fish/nr) or 40 fish (45.0 fish/nr) to study the effects of density on toadfish growth. Many food types were presented to the fish, which were fed every other day. The initial diet consisted of live, newly hatched squid (Loligo pealei). Anemia sp., and mysid shrimps. As the fish increased in size, the diet was switched to approximately 10% live food (guppies), and 90% prepared food such as chopped butterfish (Peprilus triacanthus}. oyster (Crassostrea virginica). blood- worms (Glycera capitahi). krill. and thawed squid. Due to the faster growth exhibited by the fish in Temperature A. they were placed on the prepared food diet several months earlier than the other group. Toadfish were removed bimonthly and weighed (wet weight in grams) and measured (standard length in centimeters). All 10 fish in the low-density tanks were examined, while at least 10 of the 40 were randomly chosen from the high-density tanks for measure- ment. After detachment from nests, juveniles had an average length of 1 .36 ± 0. 1 cm. After one year of culturing. fish kept at the constant 19°C temperature were significantly (/ test with Welch correction, P < 0.0001 ) larger (mean 6.10 ± 0.3 cm compared to 4.1 ± 0.4 cm) and heavier (7.70 ± 0.5 g compared to 1.7 ± 0.1 g) than conspecifics maintained on the lower temperature regime (Fig. 1 ). No differences in average length or weight were found between the two densities in each temperature regime (t test. P > 0.21). The survival rate from day 80 through day 365 was 78%. Many 248 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS *COMBINED • TEMPERATURE A •TEMPERATURES •i« <** ~*r B 50 100 150 200 250 300 350 400 TIME (DAYS) 50 100 150 200 250 TIME (DAYS) 300 350 400 Figure 1. Standard length in centimeters (At and wet weight in grams (B) are plotted against time (days) from physical detachment of larval toadfish from the nest. Fish from the nm nests were combined (diamonds) and maintained at ambient water temperatures until late October 1998. Fish (n = 50) were then subdivided into t\vo temperature regimes: Tem- perature A fish (circles) were maintained at 19.5° ± 1°C; Temperature B fish (squares) were kept at temperatures ranging IO°C to 23°C (see text for details). The Temperature A and B data represent the means ± / SEfor the fish (n = 10) kept in the low-density tanks. of the mortalities were smaller fish that showed signs of conspe- cific attack, which is consistent with the cannibalistic nature of batrachadoids (8). Future studies will test whether fish sorted by size class fare better and reduce the effects of cannibalism. A similar mariculture study conducted at approximately the same temperature range ( 15° to 20°C) on the congener Porichthyx itotatus showed slightly faster growth rates (6.2 cm at 8 months and 8.8 cm at 13 months) (8). These fish were maintained mostly on live prey, which may have contributed to faster growth. Our study has shown that full-life-cycle mariculture of Op- siinus inn may be possible. Surprisingly, growth rate did not appear to be density-dependent. It was, however, temperature- dependent as predicted. It remains to be determined how culture temperatures, which are warmer than the normal winter/spring ambient temperatures, may affect development. Concurrent be- havioral studies indicate that the cultured toadfish can accu- rately detect and capture various forms of prey (9). We also plan to increase genetic variation in future years by obtaining multiple nests from different geographical locations. At current growth rates, cultured toadfish will reach the size desired by researchers in about 3 years. We would like to thank J. Hanley and W. Mebane for assisting with this project. The project has been funded an MBL Associates Fellowship (AFM). Lawrence Scholarship (NNP), NSF grant DBI- 9605515, and NIH grant DC01837. Literature Cited 1. Ryder, J. A. 1886. Am. Nat. 20: 77-80. 2. Rome, L. C., D. A. Syme, S. Hollingworth, S. L. Lindstedt, and S. M. Baylor. 1996. Pmc. Null. Actul. Sci. USA. 93: 8095-810(1. 3. Fay, R. R., and P. L. Edds-Walton. 1997. HetiriiiK Ke*. Ill: 1-21. 4 Mensinger, A. F., J. P. Carey, R. Boyle, and S. M. Highstein. 1997. J. Comp. Neurol. 384: 71-85. 5. Mensinger, A. F., and S. M. Highstein. 1999. J. Comp. Neurol. 410: 653-676. 6 O'Neill, D. O., H. M. Wesp, A. F. Mensinger, and R. T. Hanlon. 1998. Biol. Bull. 195: 228-229. 7 Smolowitz, R., E. Wadman, and H. M. Chikarmane. 1998. Biol. Bull. 195: 224-230. 8. Mensinger, A. F., and J. F. Case. 1991. Biol. Bull. 181: 181-188. 9. Price, N. N., and A. F. Mensinger. 1999. Biol. Bull. 197: 246-247. CHEMORECEPTION AND BEHAVIOR 249 Reference: «/,./. Bull. 197: 249-250. (October 1949) Antennule Use by the American Lobster, Homarus americanus, During Chemo-orientation in Three Turbulent Odor Plumes Katrin Mjos , Frank Grasso, and Jelle Ateina (Boston University Marine Program, Marine Biological Laboratory, Woods Hole, Massachusetts) The American lobster, Homarus americanus, may use the dy- namics and spatial distribution of chemical signals for guidance to the source of a turbulent odor plume ( 1, 2, 3). Other studies have shown that lobsters orient most efficiently when they are able to use both of their lateral antennules (4, 5). These results lead us to wonder whether the use of antennules by the plume-tracking lobster varies with the type of turbulence the lobster encounters. Such variations would suggest that the lobster is actively sensing rather than merely adjusting to the mechanical stresses presented by turbulence. One way to answer this question is to measure the positions and orientations of the antennules of a lobster as it tracks different turbulent odor plumes. In this report we describe new methods for making these types of measurements and present preliminary results. We observed the plume tracking behavior of 24 locally caught lobsters (~84 mm carapace length) in a flow-through seawater flume (3 m X 0.9 m and 0.22 m water depth). During trials, a plume was injected into the background flow ( 1.5 cm/s) at one of three rates: 1,12, or 1 20 ml/min. The source was a glass tube (0.25 cm I.D.) positioned parallel to the flume floor, at antennule height (~9 cm), and in the middle of the flume. The downstream distance was selected to lie within the zone where the boundary layer had reached asymptote. A shelter was positioned 1.5 m directly downstream from the source. The lobsters (tested one at a time) were given 30 min to habituate to the flume and settle in to this shelter before the plume was turned on. If a lobster had not settled within the allotted time, it was given another 30 min to habituate. If, after this, the lobster had not habituated, it was removed from the flume, and the habituation process was begun with a different lobster. After habituation, the stimulus was turned on, and the lobster was given 20 min to track the plume. Lobsters that did not track the plume were removed at the end of 20 min and given an additional trial 8 or more hours later. Each lobster was presented with three different injection rates (i.e.. plume types) in random order. We recorded the tracking behavior of the lobsters with overhead video cameras. The dorsal surface of the lateral antennules and the anterior and posterior tips of the carapace were marked with fluorescent paint. Black lights were suspended over the testing section of the flume. This arrangement allowed us to score the positions of the antennules and the body in the X and Y coordi- nates of the flume floor. We then used the Metamorph Image Processing System™ to digitize the animals' paths from video images. The video was sampled at 6 Hz, and in each sampled frame the positions of the left and right lateral antennules were 1 Department of Biology, University of Massachusetts at Dartmouth. North Dartmouth. Massachusetts. collected in source-centered coordinates. Once calibrated to dis- tances in centimeters, these positions were used to compute the angle between the two antennules. Because the antennules are fixed in length, the angle is a measure of the bilateral sampling distance from which the lobster derives odor information. We only scored those trials in which the lobster: 1) started in the shelter or the downstream end of the behavioral section: 2) moved its ros- trum within 5 cm of the source: 3) did not contact the side walls of the flume during its tracking; and 4) did this within the 20-min time limit. Ol "3JD C = e = 11 *- = R 180- 150- 120- 90- 60- 30- 0 A. e • * • Plume 1 (IR=1 ml/min) • Plume 2(IR=12ml/min) A Plume3(IR=l20ml/min) | | 01 55 f o 45- 30- TJ Approximate Plume Boundary B. ^ • 15- 0- Source -15- *"*v -30- * -25 -50 -75 -10 Down Stream Distance (cm) Figure 1. (A) rhe an^le between the two antennules for three paths made h\ lohster 2H while trucking. The Juki taken from path* assoi iuti'tl \\-ilh three different injection rule* are shown at 6 H: sampling rate. Note the lusher inter-antennular angle tit the lowest injection rate, particularly at farther downstream distances. IBI The actual positions of the antennules from the 120 ml/min injection rate in A. Display is at 2 H;. The listens/* marks the location of the source. Each "V" tlruwn has its ir/Yn at the location of the lobster's rostrum. The tips of each "V" mark the location of the antennule tips \'iinalions in antennule length reflect the elevation «/ the antennule. 250 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS The animals met the criteria in 26 of 125 trials. However, only three animals did so for for all three injection rates. For these nine trials, we measured the animals' body orientation, walking speed, projected antennule length (an indirect measure of antennule tip elevation), the angle between the antennules. and the absolute antennular orientation from the digitized video. We report here the results for the analysis of inter-antennular angle. An analysis of variance for repeated measures (on the individual animals) indi- cated a significant primary effect of injection rate F(2,2,708) = 29.06 (P < 0.001) and a significant animal-by-injection-rate in- teraction (Fig. 1). Inter-antennular angle increased with distance from the source during tracking at the lowest of the three injection rates. The two higher injection rates are significantly different from the lowest injection rate in this analysis, but are not distinct from one another. The three animals that were analyzed held their antennules more widely at the lower injection rates, meaning that less turbulent flow leads to wider spatial sampling. Obviously, the results taken from three animals must be interpreted conservatively. However, we take these results to indicate the power of this methodology for revealing the different sampling behavior lobsters employ with their lateral antennules. If the relationship between antennular separation and plume structure demonstrated here represents lob- ster tracking behavior in general, we will continue to use these methods to further examine the relationships between sampling strategies and stimulus environment. This work was supported by NSF-REU (OCE-960599) Site Grant to Boston University and DARPA-ONR Award (N00014- 98-1-0822) to FG and JA. The authors wish to thank Mimi Sheik for valuable technical assistance and Dr. Jennifer Basil, Marjorie Steele, Kirsten Pohlman, Michael van der Waal, and Thomas Breithaupt for advice and David Sandeman for thoughtful discus- sions that suggested this approach. Literature Cited 1. Moore, P. A., N. Scholz, and J. Atema. 1991. ./. Chrin. Ecol. 17: 1293-1307. 2. Basil, J., and J. Atema. 1994. Biol. Bull. 187: 272-273. 3 Dittmer, K., F. Grasso, and J. Atema. 1995. Biol. Hull. 189: 232- 233. 4 Devine, D., and J. Atema. 1982. Biol. Bull. 163: 144-153. 5. Beglane, P., F. Grasso, J. Basil, and J. Atema. 1997. Biol. Bull. 193: 214-215. Reference: Biol. Hull. 197: 250-251. (October 1999) Temporal Correlation Between Sensor Pairs in Different Plume Positions: A Study of Concentration Information Available to the American Lobster, Homarus americaiius, During Chemotaxis John P. Hanna1, Frank W. Grasso. and Jelle Aleina (Boston University Marine Program. Marine Biological Laboratory, Woods Hole, Massachusetts) The American lobster requires both lateral antennules for effi- cient tracking of a turbulent jet odor plume (I. 2). We set out to determine what bilateral cues are available to a lobster while it tracks a "leaky" plume to its source. To do this we characterized the dynamics of a turbulent odor plume. We made video record- ings of a 5-mm thick horizontal plane through a plume at the same height above the substrate that the lobster holds its lateral anten- nules and then analyzed the spatial patterns of concentration dy- namics in that plane. The experiments were conducted in a transparent-sided recircu- lating flume (10 m long; 2 m wide; water depth, 0.44 m). We analyzed a momentum-free ("leaky") plume that issued from a length of 0.5 cm diameter Tygon tubing (terminating in a stop- pered porous section. 2 cm long) situated centrally on the floor of the flume. The tubing oozed 1 x 10~4 M rhodamine b dye (in seawater) at a rate of 15 ml/min, producing a "puddle" of dye about 10 cm in diameter on the floor of the flume. This dye was then lifted from the viscous sub-layer into the free-stream by the small amount of intrinsic vertical turbulence of our flume, which. in turn, created a plume that was transported downstream by the 9 cm/s tree M .mi current. This plume is a good model of a decom- posing can . i ither benthic odor source typical of lobster food Department of Biology. University of Delaware, Newark. Delaware. items. In addition, lobsters in our laboratory track an identical, odor-laden plume to the source under these exact conditions. We used a pulsed YAG CO-, laser (532 nm) and appropriate optics to produce a horizontal laser sheet that covered a 1 m2 area of the flume 10 cm off the flume floor. The laser light within this sheet excited the fluorescent dye that constituted the plume. The intensity of the light emitted from the excited rhodamine B (excitation 540 nm emission 625 nm) molecules (and detected by the overhead CCD camera) was proportional to their concentration. Synchronizing sig- nals were sent from an overhead CCD camera (Hitachi KP-M2U) to a 400 MHz Pentium computer running MetaMorphIM imaging soft- ware; the computer then synchronized the firing of the laser with the acquisition of images. We made seven recordings, each 2.35 min in duration, with the source positioned sequentially at 50. 100, 150, 200. 250. 300, and 350 cm upstream from the center of the recording area. The recordings were streamed directly to the computer disk at a rate of 10 frames per sec. At each recording location, we made separate recordings with the plume off to correct for background illumination, background dye accumulation from flume water re-circulation, and intensity variations in the laser sheet. Reference concentration series were also recorded so that pixel intensities could be calibrated to molar units. The individual images in each recording were corrected for background illumination and dye accumulation in the flume by subtracting the average background image. The effects from uneven light distribution in the laser sheet were corrected by using this same CHBMORECEPTION AND BEHAVIOR 251 average background image as a normalizing mask. Each image was then filtered with a 7 X 7 pixel median filter to remove speckle noise. The dye reference images were corrected in the same manner and used to create a linear calibration curve (R2 = 0.98) to convert the pixel values into molar units of dye concentration. In general, this plume resembled a series of discontinuous filaments and patches of dye. with their long axes aligned with the direction of flow. As the plume progressed downstream, these filaments became more diffuse and blended into an unhomogenous cloud at the furthest downstream recording location. At the 350-cm recording location this diffusion led to signal levels too low to analyze. Once all the recordings were appropriately calibrated (above), we extracted concentration profiles (time series) at various spatial positions within the plume. We collected from pairs of sites separated by 1. 3. and 9 cm cross-stream distances. Three centi- meters approximates the inter-antennular separation of a plume- tracking lobster. We centered these pairs at coordinates directly downstream and at 15 cm to the left and right of the source in a given image stack. One such set of six points was collected from the image stack corresponding to each distance from the source. This spatial distribution of concentration profiles provided a sam- pling, in regular x-y coordinates, of the plume concentration dy- namics within the laser sheet. We studied the cross correlation functions (CCF) of these paired concentration series (Fig. 1) to look for spatial cues that would indicate source direction and that might be available to a pair of sensors (e.g., the lobster's paired lateral antennules). For each spatial location, we measured the best con-elation magnitude. best-matched delay, peak half width, and peak asymmetry. The delay and asymmetry indicated the existence of left-right differ- ences in odor patch arrival time, half width reflected variation in odor patch size. The correlation magnitude gave an indication ot reliability of any of these cues. All 54 CCFs had significant correlations centered at 0 delay (P < 0.0001 for all CCF maximum peaks, many P < 0.0001, R- ranged between 0.99 at 1 cm and 0.25 at 9 cm inter-sensor separation). The magnitude of the correlations fell off with increased separation between sampling pairs, but did not vary systematically with the spatial location of the pairs in the plume. We also found no significant variations in CCF asymmetry or half width with spatial location. Although American lobsters are more accurate plume trackers when they use both antennules. we found no indications in this rigorous quantitative study that bilaterally relevant information exists in the plume generated by this "leaky" source. The earlier lobster studies were conducted with plumes from non-zero mo- mentum (jet) sources, where such directional cues are known to exist (3. 4, 5, 6). Since lobsters can track this exact momentumless (leaky) plume, however, we are left with two alternatives: 1) that lobsters use other bilateral cues than instantaneous cross correla- tion of concentration when tracking such plumes; or 2) that no bilateral information is required by these animals to track leaky plumes. These negative results are important because leaky plumes are common in the natural world and contrast the 'jet' sources which have received greater attention in previous physical plume characterizations (5, 6) and behavioral studies with lobsters (1, 2). These results support the idea that lobsters are more likely to use different tracking strategies in different plumes than one univer- sally effective strategy for all plumes. Right/Left Virtual Sensors 150 cm Downstream 1 cm Separation — Right Sensor — Left Sensor .2 O.OF Cross-Correlation of Above Signals -Peak r=.91 p=<.001 Half-Heighl (0.45) 0 0.5 Delay (s) Figure 1. A representative cross-correlation (n = 1411) from our data set. Only the first second and hull <>/ rlic 2.35-min range of the cross- correlation is slum-it. All nl the cross-correlations showed a zero delay and a high degree of s\mmetr\ anninil that delay. The method of determining the half-width of the cross-correlation is illustrated in the diagram. We measured half width by finding the two points of intersection between the horizontal line at the _W7r maximum correlation level and the correlogram. The half-width was the distance between these nru points projected onto the time axis. Peak asymmetry was measured by comparing the integrated areas beneath the correlograin between these nro half-widths anil the delav of the peak correlation. We thank Dr. Deborah Compton of the Boston University Aeronautical Engineering department for fluid dynamics advice, and the loan of the laser and optical equipment. We thank Doug Bowman and Neil Glickman from Universal Imaging, and Meg Steele and Carla Guenther for valuable technical assistance. This work was supported by NSF-REU (OCE-960599) Site Grant to Boston University and DARPA-ONR Award (N00014-98-1-0822) to FG and JA. Literature Cited I Devine, D., and J. Atema. 1982. Biol. Bull. 163: 144-153. 2. Beglane, P., F. VV. Grasso, J. Basil, and J. Atema. 1997. Biol. Bull. 193: 214-215. 3. Grasso, F. W.. J. H. Dale, T. R. Consi, D. C. Mountain, and J. Atema. 1996. Biol. Bull. 191: 312-313. 4 Grasso, F. VV., J. H. Dale, T. R. Consi, D. C. Mountain, and J. Atema. 1997. Biol. Hull. 193: 2l5-21h. 5. Dittmer, K., F. W. Grasso, and .1. Atema. 1996. Biol. Bull. 191: 313-314. 6. Dittmer, K., F. W. Grasso, and J. Atema. 1995. Biol. Bull. 189: 232-233. 252 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS Reference: Biol. Bull. 197: 252-253. (October 1999) Chemoreceptor Cells as Concentration Slope Detectors: Preliminary Evidence from the Lobster Nose Erik Zettler1 and Jelle Atema (Boston University Marine Program, Marine Biological Laboratory, Woods Hole, Massachusetts 02543) Chemoreceptor cells of the American lobster Ho/iiunt.s ameri- i-uniis distinguish between different chemical compounds, as well as their concentrations ( 1 . 2, 3). After an initial phasic response to an increase in stimulus concentration, chemoreceptor cells quickly adapt to the higher background and. within one to a few seconds, spike frequency reverts to zero or to a low tonic level (2. 3). The chemical signature of an odor source is dispersed through its fluid medium by generally turbulent flow resulting in patches of differ- ent sizes and shapes. As these spatial patches pass by a sensor they appear as concentration peaks over time. The onset slopes of these peaks depend on the shape and concentration of the individual patches. Despite the chaotic nature of turbulence there is a spatial gradient of patches within such an odor plume. Patches disperse in statistically describable patterns, creating "odor landscapes" such that, closer to the source, the average odor peak heights and onset slopes are greater (I, 4). If an animal could distinguish between shallow and steep odor onset slopes, it could orient itself in a turbulent plume and move towards or away from the odor source. We have investigated the response of chemoreceptor cells of the lateral antennule of the American lobster Honuinis americiiniis to odor onset slopes generated by computer-controlled piston pumps (Millipore model 510). The odor stimulus (a 0.01% aqueous ex- tract of TetraMarin fish food) was mixed with a dopamine tracer that allowed us to measure the actual stimulus concentration pro- file of each slope with high spatial and temporal resolution; the measurement was made with an IVEC-5 system (In Vivo Electro- chemistry. Harvard Apparatus). Lateral antennules were excised and inserted, with aesthetasc sensilla facing up, into an acrylic olfactometer chamber that permitted perfusion with cold oxygen- ated Ringers and use of a suction electrode to record from the proximally exposed nerve bundle. The distal section bearing the aesthetascs was bathed with a constant flow of 10 ml min ~ ' of filtered seawater into which the food odor and tracer dissolved in seawater could be injected by the piston pumps. Using a micro- manipulator, the measuring tip of the IVEC electrode was placed within the aesthetasc tuft. Details of this tracer system, the olfac- tometer. and the extracellular recording techniques for this prep- aration are found elsewhere (3). Four cells were tested with each of two stimulus slopes (Low and Medium). One of these cells was tested with an additional slope ( High) generated by suddenly opening a valve to gravity feed the stimulus to the preparation. This delivery system is similar to one used earlier (3) to achieve steep onset ramps but does not allow for easily controlled slope variation. Because all cells tested showed a similar response, we chose to show results from the cell with the greatest number of treatments (Fig. I ). Average spike frequency, calculated during the first second of stimulus concen- tration rise, was higher for steeper onset slopes, ranging from 3 ' Also at Sea Education Association. Woods Hole. Massachusetts. spikes per second for the Low slope to 60 spikes per second for the High slope. Despite this 20-fold increase in spike frequency in response to steeper slopes, the ratio of spike frequency to stimulus concentration change varied only by a factor of two. Since the length of the slopes and the peak concentrations varied between treatments and the purpose of this study was to investigate the effects of onset slopes, we also compared the spike frequency during the initial rise from background to when the dopamine tracer reached 8 juM (the highest concentration reached by the Low slope). Examined in this way, the change in concentration is identical across treatments, so differences in spike frequency 40 Low i -> 10 30 20 5 10 M i , a- ^- i 1 — -"Hi 1 J|l,l!lllli I iii i in n A -i — . — - VJ r IS TJ i *-* 40 Medium 5r 2 30 ^ 10 $ E 20 /k/-V v^/\/ J s -* ro 10 §• o- mil i 0 o o 3 ~° (A 50 15 « High ;J^ •?y T i\ A j i 40 A> V\r ^ri^'vVAi.Jx^''1^ 30 ^»Jv^ 10 20 - 5 10 II II III Illl I i n r-i III Illlllli II lllill.i i ii .1 1 ,i i i i . ill II l.ll 1 111 | 0 45678 time (seconds) 9 10 Figure 1. Duplicate odor pulses for the three slopes (top traces in each panel t, and the resulting spikes from a .single chemoreceptor cell The concentration of the dopamine tracer (measured at 200 H; using IVEC-5) i\ plotted on the left \-a\is: the dopamine was mixed in seawater with the otlor stimulus and is assumed to disperse in a manner .similar to the odor I-/). The lower section of each panel represents the occurrence of individ- ual spikes grouped into Kill ins Inns (right y-a.\is). For hulli llic stimulus trace and the spikes, the first run is shown in hlack, and the second run in gra\. Despite some variation in the pump outputs (especially after the peak concentration was reached) agreement during the initial rise in concen- tration was good bet\veen replicates. Spikes were digitized and sorted to ensure that they would represent a single receptor cell. Spike frequency mi leased with increasing odor onset slope. CHEMORECEPTION AND BEHAVIOR 253 should be due mainly to the different slopes. The spike frequency (and rise time) during this portion of each slope was 3.1 Hz (1800 ms) for the Low slope, 5.5 Hz (980 ms) for the Medium slope, and 70 Hz ( 160 ms) for the High slope. Once the concentration stopped rising, cells adapted to the constant background within a couple of seconds. We note that the lack of a response to the second slope rise in the Medium slope (middle panel between 7 and 8 seconds) of Figure 1 may be due to the large fluctuations in the output of the piston pumps preceding this rise. These periodic fluctuations (some of which exceed 5 juM) would cause cumulative adaptation in the chemoreceptor cell such that the overall response is de- pressed (5). Because spike adaptation can occur rapidly, beginning within 500 ms of the stimulus presentation, a steeper onset slope may minimize adaptation and provide the highest frequency for a given concentration. We will not discuss here the intracellular signal transduction pathways that may be involved in the excita- tion and adaptation phenomena observed. These preliminary results demonstrate the feasibility of deliver- ing measured concentration slopes and provide initial evidence that chemoreceptor cells can act as "slope detectors." Odor slope discrimination could be very useful for orienting and tracking in a fluid environment, because so much physical information about the plume and odor source is present in the distribution of slopes in the eddy field (4). In this study, single cells could discriminate be- tween a range of odor onset slopes with rise times similar to those measured in laboratory studies of jet plumes (1, 4). In general, animals may have different chemoreceptor cells "tuned" to partic- ular ranges of pulse characteristics, such as frequency, height, length, and slope (1, 2, 3). Thanks to D. Mellon and G. Gomez for helpful discussions. Supported by NSF grant IBN-9723542 to JA and a B.U.M.P. Humes alumni Award to EZ. Literature Cited 1. Atema, J. 1996. Biol. Bull. 191: 129-138. 2. Borroni, P., and J. Atema. 19S8. ./. Com/'. Physiol. A. 164: 67-74. 3 Gomez, G., and J. Atema. 1996. J. Exp. Biol. 199: 1771-1779. 4. Moore, P., and J. Atema. 1991. Biol. Bull 181: 408-418 5. Voigt, R., and J. Atema. 1990. J. Own/'- Physiol. A. 166: 865-874. Reference: Biol. Bull. 197: 253-254. (October 1999) Individual Recognition and Memory in Homarus americamis Male-Female Interactions Cristin Berkev1 and Jelle Atema (Boston University Marine Program, Woods Hole, Massachusetts 02543) Individual recognition and memory have been studied in Ho- marus americamis, the American lobster. It has previously been demonstrated, through lobster boxing matches, that males can recognize individuals. When a male encounters the same male it lost to previously, it avoids a new fight. The same animal will fight on subsequent occasions if presented with an unfamiliar opponent ( 1 ). The ability to recognize a previous opponent lasts for up to a week, and it is based on olfactory recollection of the opponents' urine (2). The same results have been obtained in female-female encounters (3). We therefore hypothesize that male-female pairs of Homarus americanus can also recognize individuals, as demon- strated by shorter periods of fighting on the second encounter with familiar individuals. Males and females were paired by carapace length, with no pair differing by more than 3 mm. It was also ensured that in all matched pairs, the animals had not previously met. Animals were kept in isolation for 24 h before their first boxing match and for the 24 h between the first and second matches. All boxing matches took place in a 240-1 glass aquarium and were videotaped. One male and one female were placed in the tank and separated by a removable, opaque divider. The lobsters were allowed to acclimate for 10 min before the divider was removed, and then allowed to interact for 20 min. There were two sets of fights, group A and group B. In group A, the same pair of animals fought twice. In group B, the animals were rotated so that each animal met a new opponent in the second fight. In both sets, the first and second fights were separated by 24 h. Any pair of animals that did not 1 Tufts University, Medford, Massachusetts. show a definite loser and winner after the first 20-min period was disqualified and the fight was not included in the data analysis. The fights were scored using a pre-established scale of agonistic levels ( 1 ) with some additions made for differences that arose as a result of the fights being between males and females. An agonistic level was assigned to each animal every 5 s. The levels ranged from —2 to 5, with -2 demonstrating fleeing behavior and 5 demonstrating claw snapping or claw ripping at the opponent. An overall agonistic scale was calculated for each animal in each fight by summing all of the agonistic values. A fight was determined to be over when one animal ceased to show an agonistic level above one. In this way, the duration of each fight was scored. To prevent bias during analysis of the behavioral tapes, the scorer was blind to whether the tapes were from a first or second fight. In group A, 10 pairs of fights were carried out. Significantly, in 9 out of 10 instances, no fighting occurred on the second encoun- ter; fighting did occur in the tenth case. In group B, 13 pairs of fights were carried out. Two sets were disqualified because there was no clear winner after the first fight. In 3 out of 1 1 instances, no fighting occurred on the second encounter. In 8 instances, fighting occurred. The fraction of times in which fighting occurred on the second encounter is significantly different between the two groups (P < 0.005 .v2 = 8.41 6 df = 1). When a fight was between familiar opponents, the aggression level of the loser was significantly lower in the second boxing match than in the first boxing match (P < 0.05 paired t test). However, for unfamiliar opponents, the aggression level of the loser was not significantly lower in the second boxing match than in the first (paired / test). The aggression level of the winner was 254 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS not significantly different between the two fights in either group A or group B (paired t test). We observed two behaviors that had not previously been seen in either male-male ( I ) or female-female (3) matches. One of these was mounting behavior, which typically occurs just prior to mating (4): the male climbs on the back of the female and attempts to turn her onto her back using his first two pairs of walking legs and his third maxillipeds. In our encounters, however, this mounting be- havior never progressed to actual mating. We observed male- female mounting behavior in five separate encounters, two of which involved the same pair of animals. A second form ol behavior not seen in same-sex boxing matches was nonreactive contact. This included any situation in which the two animals were in physical contact but did not seem to be reacting to each other. The average time spent in contact across the 42 fights analyzed was 1 79 s per 20-min fight. This value had no obvious correlation with previous exposure to the opponent. In addition to the differ- ences already mentioned, fight duration in first encounters was significantly shorter in male-female fights than in male-male fights (P < 0.05, / test). The average fight duration was 250 s (SE ± 50 s) in male-female pairs, but 457 (SE ± 73 s) in male-male pairs ( 1 ). However, there was no significant difference in fight duration between male-female and female-female first fights: the average fight duration in female-female pairs was 206 s (SE ± 73 s) (3). The similarity in fight duration between female-female and male- female pairs is consistent with the observation that the loser determines the end of the fight: in male-female pairs, females typically lost. The fact that second encounters were much shorter, to the point that no fighting occurred, supports the hypothesis that male and female lobsters can recognize each other as individuals. The results also demonstrated differences, such as nonreactive contact and mounting behavior, in the way male and female lobsters interact within the boxing match situation, compared to male-male and female-female interactions. It is possible that these differences parallel the natural interactions between male and female lobsters. Females are allowed to enter male shelters, whereas other males are not (5 ). Therefore, males and females must be capable of being near to each other without demonstrating aggressive behavior. Financial support from NSF-REU (OCE-9605099 site award to Boston University). We thank Leslie McLaughlin, Jennifer Walters, and Dr. Frank Grasso for their assistance with this project. Literature Cited I Karavanich, C., and J. Atema. 1998. Anim. Bchav. 56: 1553-1560. 2. Karavanich, C., and J. Atema. 1998. Behaviour 135: 719-730 3. Atema, J.. T. Breithaupt, A. LeVay, J. Morrison, M. Mallidis, and M. Edattukaran. 1999. Client. .SVino (in press). 4 Atema, J., and D. G. Engstrom. 1971. Nunire 232: 261 263. 5. Bushmann, P., and J. Atema. 1997. dm. J. Fish. Aqital. Sri. 54: 647-656. Reference: «;,./. Bull. 197: 254-255. (October 19l. Bull. 197: 257-258. (October I9W) Squid Axoplasm Supports the Retrograde Axonal Transport of Herpes Simplex Virus E. L. Bearer (Brown University). M. L Schlief , X. O. Breakefield2 , D. E. Sclutback2, T. S. Reese*. and J. H. LaVaif Neurotropic viruses, such as Herpes simplex virus type 1 (HSV1 1. first enter the axon terminal and are transported to the cell body where they replicate in the nucleus. Based on previous studies using whole animal assays ( 1 ). as well as assays of whole cells //; vitro (2. 3), we know that HSV enters axons and travels as an unenveloped particle — capsid plus associated tegument pro- teins— in a retrograde direction toward the cell body. The rate of transport has been estimated to be 3-5 mm/hr (2). Although these earlier studies provided important insight into the delivery of virus to the nucleus, they were limited. What has been needed is an experimental system in which HSV can be applied to a particular region of a neuron at a known concentration and time, and the transport of the virus assayed quantitatively within the axon. With such an assay one could address questions about the real time motility of the viral particle and about the viral and cellular proteins that are essential for this behavior. The giant axon of the squid, Loligo pealei, serves as a powerful model for the molecular mechanisms of axoplasmic transport. The microtubule-based motor, kinesin, was discovered in squid (4); and microtubule-based transport in both anterograde and retro- grade directions has been extensively characterized. Recently, actin-based transport of squid axoplasmic organelles has also been described (5, 6). Organelles move in either direction in the giant axon at 2-4 JLUTI/S. Organelles isolated from squid axons move towards the barbed ends of actin filaments (6. 7) at 1.6 jxm/s (8) and in either direction on microtubules at 2 ju,m/s (9). The axon contains long tracks of microtubule-actin filament bundles that appear to serve collectively as tracks for axonal transport (10). Thus, the squid axon contains all the requisite molecular machin- ery for transport. In this report, we describe the use of the squid giant axon as an alternative to previous approaches and present for the first time direct observation of the movement of HSV in living axons. To image the virus in the axon, we used a viral HSV strain in which a major tegument protein, viral protein (VP) 16, was gen- erated as a fusion protein with GFP at the C terminal. This virus was grown in Vero cells, purified, and concentrated to a titer of about 1.0 X 109 pfu/ml. Different aliquots of the viral stock were treated in various ways to remove the viral envelope. This mimics the first step in viral infection in which the viral envelope fuses with the cell membrane, releasing the capsid together with its tegument into the cytosol. In vitro removal of the envelope was necessary for these experiments because injection of virus into the axon by-passes the normal membrane fusion step. Several treat- ments were found that produced motile, fluorescent particles in the axon after injection. ' NINDS. National Institutes of Health. Bethesda. Maryland. 2 Massachusetts General Hospital. Boston. Massachusetts. 3 University of California. San Francisco. The viral preparation was co-injected with non-fluorescent oil. The oil droplet was used to determine an appropriate focal plane and as a fixed reference marker, since it remains stationary after injection (11). The movement of viral particles was recorded on a laser scanning confocal microscope. Movement was sampled ~ I frame/3 s. and transport rates were determined by analyzing se- quential frames. When we examined fields upstream from the oil droplet, i.e.. closer to the cell body, we identified rapidly moving particles. One of these moved -36 p.m in 18 s for an overall rate of 1.9 ju,m/s in the retrograde direction (Fig. 1 ). At the end of this sequence, this particle went out of the plane of focus. Of 113 moving particles examined, all were found in the region of the axon between the oil droplet and the cell body, moving in the retrograde direction. The effect on the virus of the pre-injection treatment was de- termined by electron microscopy of negatively stained prepara- tions. Of the virus in those samples that produced motile particles when injected in the axon. —90% had lost their surface envelope. Since the GFP-labeled protein. VP16, is a tegument protein, the moving particles we observed must include at least tegument, either as whole virus, tegument plus capsid. or simply as tegument aggregates. Since neither capsid nor membrane are GFP-labeled. we cannot know from these studies whether they also are capable of moving. We have demonstrated the rapid and preferential retrograde axonal transport of HSV particles in the living axon. The rate of movement of these viral particles in the squid giant axon is similar to the estimated rates of retrograde transport of this human virus in rat dorsal root ganglia in culture (2). Furthermore, this rate is consistent with the rates of retrograde transport of endogenous organelles in the squid axon and of isolated organelles on either microtubules or actin filaments. Both the direction and rate of movement suggest that a host cell motor molecule, such as the retrograde microtubule motor, dynein, may be co-opted by invad- ing virus. Thus, by combining the power of the squid axon with a biochemical and genetic dissection of the virus, we expect to be able to identify the viral proteins required for transport, as well as the cellular transport machinery that they recruit. Supported by NIH EY08773 (JHL), GM47638 (ELB), NINDS (TSR). NINDS24279 (XDB). and by the Frederik B. Bang Fel- lowship Fund and the Evelyn and Melvin Spiegel Fellowship Fund, Marine Biological Laboratory. Woods Hole. MA. and by a predoctoral NINDS fellowship (MLS). Literature Cited 1. Topp, K. S.. L. B. Meade, and J. H. LaVail. 1994. J. Neumsci. 14: 318-325. 2. Lycke, E., K. Kristensson, B. Svennerholm, A. Vahlne, and R. Zeigler. 1984. J. Gen. Virol. 65: 55-64. 258 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS muscle retrograde 1 Sodeik, B., M. W. Ebersold, and A. Helenius. 1997. J. Cell Biol. 136: 1007-1021. 4. Vale, R. D., T. S. Reese, and M. P. Sheetz. 1985. Cell 42: 39-50. 5 Kuznetsov, S. A., G. M. Langford, and D. G. Weiss. 1992. Nature 356: 722-725. 6. Bearer E. L., J. A. DeGiorgis, R. A. Bodner, A. W. Kao, and T. S. Reese. 1993. Proe. Null. Acad. Sci. USA 90: 11252-11256. 7. Langford, G. M., S. A. Kuznetsov, D. Johnson, D. L. Cohen, and D. G. Weiss. 1994. J. Cell Sci. 107: 2291-8. 8 Bearer, E. L., J. A. DeGiorgis, N. A. Medeiros, and T. S. Reese. 1995. Cell Mori/. Cytoxkeleton 33: 106-1 14. 9. Schnapp, B. J., R. D. Vale, M. P. Sheetz, and T. S. Reese. 1985. Cell 40: 455-462. 10. Bearer, E. L., and T. S. Reese. 1999. J. Neurocytol. 28: 85-98. 1 1 Galbraith, J. A., T. S. Reese, M. L. Schlief, and P. E. Gallant. 1999. Proc. Natl. Acad. Sci. USA (in press). Figure 1. Retrograde transport of a GFP-labe/ed viral particle in u living squid axon. The giant axon was dissected in Ca++ -containing seawater and injected with >IOO pfu O/GFP-VP16 labeled and extracted Herpes simplex virus. Still frames were taken from a BioRad confocal microscope sei/uence captured at 2—3 sec intervals and processed with NIH Image sojhvare. The diagram shows the orientation of the giant axon and site of injection relative to obsen-ed movements (dashed arrow). In this preparation, the cell body is to ihc right. GFP-labeled particle (oblii/ue arrowhead] moves at 1.9 fiin/s towards the cell body, contrasting with a \tati S). DNA sequences known from five other neuronal kinesin family mRNAs were used as probes in a Northern blot analysis to deter- mine whether any other kinesins were enriched in myelin. Mes- senger RNA isoforms of two other kinesins, KHC and KIF2, were found to be enriched in myelin (data not shown). The mRNA for the major KHC was quite large, 7-9 kb. whereas that for KIF2 was small, less than 2.5 kb. Northern blots were used to determine the tissue distributions and developmental appearances of these mRNAs. The low molecular weight mRNA for KIF2 had tissue distribution and developmental expression patterns similar to those of the MBP and CNP mRNAs. Unlike these "myelination-specific" mRNAs, non-neural tissues also expressed KHC and DLIC-2 (data MBP S M KIF1A DLIC-2 S M S M CNP S VI • • Figure 1. L RNA/lane) of RNA from the sttirtni^ material supernatant iSI unit myelin fraction (M) were run on ]1e agarose/ formaldeh\de Kcls and transferred to in/on membranes. The membranes were probed with "P-labeledcDNAsforMBP. CNP. and r\\-o novel cDNAs obtained b\ siibiractive hybridization (see text) according to methods used in mil lab i^i. We have not checked these membranes for eainil loading, bit! have fount! !l/' same results for each probe on several occasions. The sizes of the inKNAs are roitxhly 2 kb {MBP). 5 kb (CNP). 7 and 9 kb for the larger KIFIA hands uml more thtin 6 kb for the DLIC-2 band enriched in mvelin. The sizes for MBP. CNP and DLIC mRNAs tire the same as expected front the sizes of the cDNAs (accessed through GenBankl 260 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS Figure 2. In situ hybridization of KIF1A tnRNA in (A) the dorsal column of P10 rat spinal cord and in (B) cultured mouse OL. In both pictures the arrows point to labeled cell processes. The resolution in cultured cells is sufficient to see thai the mRNA is present in discrete granules, which would be an indicator that the mRNA was transported in granules. not shown). Their developmental expression patterns also differed from MBP and CNP mRNAs; they were expressed throughout postnatal development. In situ hybridization studies confirm the presence of these mRNAs in OL processes in vivo and in culture. We have demonstrated that the KJF1A probe recognizes mRNAs in a cluster of OLs in the dorsal column of a young rat spinal cord and in cultured mouse brain OLs (Fig. 2). In cells in vivo and in culture. mRNA is clearly seen in long cell processes, indicative of mRNA transport. Synthesis of motor proteins in OL processes indicates that complex "microtubule-based" communication sys- tems are in place to transport vesicles from sites of myelin sheath assembly back to the OL soma. This system could function to transport those proteins that must be removed from the OL plasma membrane so that the myelin sheaths will be left with their select and rather simple protein composition. We hypothesize that the appearance of KIF1A. KHC. and DLIC-2 mRNAs early in devel- opment indicates that these proteins are formed in OL processes at early developmental stages, i.e., when OLs first contact the axons they myelinate. If this is the case, the motors may play a role in transporting axon-derived material back to the OL soma. Supported by a grant (RG2944AG/1) from the National Multi- ple Sclerosis Society. Literature Cited 1 Colman, D. R., G. Kreibich, A. B. Frey, and D. D. Sabatini. 1982. J. Cell B/o/. 95: 598-608. 2. Gillespie, C. S., L. Bernier, P. J. Brophy. and D. R. Colman. 1990. J. Nfurochem. 54: 656-661. 3. Gould, R. M. 1998. J. Neurocliem. 70 Suppl. 1 S53. 4 Diatchenko, L., V.-F. C. Lau, A. P. Campbell, A. Chenchik, F. Mooadam, B. Huang, S. Lukyanov, K. Lukyanov, N. Gurskaya, E. D. Sverdlov, et al. 1996. Proc. Natl. Acad. Sci. USA 93: 6025- 6030. 5 Altschul, S. F., T. L. Madden, A. A. Schaffer, J. Zhang, Z. Zhang, W. Miller, and D. J. Lipman. 1997. Nucleic Acids Res. 25: 3389- 3402. 6. Okada, Y., V. Yamazaki, Y. Sekine-Aizawa, and N. Hirnkawa. 1995. Cell 81: 769-780. 7. Hughes, S. M., K. T. Vaughan, J. S. Herskovits, and R. B. Vallee. 1995. ./. Cell Sci. 108: 24. 8. Gould, R. M., C. M. Freund, and K. Barbarese. J. Neurocliem. 73: (in press). Reference: Biol. Bull. 197: 260-262. (October 1999) Migration Forces in Dictyosteliiun Measured by Centrifuge DIC Microscopy Yoshio Fukui1, Taro Q. P. U\eda~, Chikako Kitavanur, and Sliinya fnoiie (Marine Biological Laboraton; Woods Hole, Massachusetts 02543-1015) Amoeboid locomotion represents an important biological activ- ity involved in cell growth and development ( 1 ). Forces that underlie movement of the giant amoeba. Chaos chaos, have been estimated to be 1.5 x 102 pN/junr as measured by Kamiya's double chamber method (2). For a slime mold, Dictyosteliiun disctiideum, the forces of cell locomotion have been unknown, but the cortex resists poking with a microneedle (cortical tension) at 1.4 X K)1 pN//nirr (3). By micropipette aspiration, the cortical tension of D. discoideum has been measured as 1.55 x 103 pN//xm2 (4). In the present study, we determined the migration stalliirj forces of D. discoideum by using a centrifuge polarizing 1 Cell anil Molecular Biology. Northwestern University Medical School, Chicago. Illinois 6061 1-3008. : Biomolecular Research Group. National Institute for Advanced Inter- disciplinary Research. Tsukuha, Ibaraki 305-8562, Japan. microscope (CPM) equipped with DIC optics (5). The results demonstrated that individual wild type (NC4) amoebae (6) can crawl centripetally on a glass surface, resisting gravitational forces larger than 1 1 ,465 x g. NC4 amoebae can also undergo normal cytokinesis at forces of at least 8376 X g. Dictyostelium cells were washed with Bonner's saline solution (BSS: 10 mM NaCl, 10 mM KC1, 3 mM CaCl,) and allowed to attach to an ethanol-cleuned glass slide in a custom centrifuge chamber filled with BSS. Frozen images of the spinning micro- scopic field containing 20-50 cells were recorded onto Sony ED-Beta tape through an Olympus SLC Plan Fl 40 x (N.A. 0.55) or LC Plan Fl 20x (N.A. 0.40) objective lens and a condenser lens (LC Plan Fl 20X/N.A. 0.40). The images illuminated by a 532-nm pulsed laser were captured in real time with a Hamamatsu C5946 CCD camera equipped with an interference-fringe-free filter. The centrifuge disk rotates horizontally, and its speed was controlled in CELL MOTILITY Table I Migration stall forces in Dictyostelium 261 Measurement Maximum Rotation (rpm)t Gravity (X g) Force § (X103 pN) Strain* NC4 Ax3 HSI B. Migration stall forces of different strains and myosin mutants > II, 700 > 1 1 ,465 >8.77 ± 1.10 6,400 3,431 1.08 ± 0.42 3.500 1,025 0.28 ± 0.09 C. Migration stall forces and medium density in HSI A5 Radius (X10 4 cm) Volume (X10~10cm3) AMasst (X10~" g) A. Reduced mass of different 4.81 ± 0.95 3.78 ± 1.44 2.57 ± 0.98 strains and myosin mutants 5.47 ± 1.05 4.87 ± 1.80 3.32 ± 1.23 5.14 ± 0.83 4.26 ± 1.34 2.90 ± 0.91 5.48 ± 0.98 4.87 ± 1.69 3.31 ± 1.15 3,400 968 0.30 ± 0. 1 1 Percoll (%) 0% 10% 25% 50% 75% 100% Density (g/ml) 1.000 1.024 1.032 1.064 1.104 1.186 Maximum Rotation (rpml± 3,500 4.100 4.900 6.800 7.800 8,600 Gravity (X g) 1.025 1,408 2,011 3,873 5,096 6.195 * NC4: wild type, Ax3: axenic mutant, HSI: myosin II knock-out mutant. A5: triple (myoIA, myolB, myosin II) knock-out mutant. t AMass = (Cell density - Medium density) x Volume = (1.068 - 1.000) x Volume = 0.068 (g/cm1) x Volume (cm') t Maximum rotor rpm beyond which the amoebae were unable to crawl centnpetally. § Standard deviation each based on measurements of diameters of more than 100 cells. 100-rpm increments up to a maximum speed of I 1.700 rpm. The radius from the center of the disk to the center of the observation chamber was 7.5 cm. We determined the maximum rotation speed at which the cell's geometric center ("centroid") exhibited centrip- etal movement, i.e., movement towards the center of the rotor. The measurement was done for wild type (NC4) (6). axenic strain (Ax3) (6), and two myosin knock-out mutants. Of the two, HSI is a myosin II null mutant that does not express conventional myosin, which is responsible for production of major mechanochemical forces (7). A5 is a triple knock-out mutant that does not express myoIA. myolB, or myosin II (8). Migration forces were calculated from Newton's Force Law, i.e., F = in X a, where F is force (in pico Newton: pN). »t is mass (in grams), and a is acceleration (in centimeters per second squared). We measured cell volumes from the diameter of round cells and calculated the reduced mass by multiplying the volume by the density difference (1.068 - 1.000 g/cm') (9). The average radius, calibrated volume, and reduced mass of NC4, Ax3, HSI, and A3 are shown in Table IA. As shown, the maximum rotational speeds at which the amoebae were able to crawl centripetally were 11,700, 6400. 3500. and 3400 rpm. respectively. No centripetal migration occurred when the rotor speed was increased by 100 rpm. These values correspond to 11.465 x g. 3431 X g, 1025 X g, and 968 X g, respectively. These results showed that the gravitational forces equivalent to the migration stall forces are. respectively, >2.77 x 10' pN. 1.08 x 101 pN. 0.28 x 10' pN. and 0.30 X 103 pN (Table IB). We also examined the "maximum rotation speed" as a function of density of the medium (Table 1C). The results of these experi- ments were unexpected; the ability of the amoebae to migrate centripetally continued to increase with the density of the medium. even when it substantially exceeded the density of the amoebae themselves, so that detached amoebae would float. Since all strains exhibited the same level of adhesion up to maximum rotation (i.e.. 11.700 rpm). we propose that the capacity for centripetal move- ment in fact represents the migration forces of those amoebae. The behavior of an amoeba in a medium with a density greater than its own must signify a stalling mechanism based not on the overall buoyant density of the amoeba, but perhaps on some stratified components on or within the amoeba. This study demonstrates that the axenic strain (Ax3) is in fact a type of mutant (10) that can generate less than 39% of the migra- tion force generated by the original wild type (NC4). This study further demonstrates that a myosin II knock-out mutant (HS 1 ) can generate only 26% of the migration force that its axenic parent (Ax3) can produce. In contrast, knocking-out myoIA and IB (A5) produces no additional decrease in the generation of migration forces. The migration stall forces exhibited by those mutants are obviously not dependent on myoIA. myolB or myosin II. suggest- ing a significant contribution by other actin-based. force-generat- ing mechanisms. Literature Cited 1. Fukui, Y. 1993. Inl. Rf\: CM,,!. 144: 85-127. 2. Kamiya. N. 1964. Pp. 257-277 in Primitive Motile Systems in Cell B/o/()?v. R. D. Allen and N. Kamiya, eds. Academic Press. New York. 3. Pasternak. C., J. A. Spudich, and E. L. Elson. 1989. Nature 341: 549-551. 4 Gerald, N., J. Dai, H. P. Ting-Beall, and A. De Lozanne. 1998. J. Cell />'/»/. 141: 483-492. 5. Goda, M., S. Inoue, and R. Knudson. 1998. Biol. Bull. 195: 212-214. 262 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS ft. Raper, K. B. 1984. Pp. 19. 74-75 in The Dictyosteliads, Princeton University Press. Princeton. New Jerse> 7 Ruppel, K. M., T. Q. P. Uyeda, and J. A. Spudich. 1994. J. Biol. Cliein. 269: 18773-18780. X Kitayama, C.. J. Dai. H. P. Ting-Beall. M. A. Titus, and T. Q. P. Uyeda. 1998. Mol. Biol. Cell 9: 387a. 9. Fukui, Y. 1976. Dev. Growth Differ. 18: 145-155. 10. Kavman, S. C., and M. Clarke. 1983. ./. Cell Biol. 97: 1001-11)10. Reference: Biol. Bull. 197: 262-263. (October 1999) Dynamic Confocal Imaging of Interphase and Mitotic Microtubules in the Fission Yeast, S. pombe P. T. Tran1, P. Maddox2, F. Chang1, and S. Inone (Marine Biological Laboratory, Woods Hole, Massachusetts 02543) In the fission yeast. S. pombe, microtubules are required for multiple cellular processes, including maintenance of cell polarity, positioning of cellular organelles. and mitosis. Thus, microtubules are dynamic polymers ( 1 ), remodeling themselves within the living cell throughout the cell cycle. Unfortunately, our current view of the cytoskeletal architecture of the fission yeast microtubule conies from immunofluorescence microscopy and electron microscopy of static, fixed cells (2). However, recent technical advances in wide-field epifluorescence imaging of microtubules, made possible by fusions of green fluo- rescent protein to tubulin (GFP-tubulin), have allowed direct ob- ' Columbia University. New York. New York 10032. 2 University of North Carolina. Chapel Hill, North Carolina 27514. servation of microtubule behavior in living fission yeast cells (3). We have now applied real-time confocal microscopy to GFP- tubulin in haploid fission yeast, and can report dynamic changes in the microtubule cytoskeleton with unprecedented spatial and tem- poral resolution. A wild type haploid strain of fission yeast was transformed with a plasmid carrying the GFP-a2/tubulin gene. GFP-tubulin was therefore expressed along with endogenous tubulin. The behavior of the GFP-tubulin yeast strain is identical to that of the wild type in terms of cellular morphology and cell cycle duplication time (3). For imaging, cells were mounted on a thin layer of 20% gelatin mixed with yeast medium, between coverslip and slide. Images were digitally acquired at room temperature (23° to 26°C) with Metamorph Software (Universal Imaging Corp.) controlling a Figure 1. Real-time confocal ima^inx of GfP-nihiilin in the haploid /nwo/i \etixt, S. pombe. linages are extracted at the noted tune intervals to x/iow cell cvcle proxrcsxii'ii >/ inn lotnhnle reori>aiii.iilion and i/vnamicx. Panel A xliowx un interphiixe microtnlnile hnndle. Note the dynamic changes in microlnhnle lcn?;tli\ an, I the /" i w\/cm e nf the overlap region; o = overlap region. Panel R xhowx n initotic microtubule spindle. Note the rapid increase in \piiulle length at both emlx, a\ well i/.s nxlnil niierotiihiilex and newlv nucleated microtubules from the central region of the cell at the late Stage of mitosis; \ = spindle, a — astral microtubules. \< ale har = 5 ^m. CELL MOTILITY 263 CCD digital camera (Orca-1, Hamamatsu Corp.), mounted on a real-time confocal unit at 488 nm excitation (CSU-10, Yokogawa Corp.) attached to the Leica DMRX microscope stand equipped with a 100X/1.3N.A. oil-immersion objective (Leica Corp.). During interphase, about 4-6 bundles of microtubules extend along the long axis of the cell. Each bundle consists of 2-4 individual microtubules arranged in a characteristic manner sug- gestive of anti-parallel microtubules overlapping each other within a small region near the center of the cell (Fig. 1A). The microtu- bules are very dynamic, exhibiting a growth rate of 2.5 /nm/min and a shrinkage rate of 15 /urn/min. Switching between growth and shrinkage is frequent. Microtubule shrinkage starts at one tip of the cell, progresses to the overlap region near the center of the cell, and abruptly stops, never advancing beyond the overlap region (Fig. 1A). The behavior of the opposing microtubule in the overlap appears to be independent of the shrinking microtubule. In in- stances where both ends of the same microtubule bundle shrink completely, the overlapping region remains in the center of the cell as a small bright dot which eventually nucleates new microtubules (Fig. 1A). This suggests that the overlapping region in each mi- crotubule bundle contains secondary microtubule-organizing-cen- ters (MTOCs). whereas the SPB is the primary MTOC. At the onset of mitosis, all interphase microtubules abruptly and completely shrink, not to be renucleated. The secondary MTOCs also disappear. The SPBs appear as one bright dot, which subse- quently elongates to form the mitotic spindle. Spindle elongation appears biphasic. There is an initial slow rate of elongation of 0.2 /Lim/min until the spindle reaches a length of about 2-3 microme- ters. Then the spindle elongates rapidly, at 0.8 /nm/min. Coincident with the fast elongation phase, astral microtubules, which appear to be more or less perpendicular to the long axis of the spindle, begin to nucleate from the SPBs (Fig. IB). The ends of the astral microtubule bundle seem to interact with the cell cortex, as the spindle elongates to the length of the cell. Before the spindle has completely elongated to the tips of the cell, numerous microtuhiile nucleations can be observed at the center of the cell, where the division ring and septum eventually constrict to separate the two new cells (Fig. IB). The new microtubules undergo dynamics similar to those of the interphase microtubules. At the end of mitosis, the spindle microtubules disassemble, septation occurs, and the two new cells are once again in interphase. The complete cell cycle lasts about 2.5 h. These preliminary observations on microtubule cytoskeleton dynamics and reorganization throughout the fission yeast cell cycle were made directly on living cells at high spatial and temporal resolution. Because a myriad of mutants in fission yeast are avail- able for study with this imaging technique, important new oppor- tunities for probing and analyzing the molecular mechanisms of microtubule-dependent cellular processes and activities are now open. P.T.T. thanks Dr. Rudolf Oldenbourg for generously providing lab space and equipment during this study. This work was funded in part by NIH grants to P.T.T. and F.C. Literature Cited 1. Desai, A., and T. J. Mitchison. 1997. Anmi. Rev. Cell De\: liinl. 13: 83-117. 2. Hagan, I. M. 1988. J. Cell Sci. Ill: 1603-1612. 3 Ding, D. Q., Y. Chikashige, T. Haraguchi, and Y. Hiraoka. 1998. J. Cell Sci. Ill: 701-712. Reference: Bint. Bull. 197: 263-265. (October 1999) Dynamic Confocal Imaging of Mitochondria in Swimming Tetrahymena and of Microtubule Poleward Flux in Xenopus Extract Spindles Paul Maddox1, Arshad Desai2, E. D. Salmon1, T. J. Mitchison"'. Karen Oogema2, Tarun Kapoor3, Brian Matsuinoto4, and Slunya Inoue (Marine Biological Laboratory, Woods Hole, Massachusetts 02543) We report here the use of a real-time, spinning-disk confocal scanning unit (Yokogawa Electric CSU-10; 1 ) mounted on a Leica microscope to investigate the internal dynamics of fluorescently labeled microtubules in mitotic spindles. We aimed, in particular, to test the spatial and temporal resolution and the optical section- ing capability of the microscope setup. Live Tetrahymena were stained with the fluorescent probe, Mito Tracker Green FM (Molecular Probes, Cat#M7514). Excitation was provided by the 488-nm line from a Krypton-Argon gas laser, which was passed through the spinning disk in the CSU-10. Green fluorescence from the mitochondria was collected back through the confocal unit by a SIT (Silicon Intensified Target) camera (Dage- 1 University of North Carolina, Chapel Hill, North Carolina. 2 EMBL, Heidelburg. Germany. 3 Harvard Medical School, Boston, Massachusetts. 4 University of California, Santa Barbara. California. MTI 66) mounted at the primary image plane. Video output from the camera was recorded directly onto ED-Beta tape at 30 frames per second, i.e.. in real time. The objective lens used for this experiment was the 100X/1.3 NA Fluotar on the Leica DMRX microscope. Conventional wide field microscopy showed bright signals from all the mitochondria, so the image was blurred. With confocal observation, optical sections of the swimming Tetrahymena showed fluorescence only where mitochondria came to intersect the plane of focus. We next explored the ability of the microscope system to produce confocal images of assembly dynamics and motility of microtubules within mitotic spindles. The mitotic spindles were produced in the cell-free Xenopus extract system described else- where (2). Briefly, cytoplasm was isolated from eggs of Xenn/nis laevis. This extract, arrested by a metaphase cytostatic-factor (CSF), was driven into interphase by Ca ' ' addition, then induced 264 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS Figure 1. (Al Mitotic spindle in Xenupus egg extract. Fluorescent speckles in the microtubules were visualized by adding \-rliodamine-labeled tithulin ( ~ l'7r "I total tnhulin in extract! anil using the CSU-IO confocal unit as described in the text. Arrows point to free microtubules extending from the spindle. iBl Kymograph showing the dynamics of fluorescent speckle flux. The kymograph is made up by displaying, on the horizontal axis, the fluorescence distribution along the black line in (A) us a function of time (vertical axis). The diagonal streaks are bright spots of fluorescent speckles which move over lime within the spindle, indicating mil rombiilc poleward flux. (C) Wide field fluorescence image of a different Xenopus extract spindle (~ 5% laheled-liihiilin). Notice the hoiiiogeneitv of the \pimlU: ID) Another Xenopus extract spindle <~ 0.05% labeled-tubuliii) imaged by wide field fluorescence microscopy. Fluorescent speckles are visible, but finer details of the spindle are not visible due to fluorescence from oiit-of-focus planes. Bars: (A) and (B) = 10 P.III: 1C) and (II) = 15 /ixm. back into mitosis by addition of a small amount of CSF-arrested extract. The resulting cytoplasmic extract contained metaphasc spindles (sperm nuclei were added for a source of chromatin) that were easily observed by addition of fluorescent labels (DAPI for DNA and x-rhodamine-labeled purified tubulin for microtubules). In previous studies, photo-uncaging of fluorescently labeled lubulin in spindles was used to show that there is a bulk flow of polymer originating near the spindle mid-zone and progressing toward each pole region. This How, termed microtubule poleward lluv was found to move at about 2 /j.m/min in Xenopus extract spindles; this is the same rate as the chromosome-to-pole move- ment during anaphase in this system. Therefore, it was proposed that poleward microtubule flux could be the force generator for anaphase A chromosome separation (3). The object of this study was to image the dynamics of individual microtubules within spindles. Because the spindles are about 40-/Mm long and contain thousands of microtubules, conventional wide-field fluorescence micrographs lack tine structural detail (Fig. 1C). Lowering the ratio of labeled to unlabeled tubulin subunits in the cytoplasmic extract leads to a low density of label along the polymerized microtubule lattice (4, 5). This distribution in turn creates bright speckles of fluorescence that serve nicely as internal fiduciary marks along the lattice. One is then able to visualize either polymerization or depolymerization, as well as the flux of subunits within polymers and the movement of whole polymers (5). A problem with this technique, however, is that the small amounts of labeled tubulin (less than 0.1%) make visualization of gross spindle morphology difficult (Fig. ID). CELL MOTIL1TY 265 Mitotic Xeuo/nis extract spindles with more than 0.1% of the total tubulin pool labeled were observed with the CSU-10 real- time confocal unit, which was coupled to an Orca cooled CCD camera (Hamumatsu Photonics, Bridgewater. New Jersey). With this system, the dynamics of microtubules in the middle of spindles could be seen, as could the fibrous structure of the polymer mass (Fig. 1A). MetaMorph software (Universal Imaging Corp., West Chester. Pennsylvania) was used to control the CSU-10 shutter as well as the camera. This allowed time-lapse imaging for up to 10 min, exposing the sample for 0.75 s to the 568-nm line of the laser every 10 s, with little photobleaching. A Leica IOIK/I.3 NA objective lens was used to increase light-gathering efficiency, as well as to match magnification to the resolution limit imposed by the pixel size of the CCD chip. Analysis of the time-lapse images revealed that microtubules add subunits to their plus ends while losing subunits from their minus ends near the spindle poles. Plus ends were located through- out the half spindle, and the movement of fluorescent speckles was often seen traversing the entire spindle (Fig. IB). Preliminary measurements of the poleward movement of fluorescent speckles indicated a rate of about 2 /nm/min. corresponding nicely to previous values (4, 5). The confocal images revealed that micro- tubules were clustered into bundles within the spindle (Fig. 1A). Also clearly visible were fluorescent-speckled microtubules ex- tending out and away from the main spindle (Fig. 1A. arrows), often for great lengths (up to 50 p,m). Poleward speckle movement was detected in this population of microtubules, indicating that plus ends do not have to be within the spindle to facilitate pole- ward microtubule flux and disassembly near the poles. We thank Yokogawa Electric, Universal Imaging. Leica, Hamamatsu Photonics, and Nikon for generous support. EDS was supported in pan by the 1999 Nikon Fellowship. TJM and EDS were supported by NIH grains. Literature Cited 1. Fukui, Y., E. L. de Hostos, and S. Inoue. 1997. Biol. Bull. 193: 224-225. 2. Murray, A. W. 1991. Methods Cell Biol. 36: 581-605. 3. Desai A., P. S. Maddox, T. J. Mitchison, and E. D. Salmon. 1998. J. Cell Biol. 141: 703-713. 4. Waterman-Storer. C. M., and E. D. Salmon. 1998. Biophys. J 75: 2059-2069. 5 Waterman-Storer, C. M.. A. Desai, J. C. Bulinski, and E. D. Salmon. 1998. Ciirr. Biol. 8: 1227-1230. Reference: Biol. Bull. 197: 265-266. (October 1999) Effects of Vanadate on Actin-dependent Vesicle Motility in Extracts of Clam Oocytes Torsten Wollert1, Ana S. DePina, and George M. Liingford (Dartmouth College, Hanover, New Hampshire 03755) The effect of sodium orthovanadate on actin-based vesicle trans- port ( 1 ) was examined in extracts obtained from oocytes of the surf clam Spisula solidissima. Vanadate. an analog of inorganic phos- phate (Pi), inhibits microtubule-based motors at low concentra- tions (5-50 IJ.M) without affecting actin-based motors, and inhibits tyrosine phosphatases selectively at higher concentrations (0.5-1 .0 mM) (2). The higher concentrations of vanadate were used to determine whether myosin-dependent vesicle transport in clam oocyte extracts is regulated by tyrosine phosphatase activity. Clam oocyte extracts were incubated at 18°C for 45 min and then treated with 0.5 p.M rhodamine-phalloidin to fluorescently label aclin filaments, and 0.5 or 1.0 mM vanadate to inhibit tyrosine phosphatases. Vesicle transport on actin filaments was monitored for 60 min by video microscopy, and images of the actin filaments on the coverslip surface were recorded at regular inter- vals by epi-fluorescence microscopy. Vesicle transport on actin filaments was unaffected by vanadate during these experiments. Both motile activity and the velocity of vesicle transport in the control and the treated extracts were the same. However, the actin filament network in the extracts was altered significantly by van- adate treatment. Vanadate reduced actin filament nucleation and stimulated the formation of actin bundles. In the control samples at 15 and 60 min (C in Fig. 1). single actin filaments formed on the coverslip surface and bundles were rarely seen. In the vanadate- 1 University of Rostock, Rostock. Germany. treated samples (V in Fig. 1 ), a 3-D network of bundles formed, and the density of the network increased over time (60 min). The bundles of actin filaments in the vanadate-treated extracts sup- ported bi-directional movement of vesicles. The actin filament bundles in these extracts were disrupted by treatment with Triton X-100 (0.05 and 0.1%). indicating that actin assembly may be dependent on the presence of membranes in the extracts, as ob- served in Xenopii.i oocyte extracts (3, 4). These results suggest that some of the membranes in these extracts have the ability to nucleate actin filament assembly, although vesicle transport was driven by myosin motors. The inhibitory effect of vanadate on tyrosine phosphatases is well established (2, 5), and the actin filament bundle formation observed in these extracts was most likely due to an increase in actin cross-linking activity upon inhibition of tyrosine phospha- tases by vanadate. However, vanadate can influence many differ- ent activities in cells when used at high concentration. At milli- molar concentrations, it can inhibit the myosin motor by forming a stable complex with ADP (6), but under our assay condition, in the presence of ATP, formation of myosin-ADP-Vi is slow, with an inhibition tl/2 of 1.5 h (7). Therefore, the slow formation of the inhibitory complex explains the failure to inhibit myosin-depen- dent vesicle transport in clam oocyte extracts. Vanadate has the potential to inhibit other activities requiring ATP, including Na'/K + ATPase (8), acid and alkaline phosphatases (9. 10). phosphofructokinase (II), and adenylate kinase (12), as well as actin polymerization (13). The complexity of the vanadate effect 266 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS Figure 1. Vanadate-inditced actin filament bundles in clam oocyte extracts: the DIC (top row) and corresponding fluorescent images (bottom row) of extracts that were stained with rhodamine-phalloidin. Actin filaments were present in control samples (C> at the initial time point (15 min) as well as the final time point (60 min). In vanadate (I mM)-treated samples (V), bundles rather than filaments were detected at the 15 and 60 min time points. Concentrated extracts of clam oocytes were clarified, incubated at 1S°C for 45 min, and stained with 0.5 jiM rhodamine-phalloidin which stabilizes actin filaments and enables their detection through fluorescence microscopy. Scale bar, 5 fim. does not allow us to state categorically that the effect we observed is due solely to inhibition of protein tyrosine dephosphorylation. To resolve this potential complication, future experiments are planned to determine the level of tyrosine phosphorylation in the extract when vanadate is present, and to monitor the level of phosphotyrosine in specific actin cross-linking proteins. Literature Cited I DePina, A. S., and G. M. Langford. 1999. Microsc. Res. Tech. (In press). 2. Gilbert-McClain, L. I., A. D. Verin, S. Shi, R. P. Irwin, and J. G. Garcia. 1998. J. Cell. Biochem. 70: 141-15?. 3. Moreau, V., and M. Way. 1998. FEBS Lett. 427: 353-356. 4. Ma, L., L. C. Cantley, P. A. Janmey, and M. W. Kirschner. 1998. J. Cell Biol. 140: 1125-1136. 5. Swarup, G., S. Cohen, and D. L. Garbers. 1982. Biochem. Bio- plivs. Res. Commtin. 107: 1104-1109. 6. Goodno, C. C. 1982. Methods Enzymol. 85: 1 16-123. 7. Goodno, C. C. 1979. Proc. Nail. AcaJ. Sci. U. S. A. 76: 2620-2624. 8. Huang, W. H., and A. Askari. 1984. J. Biol. Chem. 259: 13287- 13291. 9. Vescina, C. M., V. C. Salice, A. M. Cortizo, and S. B. Etcheverry. 1996. Biol. Trace Elcm. «o. 53: 185-191. 10. Seargeant, L. E., and R. A. Stinson. 1979. Biochem. J. 181: 247- 250. I 1 Khoja, S. M., A. O. Abuelgassim, and O. A. al-Bar. 1996. Comp. Biochem. Physiol. C Phurmacol. Toxicol. Endocrinol. 115: 217-221. 1 2 Cremo, C. R., J. A. Loo, C. G. Edmonds, and K. M. Hatlelid. 1992. Biochemistry 31: 491-497. 13 Combcau, C., and M. Carlier. 1988. J. Biol. Chem. 263: 17429- 17436. CELL AND DEVELOPMENTAL BIOLOGY Reference: Bio/. Bull. 197: 267-268. (October 19"M) 267 Effects of a-bungarotoxin on Development of the Sea Urchin Arbacia punctiilata Blase Billack (Rutgers University, Piscataway, New Jersey 08854), Jeffrev D. Laskin1, Michael A. Gallo1 . and Diane E. Heck2 Recent evidence suggests that the expression of acetylcholine receptors is important in regulating growth and development ( 1 ). Normally, as sea urchins develop and progress through the pluteus stage, the postoral arms of the embryo elongate, the stomach develops into a large sack, the esophagus becomes muscular, the mouth opens and the digestive track begins to function (2, 3). During this time, the animal begins to feed, a requirement for further development (4). The purpose of the present work was to evaluate the role of a-bungarotoxin-sensitive acetylcholine receptors in the develop- ment of the sea urchin Arbacia punctiilata. For these studies, we used the fluorescent labeled drug (BODIPY-paclitaxell — which binds polymerized microtubules with high affinity (4) — to visual- ize subtle alterations in the architecture of developing structures within the sea urchin embryo. In untreated embryos, we observed polymerized microtubules appearing in the cilia, the cells lining 1 Environmental and Occupational Health Sciences Institute (EOHSI), UMDNJ-Robert Wood Johnson Medical School, 170 Frelinghuysen Road, Piscataway, New Jersey 08854. "EOHSI. Rutgers University. 170 Frelinghuysen Road, Piscataway, New Jersey 08854. the mouth, esophagus, and gut, and in distinct punctuate regions within the epithelium during development through the pluteus stages (Fig. 1, panel A). Treatment with a-bungarotoxin ( 1 /xA/K a neurotoxin known to bind a subunits of the acetylcholine receptor (5), dramatically altered this pattern of accumulation. Prior to the pluteus stage no teratogenic effects of a-bungarotoxin were evi- dent. We observed that the gross morphology, the developing internal structures that could be visualized using BODIPY-pacli- taxel, and swimming behavior were indistinguishable from un- treated animals. However, using both light and fluorescence mi- croscopy, we observed marked alterations in development from early to late pluteus induced by a-bungarotoxin. These alterations included diminished extension of the postoral arms, lack of dif- ferentiation of the gut and mouth, failure to feed and impaired swimming (Fig. 1 ). In a-bungarotoxin-treated zygotes, the pattern of microtubule accumulation within the cells surrounding the gut, which normally develops during the pluteus stage, was not ob- served. We also used rhodamine-labeled a-bungarotoxin in conjunction with confocal microscopy to study the uptake of this acetylcholine receptor antagonist into zygotes. We found that, prior to the extension of postoral arms and gut enlargement, distinct accumu- Figure 1. Fluorescence localization of polymerized microtubules bound to pacliraxel and acetylcholine receptors in the sea urchin embryo. Fertilized eggs from the sea urchin Arbacia punctula (Marine Biological Laboratories. Woods Hole. Massachusetts) were grown for three da\s in seawater, at room temperature, in the dark and in the presence or absence of I /J.M biotin-a-bungarotoxin (Molecular Probes, Eugene OR). The embryos were then treated with 1.25 ;u,M BODIPY-paclitaxel (panels A and B) and I /J.M rhodamine-labeled a-bungarotoxin (panel A. Molecular Probes, Eugene OR) or I ^g/ml rhodamine strepavldin (Sigma, panel B)for 30 min. The specimens were then fixed in 1% buffered formalin. After 1 h, embryos were washed and examined witli a BIO-RAD confocal imaging system fitted with argon and helium-neon lasers on a Nikon ES800 microscope. Panel A. normal pluteus labeled with BODIPY-paclitaxel and rhodamine-conjugated a-bungarotoxin; panel B. pluteus grown in biotin-a-bungarotoxin and labeled with BODIPY-paclitaxel and rhodamine strepavidin. 268 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS lations of a-bungarotoxin-sensitive acetylcholine receptors could be observed in these areas. Figure 1 (panel A) demonstrates this pattern in an untreated embryo. Rhodamine labeled a-bungaro- toxin binding at the distal ends of the expanding arms and within the developing gut are clearly evident. Panel B in Figure 1 dem- onstrates the altered morphology of an animal grown in biotin-a- bungarotoxin. In this animal the postoral arms are undeveloped and the skeletal structure remains immature. Finally, animals that were incubated with rhodamine strepavidin, which binds biotin-a- bungarotoxin, and observed by fluorescent confocal microscopy revealed small clusters of a-bungarotoxin in distinct areas adjacent to the developing skeletal structures. Taken together these data indicate that a-bungarotoxin disrupts zygote development during the pluteus stages in the sea urchin Arbacia punctulata. We speculate that a-bungarotoxin-sensitive acetylcholine receptors are necessary for the development of pos- toral arms and the gut in this sea urchin. Supported in part by NIH grants ES 03647, ES 05022 and ES 06897 Literature Cited 1. Lauder, J. M., and U. B. Schambra. 1999. Environ. Health Par- sped. 107: S65-S69. 2. Okazaki, K. 1975. Pp. 1 77-232 in The Sea Urchin Embryo. Biochem- istry and Morphogenesis. G. Czihak, ed. Springer- Verlag, New York. 3. Gustafson, T. 1975. Pp. 233-266 in The Sea Urchin Embryo. Bio- chemistry and Morphogenesis, G. Czihak, ed. Springer- Verlag. New York. 4 Bicamumpaka. C., and M. Page. 1998. Int. J. Moi Med. 2: 161- 165. 5. Carr, C., G. D. Fischbach, and J. B. Cohen. 1989. J. Cell. Biol. 109: 1753-64. Reference: Bi,>l. Bull. 197: 268-270. (October 1999) Leukotriene B4 as Calcium Agonist for Nuclear Envelope Breakdown: An Enzymological Survey of Endomembranes of Mitotic Cells Robert B. Silver and Nicole M. Deming (Marine Biological Lahore/ton; Woods Hole, Massachusetts 02543) The requisite calcium signal that precedes nuclear envelope breakdown (NEB) is generated, acts, and is degraded very near to the site of action (1,2). Neither calcium nor an agonist that would trigger release of calcium diffuse over distances greater than 1 micrometer from the site of agonist production and calcium release (2). In addition, this laboratory has shown that: a) leukotriene B4 (LtB4), but no other products of the arachidonic acid (AA) path- way, can evoke calcium release from endomembrane stores in vitro and in vivo in a pattern quite similar to that of the pre-NEB calcium signal (3) in sand dollar (Echinaracnius panna) eggs and mitotic cells (2-9); b) both the pre-NEB calcium signal and NEB are blocked by inhibitors of LtB4 synthesis (8, 9); c) phospholipase A, (PLA,) is present on calcium regulatory endomembranes of prophase mitotic apparatus (MA) and is selectively concentrated in the MA relative to the whole cell (9, 10); and d) phospholipase C activity, and thus production of 1 ,4,5-inositol trisphosphate, is absent from prophase MA (8-10). These four findings led Silver to hypothesize that LtB4. and not IP3, is the calcium agonist that produces the space-time patterned calcium QEDs essential for NEB (3, 8, and 9). '.eukotriene B4 is produced by a well-established mechanism (II) Phospholipase A2 acts on phospholipids to yield AA and monoai. ylglycerols. In turn. AA is converted to LtB4 through the 5'-lip<'\ygenase (5-LO) branch of the AA pathway. Leukotriene A4 (LtA4i. the precursor of LtB4, can be converted by glutathione S-transferasr (GST) to leukotriene C4 (LtC4) by the conjugation of reduced glutathione (GSH) to LtA4. Oxidized glutathione is re- duced to GSH by gluiaihione reductase through a reaction in which NADPH serves as the proton donor. The primary source for NADPH in animal and plant cells is the oxidative branch of the pentose phosphate pathway, specifically through the actions of glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphoglu- conate dehydrogenase (6PGD). We now report that: a) LtB4 is a stereospecitic agonist of endomembrane calcium release in vivo; b) observed PLA2 activity is "calcium-independent"; c) kinetic analyses show that the prophase MA endomembrane GST activity represents two distinct microsomal GST enzymes; and d) enzymes of the oxidative branch of the pentose phosphate pathway are present on prophase MA membranes. Our studies were conducted with cells from the first and second cell cycles, eggs, and isolated native prophase MA, all from the sand dollar (Echinaracnius panna) (e.g., 1, 2, 4, 12, 13). Eggs were obtained from mature females as previously described (1,2). Endomembranes were subfractionated as previously described (4, 12). Native prophase MA were isolated according to our standard methods (4, 12). Spectrophotometric assays for enzyme activities developed for these studies are adaptations of published methods for PLA, (14), PLC (15) GST (16, 17), GSR (18), creatine kinase (19). G6PD (17), lactonase (9), 6PGDU7). and pentose phosphate isomerase (9). Standard curves for identification of enzyme prod- uct were generated for each experiment at log/2 and linear/2 concentration steps (R2 typically > 0.995). Kinetic analyses were performed with GraFit™, version 4 (Erathicus Software). Protein concentration was measured with the biuret assay, where standard curves had an R2 of > 0.9985 across at least three orders of magnitude of protein concentration (e.g.. 12, 13). Triplicate assays were performed for each membrane subtraction. Specific and total CELL AND DEVELOPMENTAL BIOLOGY Table I Glutathione S-iransferase activity of calcium regulatory endomembranes 269 Km S.E. Vmax S.E. Enzyme Activity Source (mA/GSH) (±) ( mM sec ~ ' mg protein ~ ' ) (±) Total microsomal GST activity Calcium regulatory endomembranes 0.1518 0.0307 0.2246 0.0149 Microsomal GST-a activity Parsed from above data 0.0097 0.0015 0.0814 0.0052 Microsomal GST-0 activity Parsed from above data 0.2707 0.0528 0.2824 0.0188 Microsomal GST-a activity Reduced glutathione affinity column 0.0092 0.0020 0.0981 0.0052 Microsomal GST-0 activity Reduced glutathione affinity column 0.2171 0.0947 0.2335 0.0307 activities for the enzyme activities of each subtraction were deter- mined, and the values compared. Quantitative direct-pressure mi- croinjection studies were performed as previously described (1,2, 13. 20). This laboratory has demonstrated that LtB4 evokes release of calcium from MA-associated endomembranes in vivo and in vitro (3, 7-9). Intracellular microinjection of 2 — 10 picoliters of LtB4 ( 10~J to 10~K A/) evokes release of calcium from endomembranes in intact eggs and prophase cells (3, 8, 9). In contrast, microinjec- tion of similar doses of structural homologues of LtB4 such as LtA4 (3, 8. 9) LtC4 (3, 8, 9), 6-trans-UB4. or 5|S],15[S]-DiHETE do not evoke calcium release from intracellular stores. This is the first report of a stereospecific-dependence of LtB4-evoked calcium release from endomembranes in echinoderms or from mitotic cells. Thus, among the lipoxins, LtB4 is a stereospecific agonist of MA endomembrane calcium release in vivo. The PLA2 superfamily is composed of four PLA2 families, two secretory (Types I and II), and two cytosolic (Types III (calcium- dependent) and IV (calcium-independent)) (21). The activities of these enzymes have distinguishing requirements for calcium con- centration. Types I, II and III PLA-, require 5. 3 and 0.08 mM CaCK, respectively (22). Type IV PLA, requires no calcium ions for its activity and is thus considered to be calcium independent. The PLA2 activity measured as a component of the calcium regulatory endomembranes of isolated prophase MA, using meth- ods previously described (9, 10, 25), does not require addition of calcium to the reaction milieu. This is consistent with the observed MA associated PLA2 being a Type IV calcium-independent PLA2. Kinetic analyses of the GST activity in calcium regulatory endomembranes from prophase MA. measured across a substrate concentration range of 0.001 to 1.0 mM GSH. revealed values for Km and Vmax of 0.1518 mM GSH and 0.2246 mM sec"1 mg protein"1, respectively (Table 1). Lineweaver-Burke plots of the results indicated that the overall observed activity comprises two components. Parsing the data revealed two distinct microsomal GST enzyme activities designated GST-a and GST-/3 whose val- ues for Km and Vmax differ by 27.9-fold and 3.5-fold, respec- tively (Table I). Both GST-a and GST-/3 bind to immobilized GSH and, consistent with their kinetic properties, elute at 0.3 and 0.01 mM GSH, respectively. The kinetic parameters of the affinity purified GST-a and GST-/3 are consistent with values parsed from the crude microsomal GST activity (Table I). Based upon kinetic properties comparable to published values for mammalian hepatic microsomal GST (e.g., 23) and LtC4 synthase (e.g.. 24), relative associations with S-hexyl-glutathione, and their microsomal ori- gin, GST-a appears to be a LtC4 synthase, while GSH-/3 appears to be a microsomal GST. This is the first demonstration of such activities in echinoderms, mitotic apparatuses, or mitotic cells. In animal and plant cells, two dehydrogenases of the pentose phosphate pathway, specifically G6PD and 6PGD, serve as the catalytic source for NADPH. NADPH is necessary for GSR to produce GSH from oxidized glutathione. Reduced glutathione is a substrate for GST in the production of LtC4 and "deactivation" of reactive oxygen intermediates. Present experiments show that GSR and active enzymes of the oxidative branch of the pentose phos- phate pathway are present on prophase MA membranes. Specifi- cally, we have identified GSR, G6PD, lactonase, 6PGD, and pen- tose phosphate isomerase as constitutive enzymes of the calcium regulatory endomembranes proximal to the nucleus during prophase. Thus, we propose that in the presence of glucose-6- phosphate and NADP4 . the calcium regulatory endomembranes of prophase MA can produce NADPH essential for GSR-mediated reduction of oxidized glutathione. Reduced glutathione is, in turn, necessary and available for the observed microsomal GST to produce either LtC4 or glutathione-derivatized reactive oxygen intermediates. The presence of the pentose phosphate pathway enzymes also suggests a site for biosynthesis of purines, pyrimi- dines and amino acids essential for the subsequent cell cycle (e.g.. 8. 9). These results, together with earlier findings from this laboratory (e.g., space-time patterns of pre-NEB calcium signals (e.g., 1. 2). LtB4 as an agonist of pre-NEB calcium signals (3, 8, 9, 25), distribution of PLA2 and PLC activities (7-10)). reveal a network of enzymes within prophase MA that is available for regulated production of LtB4 as an agonist of the space-time patterned pre-NEB calcium signal (2, 8, 9). This is the first report of stereospecificity for LtB4 evocation of calcium release from MA endomembrane stores, of two distinguishable forms of microsomal GST (tentatively identified as microsomal GST and leukotriene C4 synthase), and of a calcium-independent PLA2 in echinoderms, mitotic apparatus, or mitotic cells. These findings support Silver's hypothesis of an intracellular control network in which LtB4 is the agonist for the space-time patterned pre-NEB calcium signal and cell ac- tivation (2, 8, and 9). Research grant support by NSF (MCB-99082680) is gratefully acknowledged. The authors are grateful to the reviewers and editor 270 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS for their many helpful and well considered suggestions made in final preparation of this manuscript. the Literature Cited 1 Silver, R. B. 1989. Dev. Biol. 131: 11-26. 2. Silver, R. B. 1996. Cell Calcium 20: 161-179. 3. Silver, R. B. 1995. Biol. Bull. 189: 203-204. 4 Silver, R. B., R. D. Cole, and W. Z. Cande. 1980. Cell 19: 505-516. 5. Bansal, V. S., and P. W. Majerus. 1990. Aniui. Rev. Cell Biol. 6: 41-67. 6. Chow, S. C., and M. Jondel. 1990. / Biol. Chem. 265: 902-907. 7. Silver, R. B., D. E. Strongin, L. R. Hurwitz, and A. P. Reeves. 1997. Biol. Bull. 193: 236-237. 8. Silver, R. B. 1999. FASEB J. (in press). 9. Silver. R. B. 1999. Science (accepted). 10. Silver, R. B., L. A. King, and A. F. Wise. 1998. Biol. Bull. 195: 209-210. 11. Samuelsson, B. 1983. Science 220: 568-575 12. Silver, R. B. 1986. Methods En;vmol. 134: 200-217. 13. Silver, R. B. 1986. Proc. Null. Acad. Sci. U.S.A. 83: 4302-4306. 14 Reynolds, L. J., L. L. Hughes, L. Vu, and E. A. Dennis. 1994. Anal. Biochem. 217: 25-32. 15. Kurioka, S., and M. Matsuda. 1976. Anal. Biochem. 75: 281-289. 16. Keen, J. H., W. H. Habig, and W. B. Jakoby. 1976. J. Biol. Chem. 251: 6183-6188. 1 7. Pabst, M. J., W. H. Habig, and W. B. Jakoby. 1976. J. Biol. Chem. 249: 7140-7147. IS. Racker, E. 1955. ./. Biol. Chem. 190: 855-865. 19. Silver, R. B., M. S. Saft, A. R. Taylor, and R. I). Cole. 1983. J. Biol. Chem. 258: 13287-13291. 20. Silver, R. B. 1997. Pp 83. 1 -20 in Cellx: A Laboratory Manual. D L. Specter, R. D. Goldman, and L. Leinwand. eds. CSHL Press. 21. Dennis, E. A. 1995. J. Biol. Chem. 269: 13057-13060. 22. Leslie, C. C. 1977. J. Biol. Chem. 272: 16709-16712. 23 Gupta, N., M. J. Gresser, and A. W. Ford-Hutchinson. 1998. Bioehim. Biophys. Ada 139: 157-168. 24 Oesch, F., and C. R. Wolf. 1989. Biochem. Pharmacol. 38: 353- 359. 25. Silver, R. B., J. B. Oblak, G. S. Jeun, J. Sung, and T. Dutta. 1994. Biol. Bull. 187: 242-244. Reference: Biol. Bull. 197: 270-271. (October 1999) Extracellular Survival of an Intracellular Parasite (Spraguea lophii, Microsporea ) Earl Weidner and Ann Findley (Biology, Louisiana State University, Baton Rouge, Louisiana) Microsporeans are intracellular parasites; they are located di- rectly in host cell cytoplasm with only a plasma membrane as an interface ( 1 ). Microsporeans have an infective spore stage that discharges the sporoplasm from a long, fine tube. The spore has but one plasma membrane which is left behind within the spore ghost during discharge. The extruded sporoplasm is surrounded by a membrane, but this structure is derived from the extrusion apparatus within the spore. Since microsporeans have not been cultured or maintained extracellularly for more than short periods, it became an objective of this study to: (a) develop a simple protocol for isolating pure populations of discharged sporoplasms; and (h) develop a procedure for maintaining pure populations of extruded microsporean sporoplasms in culture for 24 h for further biochemical investigations. To initiate spore discharge, spores of Spni^iifa lophii were incubated in 0. 1 M HEPES buffer at pH 7.0 (with 50 nM Ca4 + ) for 1 h. Subsequently. 10-100 /nl of spore suspension were transferred into a thin pool on a glass coverslip. These spores were triggered to discharge by the addition of 1-2 /j.1 of filtered (0.45 ;um pore size) mammalian or fish mucus onto the spore film, followed a few seconds later by the addition of 1-5 /*] of 0. 1 M HEPES buffer (pH 10). After several minutes, most of the spores had discharged sporoplasms that were attached to the cover glass surface. The mi: < I and discharged spore ghosts were removed from the sur- face by rapid washes with 0.1% concanavalin A (Con A) made up in Hh. (pH 7.0). The sporoplasms were transferred to a Me- dium I'- -idied with 5 mM ATP pH 7.2 ( 1 ). The supi medium that was tested at first included vitamins A ' Biology, Northea-K.-in Louisiana University, Monroe, Louisiana. and C ( 1 nM), i.-phosphatidycholine (0. 1 mg/ml), glucose (0.01%). 5% bovine serum albumen, 5% fetal calf serum, cofactors NAD and Co-A ( 1 nM) and 5 /uM concentrations of ATP and GTP. The cells were maintained at 15°C and 20°C. Although the sporo- plasms showed some stability in this medium, the cells lost much Figure 1. Spraguea lophii sporoplasms after 24 h in Medium 199 with ATP supplement and 10% Xenopus oocyte cytosol. (At Sporoplasms (ar- row} t'rei/uentl\ fuse or attach to one another. (B} Sporoplasms farrows) a/so attach or fuse with other elements in medium. Bar scale represents 4 }.un. CELL AND DEVELOPMENTAL BIOLOGY 271 of their cytosol and frequently fused during the first 6 h of incubation. When the support medium was suspended onto a 2c/c-5c/c gelatin matrix, the cells incubated within it developed a vacuolated cytoplasm. However, the sporoplastns appeared to have a more robust stability when they were added to Medium 199 made up in 0.15 M potassium phosphate buffer with 5 mM ATP. and were supplemented with 10%-30% Xenopus oocyte cytosol (Fig. 1). After 12-24 h, these sporoplasms retained cytoplasm and did not vacuolate. although the sporoplasms appeared to still attach to each other or fuse. There was no evidence of nuclear division during 24 h of incubations. Microsporean sporoplasms were clearly stabilized in Medium 199 (pH 7.1-7.2) with ATP (5 mM) on a 2% gelatin substrate onto which was added 0.01-0.02 mM cholesterol with no Xenopus cytosol. There was no evidence of vacuolation or nuclear division. However, after 12 h. the sporoplasms remained segregated and retained their cytoplasmic matrix. This significant positive effect of cholesterol addition to the medium indicates that the sporoplasm outer envelope may be devoid of cholesterol. Insertion of choles- terol into plasma membrane is an essential component of eu- karyote plasma membranes; it affects membrane fluidity and re- duces the permeability of membranes (2). Because newly extruded sporoplasms acquire an outer membrane that is believed to be derived from the extrusion apparatus (and is not the original plasma membrane of the spore), we expect that this second-hand membrane may lack a cholesterol component. This may account for the leaky condition of discharged sporoplasms when they are first entering into extracellular environs. Other primitive cells, such as mycoplasmas. also have an outer membrane that requires an external source of cholesterol from the outside environs for any level of stability. Once the cholesterol is acquired, these cells begin to regulate their internal milieu. Literature Cited Weidner, E., A. Findley, V. Dolgikh, and J. Sokolova. 1999. Pp. 172-195 in The Microsporidia and Microsporidiosis. American Society of Microbiology, Washington, D.C. Dahl, J. 1993. Pp. 167-188 in Subcellular Biochemistry: Vol. 20. Plenum, New York. Reference: Biol. Bull. 197: 271-273. (October 1999) Intense Concanavalin A Staining and Apoptosis of Peripheral Flagellated Cells in Larvae of the Marine Sponge Microciona prolifera: Significance in Relation to Morphogenesis Jane C. Kaltenbach ' , William J. Kulins2, Tracy L. Simpson3, and Max M. Burger4 (Marine Biological Laboratory, Woods Hole, Massachusetts 02543) Free-swimming larvae are released from adult Microciona sponges during a brief period in late June and early July. The larvae are covered by a layer of flagellated epithelial cells, which disappear within 24 h, at about the time of larval settlement, to a substrate such as rocks, shells, etc. (1, 2). The fate of the flagellated cells has long been discussed. As early as 1892, the inversion of these cells to form choanocytes was proposed for some species of sponge (3). However, more recent evidence (e.g.. electron micros- copy and autoradiography) indicates that, in certain species includ- ing Microciona prolifera, flagellated cells do not differentiate into other cell types but. near the time of settlement, are engulfed by large phagocytic cells presumed to be archaeocytes (4. 5). The present study addresses the fate of peripheral flagellated cells in Microciona larvae with methods other than those used in previous reports. We used lectin-based histochemical staining of surface sugars, as well as terminal LIDP, nick-end labeling, com- monly known as the TUNEL assay, and DNA gel electrophoresis, to define apoptosis. Lectins. which have binding sites for specific sugars, can be 1 Mount Holyoke College, South Hadley. Massachusetts. : Hospital for Sick Children, Toronto, Canada. 3 University of Hartford, Hartford, Connecticut. 4 Friedrich Miescher Institute. Basel. Switzerland. conjugated to markers, such as horseradish peroxidase (HRP), and used as probes to localize sites of terminal sugar residues of the glycans of membrane glycoproteins (6). To this end, larvae were fixed in 10% formalin, embedded in paraffin, and sectioned (5 /j,m). The sections were treated with H,O, to block endogenous peroxidase, and with bovine serum albumin to block non-specific staining. Sections were then incubated with HRP lectins (Table I). A brown color was developed with 3,3'diaminobenzidine (DAB)- H-.OT to indicate sites of specific sugars in the larvae. Control Table 1 Lectins and their specific affinities Lectin Sugar Concanavalin A (Con A) Wheat Germ Agglutinin (WGA) Soybean Agglutinin (SBA) and Dolichos hi floras Agglutinin (DBA) Peanut Agglutinin (PNA| Ulex europucii.t Agglutinin (UEA-1) a-Mannose N-Acetyl-Glucosamine (GlcNAc) N-Acetyl-Galactosamme (GalNAc) /3-Galactose a-L-Fucose 272 A REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS B D Figure 1. Sections of Microciona prolifera lamie: A. B: Peripheral flagellated epithelium stained intensely with eon .4 for mannose (arrows). Some large inner cells also gave a positive con A stain. Scale bars. (A): 20 fj.ni. (B) 60 /jjn. C: Peripheral epithelium stained lightly with WGA for GlcNAc. Scale bar. 60 /urn. D: Peripheral flagellated epithelium containing darkly stained apoptotic cell.', or cell fragments (TUNEL assay). Lighter background staining is due to hematoxylin counterstain. Two large phagocytic cells (arrows) have light cell membranes and contain darkly stained apoptotic cells or cell fragments within the cytoplasm I dark brown with TUNEL). sections were treated with lectin solutions containing high concen- trations of specific sugars. The TUNEL assay for apoptosis involves labeling the strand breaks of disrupted DNA (7). Sections of larvae were treated successively with proteinase K and endogenous peroxidase block- ing solution, and were then permeabilized with 0.1% Triton-X- 100. Non-specific reactive sites were also blocked. Sections were treated with TUNEL mixture followed by sheep antifluorescein- HRP conjugate. A brown color, developed by DAB-FLO,, showed sites of apoptosis. The sections were counterstained with hema- toxylin. For controls, the TUNEL mixture was omitted from the procedure. The occurrence of apoptosis was confirmed by DNA gel elec- trophoresis. Samples of DNA (3 jug) extracted from sponge cells (8) were loaded into wells prepared on 1% agarose gel and were separated electrophoretically. Standards consisted of Hind 111 (high molecular weight) DNA ladder and 100-bp low molecular weight (LMW) DNA ladder. The gel was stained with ethidium bromide and washed in several changes of water to remove excess stain. Peripheral flagellated cells showed an intense brown stain with the con A probe for mannose (Fig. IA.B). but only a weak stain with GlcNAc (Fig. 1C), and no stain at all with the other lectins tested (SBA, DBA, PNA, UEA-1 ) (not shown). This suggests that mannose was the only terminal lectin-binding sugar residue on the surface of the flagellated cells. The diagram of a high mannose N-glycan shows the two terminal mannose residues (arrows) that are able to bind to the lectin con A or to similar structures (receptors) on the surface of phagocytes. The diagram also illus- trates our notion that the weak WGA staining reflects GlcNAc residues that are covered by mannose. The negative results by other lectins indicate that the corresponding sugars were non- terminal or not present. Man* Man' Man Man— GlcNAc— GlcNAc— Asn (Glc),Man — Man — Man ' The brown staining of fragmented DNA produced by the TUNEL assay indicated that apoptosis was only located in the CELL AND DEVELOPMENTAL BIOLOGY 273 peripheral region of flagellated cells. However, brown staining was sometimes seen within large cells adjacent to the peripheral region (Fig. ID), suggesting that apoptotic cells or fragments had been engulfed by large phagocytic cells (probably archaeocytesl. Comparison of sponge DNA extracts with the standards showed a ladder of LMW DNA fragments (not shown), which is charac- teristic of apoptotic cells (indicated in larvae by the TUNEL assay). In summary, the results demonstrate that, in Microciona larvae, mannose is the only terminal, lectin-binding sugar on the surface of the flagellated cells as shown by con A staining. The cells undergo apoptosis and engulfment, indicated by dark staining of fragmented DNA (TUNEL assay). We suggest that mannose receptors are present on the surface of phagocytes in the larvae (as shown in a variety of other organisms) (9) and that such receptors recognize and bind with mannose on apoptotic flagellated cells. A mannose-mannose receptor reaction may be required for ingestion of the apoptotic cells (9, 10). Membrane surface changes are characteristic in cell development and cell death. The changes may include turnover and external- ization of high mannose N-glycans as a terminal expression in apoptotic cells and thus provide a unique method for their disposal by phagocytes (10). Other sugars that are associated with terminal differentiation and apoptosis include those that specify histo-blood group H (L-fucose) (11). Our study supports the theory that peripheral flagellated cells in Microciona larvae are terminally differentiated; their fate is apoptosis and eventually phagocytosis. Literature Cited 1- Bergquist, I". R. 1978. Sponges. University of California, Berkeley. 2. Simpson. T. L. 1984. Tin- Cell Biology of Sponges. Springer- Verlag. New York. 3. Delage, Y. 1892. Arch. Zool. Exp. Gen. 2° serie: 345-498. 4. Bergquist, P. R., and K. Glasgow. 1986. Exp. Biol. 45: 1 1 1-122. 5. Misevic, G. N., V. Schulep, and M. M. Burger. 1990. Pp. 182-187 in New Perspectives in Cell Biology, K. Riltzler. ed. Smithsonian Press, Washington, DC. 6. Faszewski, E. E., and J. C. Kaltenbach. 1995. Cell Tissue Res. 281: 169-177. 7. Kuhns. W. J., M. Ho, M. M. Burger, and R. Smolowitz. 1997. Biol. Bull. 193: 239-241. 8. Gavriela, Y., Y. Sherman, and S. A. Ben-Sasson. 1992. / Cell Biol. 119: 493-501. 9. Drickamer, K.. and M. E. Taylor. 1993. Aimu. Rev. Cell Biol. 9: 237-264. 10. Platt, N.. R. P. da Silva, and S. Gordon. 1998. Trends Cell Biol. 8: 365-372. 11. Kuhns, W. J., and C. Pann. 1972. Nat. New Biol. 240: 22-24 274 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS Reference: Biol. Bull. 197: 274-275. (October 1999) A Cuticular Secretion of the Horseshoe Crab, Limiting polyphemus: A Potential Anti-fouling Agent John M. Harrington (Biological Sciences Department, University of South Alabama, Mobile, AL 36688) and Peter B. Armstrong^ Solid surfaces placed in the ocean are targets for colonization by all manner of sessile fouling organisms. Although the carapace of the horseshoe crab presents just such an opportunity for coloniza- tion, it is usually surprisingly clean of macroscopic colonizers. The dorsal surfaces of the cephalothorax of 8 of 16 randomly chosen animals from the Marine Resources Center at the Marine Biolog- ical Laboratory were completely free of macroscopic fouling or- ganisms and in a second sample of 10 randomly chosen animals. 6.09% of the dorsal surface of the cephalothorax was occupied by macroscopic colonizers. In these samples, the commonest fouling organisms were members of the genera Crepidula (a sessile gas- tropod) and Balanns (barnacles). Some common sessile organisms, such as bryozoans, tunicates. and green algae, were not present on these animals. We have identified a viscous secretion (dermal exudate) re- leased onto the entire dorsal and ventral surfaces of the carapace of the American horseshoe crab that may have anti-fouling activity. Only a small fraction of the animals freshly collected from Chatham. Massachusetts, and brought into the Marine Resources Center show detectable traces of dermal exudate. but we found that the secretion is produced when the animal is exposed to waters containing concentrated decaying animal material. We had modest success in eliciting exudate secretion by challenging animals with dense mixed cultures of marine bacteria grown up on marine broth. A more robust and a prolonged response was achieved when the horseshoe crabs were housed in a live car in Eel Pond in the company of decaying fish parts (the head, viscera, and skeleton that remains after filleting striped bass). The dorsal surface of the cephalothorax of the pollution-challenged animal was harvested with a rubber scraper and Pasteur pipette, yielding 0.5-1 ml of dermal exudate. This material was stored at — 20°C. or with NaN,, to prevent microbial growth. The animals reconstituted the layer of secreted exudate within 2 h after its removal. The exudate is probably the product of the hypodermal glands, whose ducts pass through the dermis and terminate at the surface of the cuticle ( 1 ). This secretion is different from the material released from animals injected with lipopolysaccharide (2), which we now believe is a secretion of the gastrointestinal tract. The protein content of dermal exudate is low. approximately 20 /ng/ml. as determined by the bicinchoninic acid assay (Pierce Cat. #23225). The activities of the dermal exudate included hemolytic, hem- agglutinating, and bacteriolytic activities. Hemolysis was assayed according to standard procedures (3) with sheep red cells in a buffer containing 0.25 MNaCl, 10 mA/CaCU. 0.14 M dextrose, 10 1 Molecular and Cellular Biology, University of California, Davis, Cal- ifornia 95616, . Table I Hemolysis of sheep red cells hy dermal exudute Hemolytic agent Hemolysis (%) 10 nA/ Limulin 1:16 Dermal exudate, preparation #1 16 Dermal exudate. preparation #2 :16 Dermal exudate, preparation #3 27.5 ± 11.3 38.5 ± 7.4 13.3 ± 2.2 36.4 ±1.3 mM Tris, pH 7.3. The positive control was limulin, a sialic acid- binding lectin that is responsible for the hemolytic activity of the plasma of the horseshoe crab (4). A robust hemolytic activity was caused by a 1:16 dilution of dermal exudate (Table I). Hemagglu- tination of sheep red cells was found at a 1:8 dilution when tested in a buffer containing 0.15 M NaCl. 10 mM CaCK. 50 mM Tris, pH 7.3. Bacteriocidal activity was assayed on confluent lawns of the marine Gram-negative bacterium. Vibrio alginoh'ticus. grown on nutrient agar. Aliquoits (5 /nl) of dermal extract (azide-free) were placed at equally spaced intervals on the surface of the culture shortly after bacterial plating, and the culture was checked periodically for zones of clearance in the bacterial lawn, at sites of deposited exudate. Controls were 5 p.\ deposits of sterile 3% NaCl. Bacteriolysis was observed only in one of four separate collections of dermal exudate, so this may be a variable property of the exudate. We propose that the dermal exudate is a barrier to coloniza- tion of the cuticle by fouling organisms. Two distinct mecha- nisms probably collaborate in this function. The exudate, which is continuously secreted onto the surface of the exoskeleton, would present a mechanical barrier to contact with the solid surface of the cuticle. In addition, the exudate has antibiological properties that may kill or disable potential colonizers. Produc- tion of the exudate is stimulated by polluted water. The dermal exudate is not a perfect barrier. Green algae and cyanobacteria that succeed in establishing colonies on the cuticle slowly destroy the exoskeleton, which can prove fatal for adult horse- shoe crabs (5). Thus, the prevention of this condition clearly has important health benefits. Supported by Grant No. MCB-97-26771 from the National Science Foundation. We thank Dr. Chhanda Biswas for help with the bacteriolytic assay and Mr. Edward Enos and Dr. Norman Wainwright for suggestions on methods to elicit secretion of the dermal exudate. Literature Cited 1 . Stagner, J. I., and .1. R. Redmond. 1975. Mar. Fish. Ke\: 37: 11-19. 2. Quigley, J. P., G. Corcoran, and P. B. Armstrong. 1997. Riot. Riill. 193: 273 COMPARATIVE BIOCHEMISTRY 275 3. Armstrong, P. B., R. Melchior. S. Swarnakar, and J. P. Quisle). 1998. M,,l. Imiminol. 35: 47-53. 4 Armstrong, P. B., S. Swarnakar, S. Srinial. S. Misquith, K. A. Hahn, R. T. Aimes, and J. P. Quij-ley. 1996. J. Rial. Chem. 271: 14717- 14721. 5. Liebovitz. I... and G. A. Lewbart. 1987. Biol. Bull. 173: 430. Reference: Biol. Bull. 197: 275-276. (October Cellular Mechanisms of Hemolysis by the Protein Limulin, a Sialic-Acid-Specific Lectin From the Plasma of the American Horseshoe Crab, Limulus polyphemus Rengasamy Asokan1 and Peter B. Armstrong (Department of Molecular ami Cellular Biology, University of California, Davis, California 95616) The cytolytic destruction of foreign cells by proteins of the plasma is an important immune defense strategy of higher animals. In the American horseshoe crab. Limulus />i>l\nln'imi.\, the plasma- based cytolytic system is mediated by a single protein, the sialic acid-binding lectin. limulin ( 1 ). Limulin is a member of the pen- traxin protein family and is present in the plasma of the horseshoe crab at 30-50 nM (2). In assays using sheep red cells as the model foreign cell, the entire hemolytic activity of plasma is the province of limulin (1). Hemolysis depends on the sialic acid-binding ac- tivity of limulin. because sialylated glycoconjugates, such as fetuin and the sialic acids N-acetyl neuraminic acid and colominic acid, inhibit hemolysis, and desialylation of the target cells renders them immune to cytolysis ( 1 ). Limulin was purified from Limiilu.i plasma by sequential affinity chromatography on phosphorylethanolamine-agarose to isolate the pentraxins followed by chromatography on fetuin- Sepharose to isolate limulin. the sole pentraxin with sialic acid-binding capability. The cytolytic activity of Linnilii.\ plasma proteins was assayed with sheep red blood cells as described previously (3). The hemolytic action of purified limulin is sensitive to the ionic environment and shows a broad activity maximum at NaCl con- centrations between 0.2 and 0.35 M. These salt concentrations would be non-physiological for an immune effector from a verte- brate, but the blood of the horseshoe crab has the ionic composi- tion of seawater. 0.5 M NaCl. The curve showing the Ca + ~ dependence for hemolysis is sigmoidal, with a surprisingly sharp rise from zero activity at 0.65 mM to maximal activity at 0.85 mA/. This may reflect the Ca+~ dependency of binding of limulin to the red cell, because hemagglutination shows a similar dose-depen- dence on the concentration of Ca+2. Under optimized conditions, hemolysis is dependent upon the concentration of limulin between 2-8 nM. The macromolecular osmolites dextran-8 (Mr 8-12 kDa) and. to a lesser extent, dextran-4 (Mr 4-6 kDa) block hemolysis (Table I). This result suggests that limulin inserts into the plasma membrane to generate hydrophilic channels that allow water to flow into the cell in response to the high concentration 1 Molecular and Cellular Biology. University of California. One Shields Avenue. Davis, California 95616, . of internal macromolecular osmolites, principally the protein hemoglobin. Protection is imagined to result from the estab- lishment of a concentration of osmolites larger than the channel pore size in the external milieu that is equal to the concentration of hemoglobin in the cell so that the dextrans external to the cell exert an osmotic pressure equal to that of the hemoglobin within the cell and the cell is thus protected from osmotic rupture (4). Protection is partially reversible because red cells treated with limulin plus dextran show partial lysis when washed into limu- lin- and dextran-free buffer (data not shown). Dextran-8 fails to block hemagglutination at low Ca + 2 concentrations (data not shown) and exerts the same protective effect against hemolysis at standard (0.85 mM) and high (10 mM) Ca+2 concentrations (Table I), so dextran's activity does not involve Ca + 2 seques- tration. The molecular size of dextran-4 is approximately 1.7 nm (5). indicating an effective pore size for membrane-associ- ated limulin that is smaller than this. One of the indicators of microbial intrusion into higher animals is the plasma protein os-macroglobulm, which is activated by proteases of the intruding microbes. Furthermore, os-macroglob- ulin can be activated by small primary amines, such as methyl- amine, as well as by the reaction with proteases. In the context of this study, activated forms of Limulus os-macroglobulin inhibit the hemolytic activity of purified limulin (3). Only the activated form Table 1 Inhibition of limulin-mediated hcino/\\ix l>y high molecular mass osmolites Osmolite' Hemolysis (%)2 None 30 mM Sucrose 30 mM Melezitose 30 mM Inulin 30 mM Dextran-4 30 mM DexiM 30 mM Dextran-S + 10 mM CaCI, 47.4 ± 2.3 36.7 ± 1.6 30.2 ± 2.3 17.9 ± 1.6 13.7 ± 2.1 2.7 ± 0.9 1.5 ± 1.0 1 All sampl.". contained 7 nA/ limulin in standard hemolysis buffer. 0.25 M NaCl. 0.85 mM CaCI,. 10 mM Tns. pH 7.3. 2 Mean of 3 determinations. ± standard error of the mean. 276 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS of a,-macroglobulin inhibits limulin-mediated cytolysis; native, unreacted a2-macroglobulin has no effect (3). Treatment of methylamine-reacted Limulus a2-macroglohulin with the thiol alkylating agent iodoacetamide abolished the ability of activated os-macroglobulin to inhibit hemolysis. indicating an important role for free thiols in inhibition. Activation of a2- macroglobulin involves cleavage of an internal thiol ester bond which, in Limulus os-macroglobulin. links Cys 999 with Glx 1002. Thiol ester cleavage generates a new free thiol at Cys 999 (6). This is the only free thiol of the protein (7. 8). and. based on the effects of thiol alkylation. appears to be important for the ability of thiol ester-reacted a-,-macroglobulin to inhibit limulin-mediated hemo- lysis. Cytolysis of foreign cells is an important immune defense strategy for a broad array of animals. In Limulus, foreign cell cytolysis is produced by the plasma protein, limulin. Limulin appears to exert this action by inserting into the plasma membrane of the targeted foreign cell to establish a hydrophilic channel that compromises the integrity of the cell membrane. This cytolytic activity is modulated by a second plasma protein, a;-macroglob- ulin, and that activity appears to depend on the generation of a new free thiol group when a,-macroglobulin is activated by its reaction with proteases. This research was supported by NSF Grant No. MCB 2677 1 . Literature Cited I Armstrong, P. B., S. Suarnakar, S. Srimal, S. Misquith, E. A. Hahn, R. T. Aimes, and J. P. Quigley. 1996. J. Biol. Chem. 271: 14717- 14721. 2. Swarnakar, S., R. Melchior, J. P. Quigley, and P. B. Armstrong. 1995. Bn>l. Bull. 189: 226-227. 3. Armstrong, P. B., R. Melchior, S. Swarnakar, and J. P. Quigley. 1998. M,>l. liwinino/. 35: 47-53. 4 Hatakeyama, T., H. Nagatomo and N. Yamasaki. 1995. J. Bint. Chan. 270: 3560-3564. 5 Scherrer, R. and P. Gerhardt. 1971. J. Bacteriol. 107: 718 735 6 Ikawi, D., S.-I. Kawabata, V. Miura. A. Kato, P. B. Armstrong, J. P. Quigley, K. L. Nielsen, K. Dolmer, and S. Iwanaga. 1996. Eur. J. Binchem. 242: 822-831. 7. Armstrong, P. B. and J. P. Quigley. 1987. Biochetn. J. 248: 703- 707. X Dolmer, K., L. B. Husted, P. B. Armstrong, and L. Sottrup-Jensen. 1996. FEBS Lett 393: 37-40. Reference: «/«/. Bull. 197: 276-277. (October 1999) Identification of a Hemolytic Activity in the Plasma of the Gastropod Busycon canaliculatuni Chhanda Biswas (Inununobiology Division, Indian Institute of Chemical Biolog\, Calcutta 700 032, India), and Peter B. Armstrong1 The immune protection of higher animals from parasitic inva- sion depends on the activities of immune effector proteins in the blood. These agents must discriminate between host tissues and the surfaces of foreign cells and direct their actions only against the latter. The most effective strategy to deal with pathogenic invasion is the immediate cytolytic destruction of the pathogen. In several well-characterized examples, the agents of cytolytic destruction of invading pathogens are present in the blood, either as soluble molecules of the plasma or as molecules secreted from blood cells. In mammals, the principal agent of foreign cell cytolysis is the complement system, which consists of several dozen proteins that act either in the direct complement activation cascade or as ancil- lary regulatory molecules and receptors ( 1 ). In Limulus polyphemus, the American horseshoe crab, the plasma-based cy- tolysis of foreign cells is mediated by a single protein, the sialic acid-binding lectin limulin (2). Here we report the presence of a cytolytic activity in the plasma of the channeled whelk. Busycon cii/ui/Uii/dtiiiii, and present preliminary evidence that the cytolytic activity is mediated by hemoeyanin. the major protein of the plasma. To obtain blood, adult animals were removed from the tank and prompted to express the sea water held inside the shell. The animal was then placed in a large beaker, the shell and underlying tissue ' Molecular and Cellular Biology. University of California. Davis. Cal- ifornia 95616. were punctured adjacent to the operculum, and blood flowed from the wound into the beaker. A large animal yielded 20-25 ml of blood, which was centrifuged to remove the blood cells, and was then fractionated by differential precipitation with polyethylene glycol-8000 (PEG) (Sigma cat #P-2I39). A majority of the hemo- eyanin was precipitated at 49r PEG and gave a voluminous blue pellet upon centrifugation. PEG precipitation fractionates protein solutions largely by size, with proteins in excess of 1,000 kDa precipitating at low concentrations of PEG. Busvcon hemoeyanin exists in solution as aggregates that range in size from dimers to heptamers of the basic decameric aggregate of the polypeptide chain, yielding molecular assemblies ranging from 9,000 kDa to 35.000 kDa (3. 4). The Biisyrun hemoeyanin molecule truly is a huge protein. Plasma contains approximately 80 mg/ml of protein, the great majority of which is hemoeyanin. Precipitation by 4% PEG yielded a preparation that showed a major protein band with a subunit molecular mass in the vicinity of 300 kDa when exam- ined by SDS-PAGE in the presence of 2-mercaptoethanol. This is close to the reported subunit molecular mass of Busycon hemoey- anin. 290 kDa (5). Whole plasma and the protein precipitated by PEG were hemo- lytic when tested with horse red cells in a standard hemolysis assay (2) (Fig. 1). In this context, the mammalian red cell is a model foreign cell, albeit not a relevant parasite, and it is used because hemolysis is a convenient assay of cytolysis of foreign cells. Removal of Ca ' 2 diminished, but did not eliminate completely, the hemolytic activity of the PEG-precipitable protein (Fig. 1). COMPARATIVE BIOCHEMISTRY 277 _ o E . Camp. Immunoi 15: 251- 261. n Sugumaran, M., and K. Nellaiappan. 1991. Biochem. Biophys. Res. Coinmun. 176: 1371-1376. 7 Nellaiappan, K., and M. Sugumaran. 1996. Camp. Biochem. Plnsiol. B. 113: 163-168. COMPARATIVE BIOCHEMISTRY 283 Reference: Biol. Bull. 197: 283-284. (October 1999) The 5-AminoIevulinate Dehydratase of Marine Vibrio alginolyticus is Resistant to Lead (Pb) O. A. Ogunseitan, S. L. Yang, and E. Scheinbach (Department of Environmental Anal\sis and Design, University of California at Inine, California 926^7-7070) Vibrio algiimlyticus is a ubiquitous marine bacterium that causes vibriosis in shellfish ( 1 ). The association of V. algino- lyticus with biofilms in marine ecosystems that are contami- nated with toxic metals has led to the discovery of an inducible copper-binding protein produced by this bacterium as a mech- anism for copper detoxification through the formation of or- ganic complexes (2, 3). However, the rule of organic ligands produced by marine bacteria in trace metal cycling remains poorly understood at the molecular level. The aim of our research is to determine whether the production of certain other proteins by V. alginolyticus can be used as a biosensor for the bioavailability of lead (Pb). a potent neurotoxin in contaminated marine habitats. 8-aminolevulinate dehydratase (ALADl is a phylogenetically conserved metalloprotein that catalyses the second step in the synthesis of porphobilinogen, the universal precursor of tetrapyrroles found in hemes and chlorophylls (4, 5). It is well known that the ALAD in humans and other eukaryotic organisms is very sensitive to Pb toxicity, because the required zinc atoms in the protein are stoichiometrically replaced by Pb. In fact, the accumulation of aminolevulinic acid (ALA) due to the inhibition of ALAD activity by Pb is respon- sible for the adverse neurological effects of lead poisoning. Consequently, the excretion of ALA and the level of ALAD activity have both been used as biomarkers for lead exposure in human populations and in wildlife (6. 7). To our knowledge, this is the first report on the response of marine bacterial ALAD to Pb. Two strains of V. alginolyticus were isolated from Buzzards Bay in July 1999. The strains were purified by colony streaking and were identified through fingerprint analysis of fatty acids and methyl esters (FAME I. There was a 94.7% match between the FAME profile of a non-swarming isolate and prototype V. algino- lyticux subgroup A. whereas a swarming isolate exhibited a 91% FAME profile match with V. alginolvticus subgroup B. To deter- mine the sensitivity of ALAD activity to Pb exposure in the two strains, total protein was extracted through sonication of cell pellets from cultures grown overnight without Pb. The protein extracts were suspended in a buffer containing 20 mM Tris-CI. 1 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride (pH 7.4). Protein concentration was determined by means of the Brad- ford assay (USB Biochemicals, Cleveland, Ohio), and ALAD activity (/^mol porphobilinogen/mg protein/h) was determined ac- cording to the method of Battistuzi ft al. (4) after incubation of 1 mg aliquots of protein with 0, 10, or 500 juM of Pb++ as lead nitrate (Oguseitan et ciL. unpublished). As a control, parallel ex- periments were conducted with a freshwater strain of Pseudomo- nas putidti ATCC 700097 known to be sensitive to lead toxicity (Oguiseitan et al., unpublished). The results (Table I) show that ALAD activity in both strains of V. alginolyticus is not inhibited by Pb. The effect of Pb on Table I Effect of Pb on htu'tcrinl S-aminolevulinate delmlratase activitv ALAD Activity ( /Minol % Change in [Pb] Porphobilinogen/mg Enzyme Activity Bacterial Strain* liM protein/h)** due to Pb Control. P. putida (] 5.70 ± 0.01 ATCC 700097 1C) 4.51 ± 0.09 -20.9 500 0.41 ± 0.01 -92.3 Vibrio alginolytii /n 0 7.38 ± 0.01 Group A (Swarmer) 10 7.64 ± 0.04 + 3.5 500 8.09 ± 0.01 +9.6 Vibrio alginolyticus 0 2.37 ± 0.01 Group B 10 2.58 ± 0.04 + 8.9 (Non-swarmer) 500 2.54 ± 0.07 + 7.2 * Data in the Table are from experiments conducted with proteins extracted from cells before Pb exposure. Pb at the specified concentrations was then incubated with proteins tor I h before enzyme assay. Similar results were obtained when viable cells were exposed to the specified concentrations of Pb before protein extraction and enzyme assay. ** Experiments were replicated five times. Values reported are the means ± standard deviation. ALAD activity in protein extracts from V. alginolyticus was not significant (P > 0.05). whereas 0.5 mM of Pb caused greater than 90% inhibition of ALAD activity in the control strain, P. putida ATCC 700097. The average concentration of Pb in Buzzards Bay sediment was about 20 nig/kg (8), but the extent to which this amount of Pb is biologically available at a level that would constitute a selective pressure on the molecular evolution of ALAD is not known. The metallic component of ALAD varies according to species. Zinc is required for ALAD activity in mammals, yeast, and Escherichia coli, whereas mag- nesium is required for ALAD activity in most plants and in bacteria that are associated with plants, e.g.. Bradyrhizobiwn japonicum (9). The identity of the metallic component of ALAD, as well as the amino acid sequence of the metal-binding site of the protein, have likely evolved in response to environ- mental factors, including the presence of toxic chemicals. Fur- ther work to identify the metallic component of ALAD in V. alginolyticus is ongoing in our laboratory as an approach to the molecular basis of Pb-resistance. This research was supported by a Josiah Macy Jr. fellowship awarded to Ogunseitan. Literature Cited 1 . Elston, R., E. I.. Elliot, and R. R. Colwell. 1982. J. Fish Diseases 5: 265-284. 284 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS 2 Schreiber, D. R., F. J. Millero. and A. S. Gordon. 1990. Mar. Clirin. 28: 275-284. 3. Harwood, V. J. and A. S. Gordon. 1994. Appl. Environ. Microbiol. 60: 1794-1753. Robin, I). Boulav, N. S. Richard. C. L. Gordon, and C. E. Webber. 1998. Environ. Res. 77: 44-61. 7 Burden, V. M., M. B. Sandheinrich, and C. A. Caldwell. 1998. Environ. Polhit. 101: 285-2S9. 4. Battistuzi, G., R. Petrucci, L. Silvagni, F. R. Urbani, and S. Caiola. s shine, J. P., R. V. Ika, and T. E. Ford. 1995. Environ. Sci. Techno/. 1981. .4/»i. Hum. Genet. 45: 223-229. 29: 1781-1788 5 Duncan, R., M. A. Faggart, A. J. Roger, and N. W. Cornell. Mot. g fhauhan. S., and M. R. O'Brian. 1995. J. Biol. Chem. 270: 19823- Biol. Evol. 16: 383-396. m-,7 6. Fleming, D. E. B., D. R. Chettle, J. G. Wetmur, R. J. Desnick, J-P. Reference: Biol. Bull. 197: 284-285. (October 1999) Substituted Cyclodextrin as a Model for a Squid Enzyme that Hydrolyzes the Nerve Gas Soman Francis C. G. Hoskin (Marine Biological Laboratory, Woods Hole, Massachusetts 02543), Diane M. Steeves1, and John E. Walker1 Certain phosphorus-fluorine (P-F) compounds are powerful in- hibitors of the nerve enzyme acetylcholinesterase (AChE). and are thus termed "nerve gases." One such compound is soman. 1,2,2- trimethylpropyl methylphosphonofluoridate. An enzyme that hy- drolyzes, and thus detoxifies soman has been purified from squid nerve and immobilized on agarose resin. However, this enzyme, termed organophosphorus acid anhydrolase (OPAA), hydrolyzes the relatively non-toxic pair of diastereoisomers. C( ± )P( + ). more rapidly than the toxic pair C( ± )P( - ) ( 1 ). Cyclodextrins are donut- shaped heptahexose molecules that can also act as P-F hydrolyzing "synzymes" (2, 3); but their rates are much lower than those of the OPAAs. We now report the rapid hydrolysis and detoxication of soman by a substituted cyclodextrin: 2-O-(4-carboxy-3-iodosoben- zoyl)-/3-cyclodextrin (IBA-/3CD) (4). Dimebu (3.3-dimethylbutyl methylphosphonofluoridate) (5), an isomer of soman with only one chiral center, P( ± ), is also hydrolyzed by IBA-j3CD. Information about the catalysis of these reactions by IBA-/3CD contributes to our understanding of the active site of the naturally occurring OPAA. The hydrolysis of soman or dimebu was monitored with a fluoride-sensitive electrode ( 1 ); the loss of AChE inhibitory po- tency, by a modified Ellman reaction ( 1 ). A 5-ml solution was made 25 mM in Pipes buffer. 3 mM in soman or dimebu. and 0.03 mM in IBA-J3CD; pH 7, 22°-23°C. After approximately half hydrolysis, as measured by the fluoride electrode, 0. 1 ml of the reaction solution was removed, diluted al ice-water temperature, and tested for AChE inhibitory potency. The results of the AChE determinations, and the fluoride measurements at that point are presented in Table I. Semi-log plots of fluoride release up to that point and well beyond are shown in Figure 1. Figure 1 shows that the hydrolysis of both soman and dimebu by IBA-/3CD is catalytic. Had it been stoichiometric, about 95% of either P F compound would have remained unhydrolyzed at 200 min. Figure I further suggests that the catalyzed hydrolysis of soman involves Iwo simultaneous reactions, one with a t,/, of about 20 min, and the other with a tl/2 of 300 min or more. In contrast, dimebu is hydrolyzed by a single reaction with a (,,, of U.S. Army Nalick RD&l- Center. Natick, Massachusetts 01760. Table 1 Soman or dimebu degradation by IBA-fiCD determined by Mo methods Compound Method Degraded Soman Fluoride-sensitive electrode 34. 36 Soman AChE inhibitory loss 41,47 Dimebu Fluoride-sensitive electrode 44. 47 Dimebu AChE inhibitory loss 57, 60 about 15 min. Table I. on the other hand, shows that soman or dimebu are hydrolyzed at about the same rate at which they lose potency as AChE inhibitors. That is, when soman is about 35% hydrolyzed as determined by the fluoride-sensitive electrode, 44% has been destroyed judging by the loss of AChE inhibitory po- tency. The same values for dimebu are 46% and 59%, respectively. These observations can be explained in the following way. From the manner of its synthesis, IBA is covalently bonded to the rim of the /3CD torus. The enhanced activity of IBA-/3CD over IBA alone suggests that soman fits inside the torus and that the CH, of the 100 E 10 50 too 150 0 Time, min 50 100 Figure 1. Hydrolysis of soman and dimebu by IBA-ftCD, determined in duplicate with the fluoride-sensitive electrode. Soman hydrolysis is resolvable into tu-o reactions: (A) obsenvd values: (B) derived values. Dimebu hydrolysis appears to be a single reaction: (C) observed values. COMPARATIVE BIOCHEMISTRY 285 methylphosphono part of soman is oriented away from the IBA. No matter whether the P=O or the P-F is to one side or the other of the IBA. about equal hydrolysis of the AChE inhibitory P( - )F isomers and the relatively non-inhibitory P( + )F pair will result. However, the chiral C-CH, at the 1-propyl position of soman will, in one configuration, allow a close approach of the P-F to the IBA. but in the other configuration will increase the distance of the P-F from the IBA by 1-2A. The increased distance would cause a much slower hydrolysis rate for half of the racemic soman. The same reasoning applies to dimebu, but since there is no chiral C-CH, on the 3,3-dimethylbutyl portion, dimebu is hydrolyzed at a single fast rate. Both AChE. which is inhibited by soman, and OPAA, the enzyme that hydrolyzes soman, seem to be indifferent to the configuration around the C chiral center ( 1 ). In contrast, IBA-/3CD, in its catalytic hydrolysis of soman. shows the reverse stereospec- ificity with respect to the two chiral centers. This conclusion is supported by the use of dimebu, in which there is no C-chiral center. These findings and speculations have important implica- tions for protection against, and disposal of, the acid anhydride type nerve gases, of which soman is an important example. Literature Cited 1. Hoskin, F. C. G. 1990. Pp. 469-480 in Squid as Experimental Animals, D. L. Gilbert. W. J. Adelman, Jr.. and J. M. Arnold, eds. Plenum Press. New York. 2. Klotz, I. M., G. P. Royer, and I. S. Scarpa. 1971. Proc. Nail. Acad. Sci. USA 68: 263-264. 3. Breslow, R., and S. D. Dong. 1998. Chem. Rev. 98: 1997-2012 4. Seltzman, H. H. 1992. Pp. 24-29 in Final Report of Contract No. DAMD I7-89-C-90I2. Synthesis of Soman Scavengers. Requests for this document should he addressed to: Commander, U.S. Army Medical Research and Development Command. Attention SGRD-RM1-S. Fort Detrick, MD 21702 USA. 5. Chettur, G., J. J. DeFrank, B. J. Gallo, F. C. G. Hoskin, S. Mainer, F. M. Robbins, K. E. Steinmann, and J. E. Walker. 1988. Fiimlani. Appl. Timcol. 11: 373-380. Reference: Biul. Bull. 197: 285-286. (October 1999) Effects of Green Tea Polyphenols on Lens Photooxidative Stress Seymour Zigman, Nancy S. Rafferty, Keen A. Rafferty, and Nathaniel Lewis (Eye Research Laboratory, Department of Ophthalmology, Boston University School of Medicine, and Marine Biological Laboratory, Woods Hole, Massachusetts 02543) Our purpose was to determine whether tea polyphenols such as epigallocatechin gallate (EGCG) would protect certain functions of rabbit and dogfish lenses HI vitro against the photooxidative stress of UVA irradiation. We have asked specifically whether catalase activity was protected. New Zealand white rabbit eyes were obtained from PelFreeze Biologicals within 20 hours of death. Dogfish (Mustelus canis) lenses were obtained fresh (under conditions approved by the MBL Institutional Animal Use Committee), and kept on ice for 5 h before use. The media used to maintain the lenses were Tyrode's solution (Sigma) for rabbit tissues and elasmobranch Ringer's medium (Marine Biological Laboratory) for dogfish tissues. The HPLC-purified EGCG (one elution peak) was supplied in solid form by the Lipton Company (Englewood Cliffs, NJ), and the solid material was dissolved in Tyrode's or Ringer's solutions to make 50 ^ig/ml. Whole eyes were placed with their anterior surfaces up in small glass beakers on gauze soaked with medium. Only the corneas were protruding into the air. and the beakers were filled with Tyrode's or Ringer's. Whole eyes were exposed to the EGCG solutions as follows. Filled plastic bottle reservoirs were placed on a shelf above the beakers of eyes so that the solutions were delivered through plastic tubing connected to shortened micro-pipette tips at about 12 drops per min for 4 h. For comparison, solutions not containing EGCG were dropped onto control eyes at the same rate. Cornea epithelia and lens capsule epithelia were dissected, washed in their respective media, and homogenized in glass ho- mogenizers in 1 .5 ml of medium. Homogenates were cleared of the insoluble residues by precipitation with an Eppendorf centrifuge at 12,000 rpm for 10 min. The presence of EGCG (50 /ig/ml) was detected in the cornea and lens, which were found to exhibit the same fluorescent and absorptive spectral qualities as the pure EGCG (excited at 490 nm, emission at 540 nm). We therefore attribute the fluorescences of these ocular tissues to the presence of EGCG. Extracted lenses were pre-incubated in Tyrode's (rabbit) or elasmobranch Ringer's (dogfish) solutions containing 5 /ng/ml of EGCG. After the presoaking period, three lenses per group were placed in beakers containing 15 ml of fresh Tyrode's or Ringer's solutions without EGCG. The beakers containing lenses with an- terior surface pointing upward were placed on a rack in our UVA exposure chamber. The UVA radiation was provided by 15 Osram Sylvania 15 W BL (maximum emission at 355 nm) lamps. Total exposure was 20 J/cirr. Lenses were kept on ice until examined. When the experimental procedures were complete, the lenses were blotted, weighed analytically, and assayed for catalase activ- ity (i.e., O2 production from H2O2) with an O2 meter and probe (Microelectrodes) connected to an (XY) LKB recorder. The slope of O, generation per minute was used as a measure of catalase activity. Controls for the EGCG-treated lenses and for UV-ex- posed lenses were included in every experiment. The enzyme catalase is quite sensitive to inactivation due to UVA exposure (1. 2). This was shown to occur in lenses of 286 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS Table I Protection of lens catalase activity from UVA imictivation ftv EGCG UV exposed Dark UV % of after EGCG % of control exposed control soak control Rabbit catalase* (% O, increase) 4.0 ± 0.9 1.5 ± 0.3 38% Dogfish catalase (It O, increase) 2.78 ± 0,5 0.63 ± 0.2 23% 2.9 ± 0.5 73% 1 .38 ± 0.3 50% : Average of 3 determinations ± the Standard Deviations. numerous species. In this report, we confirm that the inactivation of catalase due to UVA exposure is significantly reduced by the antioxidant action of EGCG. The protection of lens catalase by 5 /ig/ml of EGCG ranged from 10% to 50% (Table I). This study indicates that EGCG in physiological solutions can penetrate the anterior surface of the eye in vitro so as to reach the lens. It also shows that when EGCG reaches the lens, it is incor- porated into the capsule epithelium. EGCG is known to be an antioxidant that reduces oxidative stress in non-ocular tissues (3). This report shows that it enters the eye so as to reach the lens as well and provides beneficial antioxidant effects, as shown by others in non-ocular tissues (3). Further research is needed to determine whether EGCG can reduce oxidative stress to the human lens, thus delaying some cataractous changes that are due to environmental photo-oxidants. Literature Cited 1. Zigman, S. 1997. Bi»I. Bull. 193: 253-254. 2 Zigman, S., J. Reddan. J. B. Schultz, and T. McDaniel. 1996. Photochem. Plintobiol. 63: 818-X24. 3. Mitscher, L. A., M. Jung, D. Shankel, J-H. Don, L. Steele, and S. P. Pillar. 1997. Meil. Res. Rev. 17: 327-365. ECOLOGY AND EVOLUTION 287 Reference: Biol. Bull- 197: 287-288. (October 1999) Salinity Effects on Nitrogen Dynamics in Estuarine Sediment Investigated by a Plug-flux Method Thomas Mondrup (Department of Life Sciences and Chemistry, Roskilde University, Denmark} The efficiency of nitrification and coupled denitrification of regenerated N is greater in fresh water than in the marine and estuarine environments (1). Salinity or factors related to salinity thus play a major role in determining the fate of regenerated N. Increasing salinity decreases the amount of exchangeable ammo- nium, which is thought to diminish the substrate availability for nitrifiers ( 1 ). Sulfide, which is associated with sulfate reduction in saline environments, inhibits nitrification and denitrification (2). Lastly, nitrifiers and denitrifiers presumably suffer direct physio- logical salinity stress (3). The relative significance and interaction of these relations are unclear. The purpose of this study was to investigate the effect of salinity on estuarine sediment in the absence of potential sulfide effects. We employed a plug-flux method (4) in which thin layers of sediment are incubated with a small volume of overlying water after porewater concentrations have achieved steady state in a large volume of overlying water. The advantage of the method is that steady state can be attained relatively fast, and that linear fluxes during incubation can be interpreted as production rates (4). Surface sediment (0-2 cm) was collected in April 1999 from a station with seasonally varying salinity in the Parker River Estu- ary, Massachusetts. Salinity was — 0%c at collection. Sediment was sieved ( 1 mm), kept dark at 2°C, and stirred daily for 2 weeks prior to incubations. Sediment was incubated at different concentrations of artificial seawater lacking sulfate (0, 3, 10, and 30% under oxic conditions, and 0 and 30%c under anoxic conditions). At each treatment. 14 plugs (0.8 cm deep, 4.8 cm diameter) filled with 14.5 cm1 of sediment were each placed in open 125-nil cups, and afterwards placed in a tank containing 25 1 of treatment water. The experiment was kept at 20°C. After 50 h, steady state was assumed and the cups were sealed. Exchangeable and porewater ammonium concentrations were hereafter measured to be constant in each treatment between 0 and 72 h, thus indicating that steady state had been achieved. Microelectrode measurements of oxygen concen- trations indicated that only the top 2 mm of sediment in the plugs was oxygenated. Rates of ammonification, nitrification, denitrifi- cation, and oxygen consumption were calculated from differences in final and initial concentrations in the overlying water in the sealed cups after 0, 18, 40, 48, 72. and 161 h. At every timepoint. two cups from each treatment were removed for analysis. For sediments removed after 161 h, potential nitrification was mea- sured as nitrate production in oxic conditions. The samples were shaken with treatment water enriched with 500 juM NH4+ and 200 |uA/P. The measured rates of denitrification (Fig. IE) were highly variable and not significantly different from 0 in any treatment. The measurements ranged from 0 to 100 nmol N g~' ww d~'. However, because ammonification did not differ between anoxic and oxic treatments, because there was no nitrate in overlying water at the beginning of the experi- ment, and because there was very low measured nitrification, we believe that denitrification was very low. In conclusion, there was a relative stimulation of ammonifica- tion and potential nitrification at 3%r. Neither sulfide inhibition nor the relative amount of exchangeable ammonium could explain this result, pointing to a direct physiological response of the bacterial community to salinity. This response could be due either to a larger number of active bacteria or to an increase in bacterial activity. Potential and actual nitrification was lowest at 30%r, which was expected due to severe salinity stress. Decrease in substrate avail- ability could also acount for the low actual nitrification, but not for the low potential nitrification. However, the experimental setup was not optimal for investigating actual nitrification and denitrifi- cation. First, changes in oxygen conditions influenced the condi- tions for nitrification and denitrification, so that fluxes were non- linear at several treatments. To investigate the effects on actual rates of nitrification and denitrification more thoroughly, it would be advisable to look at the two processes separately. This could have been done by investigating nitrification at thinner sediment plugs without an anoxic interface, and by investigating denitnrt- cation in plugs with an oxic anoxic interface as described above. but with a substantial addition of nitrate to the treatment water. This research was supported by an NSF-LTER Grant (OCE 9726921 ), NOAA Sea Grant, the Mellon Foundation, and Roskilde University. I thank Gary T. Banta, Anne E. Giblin, Charles S. Hopkinson. Jane Tucker, Nathaniel Weston. and Yongchen Wang for help and assistance in carrying out this research. REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS I HU T 120 x 7-> _^_ ^ ^ •! rtr> ii_ 80 b b b b b 1 **• 60 0 41 E z 40 E "o < E 20 c n AO OO O3 O10 030 A 30 AO OO O 3 O10 030 A 30 c o IS o o Q_ O3U T 300 ~r T3 _I_ v^ 200 o" 15° z 100 "*" -r£- T o T E 50 __L n Literature Cited 1. Seitzinger. S. P. 1988. l.nnnol. Oceam'Ki: 33: 702-7:4. 2. Joy, S. B., and J. T. Hollibaugh. 1995. Science 270: 623-625 3. Rysgaard, S.. P. Thastum. T. Dalsgaard, P. B. Christensen, and N. P. Sloth. 1999. Estuaries 22: 21-30. 4. Aller, R. C., and J. E. Mackin. 1989. J. Mar. Res. 47: 441-456 5 Henriksen, K., J. I. Hansen, and T. H. Blackburn. 1981. Mar. Biol. 61: 299-304. 6. Holmes, R. M., A. Aminot, R. Kerouel. B. A. Hooker, and B. J. Peterson. 1999. Can. J. Fish. Ai/uat. Sci. (in press). 7 Kana, T. M., C. Darkangelo, M. D. Hunt, J. B. Oldham, G. E. Bennett, and J. C. Cornwell. 1994. Anal. Client. 66: 4166-4170. OO O3 O10 030 DUU o T 500 •^3 "D E I 400 -^ r^- r^- r^-| | Tra 300 c ° 200 O) ° 5- I 100 O n OO O3 O10 030 60 73 50 > 40 ^ 30 o 20 1 m OO O 3 O 10 TREATMENTS 030 Figure 1. Average flux rates ± SE for each treatment (A 0 and A 30 = ano.\ic II ami Mf/,,. O II. O .\ O II). ami O 30 = oxic 0. j. In. and 30%*): IAI ammonium cfflui. (Hi nitrate effu.\. (C) potential nitrification,
UIN * IN AnunoniflcattoD H4 * INOJ Nitrification E3 Plant Uptal Plant Uptake Model Equations . i • i i le mmobUization ion r2=k2*[NH4] r3=k3*[NO3] r7=k7"[DON] r4,rS,r6=constants Microbial 1 , pr»N , Denitrificat 0 80 70 60 5^50 ~40 "" 30 20 10 0 7 • NH4 Observed • NO3 Observed A DON Observed NH4 Modeled ^ NO3 Modeled , DON Modeled A H-^-r **-*-±, • • • „ • A -» — A— -^-y^ 20 1 • • • ^ 0 60 50 40 30 River km Figure 1. (Top): A simple N-processing model shtm'inx compartments, equations, and calculated rale coefficient.*. (Bottom): Obsen-ed (symbols) and predicted (lines) concentrations of NH^. NO,~, and dissolved organic nitrogen (DON) for the Ipswich River in July 1998. 290 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS NH4 + . NO, , dissolved organic N (DON), and participate organic N (PON) and considered the processes of ammonification, nitrifi- cation, and immobilization (Fig. 1. top). First-order kinetics were assumed for all processes except for lateral inputs. Concentrations were modeled as functions of distance. The least squares approach was used to fit the model to the main channel concentration data. The concentration data from the headwater sites were not used in the model. Constraints were placed on the model so that the initial concentrations, rate coefficients (k), and uptake rates (r) were greater than zero. Uptake lengths (1/k) were calculated from the rate coefficients; an uptake length is an estimate of the average distance traveled by an element before it is removed from the water column. Phosphate concentrations were very low at the headwater sites, averaging only 0.3 p.M P (data not shown). Further downstream in the main stem of the river, PO43 was roughly the same as upstream (0.6 JJ.M). TOP concentrations showed no apparent downstream pattern and were only slightly higher than PO4?~, indicating very low DOP concentrations (0-1.5 ;u,A/). DIN con- centrations at the headwater sites were high, approaching 60 ;uM N (Fig. 1, bottom). Further downstream in the main stem of the river, DIN concentrations were markedly lower (averaging 17 /xM). TDN concentrations were much higher than DIN but, as with TDP. showed no apparent spatial pattern. Of the DIN fractions, NO, exhibited a large drop in concentration with distance down the headwater stream. Although NO," dropped, NH4 + increased slightly, suggesting denitritication of the NO," and ammonifica- tion without subsequent nitrification. In contrast, in the main channel, NH4 + and NO," concentrations were mirror images of each other but with NH4 dropping and NO, increasing. This pattern suggests nitrification. Our simple N model tracked observed data well (Fig. 1, bottom). Plots of predicted versus observed concentrations of NH4 + , NO,", and DON illustrated close agreement for all fractions. Slopes of regression lines were between 0.96 and 1 .2 for the three compo- nents. Not only was there close agreement in a general sense, but the spatial patterns were also close to observed patterns for all components. Rate coefficients determined with the model differed greatly between the various N fractions, ranging from 0.0078 km ~ ' for ammonification to 107 km"1 for plant uptake and microbial im- mobilization (Fig. 1, top). For all fractions, uptake, or transforma- tion, lengths calculated from the rate coefficients were much longer than those usually reported for more pristine stream systems (2). The uptake lengths for NH4+ and NO," were 9 km and 1 3 km. respectively. The uptake length for NO," was dominated by nitrification, as the rate of nitrification was \CT times that of plant uptake. The uptake length for DON exceeded the length of the river. NH4 ' uptake lengths reported in the literature for low- nitrogen, pristine stream systems are often between 30 and 400 in (2). Reported NO, uptake lengths for similar systems typically range from 40 to 690 m (2). The long Ipswich River uptake lengths are probably due to the relatively high concentrations of inorganic and organic N in the Ipswich River. For a given rate of processing, calculated uptake rate coefficients vary inversely with concentra- tion. The observed uptake lengths may also represent slow overall rates of N cycling in this system, which as evidenced by extremely high inorganic N:P ratios, is probably P limited. Patterns of nutrient concentration and the results of the N model suggest that an important location for N retention or loss is in the headwater streams of the Ipswich River. Inorganic N concentra- tions decrease markedly in this region. The N-cycling model indicates very long uptake lengths in the mid and lower stretches of the river. It is possible that nutrient processing is greater in the headwaters because of greater relative contact with the riverbed. There may also be more active exchange between surface and hyporheic waters in the headwater streams. We would expect high rates of denitrification in anoxic hyporheic waters. Additional studies, such as tracer-nutrient releases and 15N additions could be profitably conducted in the upper reaches. Study of nutrient pro- cessing in the Ipswich River is increasingly important because N loading is rising in this rapidly urbanizing watershed. It is unclear how long the Ipswich River will be able to continue to process the high loads of inorganic N before the uptake capacity is reached. This research was funded by NSF grants (LTER: OCE-9726921, DEB-9726862. and EAR-9807632) and a gift from the Jessie B. Cox Charitable Trust. Literature Cited 1 Ingram, K. K., C. S. Hopkinson, K. Bowman, R. Garritt, and J. Vallino. 1994. Biol. Bull. 187: 277-278. 2. Marti, E., and F. Sahatcr. 1996. Ecology 77: 854-869. Reference: Biol. Bull 197: 290-292. (October 1999) Increased Lability of Estuarine Dissolved Organic Nitrogen From Urbanized Watersheds Felisa L Wolfe1, Kevin D. Kroeger, and Ivan Valiela (Boston University Marine Program, Marine Biological Lahoraton; Woods Hole. Massachusetts 02543) Inputs of nitrogen from land can lead to eutrophication of estuaries (1-5. 6). Terrestrial N is transported as NO,", NH4 * , PON (particulate organic nitrogen), and DON (dissolved organic ' Oberlin College, Oberlin. Ohio. nitrogen), but most estimates of N loading are based on DIN (NO, + NH4 ' ). DON had been thought to be mostly refractory to organisms, but recent studies show that some portion of the DON may be labile (6, 7). Land-derived DON may thus be mineralized within estuaries, and the NH4 + released may be avail- able to organisms (4). Most calculations of N inputs to estuaries ECOLOGY AND EVOLUTION 291 are based on DIN; to the extent that DON is labile, nitrogen loads calculated on the basis of land-derived DIN inputs alone underes- timate effective N loads (6, 7). There is evidence that urbanization of watersheds alters the lability as well as the amount of DON loaded to receiving waters (6). We evaluated these issues by use of estuaries of Waquoit Bay, Massachusetts (Sage Lot Pond, Quashnet River, and Childs River), that receive different N inputs from their watersheds because of different degrees of urbanization (1,4, 5). Sage Lot Pond (SLP) has a primarily forested watershed that provides a N load of 14 kg N ha"1 y~'; Quashnet River (QR) has a watershed with an intermediate degree of urbanization and a N load of 350 kg N ha" ' y~'; Childs River (CR) has a watershed with the greatest degree of urbanization, and a N load of 601 kg N ha"1 y~' (4, 5). To gain a better understanding of how urbanization alters DON. we measured the lability of DON from the three different estuaries. In each estuary we collected surface water from a station with a salinity of 25%t. Samples were placed in acid-washed bottles and kept on ice. The water was filtered through precombusted Q.l-p.m glass fiber filters to remove larger consumers and particulates. Triplicate samples (1000-ml/flask) were incubated at 25°C in the dark with continuous shaking. Each flask was re-sampled at 1 2. 24, 36, 48, 96, and 192 h. The water samples were filtered (0.2-fxm Durapore membrane Millipore filter, prerinsed with deionized water); placed in acid-washed, high-density polyethylene bottles; and frozen for later analysis. Nutrient concentrations were deter- mined by standard methods (Lachat QuikChem 8000 Automatic Ion Analyzer, 8). DON concentrations decreased in all incubations (Fig. 1, top right) and as a percentage of the total N (Fig. 1. middle row). The loss of DON generated NH4 + , which increased in concentration during all incubations (Fig. 1, middle panel in top row, and all panels in middle row). NO," concentrations remained relatively constant during the incubations (Fig. 1, top left). The total dis- solved nitrogen (TON) did not change significantly (data not shown) within the measurement error (F values for regressions were 0.56 for SLP, 0.95 for QR. and 0.9 for CR), which implies that the transformation of TDN to paniculate forms was not significant during the incubation; the concentrations of NH4 ' , NO,", and DON accounted for measured TDN throughout. The degree of urbanization on the different watersheds did alter the lability of DON generated from the watersheds. Concentrations of DON were largest in SLP (Fig. 1. top right), the estuary with the smallest N load (Fig. 1. bottom left) and the largest ratio of forest to residential land on its watershed (Fig. 1, bottom right). Our results thus suggest that increased N loads during urbanization (Fig. 1, bottom right) are accompanied by proportionally more labile DON (Fig. 1, bottom rightl. We conclude that since DON is quantitatively a large part of inputs from land (6), a labile fraction of 20%-40% (Fig. 1, bottom left) could indeed make a significant contribution of available N to estuarine organisms. Nutrient-loading protocols should therefore include assessments of the amounts and lability of DON entering estuaries. This is particularly important in cases of eutrophication of estuaries with watersheds exposed to greater degrees of urban land use. The DON in the incubation was derived from a mix of terrestrial and marine sources. Internal sources (exudation from producers. 0 200 400 600 " 0.2 04 06 N load (kg N ha'V) Residential area/Naturally vegetated area Figure 1. Top row: concentration (mean ± SE) of nitrate, ammonium, and dissolved organic nitrogen (DON} during the incubation. Middle row: percentage composition ofN measured in each of the three estuaries (Sage Lot Pond. Quashnet River, and Childs River) during the incubations. Bottom row: relationship of total percent DON lost during the incubations vs. land-derived nitrogen load {left panel) and vs. ratio of residential to vegetated land acreage on the watersheds (right panel '). regeneration from sediments) of DON are qualitatively important (our unpublished data). In addition, we collected samples from stations at which salinity was high and marine sources could have been important. Both of these features would diminish the possi- bility of our finding differences in lability due to land-derived DON. Nevertheless, we did find such differences, suggesting that despite the potentially confounding effects of internal estuarine and marine sources of DON, we can still detect measurable influ- ences tied to land-use mosaics on watersheds. Clearly, further mass balance studies will be useful to clarify issues, but our present results point to a substantive coupling between land use on watersheds and the nature of the DON in estuarine waters. This coupling has considerable importance to the management of N loads to estuaries. We thank Caroline Plugge, Kurt Hanselmann, Gabrielle To- masky, and Jennifer Walters for analytical assistance. Also special thanks to Kenneth Foreman and The Ecosystems Center for use of the Lachat autoanalyzer and to Eric Davidson of the Woods Hole Research Center for suggestions. This research was supported by funds from the NSF Research Experience for Undergraduates program (OCE-9605099). 292 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS Literature Cited 1 Valiela, I., J. Costa, K. Foreman. J. M. Teal, B. Howes, and I). Aubrey. 1990. Bingemiiemistiy 10: 177-197. 2 Valiela, I., el al. 1992. Estuaries 15: 443-457 3. D'Avanzo, C., and J. Krenier. 1994. Estuaries 17: 131-139. 4. McClelland, J. W., and I. Valiela. 1998. Lnnnol. Oceanogr. 43: 577-585. 5. Valiela, I., G. Collins, J. Kremer, K. Lajtha, M. Geist. B. Seely, J. Brawley, and C. H. Sham. 1997. Ecol. ,4/-/>/. 7: 358-380. 6. Seitzinger, S. P., and R. W. Sanders. 1997. Mar. Ecol. Prog. Ser. 159: 1-12. 7 Bronk. D. A., P. M. Gilbert, and B. B. Ward. 1994. Science 265: 1843-1846. 8. D'Elia, C. e I al. 1977. Limnol. Oceanogr. 22: 760-764. Reference: Biol. Bull. 197: 292-294. (October 1999) Effects of Increased Nitrogen Loading on the Abundance of Diatoms and Dinoflagellates in Estuarine Phytoplanktonic Communities A. Evgenidou, A. Konkle, A. D'Ainbrosio, A. Corcoran. J. Rowcn, E. Brown, D. Corcoran, C. Decirliolt. S. Fern, A. Lamb, J. Michalowski, I. Ruegg, and J. Cehridn (Boston University Marine Program, Marine Biological Laboratory, Woods Hole, Massachusetts, 02543) Coastal and estuarine ecosystems are among the most anthro- pogenically affected ecosystems on earth ( 1 ). Increased urbaniza- tion, deforestation, and agricultural land uses are some of the main factors that cause increased nitrogen loading in the estuaries (2). The effects of this increased delivery of nitrogen on the receiving estuarine waters include increased abundance of benthic macroal- gae and phytoplankton, reduced oxygen content of the water, and deterioration of shellfish and finfish populations (2, 3). However, knowledge of how and why estuaries subject to different rates of nitrogen loading differ in the composition of their phytoplanktonic communities is not as extensive, even though many laboratory experiments have addressed this question (4, 5, 6, 7). Changes in phytoplanktonic composition may have important effects on the receiving estuaries, such as changes in the food web structure (8) or in the sedimentation rates of organic matter (9). In our study, we describe the abundance of diatoms and dinoflagellates in three estuaries of Waquoit Bay, Massachusetts, which have similar physical properties but differ greatly in their degree of urbanization and subsequent nitrogen loading rates (2). Childs River. Quashnet River, and Sage Lot Pond exhibit high (6(11 kg ha ' y ' ). medium (350 kg ha" ' y~ ' ). and low ( 14 kg ha"1 y ' ) nitrogen loading rates respectively ( 10). To describe the natural assemblages of diatoms and dinoflagellates in the three estuaries, we took water samples from one site at the mouth of each estuary, at ~2 m depth on 12 November 1998. Temperature and salinity were very similar in the three sites examined. We took six samples from Childs River and Sage Lot Pond, and three samples from Quashnet River. Natural abundance was low during the sampling period; therefore, samples were concentrated 50 times by filtering 50 1 of water through a 10-jnm-mesh filter to collect the phytoplankton. The phytoplankton was then placed in 1 1 of water and fixed with Lugol's solution. The two phytoplank- tonic groups were identified and cells were counted under com- pound microscopes. In addition, we conducted a laboratory experiment to test whether the observed differences in composition of the phyto- planktonic groups examined were driven by increased nitrogen loading. Phytoplankton from Sage Lot Pond was collected using a 10-jLim mesh, and then placed in two 35-1 tanks tilled with Childs River water that had been previously filtered through 1-jum filters. The control tank contained phytoplankton from Sage Lot Pond placed in water from the same estuary using the procedure de- scribed above. Similar quantities of phytoplankton were placed in the three tanks. All tanks were oxygenated and kept with seasonal light and temperature conditions in an incubation chamber. In each tank, three replicates were taken at 0, 3. 6, and 9 days to measure phytoplankton abundance. We used the nitrogen content in the water column as a proxy for nitrogen loading. Nitrogen concen- trations in Sage Lot Pond and Childs River water in November were 1 and 5 ^M respectively. Therefore, every 3 days we mea- sured the nitrogen concentration in all tanks and added nitrogen as needed to maintain these natural levels. Analysis of the natural abundance of diatoms and dinoflagellates showed that diatoms were the dominant group in all three estuaries (Fig. 1A). while dinoflagellates represented less than 10% of the phytoplanktonic community examined. Both diatom and dinoflagellate abundances increased from low- to high-nitrogen estuaries (Fig. 1A: ANOVA. P < 0.01 for both groups). In addition, diatoms increased to a much greater extent than dinoflagellates did and. as a consequence, the ratio of diatoms to dinoflagellates increased more than one order of magnitude from low- to high-nitrogen estuaries (Fig. IB; ANOVA. P < 0.01). Dinoflagellates represented about 10% of the total community examined in Sage Lot Pond, but were less than 1% in Childs River (Fig. IB). A further examination of the diatom community showed that both centric and pennate diatoms increased from low- to high-nitrogen estuaries (Fig. 1C; ANOVA. P < 0.01 for both types). The two groups increased in similar proportions and, con- sequently, the ratio of centric to pennate did not change signifi- cantly with higher loading rates (ANOVA. P > 0.05). The results from the experiment showed that the Sage Lot diatom community in Childs River water responded differently than the control, the same community kept in its own water (Fig. 2). Diatom abundance in Childs River water increased over the course of the experiment (t test, P < 0.01). Conversely, diatom abundance in Sage Lot water at the end of the experiment was not higher than the initial abundance (/ test, P > 0.05). in spite of the observed peak on the third day. At the end of the experiment, diatoms were more abundant in Childs River water than in Sage Lot water (t test. P < 0.01). Dinoflagellate abundance increased ECOLOGY AND EVOLUTION 293 over the experiment in both Childs and Sage Lot water (/ test, P < 0.01), reaching similar values in both types of water (/ test, P > 0.05). Results from the natural abundance survey indicate that diatoms were more abundant than dinoflagellates in the estuaries examined (Fig. 1A). Moreover, these results suggest that the dominance of diatoms is promoted by increased nitrogen loading. The results of the experiment also show that by the end of the study period diatoms were more abundant in Childs River water than in Sage Lot Pond water. However, diatom abundance in Sage Lot Pond water was inconsistent over the course of the experiment, increas- ing for the first 3 days before declining to levels not different from their initial abundance. Therefore, the results of this preliminary experiment can only suggest that nitrogen loading rates may be promoting diatom dominance in these estuaries (Fig. 2). Moreover, = 1 o j5 'a "3 ••5 gf 'S'S o c ' SLP QR CR Figure 1. Analysis of the natural phytoplanktonic communities in the three estuaries of Waquoit Bay. (A) The abundance of diatoms (open bins) and dinoflagellates (grey bins) in Sage Lot Pond (SLP), Qtiashnet River (QR}. and Childs River (CR). (B) The ratio of diatoms to dinoflagellates in the three estuaries examined. (C) The abundance of centric diatoms (open bins) and pennate diatoms (grey bins) in the three estuaries. Bins represent mean values and bars show confidence inten'als calculated from six replicates for CR and SLP and from three replicates for QR. Variables were log-transformed to comply with the assumptions of the AN OVA test employed. time elapsed (days) Figure 2. Abundance of diatoms (circles) and dinoflagellates (squares) during the laboratory experiment. Sage Lot Pond phytoplankton in Childs River water (experimental) is represented by solid symbols; Sage Lot Pond phytoplankton in Sage Lot Pond water (control) is represented by open symbols. Symbols represent mean values and bars show confidence inten'a/s calculated from six replicates for experimental tanks and from three replicates for control tanks. Lines depict the spline-smoothed trends. Variables were log-transformed to comply with the assumptions of the ANOVA test employed. there may be some other factors that could account for the increas- ing abundance of diatoms, such as differences in silica and other trace metals among estuaries. Our findings are consistent with the results of previous labora- tory manipulations. Many authors have shown that experimental nitrate enrichment results in phytoplanktonic communities domi- nated by centric diatoms (4, 5, 7). This is attributed to the higher growth rates of diatoms compared to dinoflagellates (5. 6, 7). Therefore, it is possible that diatoms could build up large stocks of biomass faster than dinoflagellates. Changes in the relative abundance of diatoms and dinoflagel- lates under increasing nitrogen loading may have important eco- logical implications for the receiving estuaries. Large centric dia- toms are the main diet of some copepods species, which in turn are preyed upon by commercial fish species (8). Therefore, increases in abundance of centric diatoms could cause changes in the food web structure of the receiving estuaries. In addition, diatoms have higher sinking rates than other groups of phytoplankton. which could lead to enhanced sedimentation rates of carbon in the estuary (9). Examination of these hypotheses is needed to assess the effects of nitrogen induced shifts in phytoplankton composition, particu- larly among diatoms and dinoflagellates. on the ecology of the receiving estuaries. Literature Cited 1. Vitousek, P. M., H. A. Mooney, J. Lubchenco, and J. M. Melillo. 1997. Science 277: 494-499. 2. Valiela, I., K. Foreman, M. LaMontagne, D. Hersh, J. Costa, P. Peckol, B. DeMeo-Andreson, C. D'Avanzo, M. Babione, C. Sham, J. Bra\vle>, and K. Lajtha. 1992. Estuaries 15: 443-457 3. Nixon, S. VV. 1986. J. Lininol. Soc. S. Afr. 12: 43-71. 294 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS 4. Davis, C. O. 1982. Pp. 323-332 in Marine Mesoct>xm.\. G. D. Gnce and M. R. Reeve, eds. Springer-Verlag. New York. 5. Takahashi, M., I. Koike, K. Iseki, P. K. Bienfang, and A. Hatlori. 1982. Pp. 333-340 in Marine Mesocosms. G. D. Grice and M. R. Reeve, eds. Springer- Verlag, New York. 6. Ishizaka. J., M. Takahashi, and S. Ichimura. 1983. Mar. Biol 76: 271-278. 7 Parsons, T. R., P. J. Harrison, and R. Waters. 1978. J. Exp. Mar. Biol. Ecol. 32: 285-294. 8. Greve, W., and T. R. Parsons. 1977. Hclt-ol. Wiss. Meeresunters 30: 666-672. 9. KiOrboe, T. 1993. ,490% of the individuals, D. Lawrence, unpublished data). A. tonsa has a high rate of popula- tion turnover and a low capacity for energy storage (3), so it is a suitable organism to use in assessing short-term responses to food supply. We expected that changes in available food supply created by the different nitrogen enrichment regimes would be reflected in the egg output of A tonsu. a variable known to respond rapidly to food supply (3). Egg production experiments were conducted in the held from K) to 20 July 1999 to measure reproductive output of A. IOIIMI in relation to chlorophyll concentration and to nitrogen load. Adult A. tonsa females were collected from each site by towing a 153-/j.m Nitex nylon net obliquely at a depth of 0.5 in. To minimize the inlluence of temperature and salinity, animals were collected from sites that ranged in temperature from 24°C to 27°C and in salinity from 25%p to 30%<. The incubation water was collected, using a Lamotte water sampler, from the same site and depth as the tows, and hltered through a 45-p.m sieve to remove all mesozooplankton and copepod eggs but retain phytoplankton. This hltered water, now devoid of eggs, was used to fill the 2-1 incubation bottles. In the laboratory, adult females were isolated and d to 10 were placed in each incubation bottle. For each site, six bottles were incubated in Waquoit Bay for 24 h. This is sufficient time to measure changes in egg production in these animals (4). Incuha- 1 Lafayette College, Easton, Pennsylvania. tions were run in situ to maintain field conditions of light and temperature. After the incubations the water was sieved twice, first through a 200-jj.m mesh to isolate the adult females and then through a 53-jmn mesh to concentrate the eggs and nauplii. After the copepods were fixed in 70% ethanol, we measured the prosome length of adult females to determine average size, and the eggs and nauplii were counted in each sample. We determined daily egg production (EP) as (E + N)/F. where E = number of eggs, N = number of nauplii. and F = number of adult females in each sample. The adult females may have cannibalized nauplii. but probably at an ingestion rate of only 1% day"1 (5). Water for determination of chlorophyll a concentration was collected on 20 July 1999 from five sites in each estuary, from near surface and bottom depths, using a Lamotte water sampler. Spec- trophotometrie measurements (6) were averaged to estimate mean chlorophyll u concentration in each incubation. Reproductive output of A. tonsa increased as chlorophyll con- centrations increased (Fig. 1, top left). Chlorophyll concentrations in the water increased at higher nitrogen loads (Fig. 1. top middle). We interpret these results to mean that increases in nitrogen supply led to increases in chlorophyll concentration, which in turn sup- plied greater amounts of food to copepods. which responded by increasing egg production. There is. therefore, an indirect correla- tion between reproductive output and nitrogen loads (Fig. I, top right). Of course, the greater reproductive production could be caused by the somewhat larger female size in the higher loaded estuaries (Fig. 1, bottom left). Nitrogen load increased both reproductive output and adult size, but egg production rates were not related to size of females (Fig. 1, bottom right). The increased reproductive output measured was therefore mediated by conditions in the estuary, not by the size of females alone. The results suggest that with increasing nitrogen loads, phyto- plankton populations increase, providing more food for A. tonsa, thus probably increasing its reproductive output. The reproductive output by this calanoid zooplankter seems therefore indirectly linked, through the response of phytoplankton to increased nutrient concentration, to the nitrogen loading rate derived from the wa- tershed land-use mosaic. This research was supported by NSF Research Experience for ECOLOGY AND EVOLUTION 295 40- S 20 D) O 10- 0. O) O) 150-1 8 o 8 o r = 0.933" 20 40 60 Mean chl a cone, (mg m") 750-1 O) .5 700 CU O w o 0.650 c 03 (1) 600 y = 0.1x + 656.1", r = 0.631 n.s. 200 400 600 Nitrogen load (kg ha~1 y"1 100- co E O) 6 § CO -F 50- o c CO 0) 40-1 y = 0.2x+ 1.2", r = 0.949" 80 100 120 "3 200 400 600 Nitrogen load (kg ha"1 y" 40- '•o - 0 0 0) « 30- o E o o o 0) 20- A O) 3- D A A -6 ° 10- dP A o CL n a CD cn LU r = 0.423 n.s. o o.1(H D) CD LU y = 9.6* ipO-OP1*", r = 0.910' 200 400 600 Nitrogen load (kg ha"1 y"1) o A D CR QR SL 640 660 680 700 720 Mean prosome length (|im) Figure 1. Top left: Egg production rate (eggs female ' day ') versus mean chlorophyll concentration (mg m ). Top middle: Mean chlorophyll concentration versus land-derived nitrogen load (kg ha I The lines represent standard error. Nitrogen loads obtained from Valiela et al. (2). Top right: Egg production rate versus land-derived nitrogen load. Bottom left: Mean prosome length versus nitrogen load. The lines represent standard error. Bottom right: Egg production rate versus mean prosome length of females (fan). One asterisk indicates significance of <0.05 and fn'o asterisks indicate significance of<0.01. Undergraduates grant. Special thanks to Felisa Wolfe, Amy Watson, Erica Stieve. and the Waquoit Bay National Estuarine Research Reserve. Literature Cited 1. Valiela, I., K. Foreman, M. LaMontagne, D. Hersh, J. Costa, P. Peckol, B. DeMeo-Anderson, C. D'Avanzo, M. Babione, C-H. Sham, J. Brawley, and K. Lajtha. 1992. Estuaries 15: 443-457. 2. Valiela, I., J. McClelland, J. Hauxwell, P. Behr, D. Hersh. K. Foreman. Ecol. Appl. (in press). 3. Kleppel, G. S., C. A. Burkart, and C. Tomas. 1998. Estuaries 21: 328-339. 4 Bellantoni, D. C., and W. T. Peterson. 1987. J. E.\r>. Mar. Bioi Ecol. 107: 199-208. 5. Lonsdale, D. J., D. R. Heinle, and C. Siegfried. 1979. J. £v/> Mar. Biol Ecol. 36: 235-248. b. Lorenzen, C. J. 1967. Lnnnol. Oceanogr. 12: 343-346. Reference: Biol. Bull. 197: 295-297. (October 1999) Long-Term Effect of Municipal Water Use on the Water Budget of the Ipswich River Basin Susannah Canfield1, Luc Claessens, Charles Hopkinson Jr., Edward Rastetter, and Joseph Vallino (The Ecosystems Center, Marine Biological Laboratory, Woods Hole, Massachusetts 02543) The Ipswich River watershed has served as a public water supply to suburban communities north of Boston since the late 1 Bates College, Lewiston, Maine 04240. 1800s. Population growth and land-use changes have affected the hydrology of the watershed by increasing the amount of water pumped from the basin and altering the land cover — a problem that is prevalent nationwide ( 1 ). In recent years, the river has suffered 296 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS from low flows in the summer (2). Low streamfiow can be detri- mental to the ecosystem of the river, the surrounding wetlands, and the estuary into which the river drains (1). The purpose of this study was to examine the effect of municipal water use on the overall water budget of the Ipswich River basin. Monthly water budgets were constructed for the period 1931- 1989 as: AS = P - ET - R - D. where P is precipitation; ET is evapotranspiration; R is streamflow; D is net diversions, including drinking water and wastewater; and AS is change in storage. Precipitation data were obtained from observations of the National Weather Service (NWS) and cooperative network. Observations from NWS first-order weather stations were used to calculate evapotranspiration using a mathematical model (3). Streamtlow data were obtained from the U.S. Geological Survey. Data on monthly water pumpage and wastewater systems were collected from individual town water departments, the Department of Envi- ronmental Protection, the Massachusetts Area Planning Council, and the Massachusetts Water Resources Authority. Public drinking water was divided into public water supply from within and from outside the watershed based on the locations of pumping stations relative to the boundary of the Ipswich River basin. Using popu- lation data and the relative distribution of urban land use for each town, we separated the total public water supply into water deliv- ered inside and outside the basin. To account for lawn and plant watering, an irrigation coefficient was calculated for the summer months by considering the difference between summer and winter pumpage. Finally, the remaining water, for commercial and house- hold use, was divided into wastewater exported out of the basin via sewer systems and wastewater retained in the watershed by on-site septic disposal. Sewered water was assumed to have a 65% infil- tration component from groundwater (4). Linear regression anal- yses were performed to examine time-dependent trends in annual, monthly, and seasonal data. On a long-term annual scale (Fig. la), precipitation, streamflow, and evapotranspiration are highly variable but do not display any significant time-dependent trends. Only diversions have increased significantly over time (/• = 0.96. P < 0.001) and currently represent 15%-20% of streamflow. One would expect that with a significant increase in diversions, streamflow would decrease sig- nificantly. It is plausible, however, that changes in land use have masked the effect of diversions on streamflow and the overall water budget; for example, the conversion of forested area to impervious land cover could lead to an increase in streamflow and a decrease in evapotranspiration (5). Our analysis of the main diversion components shows an increase in water drawn from within and outside the basin, tripling over the 59-year period (Fig. Ib). Water supply from outside the basin constitutes 30% of the total supply; of this total water supply. 71% is delivered outside the watershed. The septic wastewater component levels off after 1966. when sewer systems became more prevalent. la. 1800 -i 1500 •*• Precipitation -*- Streamflow Change in Storage Evapotranspiration Diversions -300 1930 1940 1950 1960 1970 1980 1990 Time (yr) 1C. Precipitation (1180mm/yr) Evapotranspiration (541mm/yr) Public Water Import (33mm/yr) Public Water Export (103mm/yr Ib. Id. Streamflow (538mm/yr) Sewered Wastewater (40mm/yr) PWS from within IRB -*- PWS from outside 1RB -o- Septic Wastewater -+- Sewered Wastewater — Water Delivered outside IRB 140 q 1930 1940 1950 1960 1970 1980 1990 Time (yr) -»- Precipitation -*- Streamflow -•- -*- Change in Storage Evapotranspiration Diversions ' • y \ ^^-^M A N *^ =-" ^^* f^L- -A. \ /^ V las'^^^^-^ \_ V g o -: / rn ^^ ^^ 1234567 Month 8 9 10 11 12 Figure 1. la) I93I-I9N9 annual time series of the main components of the water budget, with only diversions increasing significantly, tl'l 1 931-1989 annual time series of the components of diversions, including public water supply (PWS) from within and from outside the Ipswich River basin (1KB), water delivered outside ihe basin, septic and sewered wastewater, including infiltration, Ic) Main C(imponents of the Ipswich Ri\:er hasin water budget, including I979-I9NN annual averages. Id) 1979-I98K average nionthlv tune series of the main components o/ the water budget. ECOLOGY AND EVOLUTION 247 In the 1979-1988 water budget (Fig. Ic). which is representa- tive of current conditions, evapotranspiration (541 mm/y) and streamflow (538 mm/y) each account for about 45% of precipita- tion (1180 mm/y). Diversions leaving the basin (143 mm/y) are greater than diversions entering the basin (33 mm/y). The change in storage ( — 9 mm/y) is small. However, in this study, we ignored the absolute value of the storage component because it is highly dependent on the evapotranspiration estimate, which is the least accurate component of any large-scale water budget; only the temporal variation is considered. During the summer months (Fig. Id), the change in storage is most negative, due to increasing evapotranspiration, and it is coincident with decreasing rainfall and streamflow. Diversions remain relatively constant throughout the year, with high groundwater pumping during the summer balanced by surface water withdrawals into reservoirs during the rest of the year. The effect of diversions should be most apparent during the summer months because streamflow is lowest at this time. With increasing water demands, diversions have become a ma- jor component of the water budget — they currently represent 15%- 20% of the streamflow. Our analyses of the water budget did not reveal any significant long-term trend in change in storage or in streamflow. This suggests that to understand the impact of diver- sions on the system we ought to reduce the time step (to daily or hourly) to examine changes in streamflow; focus the study area on the upper Ipswich basin where low flows occur and the river dries up most frequently; and look at different indices of hydrological change, such as the number of days of low flow and groundwater levels. Low streamflow is detrimental not only to the river eco- systems, but also to the downstream estuary, where alterations in salinity during the summer months could increase the stress on estuarine communities, a topic that requires further research. This research has been supported by the NSF-BUMP REU program, the Cox Charitable Trust, and the NSF grants: OCE- 9726921, DEB-9726862. and EAR-9807632. We thank the water departments of those towns that provided data and assistance, the Ipswich River Watershed Association, the Department of Environ- mental Protection, and Gil Pontius and colleagues at Clark Uni- versity. Literature Cited 1. U.S. Geological Survey. 1997. Pp. 41-45 in Watershed Research in the L/.S. Geological Sumy. National Academy Press, Washington. DC. 2. USGS website (http://ma.water.usgs.gov/ipswich/). 3. Morton, F. I., F. Ricard. and S. Fogarasi. 1985. National Hydrology- Research Institute. Paper No. 24. Ottawa. Canada. 4. Allen, Scott. 1999. Boston Globe. July 16:AI6. 5. Van Patten, Peg. 1997. Nor 'Easter. Spring/Summer: 15. Northeast Sea Grant Programs, University of Rhode Island, Narragansett. Rl. Reference: Biol. Bull. 197: 297-299. (October 1999) Population Size and Summer Home Range of the Green Crab, Cardans maenas, in Salt Marsh Tidal Creeks Talia Young (Swarthmore College. Swarthmore, Pennsylvania 19081), Sharon Komarow1, Linda Deegan2 anil Robert Garritt2 The green crab. Carcinus maenas, is native to the Atlantic coast of Europe. First reported in the western Atlantic in 1817, it is abundant today in salt marshes and on rocky shores from Nova Scotia to Virginia. As a predator, it has been linked to the sharp decline of the New England soft-shell clam (Mya arenaria) industry in the 1940s (1). Since the crab was first found in San Francisco Bay in 1989. scientists and fishers have been anxiously monitoring its movement northward and its effects on the ecosystem (2). Despite interest in the extension of the species' geographic distribution, little work has been conducted on the home range of individual crabs. We examined the population size and summer home range of green crabs in a New England salt marsh tidal creek. We conducted a mark-recapture experiment in a branched primary tidal creek off of the Rowley River in the Plum Island Sound Estuary in northeastern Massachusetts. The upper 200 m of the creek has about 7274 m3 of volume and about 7128 m2 of creek bed area. Water temperature (16°-25°C) and salinity (28%e-31%o) in the creek were typical of New England salt marshes in late spring and summer. From ' Earth Systems Program, Stanford University. Stanford. California 94035. • Ecosystems Center, Marine Biological Laboratory, Woods Hole. Mas- sachusetts 02543. 29 June to 6 August 1999. crawfish traps (20 X 30 X 45 cm with 1.3-cm mesh and 8-cm opening) baited with tuna fish or dog food soaked in fish oil were laid along both branches and downstream of the confluence at seven sites 100 m apart. From 29 June to 30 July, each trapped crab measuring 40 mm or more was marked either with colored oil-based marker paint on the carapace or with a plastic loop behind the claws. The carapace width (in millimeters), sex (male or female), and carapace color (red or green) of each crab were also noted. Crabs trapped at each of the seven sites were marked with a distinct color scheme and then released at the same site. Marked crabs that were recaptured were marked a second time with the color scheme corresponding to their recapture location. Crabs trapped from 3 to 6 August were counted and removed from the creek. We used the Lincoln index and the Schnabel method to estimate population size (3). We also conducted two catch-per-unit-effort collections in five other similar-sized primary tidal creeks off of the Rowley River (Sand Creek, Shad Creek. West Creek, Club Head Creek, and Nelson Island Creek) by deploying traps from high tide to low tide (~6 h). We estimated the population of green crabs in the study creek to be 30.000-40,000 individuals (~5 crabs per nr) (Table I). Re- capture rate of marked crabs was between 5% and 11%. The average number of crabs caught over a 6-h period did not differ significantly between the study creek and the other five creeks 298 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS Table I Estimates of the population size of green crabs. Carcinus maenas, in a tidal creek in Rowley, Massachusetts Number of crabs Marking technique Marking period marked recaptured captured Recapture rate Estimated population 95% confidence interval Paint' 29 June-1 July 87 9 4095 10.35% 39585 23852-118948 Plastic tags' 12 July-16 July 240 25 3735 10.42% 35856 25636-59627 Paint2 26 July-30 July 1887 109 1848 5.78% 31992 26979-39295 All marking techniques'1 29 June-30 July 2378 120 1848 5.05% 36621 31126-44473 All marking techniques1 29 June-30 July 2629 145 4251 5.52% 32746 28082-39268 1 Estimates calculated using the Lincoln index, counting all crabs trapped in subsequent weeks as the single capture sample (6). The crabs used in these calculations was 66% of the actual number marked because approximately 33% of marked crabs in the laboratory two weeks. 2 Estimates calculated using the Lincoln index with the collection from 3 to 6 August as the single recapture sample (6). 3 Estimate calculated using the Schnabel method, treating each marking technique as one of the repeated recapture samples (6). number of marked lost their marks in sampled. We caught slightly more male crabs (/; = 1257) than female crabs (n = 1131). but the numbers were not significantly different (P = 0.32, t = 1.03, d.f. = 16). We caught five times as many green-colored crabs (n = 1921) as red-colored crabs (n = 364) (P = 0.0004, t = 4.73, d.f. = 13). As is typical of this species (4). males were larger than females in our collections (P < 0.0001. t = 21.447, d.f. = 2386). Red-colored males (mean = 56 ± 0.593 SE) were larger than green-colored males (mean = 52 ± 0.193 SE; P < 0.0001, t = 5.75, d.f. = 1187), and red-colored females (mean = 48 ± 0.269 SE) were also larger than green-colored females (mean = 47 ± 0.165 SE; P = 0.0006, t = 3.43, d.f. = 1094). We found no significant difference in average crab size between the beginning (mean = 49 ± 1.073 SE) and the end (mean = 49 ± 0.215 SE) of the study period. This stability in crab size over time suggests that we did not lose many marks to molting. About half of the 149 recaptured crabs were trapped at the same site both times (Fig. 1). As the distance from the marking site increased, the number of recaptured crabs decreased: only 5% of the crabs were recaptured 300-400 m upstream or downstream from their original marking site (the extent of the trapping area). There was no significant difference between the numbers of crabs found upstream and those found downstream (P = 0.37, t = 0.97, 50 -- £ 40 3 30 D. g JO- 'S 10 n= 1 n= I n = 2i I »J n=l 400 300 200 downstream 100 100 across 400 distance (m) and direction traveled Figure 1. Distance and direction (upstream, downstream, or across) traveled by recaptured green crabs, Carcinus maenas. in a tidal creek in Row/ev, Massachusetts. d.f. = 6). Three percent crossed from one branch of the creek to the other, either through the mosquito ditch network connecting the two branches (about 150 m of travel), or down to the conflu- ence and back up the other branch (about 300 m). Our population density estimate of 5 crabs per m2 is comparable to previous estimates for green crabs on rocky shores in Wales (4). The results of the catch-per-unit-effort comparison for the six creeks suggest that this value may be a good estimate of green crab populations in the Rowley River. Green-colored crabs are found throughout the molt cycle, but some crabs become red-colored during prolonged intermolt stages (5, 6). Green-colored crabs are more tolerant of low salinities than are red-colored crabs (6): the green form has been found primarily in the intertidal zone on Welsh shores and the red form primarily in the subtidal zone (7, 8). The dominance of green-colored crabs in our collection may be a result of the large tidal range (>3 m) and narrow subtidal zone in the study creek. McGaw (5) found red-colored males to be larger than green-colored males and hy- pothesized that this color change may be partly associated with sexual maturity. The recapture data suggest that green crabs can move at least 400 m upstream or downstream, but that for the most part they remain within a 400-m range during the summer. Frequency of distance traveled was calculated only from crabs recaptured 1 to 3 weeks after each marking period in order to allow marked crabs to remix with the general population of the creek. But we also recaptured several crabs 300-400 m away from their original marking site within 4 days of being marked, indicating that they can move at least 400 m in a matter of days; distance traveled may thus not be directly related to time. A study of the movement of crabs within the creeks in relation to tidal cycles would expand on previous research showing that these crabs follow tides up and down rocky shorelines in Wales (4, 7, 8). Warman (7) and Crothers (4) suggest that green crabs move offshore in the winter; an investigation of the winter range of green crabs would also add to information on the annual range of individual green crabs. Such research would contribute to the understanding of the role of this invasive species in coastal ecosystems. This research was funded by the NSF Research Experience for ECOLOGY AND EVOLUTION 299 Undergraduates through the Boston University Marine Program and also by the Plum Island Sound LTER Program. We thank Simon Panall, Joao Feliciano Salgado, Susan Oleszko. Jaimie Champagne, Nate Tsao. Marc McDonnell, Jeff Hughes, and Jesse Young for their help. Literature Cited 1. Ropes, J. VV. 1968. Fisheiy Bull. Fish Wildl. Sen: U.S. 67: 183-203. 2. Cohen, A. N., J. T. Carlton, and M. C. Fountain. 1995. Mar. Biol. 122: 225-237. 3. Tanner, J. T. 1978. Ciu'ule to the Stinlv of 'Animal Po/iitlatiiwi. The University of Tennessee Press, Knoxville. 4. Crothers. J. H. 1967. Field Stud. 1: 407-434. 5. McGaw, I. J., M. J. Kaiser, E. Naylor, and R. N. Hughes. 1992. J. Zool. Loud. 228: 351-359. 6 Reid, I). G., P. Abello, I. J. McGaw, and E. Naylor. 1989. Pp. 40-60 in Phenotypic Kes/mnxcx and Individuality in Aquatic Eclo- therms, J. C. AlcJrich, ed. Japaga, Co. Wicklow, Ireland. 7. Warman, C. G., D. G. Reid, and E. Naylor. 1993. ./. Mai: Biol. Assoc. U.K. 73: 355-364. 8. Edwards, R. L. 1958. J. Aiiim. Ecu/. 27: 37-45. Reference: Biol. Bull. 197: 299-300. (October 1999) Influence of Marsh Flooding on the Abundance and Growth of Fiinduliis heteroclitm in Salt Marsh Creeks Sharon Komarow {Earth Systems Program, Stanford Umversirv, Stanford, California 94305), Talia Young1, Linda Deegan2, and Robert Garritt2 Like many other estuarine fish and crustaceans, Fitndulus hct- eroclitus (mummichog) regularly makes use of the marsh as a foraging area, nursery habitat, and refuge from predators. Mum- michogs are known to follow flooding tides onto the intertidal marsh to forage (1, 2). Through this behavior, they provide an important trophic link between salt marsh and open estuary (3). Previous research indicates that access to the intertidal flooded marsh has significant effects on the growth rate of F. hetemclitus. Weisberg and Lotrich (4) showed that foraging exclusively on subtidal food sources was not sufficient to support normal growth rates of mummichogs. Javonillo et al. (5) found that mummichogs denied access to the marsh had lower growth rates than those that were allowed entree to the marsh surface. Both of these studies employed caging techniques on a relatively small scale. Our goal was to examine the importance of marsh flooding to mummichog growth and abundance in a natural environment without enclo- sures. Tidal creek flooding onto the marsh determines the vegetation in the area surrounding the creek. Spartina alterniflora grows on the marsh adjacent to the creek that floods on every high tide, whereas S. patens grows on the higher marsh that floods less frequently. We measured the length from the creek edge to the transition between S. alterniflora and S. patens at increments along the creek. The mean of these measurements multiplied by the length of the creek was considered the area of marsh accessible to mummichogs at high tides. This area is equivalent to the area of marsh adjacent to the creek covered by S. alterniflora. A comparison of the regularly flooded area in the 5 tidal creeks that were part of our study is shown in Figure la. We measured the abundance and growth of F. heteroclitnx in tidal salt marsh creeks of the Rowley River in the Plum Island Estuary in northeastern Massachusetts (42°44' N X 70°50' W). 1 Swarthmore College, Swarthmore, Pennsylvania 19081. 2 The Ecosystems Center, The Marine Biological Laboratory. Woods Hole. Massachusetts 02543. Over 6 weeks, catch-per-unit-effort (CPUE) was measured three times in each of five salt marsh creeks. Ten minnow traps (6.35-mm mesh), spaced evenly in the primary tidal creeks, were set at high tide and retrieved about 5 h later during low tide. In two ot the creeks, we measured growth of mummichog young-of-the- year. the life stage in which the most dramatic growth occurs. Four times during the 6 weeks (first three times coincided with CPUE measurements, plus one additional growth measurement), the total lengths of between 275 and 1000 fish from each creek were measured, and length-frequency histograms were constructed. Probability paper was used, according to the method described by Harding (6), to identify the young-of-the-year cohort from the length-frequency histograms. Mean values from each set of mea- surements were plotted to evaluate growth. Catch-per-unit-effort measurements indicated that mummichogs tended to be more abundant in creeks with greater areas of fre- quently flooded marsh (con-elation coefficient = 0.83, P = 0.09) (Fig. Ib). This relationship suggests that creeks with increased marsh flooding are able to support a larger population of mummi- chogs by providing greater regularly flooded areas for foraging, or that creeks with increased flooding offer greater refuge from predation. Mummichogs that follow the high tide onto the marsh surface become more exposed to predation by shorebirds. but they gain protection from predation by larger fish, the more likely predator. Although the creeks are very similar, properties other than regularly flooded area — including dimensions, water volume, temperature regime, productivity, and food availability — may af- fect the abundance of mummichogs in a creek. The pattern of growth was the same for young-of-the-year mummichogs in Sweeney Creek and Club Head Creek (Fig. Ic). However, the mean total length values of mummichogs from Sweeney Creek were significantly greater than mean total length measurements from Club Head Creek (Complete Randomized Block ANOVA P < 0.05). Though statistically significant, the very small mean difference between measurements of 1.25 mm is unlikely to be of ecological significance, especially since the pattern of growth did not differ between the creeks. Unlike in 300 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS SW Creek 1B / «J 60 50 40 4 t 4 t 30 T I 20 4 > y 1 10 n 1000 2000 3000 4000 5000 6000 Accessible marsh area (m2) 1C 0) £ 50 1) 40 6 * * o?30 >• E * o .- 20 f1*" 10 V 1 o ™ R/T 1 1 1 7IQQ 7/1 1 /QQ V/OC/OO Q /Q Date enclosure experiments in which mummichogs were denied access to the marsh (4, 5), all fish in our study could access the marsh, though the extent of this access differed among creeks. Thus, because the lower marsh is accessible at every high tide, differ- ences in regularly flooded accessible marsh area may not be great enough to cause a large difference in mummichog growth. This work was funded by NSF Research Experience for Under- graduates through the Boston University Marine Program and the Plum Island Sound Long-term Ecological Research Program. We thank Simon Panall, Nate Tsao, Marc McDonnell, Susan Oleszko, Joao Salgado, Jaimie Champagne, and Jeff Hughes for their help with the project. Literature Cited I Itiiinrr. A., and B. H. Brattstrom. 1960. Copcia 1960: 139-141. 2. Weisberg, S. B., R. Whalen, and V. A. Lotrich. 1981. Mar. Biol. 61: 243-246. 3. Kneib, R. T., and A. E. Stiven. 1978. J. E.v/>. Mar. Biol. Ecol. 31: 121-140. 4. Weisberg, S. B., and V. A. Lotrich. 1982. Mar. Biol. 66: 307-310. 5. Javonillo, R., L. Deegan, K. Chiaravalle, and J. Hughes. 1997. Bi»l. Bull. 193: 288-289. h. Harding, J. P. 1949. J. Mar. Biol. Assoc. U.K. 28: 141-153. Sweeney o Club Head Figure 1. (.4) Regularly flooded accessible marsh area (m:) bv tidal creek, ordered from upstream to downstream: SW, Sweeney Creek: SA, Sand Creek: WE, West Creek: CL. Club Head Creek: and NE. Nelson Island Crefk. (B) Mean catch-per-unit-effort (number of fish ± / standard error) plotted against regularly flooded accessible marsh area (m~l (O Mean total length measurements (mm ± / standard error, standard errors a/I < O.I ) at Sweeney Creek and Club Head Creek plotted against date of measurement. Reference: Biol. Bull. 197: 300-302. (October 1999) Decline of a Horseshoe Crab Population on Cape Cod Justin W. Wiclener ami Robert B. Barlow (Marine Biological Laboratory, Woods Hole, Massachusetts 02543) The American horseshoe crab, Liiiiuhix polyrihemiis. inhabits coastal estuaries of North America from Northern Maine to Flor- ida, as well as the region around the Yucatan peninsula. Delaware Bay contains the largest known population, but surveys of the New Jersey beaches that border the Delaware Bay show a decline of about 50% in the spawning population since 1990 (ref. 1 and B. L. Swan, pers. comm.). Trawl surveys of Delaware Bay from 1990 to 1997 yielded a 74% decline in crabs caught per tow (2). These declines do not appear to be isolated events; the populations of crabs spawning on the beaches of Cape Cod have also declined. We report here the results of a longitudinal study of the spawning population at Mashnee Dike. Bourne, Cape Cod, Massachusetts. In ECOLOGY AND EVOLUTION 301 the 15 years from 1984 to 1999 the population declined more than 80%, and its spawning activity decreased 95%. We surveyed the horseshoe crab population at Mashnee Dike in parallel with studies of their visually guided behavior (3. 4). We selected Mashnee Dike because its south-facing beach was an active spawning area for horseshoe crabs when we began our studies in the early 1980s, and because no humans inhabit the area bordering the spawning beach (length: —1 km). Mashnee Dike is under the jurisdiction of the U.S. Army Corps of Engineers, which does not permit alteration or development of the Dike. Each spring animals migrate to the beach from offshore as the moon ap- proaches new and full phases. Maximum migration is coordinated with the higher of the two daily high tides that occurs in the late afternoon and throughout the night (5). To quantify the dynamics of the crabs' migratory behavior we followed a surveying proce- dure published elsewhere (5). In brief, we mapped out three 10-nr quadrats with 10-m spacings along a 50-m transect at the water's edge (longitude: 70° 37'46" W and latitude 41 :'46'34" N). We then counted the number of animals in each quadrat at 30-min intervals for a period of 2 to 3 h as the tide flooded and ebbed. The 10-m spacings of the quadrats, together with the animals' slow move- ments and the insignificant tidal flow, precluded the crabs being counted twice in an observation period. Summing the data from the three quadrats for an observation period yielded a measure of the density of spawning animals for that period of the tidal cycle. Comparison of the data over the 2- to 3-h tidal cycle revealed the dynamics of the animal's migratory behavior. In general, animal density increases as the tide floods, peaks about 1 h after high tide, and decreases as the tide ebbs. Frequent surveys outside the quadrats during a tidal cycle indicated that the density of animals in the quadrats was representative of that along the appoximately I km of spawning beach. On this basis we assume that the maximum density per tidal cycle observed in a spawning season is proportional to the size of the spawning population in the vicinity of Mashnee Dike for that year. The sum of the maximum densities per tidal cycle over an entire spawning season is a measure of the spawning activity for that year. Figure I shows a dramatic decline in the spawning population of horseshoe crabs at Mashnee Dike from 1984 to 1999. We counted a maximum of 247 crabs in the three quadrats near the time of the new moon in late May of 1984. Six years later, in 1990. near the full moon at the beginning of June, the maximum count was 73 crabs. In 1999 the maximum number of crabs was also observed near the time of full moon at the beginning of June, but it was considerably less (total of 42) than those in 1984 and 1990. Taken together the results from 1984 to 1999 reveal an 83% decline in the maximum density/transect/season, implying a comparable decline in the spawning population all along Mashnee Dike. The decline in spawning activity was greater, decreasing from 3171 in 1984 to 736 in 1990 and finally to 148 in 1999— a remarkable 95.39r decline. The decline in spawning activity at Mashnee Dike accompanied a shortening of the spawning season. In 1984. crabs began spawn- ing on the nighttime high tide of 13 May and were last seen in the transects on 7 July — a total of 56 days. In 1999 the spawning season lasted only 1 1 days — 28 May to 7 June. The 5-fold short- ening of the spawning season and concomitant decrease in spawn- ing activity may be related to the decrease in the spawning pop- Full Moon o New Moon Full Moon O New Moon 200 1984 O 200 100 1990 . ..... ilh.. ... i i E 3 o o 200 100 1999 15 20 25 31 5 10 15 20 25 1 5 May June July Figure 1. Maximum number of Limulus counted in three 10-nr i/ntiti- rats during the 1984. 1990. and 1999 spawning seasons at Mashnee Dike. The height of each bar gives tlic maximum number of males and females in a single half-hoiir/v survey each day. The open and filled circles indicate the times of full and new moonx. Suireys were not done on 28 and 30 May 1990 because of bad weather. Data were interpolated for these two dayx to calculate spawning activity. Observation periods were 14 May to 8 July 1984: 19 May to 22 June IWO. and 24 May to 16 June 1999. ulation. The data shown in Figure 1 are representative of the overall trend we observed of high densities in the early 1980s, intermediate densities from the mid-1980s to the mid-1990s, and low densities in the late 1990s. We have surveyed systematically only the crab population near Mashnee Dike. Because of the decline in this population we transferred our studies of horseshoe crab vision in 1994 to a spawning beach in Stage Harbor (Chatham, Massachusetts). Over the 5-year period, we observed a substantial decrease in the Stage Harbor population, suggesting that the decline of horseshoe crab populations on Cape Cod may be widespread. The causes of the declines in the horseshoe crab populations in Delaware Bay. Mashnee Dike, and Stage Harbor are not known. Possibities include loss of habitat, loss of food source, change in water conditions, and increase in predation. Mashnee Dike is an exemplary site, for it has been under the jurisdiction of the U.S. 302 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS Army Corps of Engineers since its construction over 50 years ago, and consequently no man-made alteration or development of the beach has occurred. Also no known changes have occurred in the availability of food sources at Mashnee Dike. The crabs are pri- marily scavengers with a diet of polychaetes, seaweed, nematodes, soft-shell bivalves, and detritus (6). In addition, no changes in chemical or other characteristics of water conditions of Phinney's Harbor have been reported. There is. however, clear evidence of increased predation. Each year since the mid-1980s, fishermen have been observed removing horseshoe crabs from the entire length of the Dike at high tide during the spawning season (R.B. Barlow, unpubl. obs.). On two occasions during the 1990 and 1995 spawning seasons, we succeeded in intercepting some fishermen, purchased the harvested animals (total: 1376). and returned them to their habitat. The yearly harvesting of crabs at the Dike has intensified since the mid-1990s, and activity correlates well with the large decline in the spawning population we observe. Also, the recent decline in the spawning population at Stage Harbor corre- lates with intensified harvesting in that area. It is not certain that harvesting caused the observed population declines in either area. Note however that the population at Mashnee Dike appears to be a local one. Animals tagged during a spawning season return the following seasons, and pairs of horseshoe crabs (some tagged) have been found during winter scuba dives buried in the bottom of Phinney's Harbor 100 to 300 m offshore (R. B. Barlow, unpubl. obs.). Harvesting large numbers of animals from such a local population would have a significant impact on its size. Delaware Bay has also experienced increasingly large annual harvests of horseshoe crabs for use as bait in the eel and conch fisheries (7). Expansion of this bait fishery has led to near historic peaks in annual harvests in Delaware Bay (8) concomitant with the decline in population noted above. Although the spawning popu- lalion in Delaware Bay is the largest on the East Coast, local populations appear to inhabit various beaches along the Bay (B. Swan, personal communication), as they do at Mashnee Dike. In an attempt to preserve the horseshoe crab fishery, the states of Delaware. Maryland, and New Jersey that border Delaware Bay have established regulations for harvesting the animals (9). Vir- ginia, New Hampshire, and South Carolina have also established regulations, with those of South Carolina being the strictest. No regulations exist in Massachusetts. What is the impact of a declining horseshoe crab population? In Delaware Bay it may have an important effect on the survival of shorebirds that migrate 3000 to 4000 miles from South America to the Arctic. The Bay provides an essential stopover for nourishment and rest for perhaps as many as one million shorebirds ( 10). Their springtime arrival coincides with the intense spawning activity of horseshoe crabs, and upon landing they devour crab eggs, with some birds consuming more than 100.000 in 2 weeks before continuing on to the Arctic for their own spawning activity (11). There is concern that the current density of crabs in Delaware Bay is not sufficient to provide nourishment for the migratory shore- birds. Indeed, Joan Walsh of the Cape May Research Observatory has been quoted as saying that "the density (of horseshoe crabs) is not great enough to provide food for shorebirds." She also noted that birds are arriving at their Arctic destinations "underweight" (12). The effect of declining spawning populations of horseshoe crabs on Cape Cod is not known. We thank N. Buelow, S. Gibson, E. Herzog, L. Kass, M. Kelly-Manglapus. J. Marler, M. Parsley, J. Pelletier, M. Powers. and T. Thiele for assistance in transect counts at Mashnee Dike. We also thank the U.S. Army Corps of Engineers for providing access to Mashnee Dike. Study was supported in part by the National Science Foundation and the National Institutes of Mental Health. Literature Cited 1 Swan, B. I... \\. R. Hall, Jr., and C. N. Shuster, Jr. 1996. Pages 35—39 in Proceedings of the Horseshoe Crab Forum: Status of the Resource, J. Farrell and C. Martin, eds. University of Delaware, Sea Grant Program, Lewes, Delaware. 2. Atlantic States Marine Fisheries Commission. 1999. Stock As- sessment Report No. 98-01 (Supplement) page 47, Washington. DC. 3. Barlow, R. B., Jr., L. C. Ireland, and L. Kass. 1982. Nature 296: 65-66. 4 Herzog, E. H., M. K. Powers, and R. B. Barlow. 1996. Visual Neiirosci. 13: 31-42. 5. Barlow, R. B., Jr., M. K. Powers, H. Howard, and L. Kass. 1986. Iliol. Bull. 171: 310-329. 6. Botton, M. L., and H. H. Haskin. 1984. Fish. Bull. 82(2): 383-389. 7. Shuster, C. N., Jr. 1996. Pages 5-14 in Proceeilintis of the Horse- shoe Crah Forum: Status of the Resource, J. Farrell and C. Martin, eds. University of Delaware, Sea Grant Program, Lewes, Delaware. 8. Loveland, R. E., M. L. Botton, and C. N. Shuster, Jr. 1996. Pages 15-22 in ProceeJiiif>s of the Horseshoe Crah Forum: Status of the Resource. J. Farrell and C. Martin, eds. University of Delaware, Sea Grant Program, Lewes, Delaware. 9 Schrading. E.. T. O'Connell, S. Michels, and P. Perra. 1998. Fishery Management Plan for the Horseshoe Crab Uinulus polyphemus. Fisheries Management Report oj the Atlantic Stales Ma- rine Fisheries Commission. 7-9. Washington. DC. 10. Clark, K. E., L. J. Niles, and J. Burger. 1993. Comlor 95: 694- 705. 11. Myers. J. P. 1986. Nut. Hist. 95(5): nS-77. 12. McDonald, G. G. 1999. The Cape Codder. June IS, 1999, p. 3. ECOLOGY AND EVOLUTION 303 Reference: Bid. Bull. 197: 303-306. (October Evaluation of a Reporter Gene System Biomarker for Detecting Contamination in Tropical Marine Sediments Lisa M. Kerr (Biologv Department, University of Massachusetts, 100 Morrissey Blvd., Boston. Massachusetts 02125}, Phillip S. Lobe!1, and J. Mark Ingoglia2 A major challenge in conducting field assessments of potential ecological impacts is optimizing the number of samples and the costs. This is especially important in light of the growing concern over the presence of persistent organic contaminants, such as PCBs. dioxins, furans, and PAHs in sediments. A reporter gene system (RGS) assay that measures induction of the CYP1 Al gene and transcription of P450 enzyme systems is often used to assess potential toxicity of these compounds in environmental samples ( I. 2. 3). RGS has gained acceptance as an inexpensive, rapid method for screening environmental samples for contaminants (4. 5). The RGS approach has been validated in the laboratory with pure compounds, known chemical mixtures, and from field-collected sediments by comparing RGS system response and chemical con- centrations (1,2. 3. 6). Our study differed from other validation studies in two respects: we used field-collected sediments over a wide range of contaminant concentrations and evaluated RGS response to sediment samples containing a mixture of dioxins. furans, and PAHs. With few exceptions, the previous validation studies using field-collected samples used fairly small sample sizes and generally evaluated one chemical group (3. 6). Few studies have compared large numbers of samples containing both PAHs and 2.3.7.8 tetrachlorodibenzo-p-dioxin (TCDD) over the range reported here. The purpose of this study was to determine if there is a high correlation between RGS response and chemistry results from the same samples. If this proves to be the case, the assay could be used to screen large areas at a relatively low cost. Samples exhibiting high responses could be targeted for further characterization using more precise, but costly, GC/MS methods. Matched sediment samples (n = 31) were collected off the northwestern shore of Johnston Island, adjacent to potential sources of PAHs and 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDDl (Site 1), and at sites farther removed from the potential contami- nant sources (Sites 3, 4). To ensure blind analysis of the samples, P450 RGS assays were conducted by MEC Analytical Systems (Carlsbad, California) and Columbia Analytical Services (Vista, California) while chemical analyses were completed by the Toxic Contaminant Research Laboratory at Wright State University (Dayton. Ohio). The RGS assay used in this study was developed by Anderson c/ «/. ( I ) and detailed methods have been described (1. 7). Induc- tion in the assay is dependent upon the aryl hydrocarbon receptor (AhRl activation pathway. AhR ligands. including planar PCBs, PAHs. and TCDD, bind to AhR, activating it and resulting in its translocation to the nucleus of the cell. In the nucleus, the activated ' Boston University Marine Program. Marine Biological Laboratory, Woods Hole, Massachusetts 02543. 2 United States Air Force, Environmental Restoration. Hickam AFB, Hawaii 96853. AhR complex hinds to the xenobiotic responsive element in the promoter region of the CYP1 Al gene, resulting in its transcription. Compounds that are not AhR ligands, such as metals and pesti- cides, do not cause induction in the system (1,7). Briefly, the RGS methodology involves exposing human 101L cells to 10 p.1 of solvent extracts for 16 hours (1, 5). Sediment extracts were pre- pared according to EPA method 3540. The cells are stably trans- fected with a plasmid containing firefly luciferase linked to the human CYP1 Al promoter sequence. This promoter sequence con- tains 1800 bp of flanking regulatory DNA with three xenobiotic responsive elements ( 1 ). Exposure to Ah receptor ligands induces luciferase activity which is quantified with a luminometer (7, 8). Previous studies have determined that RGS detection limits in sediment are 8 pg/g for dioxin. 63 pg/g for furan. 6.2 to 7500 ng/g for specific PAHs, and 250 ng/g for a mixture of PAHs (7). Solvent blanks were used as negative controls while extracts from sedi- ments spiked with 2 ng/g TCDD were used as positive controls (/; = 6). From the level of induction generated by each sample, equivalent doses of TCDD or benzo(a)pyrene (BaP) that would result in the observed level of induction were calculated. While the level of induction is the same in each sample, the amount of pure compound (either TCDD or BaP) required to produce the observed induction level differs between the compounds. Equivalent doses for total PAHs and dio.xins/furans calculated from RGS induction are designated as "RGS BaPEQ" and "RGS TEQ" respectively. For the GC/MS analyses, sediments were extracted using EPA Method 3540. PAHs and dioxins/furans were measured using EPA Methods 8270 and 8290. Detection limits were from 0.0638 to 0.777 pg/g for the 17 individual dioxin or furan congeners mea- sured. Total PAHs measured included 14 individual compounds with detection limits ranging from 19.1 to 40.7 ng/g. Toxicity equivalents (TEQs) based on the 17 dioxin and furan congeners measured were calculated using human/mammalian toxic equiva- lency factors (TEFs; 9). TEQs calculated from the chemical anal- ysis of dioxins and furans are designated as "Chem TEQ." Total PAHs measured by chemical methods are referred to as "Chem PAHs." Linear regression analysis was used to determine if signifi- cant relationships existed between the RGS TEQ and Chem TEQ. Similarly, the relationship between Chem PAH and RGS BaPEQ was determined. Data were log transformed to equalize variances. There was a significant statistical relationship between Chem TEQ and RGS TEQs I)-2 = 0.774; P< 0.001) (Fig. 1 ). However. 4 1.9% of the points fall outside the 95% confidence intervals. Additionally. there was a significant statistical relationship between Chem PAHs and RGS BaPEQ (r = 0.564; P < 0.001 ). In this case, 35.5% of the points fall outside of the 95% confidence intervals. While there was significant covariance between Chem and RGS values, the actual 304 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS A. 10000 I o LU oo >f> o 1000 100 CHEM PAHs (ng/g) 10000 Ol 01 Ct 1000 100 XX CHEM TEQ (pg/g) Figure 1. (A) Comparison of chemically derived total PAH concen- tration (Chem PAHs) to RGS estimates oj benzo(a)pyrene equivalents (RGS BaPEQs) r = 0.564 P < 0.007. (B} Comparison of chemically- derived TEQs (Chem TEQ) to P450 RGS estimated TEQs (RGS TEQ) r = 0.564 P < 0.001. Best fit regression lines are shown with 95% confidence intervals. magnitude of the values differed. RGS TEQs were 10~ to 104 times higher than Chem TEQs (Table I). Similarly. RGS BaPEQs were 10 to 104 times higher than Chem PAHs. The levels of induction and the resultant estimates of RGS TEQ and RGS BaPEQ decreased with distance from shore (Table I). This pattern also held for Chem TEQ and Chem PAHs, with those samples with the highest concentrations being the closest to shore (Site 1 ). Three samples exceeding ecological screening levels were found closest to shore. One sample exceeded the effects range low (ER-L) screening levels for Chem PAHs, including total PAHs. low and high molecular weight PAHs, and nine individual PAHs 1 10). Two additional samples from site I had contaminant concen- trations exceeding ecological screening levels. These samples with Chem TEQs of 68.24 pg/g and 901 .27 pg/g exceeded the low and high risk to aquatic life screening values respectively (II). When using the RGS assay as a screening method, the samples with the highest responses or induction levels might be "chosen" as the samples expected to contain the highest contaminant con- centrations. In this study, this was the case, and the three samples with the highest contaminant concentrations would have been detected. However, there are two important points: 1 ) induction was higher than that of the positive controls (around 100-fold) in eight samples (129- to 316-fold), and 2) the samples with the highest overall induction levels were not necessarily the samples with the highest dioxin or PAH concentrations. Additionally, some samples exhibited significant RGS induction, although very low levels of PAHs. dioxins. or furans were detected. These responses could be considered false positives. A false negative could be identified only if arbitrary limits were set on the number of samples further characterized. For example, if budget constraints limited the number of samples that could be chemically charac- terized to five samples and we chose to characterize only those samples with the highest RGS response, the sample with the highest concentration of PAHs would not have been detected, resulting in a false negative. One potential explanation for the variable RGS response in this study is induction by other compounds not measured by GC/MS. Other chemicals that may induce the RGS system include planar PCBs as well as some dioxin congeners not measured in this study. The contribution of PCBs to induction is unlikely since previous sampling events at the same sites found total PCB concentrations below the 10 ng/g RGS detection limit. Another explanation suggests that in tropical environments. P450 activity is higher in some fishes and may be related to a herbivorous diet (12). This suggests further consideration of the presence of naturally occurring toxins such as ciguatoxins or other allelochemicals potentially inducing the RGS assay or interfering with cell function. Furthermore, coral reef environments may have different sediment characteristics due to physical and chemical factors that may affect bioavailability. The RGS assay detects compounds that may or may not be specifically measured by GC/MS and assesses the synergistic and/or antagonistic effects of the constituents in the mixture. This study found significant co-variance between Chem TEQ and RGS TEQ as well as between Chem PAHs and RGS BaPEQs. However, there was a high level of variability (more than 35% of values fall outside the 95% CD; and of the samples with the highest RGS response (greater than positive controls), a high percentage (5 of 8) could be identified as false positives. Qualita- tively, the RGS assay revealed a contaminant gradient on a scale of hundreds of meters. We conclude that this method is useful as a broad area assessment tool tor screening purposes, provided that data are interpreted carefully. Use of this screening method is substantially complicated by the presence of multiple compounds, as at Johnston Atoll. This may include natural or other anthropogenic compounds that were not chemically measured in the GC/MS analysis. While the response to these various compounds may be complex, the value of the RGS assay is that it gives an integrated and more biologically relevant response than chemistry alone. Additionally, given the expense of GC/MS analysis of dioxin congeners (>$1000/sample). use of the ECOLOGY AND EVOLUTION 305 Table 1 Conitimiiuint concentrations in sediment samples from the west end of Johnston Atoll compared tu ;o/>n;«fs generated h\ the RGS assa\ Sample Nu m he i Site Chem TEQ pg/g RGS TEQ pg/g Chem PAH ng/g RGS BaPEQ ng/g Induction 030 1' 0.01 2400 21.7* 2000 25 064 1 0.95 18200 158.5* 15200 183 074 1 0.40 1700 1.3* 1400 17 076 1 0.02 1X00 77.1* 1500 18 086 2.04 7800 937.9 6500 79 088 7.29 31200 1125.0 26000 316 095 0.00 1200 11.6* 1000 12 104 13.63 6400 8.0* 5300 64 108 0.00 1100 2.1* 900 11 120a 2.35 13200 9711.0 11000 133 217 0.04 3400 151.7* 2800 34 225b 68.27 21300 64.9* 17700 218 233 0.78 4400 124.5* 3700 45 234 1 4.44 30700 1988.0 25600 308 238 1 6.43 7400 41.5 6100 74 242 1 2.38 5200 343.2* 4400 53 248 1 0.05 5500 174.8 4600 55 249 1 1.26 7300 rid 6100 73 254 1 9.13 18700 906.3 15600 188 256" 1 901.29 14700 50.5* 12300 147 257 5.87 12800 2383 10600 129 307 2.14 2600 21.4* 2200 26 013 3- 0.00 700 2.4* 600 7 027 3 0.00 600 1.4* 500 6 043 3 0.00 300 2.8* 300 3 118 3 0.00 200 2.4* 100 2 243 3 0.00 200 1.4* 200 2 038 4 0.00 200 3.2* 200 2 124 4 0.00 400 2.4* 400 5 105 4 0.00 200 0.8* 400 2 119 4 0.00 200 nd 200 2 Control 2000.00 79 Control 2000.00 101 Control 2000.00 90 Control 2000.00 77 Control 2000.00 81 Control 2000.00 137 1 Site 1 is adjacent to the former herbicide orange storage site and burn pits on Johnston Island. " Sites 3 and 4 are across the shipping channel from site 1. approximately 1000 m from shore. a Exceeds screening level for PAHs (Effects Range-Low for total PAHs = 4022 ng/g). h Exceeds low risk screening limit for TCDD (60 pg/g). c Exceeds high risk screening limit for TCDD (100 pg/g). d Positive Controls. [PAHs] below quantifiable limit, nd-none detected. RGS assay ($150/sample) shows potential for possible cost sav- ings through incremental, phased use in combination with GC/MS methods. As site specific complexities and relationships between the two methods and the matrix are further defined, the under- standing of and confidence in the RGS results can improve. This can lead to reduced need for GC/MS analyses and increased emphasis on RGS results. Annual long-term monitoring, which may span decades, lends itself to such incremental cost savings through the development of large data sets and cumulative expe- rience gained over time. This research supported by Army Research Office grant DAAG 55-98-1-0304 for the Johnston Atoll Reef Study. Literature Cited 1 Anderson. J. W., S. S. Rossi. R. L. Tuke>, T. Vu, and L. C. Quattrochi. 1995. Environ. Toxicol. Chem. 7: 1159-1169. 306 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS 2. Murk, A. J., J. Legler. M. S. Denison, J. P. Giesy, C. van de Guchte, and A. Brouwer. 1996. fiimlam. A/t/il. Taxicol. 33: 149- 160. 3. Jones, J. M., and J. W. Anderson. 1998. Pp. 1 -6 in Risk. Resource, and ReKiilnttny As»f.v, G. B. Wickramanayake and R. E. Hinchee. eds. Battelle Press, Columbus, Ohio. 4. APHA. 1996. Pp. 24-25 in Standard Methods for the Examination of Water ami Waste Water. 19th ed. Supplement. American Public Health Association. Washington. DC. 5. ASTM. 1997. Pp. [392-1391 in Biological Effects and Environmen- tal fate: Biotechnology; Pesticides. IW7 Annual Book of ASTM Standards. Vol. 11.05. American Society for Testing and Materials. West Conshohocken, Pennsylvania. 6. Kim, G. B., J. W. Anderson, K. Bothner, J. Lee, C. Koh, and S. Tanahe. 1997. Biomarkers 2: 1X1-188. 7 Anderson, J. \V., K. Bothner, D. Edelman, S. Vincent, T. P. Vu, and R. H. Tukey. 1996. Pp. 150-lhX in field Apnluution\ of Biomaikers for Agrochemicals and Toxic Substances, J. Blancato. R. Brown. C. Dary. and M. Saleh. eds. American Chemical Society. Washington. DC. S. Jones, J., and J. W. Anderson. 1999. Em-iron. Toxicol. Pharni. 1: 19-26. 9. Van den Berg, M. L. Birnbaum, A. T. C. Bosveld, B. Brunstrom. P. Cook, M. Freely, J. P. Giesey, A. Hanberg, R. Hasegawa, S. W. Kennedy, T. Kubjak, J. C. Larsen. R. X. R. van Leeuwen, A. K. D. Liem, C. Nolt, R. E. Peterson, L. Poeliinger, S. Sale, D. Schrenk, D. Tillitt, M. Tysklnd, M. Vounes, F. \\aern, and T. Zacharewski. 1998. Environ. Health Perspecl. 106: 77S-792. 10 Long, E. R., D. D. MacDonald, S. L. Smith, and F. D. Calder. 1995. Environ. Manage. 19: 81-97. 11. U.S. EPA. 1993. P. \x in Interim Rc/'ort on Data and Methods of 2.1,7,8 tetrachlorodibenzo-p-dioxin Risks to Ai/uatic Life and Associ- ated Wildlife. EPA-600-R-93-089. Environmental Protection Agency. Washington, DC. 12. Stegeman, J. .)., B. R. \\oodin, H. Singh, M. F. Olesiak, and M. Celander. 1997. G-;»/>. Binchem. Plminl. 116C(1): 61-75. Reference: Biol. Bull 197: 307. (October IW4) Published By Title Only Ku/ii i.in. Alan, and John Clay Vang, Stacy, and Oladele Ogunseitan Isolation of K+ channel containing vesicles from squid Cell swarming and ALAD activity in Vibrio alginolyticus: giant axons. testing an environmental selective pressure hypothesis. Simpson, Tracy, Max M. Burger, and William J. Kuhns Furlong, Christopher, David Lawrence, and Ivan Valiela Localization and selectivity of CD44 antigen for a cell Impact of anthropogenic nitrogen loading on phytoplank- population from the marine sponge Microciona prolifera. ton production in Waquoit Bay and Popponessett Bay. 307 MARINE BIOLOGICAL i . -^^ Substantial financial assistance is available for many programs! 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For further information, contact: Carol Hamel, Admissions Coordinator / Marine Biological Laboratory / 7 MBL Street, Woods Hole, MA 02543 email: admissions@mbl.edu / phone: 508-289-7401 THE BIOLOGICAL BULLETIN DECEMBER 1999 DEC 27 1999 Editor Associate Editors Section Editor Online Editors Editorial Board Editorial Office MICHAEL J. GREENBERG Louis E. BURNETT R. ANDREW CAMERON CHARLES D. DERBY MICHAEL LABARBERA SHINYA INOUE. Imaging and Microscopy JAMES A. BLAKE, Keys to Marine Invertebrates of the Woods Hole Region WILLIAM D. COHEN, Marine Models Electronic Record and Compendia PETER B. ARMSTRONG ERNEST S. CHANG THOMAS H. DIETZ RICHARD B. EMLET DAVID EPEL GREGORY HINKLE MAKOTO KOBAYASHI DONAL T. MANAHAN MARGARET McFALL-NcAi MARK W. MILLER TATSUO MOTOKAWA YOSHITAKA NAGAHAMA SHERRY D. PAINTER J. HERBERT WAITE RICHARD K. ZIMMER PAMELA CLAPP HINKLE VICTORIA R. GIBSON CAROL SCHACHINGER PATRICIA BURNS The Whitney Laboratory, University of Florida Grice Marine Biological Laboratory, College of Charleston California Institute of Technology Georgia State University University of Chicago Marine Biological Laboratory ENSR Marine & Coastal Center, Woods Hole Hunter College, City University of New York University of California, Davis Bodega Marine Lab., University of California, Davis Louisiana State University Oregon Institute of Marine Biology, Univ. of Oregon Hopkins Marine Station, Stanford University Cereon Genomics, Cambridge, Massachusetts Hiroshima University of Economics, Japan University of Southern California Kewalo Marine Laboratory, University of Hawaii Institute of Neurobiology, University of Puerto Rico Tokyo Institute of Technology, Japan National Institute for Basic Biology, Japan Marine Biomed. Inst., Univ. of Texas Medical Branch University of California, Santa Barbara University of California, Los Angeles Managing Editor Staff Editor Editorial Associate Subscription & Advertising Secretary Published by MARINE BIOLOGICAL LABORATORY WOODS HOLE, MASSACHUSETTS Cover The polychaete worm Chaetoptenis is found worldwide in littoral sediments, where it builds and inhabits a U-shaped, parchment-like tube. Seawater is pumped through the tube by "fans" — modified appendages on three adjacent mid-body segments. Microscopic food particles suspended in the seawa- ter current are caught in a mucous net that the worm secretes, deploys, and then (when the net is full) ingests. Chaetoptenis has highly specialized seg- ments that are grouped in three functionally distinct regions, so the morphology is more complex than that of most annelids. This complexity is evident in the two adults shown in the large photograph on the cover; note that because the worms are designed to fit within the curve of the tube, they curl up when removed from it (anterior is up). Chaetoptenis is well-known for studies of its feeding behavior, its intense luminescence, as well as its development. In particular, this worm is a classic model for the study of early embryogenesis because it can be readily spawned in vitro and also cultured through its larval development. As a rule, annelidan segments are generated from a posterior growth zone, arising and developing sequentially, from anterior to posterior. But morphogenesis in Chaetoptenis does not follow this canonical pat- tern. Rather, an examination of larval ontogeny- reported by Steve Irvine and his colleagues in this issue (p. 319) — reveals that those segments destined for the mid-body region develop precociously and more rapidly than the anterior segments. This ac- celerated development (or heterochrony) is not seen in other polychaetes — not even in very closely re- lated families. Thus it must have arisen within the chaetopterid lineage. [Segmental development is shown in the small photographs on the cover; they depict (left to right) an early larval stage (4 days old, 180 jLim), a mid-stage larva (about 30 days old. 400 jam), and a newly metamorphosed juvenile (about 60 days old; 1.2 mm).] Further information on the laboratory culture of Chuetoptenix larvae and those of related spionidan polychaetes is available in a companion article by S. Q. Irvine and M. Q. Martindale published in the Marine Models Electronic Record (MMER) at: www.mbl.edu/html/BB/MMER/IRV/IrvTit.html.In addition, a general review of Chaetoptenis as a system for studying developmental biology, includ- ing methodology and a comprehensive bibliogra- phy, is set out by W. R. Eckberg and S. D. Hill (1996) in another MMER paper at; http://www. mbl.edu/html/BB/MMER/ECK/EckTit.html. Cover h Beth Liles CONTENTS VOLUME 197, No. 3: DECEMBER 1999 RESEARCH NOTE Kelrnan, Dovi, and Richard B. Emlet Swimming and buoyancy in ontogenetic stages of the cushion star Ptemxtn' tessflatus (Echinodermata: Aster- oidea) and their implications for distribution and movement 309 Yusa, Yoichi, and Shigeyuki Yamato (.topping of sea anemone tentacles by a symbiotic barnacle 315 DEVELOPMENT AND REPRODUCTION Irvine, Steven Q., Oleg Chaga, and Mark Q. Martindale Larval ontogenetic stages of Chai:ll the response of rock crabs, Canrer imtratus, to prey odors following feeding experience 36 PHYSIOLOGY Silverman, Harold, John W. Lynn, Peter G. Beninger, and Thomas H. Dietz The role of latero-frontal cirri in particle capture bv the gills of A/V/////S i'ltr>nii\ 275 Biswas, Chhanda, and Peter B. Armstrong Identification of a hemolytic activitv in the plasma of the gastropod HIO\I'IHI nntnUiiiliitniii 276 Kuhns, William J., Max M. Burger, and Eva Ttirley Hvaluronic acid: a component of the aggregation factor secreted by the marine sponge, Micnn IOIKI /n<>- li/i'i/i 277 Popescu, Octavian, Rey Interior, Gradimir Misevic, Max M. Burger, and William J. Kuhns Biosynthesis of tyrosine O-sulfate bv cell proteoglycan from the marine sponge, Mii'iiniiiuii /im/i/nti 279 Vasse, Aimee, Alice Child, and Norman Wainwright Prophenoloxidase is not activated b\' microbial sig- nals in I.iimiliis polyphemus 281 Ogunseitan, O.A., S.L. Yang, and E. Scheinbach The 8-aminolevulinate dehydratase ol marine \'ilnn> i//tf/i/ii/\//i n\ is resistant to lead (Pb) 283 Hoskin. Francis C.G., Diane M. Sleeves, and John E. Walker Substituted cvdodcxtrin as a model lor a squid en- /vme lli.it hyclrolyzes the neiTe gas soman 284 Zigman, Seymour, Nancy S. Rafferty, Keen A. Rafferty, and Nathaniel Lewis Effects of green tea polyphenols on lens photooxida- liu- si i ess . . 285 CONTENTS: VOLUME 197 ECOLOGY A\I> EVOLUTION Mondnip. Thomas Salinity effects on nutrient dynamics in estuarine sediment investigated bv a plug-flux method 287 Pease, Katherine M., L. Claessens, C. Hopkinson, E. Rastetter, J. Vallino, and N. Kilham Ipswich River nutrient dynamics: preliminary assess- ment of a simple nitrogen-processing model 289 Wolfe, Felisa L., Kevin D. Kroeger, and Ivan Valiela Increased lability of estuarine dissolved organic ni- trogen from urbanized watersheds 290 Evgenidou, A., A. Konlde, A. D'Ambrosio, A. Corcoran, J. Bowen, E. Brown, D. Corcoran, C. Dearholt, S. Fern, A. Lamb, J. Michalowsky, I. Ruegg, and J. Cebrian Effects of increased nitrogen loading on the abun- dance of diatoms and dinoflagellates in estuarine phytoplanktonic communities 292 Cubbage. Andrea, David Lawrence, Gabrielle Tomasky, and Ivan Valiela Relationship of reproductive output in Amrtiii tonsa, chlorophyll concentration, and land-derived nitrogen loads in estuaries in Waquoit Bav, Massachusetts 294 Canfield, Susannah, Luc Claessens, Charles Hopkinson Jr., Edward Rastetter, and Joseph Vallino Long-term effei I ol municipal water use on the watei budget of the Ipswich River Basin 295 Young, Talia, Sharon Komarow, Linda Deegan, and Robert Garritt Population si/e and summer home range of the green crab, < .nniiim iiiciiiin.'t, in salt marsh tidal creeks 297 Komarow, Sharon, Talia Young, Linda Deegan, and Robert Garritt Influence of marsh flooding on the abundance and growth of Eiiinliilin In-trim lilu.', in salt marsh creeks . . . 299 Widener, Justin W., and Robert B. Barlow Decline of a horseshoe crab population on Cape Cod. 300 Kerr, Lisa M., Phillip S. Lobel, and J. Mark Ingoglia Evaluation of a reporter gene system biomarker for detecting contamination in tropical marine sediments. 303 ORAL PRESENT A T/U.\\ Pi 'Hl.KHED BY TlTUC (>\l > . 307 No. 3, DECEMBER 1999 RESEARCH NOTE Kelman, Dovi, and Richard B. Emlet Swimming and buoyancy in ontogenetic stages of the cushion star Pteraster tesselatus (Echinodermata: Aster- oidea) and their implications for distribution and movement 309 Yusa, Yoichi, and Shigeyuki Yamato Cropping of sea anemone tentacles by a symbiotic barnacle 315 DEVELOPMENT AND REPRODUCTION Irvine, Steven Q., Oleg Chaga, and Mark Q. Martindale Larval ontogenetic stages ot Chaetopterus: develop- mental heterochronv in the evolution of chaetop- terid polvchaetes 319 Degnan, Bernard M., and Craig R. Johnson Inhibition of settlement and metamorphosis of the ascidian Hrrdmania cunxitn by non-geniculate coral- line algae 332 Kanungo, Jyotshna, Ruth M. Empson, and Howard Ras- mussen Microinjection of an antibody to the Ku protein ar- rests development in sea urchin embryos 341 NEUROBIOLOGY AND BEHAVIOR Lindsay, S.M., T.M. Frank, J. Kent, J.C. Partridge, and M.I. Latz Spectral sensitivity of vision and bioluminescence in the midwater shrimp Sergestes similis 348 Ristvey, Andrew, and Steve Rebach Enhancement of the response of rock crabs, Ciiim-i itroratus, to prey odors following feeding experience 361 PHYSIOLOGY Silverman, Harold. John W. Lynn, Peter G. Beninger. and Thomas H. Dietz The role of latero-frontal cirri in particle capture by the gills of M\tilii-'i rdulis 368 Bayne, Brian L., Susanne Svensson, and John A. Nell The physiological basis for faster growth in the Svd- ne\r rock oyster, Sarrostrea comint'rcittlif> 377 Siebenaller, Joseph F., and Thomas F. Murray Hydrostatic pressure alters the time course of GTP[S] binding to G proteins in brain membranes from two congeneric marine fishes 388 CELL BIOLOGY Lema-Foley, Christine, Kyeng G. Lee, Tchaiko Parris, Zoya Koroleva, Nishal Mohan, Pierre Noailles, and Wil- liam D. Cohen Reversible alteration of morphology in an inverte- brate erythrocyte: properties of I he natural inducer and the cellular response 395 Index for Volume 197 415 CONTENTS: VOLUME 197 Notice to Subscribers 2000 SUBSCRIPTION RATES FOR THE BIOLOGICAL BULLETIN Per year (six issues, two volumes): Per volume (three issues): Back and single issues: (subject to availability) Libraries $225.00 $112.50 $ 40.00 Individuals $95.00 $47.50 $20.00 For additional information, please contact our subscription manager at the Marine Biological Laboratory, 7 MBL Street. Woods Hole. MA 02543: tel: (508) 289-7402; e-mail: subscriptions© mbl.edu. Reference: Biol. Bull. 197: 309-314. (December 1999) Swimming and Buoyancy in Ontogenetic Stages of the Cushion Star Pteraster tesselatus (Echinodermata: Asteroidea) and Their Implications for Distribution and Movement DOVI KELMAN1 AND RICHARD B. EM LET2 * 1 Department of Zoology, George S. Wise Faculty of Life Sciences. Tel-Aviv University, Ramat Aviv. Tel Aviv 69978, ISRAEL: and : Institute of Marine Biology and Department of Biology, University of Oregon, Charleston, Oregon 97420 The eggs of some marine fish (1) and benthic inverte- brates such as many corals (2. 3) and lecithotrophic echi- noderms (4, 5) are positively buoyant at time of release front the parent, and density increases later in ontogeny. How these eggs and lan'ae are distributed in the water column and eventually reach suitable habitat for settlement will depend, in part, on their vertical velocity and on the turbu- lence in the water (i.e., the eddy diffusivity). For eggs and unhatched stages, vertical velocity is passive and depends on egg or embryonic volume and density relative to the seawater (6. 7). For motile stages, vertical velocity depends on relative density, swimming ability, and behavior of the lan'ae (8, 9). We have measured the vertical velocity of eggs and lan'ae of the sea star Pteraster tesselatus Ives, which spawns floating eggs (1.1 to 1.5 mm diameter) that develop into nonfeeding larvae and spend several weeks in flic plankton before settling to the benthos (10). Because of the simple shapes of eggs and lan'ae, we used force balance equations for drag and buoyant forces to determine the density of eggs and t\ro lan'al stages. Initially the eggs were positively buoyant and floated upwards at about I mm/s. Even formalin-fixed eggs floated in seawater, so concentra- tions of light ions were not responsible for the buoyancy. The density of the lan'ae increased in the first 10 to II days, but it varied considerably between the three larval cohorts examined. Ten-day-old lan'ae that were negatively buoyant swam downward at mean speeds as high as 1. 7 mm/s, while Received 24 May 1999; accepted 16 August 1999. * To whom correspondence should be addressed. E-mail: remleKs1 oregon.uoregon.edu positively buoyant lan'ae of the same age swam upward in about I mm/s. These patterns of buoyancy and swimming velocity should initially facilitate dispersion and later pro- mote settlement into subtidal habitats. Position in the water column and relative to the sea bottom will determine the amount of advection and the likelihood of encountering suitable habitat for settlement; variation in vertical position between related propagules will increase the spread of siblings or species (9. 11. 12). Predicting the depth distribution of eggs and larvae and other planktonic organisms requires information on their vertical velocity and on the turbulence structure of the water column (6-9). Alternatively, if the vertical velocity and distribution of propagules are known, one can estimate the turbulence structure (6). Many studies examine the density offish eggs, and some relate this to their vertical distribution (e.g., 6, 7, 13, 14). For larvae of benthic invertebrates, studies on swimming behavior and responses to environ- mental cues dominate discussions of vertical distribution (see reviews 15-17). With the exception of numerous stud- ies on crustacean meroplankton (e.g., 18, 19), there are few observations on vertical distribution as a function of stage of development or on density of the eggs and larvae of benthic invertebrates. Medeiros-Bergen et al. (20) showed that the lecithotrophic larvae of several sea cucumber species, which are released as positively buoyant eggs, can be distributed to depths exceeding 50 m, though they were most common in the upper 20 m of the water column. Young and Cameron (21) measured the rate of rise of positively buoyant eggs of the bathyl echinoid Phormosoma placenta, calculated the density of the eggs, and predicted that these embryos would 309 310 D. KELMAN AND R. B. EMLET u I P •* k Cohort A Cohort B Cohort C -r 1 f -r ft s* C 1 ^N 0 I •± ^i II S E 1 £ Dg b II ^i ll b. * >s 0 fS;i | II -p TT |:|x|: j o o e g b e T e g bu (U T ^ 1 ** -1 I \ ( ! Down _2 - Figure 1. Rising and sinking rates of eggs with jelly coats (e), hatched gastrulae (g), and bilobed larvae (b) from three cohorts of Pterasler tesselatus. The open bars represent passive movement of eggs or deciliated larvae, and the shaded bars represent swimming larvae. The value on each bar represents the mean from 10 or 12 eggs, gastrulae, or larvae. Error bars are 1 S.E. bu, bilobed larvae swimming up; bd, bilohed larvae swimming down. We collected adult Pteraste r tesselatus Ives near San Juan Island. Washington (USA), in May 1998. Adult sea stars were induced to spawn by intracoelomic injection of 2-3 ml of 100 pM 1-methyladenine (4). We collected the spawned eggs from each of three females and washed them in filtered seawater (FSW). Larval stages that we studied were from eggs exposed to sperm at low concentrations. We do not know whether the sperm fertilized the eggs or was necessary for development, as oocytes of this species have been reported to develop without sperm (see Ref. 10). Vertical speeds were measured in a cylindrical chamber (10 cm in diameter, and 15 cm tall) filled to within 2 cm of the top with 1 liter FSW (salinity. 289fr) and marked with graduations that circled the chamber at 2-cm intervals. The chamber was covered and immersed in an insulated aquarium maintained by a circulating water bath at a temperature of 12.2° ( ±0. 1°C) to minimize convection currents that might affect the movement of the eggs and larvae in the chamber. Positively buoyant eggs and larvae that floated or swam up were introduced to the center of the chamber bottom. Negatively buoyant larvae and those that swam down were released at the surface in the center of the chamber. Rising and sinking times were measured for two successive 2-cm intervals in the middle of the water column where wall effects were minimal. Measurements that differed more than 10% were discarded. The individual was then repositioned at the bottom (or top) and allowed to rise (or sink) again; the new times were recorded. The average time to rise or sink 2 cm was calculated and used to determine speed. rise into the warmer surface waters where their rate of development would be increased. Measurement of vertical velocities of invertebrate eggs and swimming larvae is relatively simple and can be used to determine ontogenetic changes in velocity and density as well as to predict distributions of propagules in the field. Measurements on multiple cohorts can reveal variation among offspring from different parents. Among echino- derms, lecithotrophic larval development has evolved re- peatedly from planktotrophic larval development (22-24), and many of the lineages with derived, nonfeeding larval development also have large, positively buoyant eggs that have evolved from negatively buoyant ones (5). Shifts in egg and embryonic buoyancy may require functional changes in swimming that are revealed by observations on vertical movement and orientation. For three cohorts (from separate female parents), we measured the vertical velocities of unfertilized eggs, newly hatched gastrulae (5 d), and older, bilobed larvae ( 10-1 1 d). After swimming velocities were measured for individual larvae, they were deciliated in hypertonic seawater and their vertical velocity was measured again and used to determine the density of these stages. Eggs (with intact jelly coats) always floated up, rising at speeds between 0.2 and 1.3 mm/s. The mean rising rates of eggs were 1.2, 0.4, and 1.0 mm/s for cohorts A, B, and C, respectively (Fig. 1 ). An ANOVA followed by a multiple comparisons test indicated that the rising rates of eggs were SWIMMING AND BUOYANCY IN A YOLKY LARVA 311 significantly different among cohorts (F = 135.6, df 2,27, P < 0.001; Tukey HSD test./) < 0.001 for all three pairwise comparisons). Differences in egg rising rates among cohorts were due to differences in egg density and in part to differences in the egg and jelly coat volumes (Fig. 2a). Cohorts had signifi- cantly different egg densities ( ANOVA. F = 1 25.6, df 2.27. P < 0.001; Tukey HSD test, P < 0.001 between all three pairwise comparisons). Eggs of cohort A, with the fastest mean rising rate, also had the lowest density (1020.2 kg/ m3); eggs of cohort B, with the slowest rising rate, had the highest density (1021.4 kg/m3). Comparisons of the egg diameter and jelly coat thickness revealed significant vari- ation among the cohorts in each trait (egg diameter ANOVA, F ' 125.3. df 2,27, P < 0.001; jelly coat ANOVA, F = 23.02. df 2.27, P < 0.001 ). However, mul- tiple comparisons (Tukey HSD tests) revealed that cohorts A and B each differed from cohort C in egg diameter (P < 0.001 ) and jelly coat thickness (P < 0.001 ). but A and B were not significantly different from each other in egg diameter (P = 0.76) or jelly coat thickness (P = 0.15). Thus differences in rising rates between cohorts A and B were due to differences in egg density, whereas differences be- tween cohorts A and C and cohorts B and C were due to both density and dimensions of the eggs. Though eggs of cohort C had an intermediate density, their relatively large diameters and thin jelly coats contributed to their rising rates, which approached those of cohort A. To determine whether active metabolic concentration of light ions was a possible buoyancy mechanism in the eggs of Pteraster, we fixed eggs in 3% formalin in filtered seawater (FSW) for several hours and then transferred them to fresh FSW. Formalin-fixed eggs floated, indicating that their positive buoyancy did not involve the metabolic mech- anisms that maintain ion gradients (e.g., 25, 26). Positive buoyancy may also be due to the presence of buoyant lipid reserves. According to Jaeckle (27), lipid content in leci- thotrophic eggs of echinoderms ranges between 34% and 50% of total organic weight and is twice that found in planktotrophic eggs. A high lipid content could reduce the densities of the eggs and cause them to float. No data are available on the biochemical composition of P. tesselatus eggs and larvae or on the possible importance of lipid content in floating. For another estimate of egg density, we assumed that the jelly coats were neutrally buoyant in seawater (Fig. 2a). Because the jelly coat has a very high water content, we believe this assumption was reasonable. Furthermore, the jelly coats of eggs of the sand dollar Dendraster cxcentricits are neutrally buoyant (28). The estimated densities of eggs without jelly coats were also significantly different among cohorts (ANOVA. F = 159.4, df 2.27. P < 0.001; Tukey HSD test, P < 0.001 between all three pairwise compari- sons). Differences in egg density may be due to variation in biochemical composition and could also reflect variation in egg or maternal nutritional state. The estimated densities of the eggs without jelly coats were lower by 0.4 kg/m3 (range. 0.7 to 0.2 kg/m1) than those for the eggs with jelly coats. If the jelly coat is neutrally buoyant, it slows the rising rate of the buoyant egg. We calculated that the mean rising rates of eggs without jelly coats would be 12%, 15%. and 7% faster than those measured for intact eggs of cohorts A, B, and C respectively. Eggs hatched after 3 to 4 d, and by day 5 swimming gastrulae were either at the water surface or at the bottom of their mesh-bottom culture vessels, depending on the female of origin. Gastrulae in cohorts A and C swam up at mean speeds of 1 .4 and 1 .2 mm/s respectively. When deciliated. all gastrulae in these cohorts were positively buoyant, rising at mean speeds of 0.9 mm/s (cohort A) and 0.3 mm/s (cohort C). In contrast, all gastrulae of cohort B swam down at a mean speed of 1.1 mm/s; when deciliated. 7 of 10 gastrulae were negatively buoyant and three others were slightly positively buoyant. The mean sinking speed of deciliated gastrulae from cohort B was 0.6 mm/s (see Fig. I ). Larvae swam up or down, usually along a straight, ver- tical path, and rotated around their anterior-posterior axes as they swam. Regardless of its direction of vertical motion, a gastrula always had its anterior end up and its posterior end, with blastopore, down. Deciliated gastrulae also showed this orientation whether they rose or sank. This posture was assumed as soon as a swimming gastrula was placed in the stable water column and was maintained, without exception. as long as the larva was moving in the water column. This orientation appeared to result from an uneven distribution of buoyancy, with the anterior end being less dense than the posterior end. Because swimming speeds downward ex- ceeded sinking speeds, the cilia must have produced cur- rents that moved water from the posterior end toward the opposite end. The downward movement was sustained and was not likely to result from the transient reversal of ciliary beat that is known for planktotrophic larvae (29; Emlet, pers. obs.). The consistent and sustained downward swim- ming by gastrulae of cohort B, with the blastopore leading, indicates that the coordination of their cilia was different from that of negatively buoyant gastrulae that swim up. anterior end first, by moving water from anterior to poste- rior. This change in ciliary coordination from that typical of planktotrophic species has also been observed in down- ward-swimming, positively buoyant, lecithotrophic larvae of the echinoids Heliociilaris ery thro gramma and Holo- pneustes piirpurascens (Emlet, pers. obs.). Larvae developed into a bilobed stage, with a circumfer- ential groove that divided the larval body into anterior and posterior regions. By 10-1 1 d after fertilization, podia were beginning to form within the circumferential groove, but most larvae were still shaped like prolate spheroids and 312 D. KELMAN AND R. B. EMLET 1022 -i 1021.4 1021 1 1020.9 10207 Cohort Figure 2. Density of unfertilized eggs (a) and eggs and larvae (b) for three cohorts of Pteraster tesselatus. (a) Two estimates of density: open bars are for eggs with jelly coats; dark bars are for eggs only, assuming the jelly to be neutrally buoyant. The value on each bar represents the mean for 10 eggs. Error bars are 1 S.E. (b) Ontogenetic changes in density from eggs and two larval stages for three cohorts. The bars represent the means for eggs with jelly coats (e). hatched gastrulae (g). and bilobed larvae (b). All sample sizes were lOeggsor 10-12 larvae. Error bars are 1 S.E. The dotted line shows the density of seawater at 12.2°C and 28f?< salinity. The Reynolds numbers (Re) were £0.8 for all eggs and <0.5 for all passively moving larvae, so we used the low-Re equation for terminal velocity and solved for the density of the egg or larva (see e.g., 31, equation 15.1 I. p. 340). We treated eggs as spheres and larvae as prolate spheroids; in the latter case we included a shape-correction term (see 32). We measured the diameter of each egg and the thickness of the jelly coat on a microscope with a 4x objective, after the egg rising time was measured. A suspension of India ink was added to reveal the edge of the translucent jelly coat. The length and width of gastrulae and larvae at their widest point were measured after larval swimming times were measured. Each larva was then deciliated by placing it in double-strength seawater for 10-15 s, then immediately rinsed three times in normal-strength seawater. This method has been used to collect cilia from echinoderm larvae and does not kill the larva; in fact, cilia are regenerated in a matter of hours (e.g.. 33; Emlet. pers. obs.). After the deciliated larvae were equilibrated in seawater at 28%c, we placed them individually in the chamber and measured their passive rising or sinking rates. The seawater in the chamber was at 12.2°C and had a salinity of 2X'..'.; from tables (34) we determined its viscosity to be 0.0013 N s/nr and Us density to he 1022 kg/m1. Sources of error in our calculations of the densities of eggs and larvae include our measurements of dimensions (including jelly coat for eggs) and our assumptions of shape (prolate spheroid or spheres). Measurement errors of 100 /j.m (7%-10% of egg + jelly diameters) would result in deviations of density of £0.5 kg/m'. These errors would be random and were not as advanced as the 8-day larva in figure 3 of McEdward (10). The swimming direction and buoyancy of larvae varied among the cohorts (Fig. 1). All larvae of cohort A swam up and were positively buoyant after they were deciliated, and all those of cohort B swam down and were negatively buoyant after they were deciliated. Cohort C was highly variable, with 7 of 12 larvae swimming up and 5 of 12 swimming down. Five of the seven larvae of cohort C that swam up were either positively or neutrally buoyant after being deciliated, whereas all of those that swam down were negatively or neutrally buoyant after being deciliated. The exceptions were two larvae that swam up opposite the direction of their passive motion. Compared to gastrulae, the bilobed larvae swam up at slower speeds or down at higher speeds, reflecting the generally increased density of these later stages (Figs. 1 and 2b). Bilobed larvae showed the same orientation that gastrulae did with larval anterior (adult oral) up and posterior (adult aboral) down regardless of their direction of swimming. During development, the density of gastrulae and larvae increased relative to that of the eggs, though cohorts varied in the extent and timing of increase (Fig. 2b). Cohorts A and C showed consistent increases in density, with the bilobed larvae of cohort C obtaining a mean density not different from that of seawater (t test, t = 0.95, df = 1 1. P = 0.362). In contrast, the mean density of gastrulae of cohort B exceeded that of seawater (/ = 3.0. df = 9, P = 0.015), and some of these individuals had higher densities than any other stages across all cohorts (Fig. 2b). Differences in density among eggs (without jelly), gas- trulae. and bilobed larvae were analyzed separately for each cohort because of significant heterogeneity of variances within cohorts A and B but not cohort C (Cochran's C tests, P < 0.01 for cohort A. P < 0.001 for cohort B, P = 0.1 1 for cohort C). Cohorts A and C had significantly different densities among all stages. (Cohort A: Kruskal-Wallis test, H --- 21.8: multiple comparisons with a nonparametric equivalent of the Tukey test (Ref. 30), P < 0.05; cohort C: ANOVA, F = 91.9 df 2.29. P < 0.001: Tukey HSD, P < 0.002 for all comparisons). Cohort B also had significant differences in density among stages (Kruskal-Wallis test, H = 19.9, P < 0.001). Nonparametric pairwise compari- sons indicated that density differed significantly between eggs and gastrulae (P < 0.001 ), between eggs and bilobed larvae (P < 0.005), but not between gastrulae and bilobed larvae (P > 0.5). McEdward (10) observed that eggs and embryos of P. tesselatus were positively buoyant until the time close to should inflate the variation without biasing the means. Our assumption of shape could systematically bias the means through estimates of drag force or volume, but again the magnitude of the error would be about 0.2 kg/m'. We do not think that any of these biases created erroneous trends in the data. SWIMMING AND BUOYANCY IN A YOI.KY LARVA 313 settlement, when larvae swam near the bottom. The present study confirms that observation for two cohorts, but a third cohort was negatively buoyant soon after hatching. The rising speeds of eggs and the swimming speeds of gastrulae and larvae of Pteraster usually exceeded 1 mm/s. Although still slow, these rates are about five times the sinking speeds of planktotrophic eggs and two to three times the swimming speeds of planktotrophic larvae of echinoderms (5). Our measurements showed significant increases in density as development progressed (Fig. 2b), with bilobed larvae ap- proaching or exceeding the density of seawater. Becoming less positively buoyant or negatively buoyant may increase downward swimming speed and assist larvae in the search for suitable settlement habitats. The mean downward swimming speed of 1.7 mm/s for bilobed larvae of cohort B should allow them to overcome resuspension by turbulent mixing during some parts ot the tidal cycle. Gross et al. (8) modeled larval settlement in tidally dominated flows found in estuarine and shelf condi- tions, exploring how larval swimming speed influenced the probability of settlement. For a water depth of 50 m, the model predicted that a doubling of fall velocity from 0.8 to 1.6 mm/s resulted in a 12-fold increase in the probability of contacting the bottom throughout the entire tidal cycle. During periods of weak tidal flows, the model predicted that up to 40% of larvae swimming at 1.6 mm/s could interact with the bottom and possibly settle, while 1 % to 4% of those swimming at 0.8 mm/s would be capable of settling (8). Our studies were conducted at a salinity of 28%c and a temperature of 12.2°C, conditions typical of the surface waters near San Juan Island, Washington. Though seawater higher in salinity (and hence more dense) is found in other coastal settings, we believe that our results are applicable to these regions as well. Preliminary observations showed that when deciliated larvae of Pteraster that were negatively buoyant at 28%t were placed in water with a salinity of 30%(, they would initially float for a few minutes before sinking at constant speed. A possible explanation is that the osmotic difference resulted in water loss and salt gain by larvae, restoring the relative density of the larvae in seawa- ter. Further studies that vary salinity would be necessary to determine if the absolute magnitude of the differences be- tween the density of the larvae and the seawater are the same when the salinity changes. Other studies (Emlet, un- pub. data) of buoyancy and swimming in ontogenetic stages of two echinoids at a salinity of 35%c found patterns similar to those reported here. This study has documented ontogenetic changes in buoy- ancy from positive to either neutral or negative buoyancy and found changes in swimming direction and speed that correlate with buoyancy for one species of sea star with positively buoyant eggs. The eggs and larvae exceeded 1 mm in diameter and departed from the density of seawater by as much as 2.0 kg/m3. (For comparison, the density of seawater changes by 2 kg/m3 for each 10°C change in temperature.) Though small, the resulting difference in den- sity caused the eggs and larvae to rise or sink relatively rapidly compared to planktotrophic larvae, and rates of movement were augmented later in development by ciliary swimming. These patterns should initially facilitate disper- sion from the site of egg release and then promote settle- ment. Buoyancy and swimming for specific stages were also found to vary within and between cohorts, which should increase the spread of siblings as well as offspring of conspecifics (12). Finally, the swimming orientation of an- terior up, posterior down that was maintained during devel- opment suggests that the ciliary coordination of positively buoyant larvae has been changed from that of planktotro- phic ancestors. Acknowledgments A. O. D. Willows. Director, provided space and facilities at the Friday Harbor Laboratories. We thank R. Strathmann and J. Hoffman for contributing ideas throughout this study and for valuable comments that improved the manuscript. The manuscript was also improved with helpful comments by O. Mokady and anonymous reviewers. Funding was provided by the Friday Harbor Laboratories Marine Science fund (# 63-3972), an Aharon Katzir Foundation travel grant to D.K, and NSF grant OCE-9416590 to R.B.E. Literature Cited 1 Craik, J. C. A., and S. M. Harvey. 1987. The causes of buoyancy in eggs of marine teleosts. J. Mar. Dial. Assoc. UK 67: 169-182. 2. Harrison, P. L., R. C. Babcock, G. D. Bull, J. K. Oliver, C. C. Wallace, and B. L. Willis. 1984. Mass spawning in tropical reef corals. Science 223: 1 1 86-1 189. 3. Harrison, P. L., and C. C. Wallace. 1990. Reproduction, dispersal, and recruitment of scleractinian corals. Pp. 1 33-207 in Coral Reefs. Z. Dubmsky, ed. Elsevier, Amsterdam. 4. Strathmann, M. F. 1987. Reproduction and Development of Marine Invertebrates of the Northern Pacific Coast. University of Washington Press, Seattle. 5. Emlet. R. B. 1994. Body form and patterns of ciliation in nonfeeding larvae of echinoderms: functional solutions to swimming in the plank- ton? Am. Zoo/. 34: 570-585. 6. Sundbv, S. 1983. A one dimensional model for the vertical distri- bution of pelagic tish eggs in the mixed layer. Deep-Sea Res. 30: 645-661. 7. Sundby, S. 1997. Turbulence and ichthyoplankton: influence on vertical distributions and encounter rates. Sci. Mar. 61 (Suppl. 1): 159-176. 8. Gross, T. F., F. E. Werner, and J. E. Eckman. 1992. Numerical modeling of larval settlement in turbulent bottom boundary layers. J. Mar. Res. 50: 611-642. 9. Eckman, J. E., F. E. Werner, and T. F. Gross. 1994. Modelling some effects of behavior on larval settlement in a turbulent boundary layer. Deep-Sea Rex. II Top. Stud. Oceiiii»t>r. 41: 185-208. 10. McEdvvard, I,. R. 1992. Morphology and development of a unique type of pelagic larvae in the starfish Pteraster tesselatus (Echinoder- mata: Asteroidea). Bio/. Bull. 182: 177-187. 314 D. KELMAN AND R. B. EMLET 1 I Okubo, A. 1980. Diffusion and Ecological Problems: Mathematical Models. Springer- Verlag. Berlin. 12. Strathmann, R. R. 1974. The spread of sibling larvae of sedentary marine invertebrates. Am. Nut. 108: 24-44. 13. Coombs, S. H., C. A. Fosh, and M. A. Keen. 1985. The buoy- ancy and vertical distribution of eggs of sprat (Sprattus sprattus} and pilchard (Sardina pilc/uirdiis). J. Mar. Bio/. Assoc. UK 65: 461-474. 14 Bullman. C. M.. and J. A. Koslow. 1995. Development and depth distribution of the eggs of orange roughy. Holoslethus atlanticiis (Pisces: Trachichthyidae). Mar. Fresh\\: Res. 46: 697-705. 15. Sulkin, S. D. 1984. Behavioral basis of depth regulation in the larvae of brachyuran crabs. Mar. Ecol. Prog. Ser. 15: 181-205. 16. Young, C. M., and F. S. Chia. 1987. Abundance and distribution of pelagic larvae as influenced by predation. behavior, and hydrographic factors. Pp. 385-463 in Reproduction of Marine Invertebrates. Vol. 9, A. C. Giese. J. S. Pearse. and V. B. Pearse, eds. Blackwell Scientific. Palo Alto. California. 17. Young, C. M. 1995. Behavior and locomotion during the dispersal phase of larval life. Pp. 247-277 in Ecology of Marine Invertebrate Larvae. L. McEdward. ed. CRC Press, Boca Raton. Florida. IS. Shanks, A. L. 1986. Vertical migration and cross-shelf dispersal of larval Cancer spp. and Randallin ornata (Crustacea: Briichyu.ru) off the coast of southern California. Mar. Riol. 92: 189-199. I1). DeVries, M. C., R. A. Tankersley, R. B. Forward, Jr.. W. W. Kirby-Smith. and R. A. Luettich. Jr. 1994. Abundance of estua- nne crab larvae is associated with tidal hydrologic variables. Mar. Biol. 118: 403-413. 20. Medeiros-Bergen, IX E., R. R. Olson, J. A. Conroy, and T. I). Kocher. 1995. Distribution of holothurian larvae determined with species-specific genetic probes. Limnol. Oceanogr. 40: 1225-1235. 21. Young, C. M., and J. L. Cameron. 1987. Laboratory and in xttu floating rates of lecithotrophie eggs from the bathyl echinoid Phormo- xo/na placenta. Deep-Sea Res. 34: 1629-1639. 22. Strathmann, R. R. 1978. The evolution and loss of feeding larval stages of marine invertebrates. Evolution 32: 894-906. 23. Emlet, R. B. 1990. World patterns of developmental mode in echinoid echinoderms. Pp. 329-334 in Ativancex in Invertebrate Reproduction Vol. 5, M. Hoshi and O. Yamashita. eds. Elsevier, Amsterdam. 24. Wray, G. A. 1995. Evolution of larvae and developmental modes. Pp. 413-447 in Ecology of Marine Invertebrate Lunae, L. McEd- ward. ed. CRC Press, Boca Raton. Florida. 25. Lambert, C. C., and G. Lambert. 1978. Tunicate eggs utilize ammonium ions for floatation. Science 200: 64-65. 26. Newton, C., and W. T. W. Potts. 1993. Ionic regulation and buoy- ancy in some planktonic organisms. ./. Mar. Biol. Axxoc. ('A' 73: 15-23. 27. Jaeckle, W. B. 1995. Variation in the si/e, energy content, and composition of invertebrate eggs: correlates to the mode of larval development. Pp. 49-77 in Ecology of Marine Invertebrate Larvae. L. McEdward. ed. CRC Press, Boca Raton. Florida. 28. Podolsky, R. D. 1995. Consequences of temperature, viscosity, and small size for early life-history processes in the sand dollar Denclraster excenlricus. Ph.D. dissertation. University of Washington. Seattle. 29. Stralhmann, R. R. 1971. The feeding behavior of planktotrophic echinodcrm larvae: mechanisms, regulation and rates of suspension- feeding. J. E.\p. Mar. Biol. Ecol. 6: 109-160. 30. Zar, J. H. 1984. Biostali.stical Analysis. Prentice Hall. Englewood Cliffs, New Jersey. 31. Vogel, S. 1994. Life in Moving Fluid*,. The Physical Biology of Flow. Princeton University Press. Princeton. 32. Happel. J., and Brenner, H. 1986. Low Reynolds Number Hvdro- dynamicx. Martin Nijhoff. Dordrecht, The Netherlands. 33. Stephens, R. E. 1986. Isolation of embryonic cilia and sperm fla- gella. Pp. 217-227 in Methods in Cell Biology. Vol. 27: Eclunodcnii Gametes and Embryos, T. E. Schroeder. ed. Academic Press. Orlando, Florida. 34. Walton Smith, F. G., and F. A. Kalber, eds. 1974. CRC Handbook of Marine Science. CRC Press. Cleveland. Ohio. Reference: Binl. Bull. 197: 315-318. (December 1499) Cropping of Sea Anemone Tentacles by a Symbiotic Barnacle YOICHI YUSA* AND SHIGEYUKI YAMATO Seto Marine Biological Laboratory, Kyoto University. Shirahama, Wakayama 649-221 1, Japan As sessile animals, barnacles (Cirripedia: Tlwracica) are generally suspension feeders, extending their cirri into the surrounding water to collect food particles ( 1 ). Although it has been suggested that some symbiotic barnacles obtain nutrients directl\ from their hosts, either b\ absorbing body fluids (2 — t) or by rasping the host's tissue (4-6), most of these cases are inferred from their morphology. Direct evidence, such as gut content analysis, has been limited (for an exception, see ref. 5), and no actual feeding on their hosts has been absented. Koleolepas avis (Hiro, 1931} is a pedunculate barnacle symbiotic with the sea anemone Cal- liactis japonicu, which lives on gastropod shells occupied b\ large hermit crabs ( 7), mainly Dardanus arrosor. Symbiotic- relationships bet\\'een various hermit crabs and sea anem- ones have been well documented (8), but the relationship benreen the barnacle and its host sea anemone has been virtuallv unknown. From February to April 1996, we col- lected living individuals o/K. avis from lobster nets landed at Minabe Fishery Port, southwestern Japan (33° 44' N, 135° 20' E). On the basis of behavioral obsen'ations in the laboratory and analyses of fecal pellets and gut contents, we concluded that this barnacle feeds actively on its host's tentacles. The shape of Koleolepas avis (Fig. 1 ) differs from that of typical pedunculate barnacles like Lepas spp. in many ways (7). First, K. avis, like its two congeners K. willevi Stebbing (9) and K. tinkeri Edmondson (10), has a sheath-like struc- ture extending from the base of the peduncle and covering the main body (the attachment disk). Second, at the orifice, A', avis has a chitinous, bill-like projection that is developed only in this species. Hiro (7) stated that this projection gives Received 12 April 1999; accepted 28 September 1999. * Author to whom correspondence should be addressed. Present address: Laboratory of Insect Ecology, Kyushu National Agricultural Experiment Station, Nishigoshi. Kumamoto 861-1192, Japan. E-mail: yusa@knaes. affrc.go.jp "the cirriped a bird-bill-shape." These two characteristics are reflected in the generic and specific names, respectively ("koleo" means sheath and "avis" means bird). Third, the peduncle is highly distensible, being about three times as long as the length of the capitulum. In addition to these external characteristics, the internal morphology is also distinct. The cirri are short, with sparse setae, and thus unsuitable for filter feeding, and the cutting edge of the mandible is peculiarly serrate (7). Observations on living individuals showed that the at- tachment disk of K. avis was interposed between the pedal disk of its host anemone and the gastropod shell. Most of the barnacle's main body was retracted into the attachment disk, and only the upper part of the capitulum was visible from outside of the host (Fig. 2 A). A rhythmical cirral movement could be seen through the semitransparent capitulum, but otherwise the barnacle usually remained motionless. When tentacles of the sea anemone touched K. avis (presumably its cirri), the capitulum of the barnacle imme- diately came out from the gap under the host and began to follow the tentacles (Fig. 2B). This tentacle-following be- havior lasted 89 ± 122 s (mean ± SD of 46 observations on 10 individuals). During this period, the cirri were widely spread and almost motionless. When the cirri of K. avis touched a tentacle of its host again, the barnacle grabbed it with its cirri, pulled part of it into the capitulum, and closed the orifice firmly. At the same time the peduncle shrank and bent frontally (to the direction of the orifice). As a result, the tentacle became pinched by the upper ridge of the bill-like projection of A", avis (Fig. 2C). In response to the pinching, the sea anemone contracted its tentacles. Pulled from the both ends, the pinched tentacle tore off (Fig. 2D). This tentacle pulling lasted 109 ± 95 s. A barnacle cropped a tentacle 1.4 ± 1.4 times a day (mean ± SD of 10 individuals). We did not observe the barnacles eat other parts of the host, nor other possible foods 315 316 Y. YUSA AND S. YAMATO M Figure 1. A'»/<'o/ty>. Bernice P. Bishop Mus. 20: 183-243. 1 1 . Fautin, D. G., C-C Guo, and J-S Hwang. 1995. Costs and benefits of the symbiosis between the anemoneshrimp Periclimenes brevicar- pulis and its host Entacmaea i/nadricolor. Mai: Ecol. Prog. Ser. 129: 77-84. 12. Mercier, A., and J. F. Hamcl. 1994. Deleterious effects of a pyc- nogonid on the sea anemone Bartho/omea annulata. Can. J. Zool. 72: 1362-1364. 13. Bieri, R. 1966. Feeding preferences and rates of the snail, lanthina prolongata, the barnacle, Lepas anserifera, the nudibranchs. Glaums ».-•• • H \ , Ihc 1 — -pyg hg \ pyg at st mg -mt hg- — pyg Figure 2. Larvae, stages LI through L3. Anterior is toward the top of the page in all plates, unless noted, (a) Stage LI protrochophore at 24 h. (b. c) Stage L2 metatrochophor.es at 4S h in ventral and left-lateral views respectively, (d-f) Early stage L3 larvae at 4 days in dorsal, lateral, and ventral views respectively, (g-i) Late stage L3 larvae at 18 days in dorsal, left-lateral, and ventral views respectively. Medial pair of eyes is slightly out of focus in (g) and at dorsal surface in (h) (open arrowheads). Unicel- lular ingested algae are visible in the midgut at all stages, at, apical tuft; hg, hindgut: Ih. lateral bristle; Ihc, lateral hooked cilia; lo, larval ocellus; mg, midgut: mt. metatroch; pyg. pygidium; st. stomndeum. Scale bars are 50 /am. typical of early polychaete trochophore larvae formed (Henry, 1986; Eckberg and Hill, 1996). Stage L2— 36-72 hours (Fig. 2b-c): Metatrochophore Size: 180 X 90 Mm Ciliary structures: apical tuft present; one pair of lateral bristles form; trochal band absent CHAETOPTERUS LARVAL ONTOGENY 323 Ocelli: 2; one lateral pair Reference*: Henry ( 1986) fig. 3b-c; Bonch-Bruevich and Malakhov (1987) fig. 1 By this stage gastrulation is complete, forming a tripartite gut. A ventrally opening ciliated stomodeum is visible an- teriorly. The more medial stomach occupies about half the volume of the larva, and a much smaller intestine is located just anterior to the pygidium. The anus opens dorsally. The gut is functional at this time, as evidenced by algal particles in the stomach. A pair of stiff lateral hooked bristles, com- posed of hooked cilia, take the place of the lateral hooked cilia (Henry, 1986). A distinct trochal band is not present at this stage. Stage L3— 3-30 days (Fig. 2d-i): Size: 180-320 X 90-180 /am Ciliary structures: apical tuft present; mesotroch present; lateral bristles persist Ocelli: early: 2; one lateral pair: late: 4; one lateral pair and one medial pair References: early: Cazaux (1965) pit. 2. fig. 5; late: Cazaux (1965) pit. 3, fig. 6; Enders (1909) pit. II. fig. 9 Earl\ period, 3-10 days. In this period the relative size of the stomach enlarges to occupy most of the larva. A more distinct pygidial papilla forms. A distinct trochal band is first visible at the level of the intestine, here referred to as a mesotroch, following the terminology of Okada (1957). The lateral bristles of Stage L2 persist. In histological sections the neuropil of the cerebral gan- glion is visible anterior and dorsal to the stomodeal opening (Fig. 3a, b). At this stage other neural tissues were not visible in section, although a ventral nerve network has been reported in slightly younger specimens examined with transmission electron microscopy (Bonch-Bruevich and Malakhov, 1987). The stomodeum itself has three dorsal diverticulae and opens to the stomach through a pharyngeal valve in the midposterior floor of the stomodeal cavity (Fig. 3b). The midgut endoderm consists of relatively large cells, especially at the anteroventral side, whereas the endoderm of the intestine forms a much thinner epithelium. The anus opens from the intestine dorsally, just anterior to the py- gidium (not shown). Mesoderm-lined coelomic compartments are visible an- terior to the stomodeum and along the ventral midgut and hindgut (Fig. 3a, b). These observations are consistent with those of Bonch-Bruevich and Malakhov (1987), who report one unpaired preoral coelom and three pairs of trunk coe- loms, although we were unable to locate with certainty the boundaries between the trunk cavities. Identifiable cell types present at this stage include neu- rons, secretory digestive cells, trochoblasts. and muscle > he' mg *r*'"~ pv vbv •<*%£*& ^MliP* ' ~'-^^«y^--v Figure 3. Semithin plastic sections of stages L3 and L4 larvae stained with hematoxylin, eosin. and alcian blue, (a) Early stage L3 larva in sagittal section; ventral is to the left and anterior toward the top of the page, unless noted. Arrow points to algal particle entering stomodeum. Roman numerals denote locations of three trunk coelomic spaces, (b) Higher magnification view of same stage larva as in (a), (c) Oblique frontal section of a late stage L3 larva. Arrow points to an anterior septum dividing one of the region A segments, (d) Oblique transverse section through head of stage L4 larva with dorsal side up. (e) Frontal section through palette rudiments (segments B2-B5). (f) High-magnification view of transverse section through ventral body wall of stage L4 larva midway between the mouth and anterior mesotroch. Arrowhead points to the ventral nerve cord. Arrow indicates the ventral mesentery, (g) Transverse section, with ante- rior to the left, through posterior metatroch (setiger B2) and palette rudi- ments (setigers B3-B5). Arrowhead points to trochal cell. Ih. i) Sections through trochal bands at stage L4 tangential to body wall. Arrow points to line of basolateral trochal cell nuclei, ce, circumesophogeal connective; eg, neuropil of cerebral ganglion; e, endodermal cell of the midgut; he, head coelom; mg. midgut: ml, mesotroch; pal. pallette rudiment; pv, pharyngeal valve; se, stomodeum: vbv. ventral blood vessel; vnc, neuropil of ventral nerve cord. 324 S. Q. IRVINE ET AL cells. Also visible are light-emitting photocytes ventrolat- eral to the intestine; these, described by Henry (1989). are functional by stage L2. Staining with alcian blue (not shown) reveals large mucosal cells dorsal and lateral to the stomodeum. Late period, ll-30da\s. This period is morphologically similar to the preceding, the most obvious difference being the addition of a pair of dorsomedial eyes. The stomach becomes still larger relative to the overall body, and the mesotroch widens with it. The pygidial papilla becomes longer and more distinct. The apical tuft is still present along with the lateral bristles, both of which are lost by the end of this stage. These observations correlate well with previous descriptions (Enders. 1909; Cazaux, 1965). //; situ hybridization with a (3-titbulin riboprobe reveals cells with extensive ciliation in the apical tuft, stomodeum, and mesotroch (Fig. 4a). However, at this stage our probe does not reveal neural elements. Stage L4— 30-60 days (Fig. 5a, b): Size: approx. mean 600 X 400 /MIII Cilian- structures: apical tuft lost; two mesotrochs Ocelli: 6; 2 pairs lateral, 1 pair medial References: Cazaux (1965) pit. 4, rigs. 7-8; Enders (1909) pit. II, rig. 10 Two major changes from stage L3 are evident in this stage. The first is the appearance of a second trochal band just anterior to the existing mesotroch. The second change is the advent of overt segmentation in the region between the posterior mesotroch and the pygidium. Three distinct annu- lar bulges are visible in this region; as becomes evident in later stages, these are rudiments of setigers B3-B5. The segmental anlage of the anterior 1 1 setigers are not apparent by visual inspection, but the prospective cell populations of the parapodia are present. This was shown by staining with an anti-Distal-less antibody that recognizes the prospective apical cells of body wall outgrowths (refer to Panganiban et til., 1997). The basic structure of the gut present from stage L2 persists, with the intestine occupying the postmesotro- chal segmented region. A second pair of lateral ocelli de- velop at this stage, making a total of six ocelli in three bilateral pairs. The central nervous system now has the basic compo- nents of the juvenile. The cerebral ganglion forms a disk just beneath the most rostral epidermis (Fig. 3d). The circum- esophogeal connectives flank the stomodeum (Fig. 3c, d) and join in the ventral midline at the anterior midgut level (Fig. 3f). The ventral nerve cord ( VNC) remains paired as it travels toward the posterior, with numerous commissures connecting the bilateral segmental ganglia. In the overtly segmented posterior region (setigers B3-B5), distinct paired segmental ganglia are visible (Fig. 3e). Late in this stage the two hemilateral cords of the VNC diverge anterior to the mesotrochs. This splitting of the paired nerve cords results in the adult arrangement of the CNS: laterally placed nerve cords in setigers A1-A1 1 join at setiger Bl and run at the ventral midline more posteriorly (Martin and Anctil, 1984). The lateral divergence of the anterior nerve cord is visible in a Figure 4. Whole mount in xitit hybridi/ation to a digoxygenin-labeled antisense p-nihiilin nhoprobe. Anterior is toward the top of the page in each view, (a) Stage L3 larva viewed from the ventral side in optical section. Slaining is visible at the base of the apical tuft (arrow), around the posterior stomodeum (open arrowhead), and in the trochoblasts of the me.sotroch (arrowhead), (b) Ventral view of a stage L4 larva. The dorsal body wall and head have been dissected open for photography. Strong staining is seen in the trochoblasts of both mesotrochs (arrowheads), and in the anterior [open arrowhead) and posterior (double arrowhead) ventral nerve cord. Setiger numbers of posterior ganglia are labeled on the right, (c) Higher magnification view of anterior ventral nerve cord. The axon tract of the nerve cord (arrows) is visible just subjacent to serially iterated blocks of staining ectodermal cells (arrowheads). CHAETOPTERUS LARVAL ONTOGENY 325 Figure 5. Larvae, stages L4 and L5. Anterior and posterior mesotrochs are marked by arrowheads, (a. h) Stage L4 larvae at 30 days in dorsal and ventral views, (c-e) Stage L5 larvae at 60 days in dorsal, lateral, and ventral views respectively. The locations of adult setiger rudiments are labeled in (d). Note the appearance of the red adult ocelli, visible in (e) along with the persistent larval ocelli, ao, adult ocellus; hg. hindgut; lo, larval ocellus; mg, midgut; nr. notopodial rudiment of setigers A1-A9; pa, palp rudiment; pal, palette rudiment of setigers B3-B5; pol, post-oral lobe; pp. papilla; prl, pre-oral lobe; st, stomodeum. Scale bars are 100 ftm. the expression pattern of ft-titbulin visualized by in situ hybridization (Fig. 4b, c). The fact that in situ hybridization to p-tubiilin transcripts failed to detect a distinct VNC prior to this stage suggests that the VNC had not yet formed. Segmental boundaries are not distinguishable, by conven- tional microscopy, anterior to the mesotrochs in any tissue. However, in situ hybridization to jB-tubii/iii transcripts re- veals that reiterated ganglionic cell populations, presumably segmental, are present at this stage (Fig. 4b, c). Bilateral ganglionic cell populations are also visible in the overtly segmented anlagen of setigers B2-B5 (Fig. 4b). Capacious coelomic cavities with distinct septa surround the larval foregut, as seen in frontal section (Fig. 3c). Transverse sections at the middle of the larva reveal bilat- eral coelomic cavities, separated by a ventral mesentery, medial to the nerve cord. Between these cavities and the gut, the ventral blood vessel is located at the midline (Fig. 3f). A particularly distinctive cell type is that of the ciliated cells of the trochal bands. These are large prismatic cells with a uniform granular cytoplasm (Fig. 3g). They are extended along the anterior-posterior axis, and packed reg- ularly in a continuous circumferential ring (Fig. 3h). The cell nuclei are positioned at the basilateral ends of the cells (Fig. 3i). Stage L5 — approx. 60 days (Fig. 5c-e): Competent to metamorphose Size: approx. mean 800 X 400 ju,m Ciliary structures: apical tuft absent; two mesotrochs Ocelli: 8; 4 lateral, 2 medial, 2 lateral adult ocelli References: Ca/atix (1965) pit. 5, tig. 10; Enders (1909) pit. II, rigs. 11-12 At this stage larvae are competent to metamorphose — in fact, we observed one specimen from this stage that had reached late stage L7 within 6 h after transfer from mass culture to a pctri dish with fresh seawater. Specimens from this stage routinely passed completely through metamor- 326 S. Q. IRVINE ET AL. phosis overnight, indicating that the rudiments of all juve- nile structures are present. As compared with stage L4. the postoral lobe grows disproportionately with respect to the preoral lobe and folds towards the posterior. Many eosin-reactive secretory cells are visible in section in the epidermis of this organ (Fig. 6b). Appearing at this stage are visible palp and anterior parapo- dial rudiments. A pair of red adult ocelli appear at the most lateral margin of the preoral lobe. The smaller dark larval ocelli remain throughout the stage. In section, the setal sacs and septation of segments A1-A9 are apparent (Fig. 6a, b). The epidermis in the region of prospective setigers B2-B5 develops deeper infolding, creating distinct annuli anchored at the ventral midline (Figs. 3g, 6b). However, the region around the two mesotrochs has yet to exhibit any segmental character visible either in the exterior morphology or in section. A pair of lateral outgrowths emerge just anterior to the pygidium. which Enders (1909) identifies as the notopo- dia of segment Cl . These bear stout setal sacs (not shown). Based on the locations of ganglia in the B and C regions and the developing parapodia of the A region, it is possible to locate the primordia of the first 1 5 adult setigers at this stage (Fig. 5d). The identity of the posterior mesotroch with the aliform notopodia of setiger Bl can be inferred from Hox gene expression patterns (Irvine, 1998; Irvine and Martin- • »'ff'5t/"~-* aS^-s- i,v - ;j JK9. #p •-• | V '••? '. : i j£ _M . *«_. * iffefei TOfe4 Pal m& «? w-*< Figure 6. Senuthin sections of stage L5 larvae stained as in Figure 7. (a) Oblique transverse section through region A. Dorsal is towards the top of the page. Open arrowhead points to a typical anterior seta. Arrow points to an antciuir septum, (h) Parasagittal section with ventral to the left and anterior up. cutting through several anterior setal sacs (arrow) and both mesotrochs (arrow heads). Bars denote approximate plane of section shown in (a), ere. eosin-rcactive cells; fg, foregut; mg, midgut; pal. palette rudiment of setiger B2; vnc. axon tract of ventral nerve cord. dale, 1999b). Ironically, the longest adult segment, B2, forms from the shortest, most cryptic of the larval setigers. The hemilateral cords of the anterior ventral nerve cord have continued to diverge from the ventral midline to ap- proach the ladderlike form of the adult nervous system in setigers A1-A9 (Martin and Anctil. 1984). The basic struc- ture of the nervous system more posteriorly persists, as described for stage L4. Stage L6 — approx. 60 days (Fig. 7a-d): Mid-metamorphosis Size: 1-2 mm Ciliary structures: apical tuft absent: two mesotrochs Ocelli: 8; 4 lateral, 2 medial, 2 lateral adult ocelli References: Cazaux (1965) pit. 5, fig. 10; Enders (1909) pit. II. figs. 11-12 This transitory stage is characterized by the transforma- tion of larval to adult structures (Fig. 1). The prostomium and peristomium form by the retraction of the preoral lobe and the folding rostrally of the postoral lobe. The pair of dorsomedial ocelli disappear during this stage (compare Fig. 7a and 7c). The two larval pairs of lateral ocelli persist, with the adult ocelli roughly coincident with the most ven- trolateral pair of larval ocelli. The parapodial rudiments of setigers A1-A9 emerge laterally, correlated with a dorso- ventral flattening of region A. The anterior mesotroch de- generates, and the posterior mesotroch becomes incorpo- rated into the aliform notopodia of segment Bl, which appear dorsolaterally. This fate is confirmed by /'/; situ hybridization to Hox segmental markers (Irvine, 1998: Ir- vine and Marti ndale, 1999b). Just caudally, the digestive and accessory feeding organs of setiger B2 appear along the dorsal surface with a swelling of this portion of the larva. The three annular bulges evident at stage L4 expand to take on the shape of the palettes of segments B3-B5. The no- topodia of segment Cl continue to project ventrolaterally. Stage L7 — approx. 60 days (Fig. 8a-b): Juvenile Size: 2-3 mm Ocelli: 2 lateral adult ocelli (2 pairs of larval ocelli degenerate) References: Cazaux (1965) pit. 6, figs. 11-14 At the completion of metamorphosis, the juvenile worm has taken on the general form of the adult for the head and anterior 15 setigers. The most conspicuous change from stage L6 is the extreme extension of the body axis from setigers B1-B5. In addition, the aliform notopodia in setiger B2, the accessory feeding organ in setiger B2, and the palettes in setigers B3-B5 all extend out from the body wall and assume their adult form. The remaining two pairs of larval ocelli degenerate, leaving the larger red adult ocelli. The remainder of the roughly 40 abdominal gametogenic CHAETOPTERUS LARVAL ONTOGENY 327 Figure 7. Stage L6 larvae, (a, b) Early stage L6 larva in dorsolateral and ventrolateral views, (c, d) Late stage L6 larva in dorsolateral and ventrolateral views. Note the rotation of the postoral lobe to an anterior- facing direction in comparison with the orientation at stage L5. afo. accessory feeding organ rudiment: an. aliform notopodium rudiment; ao, adult ocellus: lo. larval ocellus: nr. notopodial rudiment of setigers A1-A9; pa. palp rudiment: pal. palette rudiment of setigers B3-B5; pol. postoral lobe: prl. preoral lobe. Scale bar is 100 /j.m. segments have yet to be produced. This occurs by interpo- lation between setigerCl and the pygidium (Cazaux, 1965). Tube construction was never observed in these cultures. This is probably because that substrate was never provided, the stage L7 juveniles always being kept in glass or plastic vessels without mud or sand (Irvine and Martindale, 1999a). Discussion Developmental variation within the genus Chaetopterus The genus Chaetopterus has several species that show variation in overall adult size, tube morphology, and details of parapodial and setal form. Published descriptions of Chaetopterus development differ from our results in some respects. Cazaux' s ( 1965) figure 5 is a drawing of a 48-h larva corresponding in part to our observations. However, our cultures and those described in Henry (1986) do not reach the general morphology depicted until at least 72 h. despite higher culture temperatures. Four other differences are apparent between our results and those in figure 5 of Cazaux (1965): (i) we do not detect trochal bands around the stomodeum; (ii) a distinct intestine is visible, rather than the extension of the posterior stomach shown: (iii) the mesotroch is more posterior in our preparations: (iv) only one pair of laterally placed eyes are visible rather than the two pairs depicted. Since Cazaux's specimens came from the Atlantic coast of France, he may have been observing another species of Chaetopterus — neither C. variopedatus Renier, 1804, nor C. variopedatus sensu Enders. 1909, but possibly C. valencinii Quatrefages, 1866 (M. E. Petersen, Copenhagen Museum, pers. comm). Trochal bands As mentioned, a circumferential ciliary band appears midway along the anterior-posterior body axis at Stage L3; following Okada (1957). we have termed this band a me- sotroch. At Stage L4 another trochal band, which we also call a mesotroch, forms just anterior to the first band. Rouse (1999) characterizes this younger band as a metatroch. following early work of Wilson (1882) that depicts a larva resembling our Stage L3. However, tracing the fate of both these trochal bands ahead in ontogeny reveals that they come to lie well within the segmented trunk of the larva, contrary to the definition of a metatroch as a presegmental structure lying on the peristomium (Rouse, 1999). Thus, rather than a metatroch having evolved within the Chaetop- teridae lineage, as concluded in the transformations of the Rouse (1999) analysis, a different type of trochal band arose, not strictly homologous to a metatroch. Although the Chaetopterus mesotrochal bands are not metatrochs, as defined above, they may be homologous to other types of trochal bands at the level of their develop- mental pathway. Rouse's (1999) analysis indicates that the various types of larval trochal bands can appear and be lost independently in different lineages; i.e., as characters they exhibit a high degree of homoplasy. This finding suggests that trochal bands share a developmental pathway that can be activated at various levels along the anterior-posterior axis. 328 S. Q. IRVINE ET AL. Figure 8. Juvenile worms within 1 day of metamorphosis at about 60 days. Anterior is to the left, (a) Dorsal view. Letters and numbers indicate adult setiger locations. Asterisk denotes the ciliary groove of the aliform notopodium of setiger B I , derived from the posterior mesotroch. Arrowhead points to a parapodium of the first 'abdominal' setiger Cl. (b) Two newly metamorphosed specimens. Upper specimen is in ventrolateral view and lower is in dorsolateral view. Arrowheads as in (a) above, afo. accessory feeding organ rudiment; an. aliform notopodium rudiment; ao. adult ocellus; eg. ciliated groove; lo, larval ocellus; m, mouth; nr, notopodial rudiment of setigers AI-A9; pa, palp rudiment; pal, palette rudiment of setigers B3-B5; pol, postoral lobe; pyg. pygidium. Scale bar is 100 juni. Larval segmentation and relationship to adult bod\ plan The most commonly described form of larval develop- ment in polychaetes is the production of a trochophore larva that adds segments sequentially from a posterior growth zone to produce a nectochaete larva (Okada, 1957; Ander- son, 1966). There is some controversy over whether the first three larval segments develop in the same sequential man- ner as subsequent segments, but in typical cases the demar- cation of each of the segmental boundaries is evident in the external form of the larva from a very early stage. Chaetopterus represents a distinct departure from this gen- eral pattern. The first external signs of segmentation are the rudiments of segments B3-B5 visible at stage L4, at an age of 30 days. At no point does the metatrochophore take on the overtly segmented form of the typical nectochaete larva. However, some incipient segmentation is present before it becomes visible externally. Bonch-Bruevich and Malakhov (1987) describe three trunk coeloms existing at stage L2, which is consistent with our stage L3 sections (Fig. 3a). If we use the trochal bands as landmarks, the anterior trunk coelom (']' in Fig. 1 stage L3) roughly corresponds with the position of adult segments A1-A9, the middle coelom ('II') with segment Bl or segments Bl and B2. and the posterior coelom ('III') with juvenile segments B3-B5. By stage L4 the segmental character of the ventral nerve ganglia is apparent in fi-tubulin expression, even though no segmental divisions are visible by conventional microscopy (Fig. 4b, c). Expression of Distal-less protein also reveals segmen- tally iterated structures, the parapodia of setigers A1-A9, at stage L4. before they are evident morphologically (Panga- CHAETOPTERUS LARVAL ONTOGENY 329 niban et Hydrolithon onkodes > L. prolifer. Non-geniculate coralline algae cause sloughing of larval sensory papillae and necrotic cell death Unlike normal 14-16 h posthatch larvae, which have extended papillae, larvae cultured in the presence of NCA had rounded trunks and lacked papillae (Fig. 3). Normal larvae were evident only in the treatment containing small and medium shards of L. prolifer (Fig. 2A). Microscopic inspection of larvae revealed that all three NCA species appeared to induce this same effect on larval morphology, although the amount of NCA required to affect larval mor- phology differed (Fig. 2 A). For these reasons, only the effects of L. prolifer, the least potent of the NCA, on H. cun'ata larvae were documented further. Large shards of L. prolifer (Fig. 2 A) induced a range of changes in H. curvata larval morphology that appeared to be related but differed in severity. Slightly abnormal larvae had normal axial structures (notochord. neural tube, and muscle) and trunk structures (sensory vesicle containing otolith and ocellus, and endoderm rudiment) but lacked projecting pa- pillae and had a slightly rounded trunk (Fig. 3C, D). Some of these larvae still had papillae associated with them; however, the papillae were no longer attached to the anterior trunk and appeared to be in the process of being sloughed off (Fig. 3E). In the most severely altered larvae, the trunk was small and rounded, the tail was kinked, and the muscle cells had lost integrity and their usual columnar shape (Fig. 3F). Between these extremes of NCA-induced abnormali- ties was a continuum of morphological defects. To determine whether the inhibitory factor or factors associated with L. prolifer or the other NCA were inducing general cell death, we investigated the structure of the nuclei of larvae that exhibited an intermediate abnormal morphol- ogy (i.e.. rounded trunk, straight tail with slightly rounded muscle cells). Nuclei were stained with propidium iodide and analyzed by laser scanning confocal microscopy. Opti- cal sections were taken through the trunk epidermis and mesenchyme of normal and abnormal larvae, and compared. In both tissues, the nuclei of larvae exposed to L prolifer were larger Ihan those of normal larvae (Fig. 4). Mesenchy- mal nuclei of normal larvae were circular (diameters be- tween 3.5 and 4.3 jam) and appeared granular when stained 336 B. M. DEGNAN AND C. R. JOHNSON 80 i.8 T •£ 60 E V "CD CO CD 4° 0) CD 5 (N. brassica-florida = H. onkodes = L. prolifer)]. with propidium iodide (Fig. 4 A). The mesenchymal nuclei of larvae cultured with L prolifer were oval, larger (4.3-7. 1 jum), and more diffusely stained with propidium iodide (Fig. 4B). Normal epidermal nuclei were similar in appear- ance to normal mesenchymal nuclei (Fig. 4C). Larvae ex- posed to L. prolifer had irregularly shaped epidermal nuclei that stained intensely with propidium iodide and were about the same size as normal nuclei (Fig. 4D). There was addi- tional, non-nuclear staining of these epidermal cells. Metamorphosis cannot occur in lan-cie previously exposed to non-geniculate coralline algae To determine whether the effect of the NCA on H. cnrvata larvae was transient, we transferred larvae that were cultured with either H. onkodes, N. brassica-florida or L. prolifer into culture chambers containing 40 mM KC1- elevated FSW. Because the larvae exhibited a range of abnormalities, we transferred only those showing slight abnormalities (i.e., lost papillae and rounded trunk; e.g., Fig. 3C, D). Larvae from the same fertilization batch that were not exposed to NCA and had not metamorphosed in the plastic chambers acted as controls and were transferred to the KCl-elevated FSW or FSW. We monitored and scored the number of larvae that had initiated metamorpho- sis (i.e., began tail resorption) every hour for 3 h. Although most of the untreated larvae in both FSW and KCl-elevated FSW began metamorphosing over this period, only 2 of a total of 180 larvae previously exposed to any one of the algae initiated metamorphosis (Fig. 5). Analysis of these cultures after 24 h revealed that control larvae were meta- morphosing normally and that larvae previously cultured with NCA had not metamorphosed and died. The two post- larvae previously exposed as larvae to the NCA were also dead after 24 h. Discussion Most competent Henlnutnia cnn-ata larvae will normally settle and metamorphose in seawater, FSW, and FSW with antibiotics within 24 h of hatching (Degnan el al., 1997; unpub. data). The percentage of larvae that will spontane- ously settle varies between cohorts, with those cultured in until tered seawater generally settling at a greater rate than those cultured in FSW. To assess the extent of any inhibi- tory effects of NCA on larval settlement and metamorpho- sis, we cultured the larvae and NCA in untiltered seawater. The high percentage of larvae that settled in chambers containing seawater demonstrated that H. ciin-iitu will settle spontaneously under these culture conditions. The signifi- cant reduction in settlement rates of larvae cultured in the presence of the different NCA demonstrates that these algae are inhibiting settlement in this tropical ascidian. H. ciirrata larvae respond differentially to a range of epitloral and faunal substrata associated with the cryptic INHIBITION OF ASCIDIAN SETTLEMENT 337 A 100 a. Neo (sml) -, b. Neo (med) -, c. Neo (Ig) ns ns .BL ns 50 1 ' - met Pl nca ^ nca -I-, met nca pl met ^ d. Hydro (sml) e. Hydro (med) f. Hydro (Ig) S 50 pl T ns ns ns <5 Q. met - pl — i — met - met nca C nca nca Pl D . 0 g. Litho (sml) h. Litho (med) i. Litho (Ig) - Pl — i— - Pl ~M met 50 - JL - met P met ns n nca L nca ns nca H" B (i) Total settlement Litho Litho Litho Hydro Neo Hydro Neo Hydro Neo sml tge med sml sml med med Ige Ige largesl mean smallest mean (ii) Metamorphosed but not settled Litho Litho Litho Hydro Hydro Neo Hydro Neo Neo med sml Ige Ige sml sml med med Ige smallest mean (iii) Neither metamorphosed nor settled Neo Neo Hydro Hydro Neo Hydro Litho Litho Litho Ige med Ige med sml sml Ige sml med largest mean smallest mean Figure 2. Dose-dependence of inhibitory effect of non-geniculate coralline algae on settlement and meta- morphosis of Herdmania cumita in a "no-choice" experiment. (A) Mean response ( + SE) of H. ciimita larvae in the presence of small, medium, and large (= "sml," "med," "Ige," respectively) shards of the NCA Neogoniolithon brassica-florida ( = "Neo"), Hvdrolithon onkodes ( = "Hydro"), and Lithothamnium prolifer ( = "Litho"). "nca" = settlement and metamorphosis on NCA shards; "pi" = settlement and metamorphosis on the sides of the plastic settlement chambers; "met" = larvae metamorphosed but not settled, and "ns" = larvae neither settled or metamorphosed; n = 6 for all treatments. Greatest total settlement occurred in controls (68.72% ± SE = 5.11; not shown on figure). Tukey groupings (P = 0.05) indicated settlement in controls was significantly greater than in treatments containing medium- and large-sized shards of N. brassica-florida and H. onkodes (one-way ANOVA, F(9, 50) = 15.05. P < 0.0001, transformation y""'). The pattern of settlement in control chambers (n = 6) containing seawater only (not shown) was not significantly different from the treatment containing small shards of L. prolifer (panel g). (B) Tukey groupings following detection of significant interaction between NCA species and size of shard ( = "dose") for total larvae settled, larvae metamorphosed but not settled, and larvae neither settled nor metamorphosed (two-way ANOVA, species x size interaction; total larvae settled and metamorphosed, F(4. 45) = 3.31. P = 0.018, transformation V'551; larvae metamorphosed but not settled, Fl-t. 45) = 5.02, P = 0.002. transformation = arcsin Vy; larvae not settled or metamorphosed. F(4. 45) = 4.96, P = 0.002, no transformation required). These results show that the inhibitory effect of N. brassica-florida and H. onkodes on larval settlement and metamorphosis was greater than that ot L. prolifer, and that the dose-response relationship was significantly steeper for N. brassica-florida and H. onkodes than for L. prolifer. community of the reef crest and slope of the Great Barrier Reef: metamorphosis is induced by some substrata and not induced or inhibited by others in the laboratory. Manual removal of the larval trunk anterior of the otolith and ocellus prevents the posterior part of the larva from being induced to metamorphose with KCl-elevated FSW or natural indue - ers (Degnan et ai, 1997), suggesting that responsiveness to inductive substrata in H. cun-ata is mediated by the che- mosensory papillae and an anterior signaling center. The Hemps gene, which encodes a protein with a putative se- cretion signal sequence and epidermal growth factor (EGF)- like repeats, is expressed in this region and has been shown to regulate the induction of metamorphosis (Arnold et til.. 1997; Eri et ai, 1999). The NCA investigated in this study (Neogoniolithon brassica-florida, Hydrolithon onkodes, and Lithothamnium prolifer) appear to be toxic to H. curvata larvae, inhibiting settlement on the surface of the algae, significantly lowering the level of spontaneous settlement, and preventing the future ability of larvae to respond to inductive cues. Trunk ectodermal and mesenchymal nuclei of larvae cultured in the presence of L. prolifer were bloated and irregular in shape respectively, both features of necrotic cell death (Kerr and Harmon, 1991). Induced morphoge- netic changes during normal metamorphosis do not include this form of cell death (see Degnan et ai, 1996. 1997; Hinman and Dennan, 1998). H. curvata larvae that were 338 B. M. DEGNAN AND C. R. JOHNSON B E \ Figure 3. The effect of Liilintlmiiiniiini I'mlifer on normal develop- ment and larval structures of Hcn/iminui curnitu. Larval anterior is to the right in all micrographs. (A) A normal postlarva approximately 12 h after initiating metamorphosis; degenerating larval muscle cells (Imc) and pro- jecting ampullae (amp) are evident. (B) Normal tadpole larva with sensory papillae (arrow). (C-F) Larvae cultured with L firnlifcr. (C, D) Larva with a rounded trunk and no papillae; arrows point to region where papillae are normally located. (E) Larva in the process of shedding a papilla (arrow). (F) Larva with rounded trunk, no papillae, kinked tail, and necrosing muscle cells (arrow). Scale bars: A, B, C, F, 100 mm); D. E, 50 /urn. cultured with NCA and had not settled lacked papillae and had rounded anterior trunks. In some of the least morpho- logically disturbed individuals, the palps were observed Figure 4. Degeneration of the nuclei of Herdmania cun-aia larvae cultured in the presence of Lillinilitiiiiiiiiini />/«///<•/•. Confocal micrographs of nuclei stained with propidium iodide. (A) Normal larval trunk mesen- chyme (trunk ventral cells; Satoh. 1996). (B) Trunk mesenchyme of larva exposed to L. pmlifer. (C) Normal larval Irunk epidermis. (D) Trunk epidermis of larva exposed to L. pmlifcr. Scale bar, 10 /j.m. 1 2 h post-treatment Figure 5. Percentage of Herdmania cun-ata larvae metamorphosing when treated with 40 m/W KCl-elevated FSW. Prior to treatment with KCl-elevated FSW, larvae were cultured either in FSW. or in the presence of Nei>K»niolitlmn brassica-florida. Hydrolithon onkodes. or Litlinllhiiii- niiini /uvi/j/c; for 12-14 h. Filled triangles, larvae cultured in seawater and then transferred to KCl-elevated FSW; open circles, larvae cultured in seawater and then transferred to FSW; open squares, larvae exposed to Neogoniolithon brassica-florida and then transferred to KCl-elevated FSW; diamonds (hidden behind open squares), larvae exposed to Hydro- liihuii (inkoiU's and then transferred to KCl-elevated FSW; half-filled squares (hidden behind open squares), larvae exposed to iithothamnium prolifer and then transferred to KCl-elevated FSW. Data are means ( ±SE). being sloughed from the trunk of the larva, suggesting that the toxic effect of the NCA upon the larva first disables the chemosensory and primary signaling system. Hemps is ex- pressed in the papillae and the papillae-associated tissue (PAT) which is located in the anterior epidermis between the three papillae and consists of about 5 cells (Eri ct . Biol. 39: 483-490. 340 B. M. DEGNAN AND C. R JOHNSON Draper, N. R., and H. Smith. 1991. 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Massachusetts 02543: 3 University Department of Pharmacology. University of Oxford, Oxford, OX] 3QT, UK Abstract. Ku is the regulatory subunit of the DNA-depen- dent protein kinase (DNA-PK). This enzyme plays a role in DNA repair, recombination, and transcription. It is composed of a large catalytic subunit (p460), and a regulatory het- erodimer. the Ku protein, which consists of 86-kDa and 70- kDa subunits. These various components of the enzyme have been found in both eggs and embryos of the sea urchin. When variable amounts of a specific monoclonal antibody to the Ku protein (Ku 162) were injected into one cell of a 2-cell embryo of L\techimis /rictus, they caused a dose-dependent develop- mental arrest of the injected cell. The non-injected cell contin- ued to develop normally. In contrast, injection of an antibody (N3H10) raised against the 70-kDa subunit of the Ku protein had no effect on development when injected into 2-cell-stage embryos. Co-injection of purified DNA-PK with the antibody reversed the antibody-mediated inhibition of development. In the fertilized egg and during the early stages of development, the DNA-PK was localized largely in the cytoplasm, but in later developmental stages, it assumed a nuclear location. On the basis of these results, we postulate that the injection of the Ku antibody either prevents the translocation of the DNA-PK into the nucleus or interferes with its enzymatic activity either in the nucleus or in the cytoplasm. In either case, the results suggest that DNA-PK plays an important role in regulating the early stages of embryogenesis in this primitive organism. Introduction The DNA-dependent protein kinase, DNA-PK, was first detected in the nuclei of mammalian cells (Walker et al.. 1985: Carter et al., 1990). Studies in the last several years Received 24 March 1999; accepted 16 August 1999. * Author to whom correspondence should he addressed. E-mail: hrassmus@mail.mcg.edu have provided evidence that this enzyme is involved in DNA repair, and in the activation of specific transcription factors (reviewed by Anderson and Lees-Miller. 1992; Weaver. 1995). It may also be involved in regulating DNA synthesis (Brush et al.. 1994). Ku-associated ATP-depen- dent helicase activity (Tuteja et al.. 1994). and DNA-de- pendent ATPase activity (Vishwanatha and Baril, 1990: Cao et al., 1994). The enzyme consists of a large catalytic subunit. p460. and a heterodimeric regulatory component, the Ku protein. The latter consists of subunits of 86 and 70 kDa (Anderson. 1993: Gottlieb and Jackson. 1993). The Ku protein possesses a DNA-binding domain, and enzymatic activity is thought to be induced when this domain binds double-stranded DNA. (ds)DNA (Anderson and Lees- Miller, 1992; Dvir et al.. 1992; Anderson, 1993; Gottlieb and Jackson, 1993). However, the catalytic subunit of DNA-PK can be activated by another DNA-binding protein, heat shock transcription factor 1 (Peterson et al, 1995). Additional complexity of control may be involved in its catalytic function, because the p86 subunit of Ku is a somatostatin receptor protein that can regulate the activity of protein phosphatase 2A (Le Romancer et al.. 1994). In addition, it has recently been reported that in cultured mam- malian cells, a significant amount of the Ku protein is present in the cytoplasmie portion of the cell (Fewell and Kuff, 1996). These findings have led to the proposal that one or both of the Ku subunits may function in ways other than through the activation of nuclear DNA-PK. or that DNA-PK can be activated by factors other than (ds)DNA and function to regulate events in the cytoplasmie domain. More recent work has shown that the enzyme is present in lower organisms, and particularly in the oocytes. eggs, and embryos of several marine invertebrates (Walker et al.. 1985; Kanungo et al.. 1996a, b) as well as in mice, frogs, and 341 342 J. KANUNGO ET At. Drosophila (Finnic et al, 1995). Thus, DNA-PK may have one or more evolutionarily conserved functions. Transgenic mice that are deficient in the Ku 86 subunit exhibit severe combined immunodeficiency (Nussenzweig et al., 1996; Zhu et al., 1996). Such mice are considerably smaller (40%- 80% ) than their normal litter-mates, and cultured cells derived from these mice display variable degrees of delay in cell cycle progression (Nussenzweig et al., 1996). That high concentrations of DNA-PK exist in the eggs of sea urchins (Kanungo et ill., 1996a, b) and frogs (Kanungo et nl., 1997) raises the interesting possibility that this en- zyme may also play a regulatory role in early embryogen- esis in these species. To explore this possibility, we initially undertook studies of DNA-PK localization and activation before and after fertilization of the eggs of Arbacia punctii- liitu, the purple sea urchin. The unfertilized egg of this organism contains considerable amounts of both Ku and p460, as measured by western analysis, and by immunocy- tochemistry (Kanungo et al., 1996b). These enzyme sub- units are located largely, if not entirely, in the cytoplasmic compartment of the egg. Nonetheless, in the unfertilized egg, the DNA-PK cannot be activated by the addition of (ds)DNA to cytoplasmic extracts; i.e.. the enzyme is present in the cytoplasm in some cryptic form (Kanungo et al.. I996b). Within minutes of fertilization, and without evi- dence of new protein synthesis, the enzyme is still in the cytoplasmic fraction, but can now be activated when (ds)DNA is added to this fraction. Of equal interest is that, if one coats Protein A Sepharose (PAS) beads with a spe- cific monoclonal antibody to the Ku protein, Ku 162, and then uses these beads to isolate the Ku proteins from the cytoplasmic extracts of fertilized eggs, the beads bind the holoenzyme, and in this bound form the enzyme is active in the absence of (ds)DNA. As the fertilized egg develops, there is a progressive shift of the various protein components of DNA-PK from a cytoplasmic to a nuclear location within the cell (Kanungo et ul.. 1996b). The enzyme is located exclusively in the nuclear domain from the blastula stage onward, but in embryos at the 2- and 4-cell stages the enzymatic activity remains largely in the cytoplasm (Kanungo et al., 1996a, b). In this report, we demonstrate that microinjection of the antibody, Ku 162, into one cell of a 2-cell embryo of Lytechinus pictns, the white sea urchin, inhibits the further development of the injected cell, but has no effect on the non-injected cell. This species, rather than Arbacia punctu- lata. was employed in the present experiments because the microinjection experiments were more easily performed in its embryos. Materials and Methods Collection of eggs and embryos. Male and female Lyte- chinus pictns were obtained from the Marine Resources Department of the Marine Biological Laboratory. Woods Hole, Massachusetts. Shedding of eggs and sperm was induced by injecting 0.5 ml of 0.5 M potassium chloride into the coelom. Batches of eggs were inseminated by mixing them with diluted sperm. Embryos were collected at sched- uled times. Kinase assay. Cytoplasmic extracts from the 2-cell em- bryos of L. pictns were prepared following the procedures already described (Ballinger et al., 1984; Kanungo et al., 1996a). Twenty-five units of purified human DNA-PK (Pro- mega) or extracts prepared from L. pictns 2-cell embryos (from 250 embryos) was added to the antibody-coated PAS beads (Pharmacia) in a 500-fil volume made up by kinase assay buffer, and incubation was carried out at 4"C with constant mixing for 2 h. The PAS beads were washed six times with kinase assay buffer. The assay consisted of 12 /ul of kinase assay buffer containing DTT (dithiothreitol) at a final concentration of 1 mM, 200 ;u,M of peptide substrate (EPPLSQEAFADLWKK) (Anderson, 1993). 2 mM of MgCK, 130 mM of ATP, and 10 /iCi of gamma-[32P]ATP (3000 Ci/mmol) (NEN, Du Pont). The assay was carried out at 25°C for 30 min and stopped by adding glacial acetic acid to a final concentration of 30%. The reaction product was spotted onto p81 phosphocellulose discs (Whatman). Sev- eral washes with 15% acetic acid following a 30-min wash with 30% acetic acid were carried out. The discs were finally washed for 5 min in acetone, air-dried, and the uptake of radioactivity was assessed by scintillation count- ing. The counts obtained from the control beads, not coated with any IgG but treated with either the purified human DNA-PK or extracts of L. pictns embryos, were used as a means of measuring nonspecific background radioactivity. These values were subtracted from the counts obtained from reactions using mouse IgG- and Ku 162-coated PAS beads. Immnnoprecipikition. DNA-PK holoenzyme was iminu- noprecipitated with PAS beads that had been coated with the Ku antibody as follows: PAS beads were pre-swollen in kinase assay buffer (50 mM HEPES, pH 7.4; 10 mM EGTA. 40 mM NaCl, 100 mM potassium acetate, 8.5 mM CaCl:, 2.29 mM MgCK, 277 mM glycerol). Two micrograms of preimmune mouse IgG or of a monoclonal antibody to human Ku (Ku 162) that recognizes a conformational epitope of the Ku protein (Neomarkers, CA) was added to a 10-jixl packed volume of PAS beads. After 1 2-h incubation at 4°C with constant mixing, the beads were washed four times with kinase assay buffer. Innnnnoblotting. The immunoprecipitates from PAS beads were eluted by boiling in SDS-PAGE sample buffer and resolved on a 7.5% SDS-PAGE column (Laemmli. 1974). Duplicate gels were run. One was employed to prepare autoradiographs. The proteins from the other gel were transferred to nitrocellulose membrane and immuno- blotted (Towbin et al.. 1979) with anti-p460 antibody as previously described (Kanungo et al., 1996b). KU ANTIBODY INHIBITS Oll.L DIVISION 343 Preparation of antibodies for microinjection. The IgG antibodies (Ku 162 and N3H10) and the control IgGs were used for the microinjection experiments (Wang el al.. 1993). They were a generous gifts from Dr. Westley Reeves of the University of North Carolina, Chapel Hill. Antibodies against RNAP II were obtained from Promega. and preim- mune mouse IgG was purified using a Pierce immunoglob- ulin purification kit (Cat # 44667). The purified IgGs were dialyzed against Ca2 + -. Mg2+-free PBS (Silver. 1986) and concentrated to 3 /Mg//u.l. The antibody-enzyme complex was prepared using 6 jag of Ku 162 antibody and 10 /^g of purified human DNA-PK (Promega) and incubated on ice with intermittent mixing for 2 h. The mixed aliquot was diluted to 1 ml with Ca2 + -, Mg2 + -free PBS. then dialyzed (4X2 liters) overnight. The dialyzed antibody-enzyme complex was collected and concentrated using an Amicon filter concentration unit. The final concentration was brought back to the original volume of the antibody solution used (10 /id), giving a final concentration of 600 ng/ju.1 Ku 162 and 1000 ng/jul DNA-PK. Microinjection. Specimens of L. pictus were obtained from Marinus Inc. (Long Beach, CA). To induce a female to shed eggs, about 0.5 ml of 0.5 M KC1 was injected into the intracoelomic cavity. The eggs were then passed through an 80-/J.M diameter Nitex membrane to remove the jelly that surrounded them and washed once in Ca2 + -containing sea- water. A 0.5-ml sample of eggs was placed onto a glass coverslip previously coated with poly-L-lysine. and the eggs were exposed to sperm. Low densities of activated sperm (1/40.000 dilution of dry collected sperm) were used to prevent the occurrence of polyspermy. If fertilization pro- ceeded with an efficiency of at least 90%, we continued the experiment. After fertilization, the dishes were covered and left at 17°-19°C for 1 h, at which time the embryos begin first division. Solutions of antibodies that had been dialyzed and concentrated to known protein concentrations (3 jug//u.l in the pipette, estimated as a final amount of 15 X 10~'~ g in the cell) were microinjected into one cell of the 2-cell- stage embryo shortly after first cleavage and before aster formation of the second cleavage. We typically injected l%-2% of the volume of the embryo. An occasional em- bryo was damaged by the microinjection procedure: such clearly damaged embryos were discarded. The injected em- bryos were again covered and remained on the microscope stage for a further 2 h; we then determined the incidence of continued or arrested division. In most experiments, digital images of the eggs were recorded using either an integrating cooled CCD camera (Hamamatsu, USA) or an intensified CCD camera (Photonics Science. Robertsbridge. UK). The camera was attached either to a Metamorph (Universal Imaging. USA) system controlled by a Pentium PC or an lonvision (Improvision, Coventry. UK) system run by a Macintosh. In all experiments we used the uninjected cell as an internal control. Immunocytochemistry. Embryos at the 2-cell stage im- mediately after antibody injection were fixed in 3% form- aldehyde (Kanungo et al., 1996b) and processed for indirect immunofluorescence by incubating with fluorescein conju- gated anti-mouse IgG (Sigma Cat # F 0257). Embryos were then whole-mounted onto glass coverslips and viewed with epiftuorescence and appropriate filters using a Zeiss 135 inverted microscope (Zeiss. Oberkochen. Germany). Pic- tures were recorded using a cooled CCD camera and col- lected digitally with Metamorph (Universal Imaging, West Chester. PA). The DNA of some of the injected cells ar- rested as a consequence of Ku 162 administration were counter-stained with a DNA stain. Hoechst 3342 (0.001 mg/ml. Molecular Probes. Oregon) to determine the struc- ture of the DNA. and specifically to answer the question of whether the failure of these cells to undergo cell division could be due to the induction of apoptosis as a result of the antibody. Results and Discussion To determine whether DNA-PK plays a unique role dur- ing early embryogenesis, we microinjected two anti-Ku antibodies, both raised against human Ku, into one cell of 2-cell embryos, and examined their effects on subsequent development. For technical reasons, we employed Lytechi- nus pictus in these studies rather than Arbacia punctulata. Hence, it was necessary to demonstrate that the fertilized eggs and early embryos of this species would express DNA- PK, and that this enzyme could be activated by the same means as those employed in the Arbacia. The first mono- clonal antibody against the Ku protein, Ku 162, is an IgG that recognizes a conformational epitope on the Ku het- erodimer. It has been used successfully to immunodeplete the DNA-PK activity from Xenopus oocytes (Kanungo et al.. 1997). When PAS beads are coated with this antibody, the enzyme in cytoplasmic extracts of L. pictus 2-cell em- bryos associates with the beads and is catalytically active in the absence of (ds)DNA (Fig. 1 A). This antibody could not be employed in western analysis because it detects the Ku heterodimer, but neither of its subunits. However, a western blot of the proteins eluted from the PAS beads after labeling with [32P]-gamma ATP shows that the interacting catalytic subunit of DNA-PK (p460) is present and can undergo autophosphorylation (Fig. IB). An immunoblot of the im- munoprecipitates made with a monoclonal antibody raised against the human p460 shows that a polypeptide identical to p460 is co-immunoprecipitated by Ku 162 antibody from the cytoplasmic extracts prepared from 2-cell embryos of L pictus (Fig. IB. lanes 5 and 6). Based on these results, initial experiments were carried out to determine the effect of the injection of the antibody. Ku 162, or purified DNA-PK, into one cell of a 2-cell embryo. The injection protocol is illustrated in Figure 2A. A 344 J. KANUNGO ET AL. I I 1 u £ Mouse IgG Ku 162 Mouse IgG Ku 162 human sea urchin B 1234 56 p460 Figure 1. (A) DNA-PK activity of immunoprecipitates of human purified DNA-PK. and of the cytoplasmic extracts of 2-cell embryos of Lytechinus pictus. The DNA-PK assay was performed on washed PAS beads coated with the Ku 162 antibody. Reactions for a single experiment were run in duplicate. A representative assay (of five assays) is shown. Note that specific peptide phosphorylation activity is present in immuno- precipitates of cytoplasmic extracts of the sea urchin embryo. The activity from 250 embryos was a little less than half of that obtained from the immunoprecipitates of 25 units of purified human DNA-PK. PAS beads coated with preimmune mouse IgG did not immunoprecipitate any DNA-PK activity. (B) Autoradiograph of proteins recovered from the PAS beads coated with the Ku 162 antibody (lanes 1-4) showing autophospho- rylated p460. Preimmune mouse IgG immunoprecipitated no labeled pro- tein from purified human DNA-PK (lane 1 ) or from cytoplasmic extracts prepared from the two-cell-stage embryos of L. pictus (lane 2). The autophosphorylated DNA-PK catalytic subunit (p460) was present in Ku 162 immunoprecipitates of the human en/yme (lane 3), and it was present in Ku 162 immunoprecipitates of cytoplasmic extracts from 2-cell embryos of L pictus (lane 4). Immunoblots of proteins eluted from lanes 3 and 4 are shown in lanes 5 and 6. The p46() polypeptide, eluted from Ku 162 immunoprecipitates of purified human DNA-PK on the immunoblot, was recognized by a monoclonal antibody. mAb 42-26 (Carter ct ai, 1990) against human p460 (lane 5); a similar protein, from the Ku 162 immu- noprecipitates of cytoplasmic extracts of L />/V///.v 2-cell embryos, was also recognized by this antibody (lane 6). concentration of Ku 162 was chosen (2.5 pg) that com- pletely arrested cell division in about one-half of the in- jected cells in 2-cell embryos 1 h after fertilization (/( =17). At that time, most of the uninjected embryos were at the 16/32 cell stage of development. On the other hand, further progression of the injected cell was completely arrested in one-half of the embryos (/; = 17) injected with 2.5 pg of Ku 162 (Fig. 2B). Some cells were injected with a standard, but not maximally effective, concentration of purified DNA-PK either in the presence or absence of Ku 162. Injection of purified DNA-PK (the holoenzyme) alone had no effect on the development of the injected cells (n = 12. data not shown). However, when the holoenzyme was co-injected with Ku 162, the normal Ku 162-dependent inhibition of cell development was overcome, and 90% (n = 10) of the co-injected cells developed normally (Fig. 2C). In other cells, injection of either 0.5 M KC1 (n = 13) or anti-RNA polymerase II antibody (Promega) (n = 15) had no effect on the ability of the antibody, Ku 162, to induce developmental arrest. The further effects of the antibody, Ku 162. on embryonic cell development were analyzed in several ways: serial dilutions of Ku 162 were used to construct a dose-response curve; the effect of the second antibody, N3H10, was de- termined; and that of preimmune mouse IgG was examined. In addition, the possibility that Ku 162 was inhibiting cell division by causing cell necrosis or apoptosis was evaluated. As shown in Figure 3A, the injection of standard aliquots of serially diluted Ku 162 antibody solution into one cell of a 2-cell embryo produced two effects on cell development. First, the number of cells that are completely arrested de- creased as the amount of injected Ku 162 decreased (Fig. 3A. B. C). Second, at the highest concentration of Ku 162 (5 pg/cell) the development of all injected cells (n = 21 ) was completely arrested at the single-cell stage (Fig. 3A). The injection of 2.5 pg/cell of the Ku 162 antibody completely arrested development of 40% of the cells (/; = 18) (Fig. 3A. right), and the remaining 60% of injected cells (n = 17) displayed variable rates of development (Fig. 3B, left). The injection of 1 pg of Ku 162 had no discernible effect on 50% of cells; i.e., there was no evidence of developmental arrest (Fig. 3C, left). In this case, only 10% of cells showed a complete inhibition of cell division, and 40% displayed variable degrees of retarded development (Fig. 3C, right). To determine if Ku 162 antibody acts by inhibiting cell division, and not by causing cell necrosis or apoptosis, a number of the cells of embryos (n - 12) that displayed complete arrest were counter-stained with Hoechst 33342. Evidence of apoptosis was not seen in any of the arrested cells (Fig. 3 A, right). In contrast to the results with the Ku antibody 162, injection of comparable amounts of N3H10, a Ku antibody directed against the 70-kDa subunit of Ku, had no apparent effect on the development of the injected cell of a 2-stage KU ANTIBODY INHIBITS CELL DIVISION 345 A. Inject antibody + purified enzyme 1 hr /A /gatf\ 2 hrs Embryo 1st cell division 16/32 cell stage B. Ku 162 arrest C. Ku 162 + Enzyme Figure 2. The effect of the antibody, Ku 162, with or without the simultaneous injection of the DNA-PK holoenzyme, on the ability of the injected cell in a 2-cell embryo of Lytechinus pictiis. Injected cells are identified with an arrow. (A) A diagram of the experimental protocol. (B) The injection of the Ku 162 antibody (2.5 pg/cell) into one cell of a 2-cell embryo caused complete arrest of cell division in the injected cell, but not in the non-injected cell. (C) Two cells, one in each of a 2-cell stage embryo, microinjected simultaneously with Ku 162 antibody (2.5 pg/cell) and purified DNA-PK holoenzyme (4 pg/cell). In neither embryo was arrest or delay of cell division seen in the injected cells, or in the uninjected cells. embryo (n = 15). It is of interest that one of the subunits (Ku 86) of Ku, but not the other (Ku 70). affected the postfertilization cleavage in sea urchin. This differential function specific to Ku 86 is consistent with the observation that potential functions of DNA-PK. such as X-ray sensi- tivity, is restored by Ku 86 even when Ku 70 is absent (Smider et <;/.. 1994; Taccioli et : 10", 3.45 x 10", 6.41 X 10", 3.95 X 1012, 1.01 X 1013, 2.24 X 1013. 3.40 x 1013, 3.87 x 1013, 4.98 x 1013. 7.65 X 1013, 9.72 X 1013. Spectral sensitivity: To measure the spectral efficiency of luminescent countershading. dark-adapted animals were first exposed to a 490-nm stimulus (at 2.24 X 1013 photons m~2 s~ ' ) for 25 min to induce counterillumination. Animals were then exposed to light stimuli at 20-nm increments from 400 to 640 nm. following the general protocol described above: 60 s darkness, 60 s test wavelength stimulus, 60 s darkness, 5 or 10 min of the standard illumination to main- tain the induced countershading state. This process was repeated for each wavelength tested. The average duration of a complete trial was 3 h. The order of test wavelengths was randomized. Because shrimp eyes operate as photon counters, and the total number of photons in a given stim- ulus is a function of both light intensity and wavelength, stimulus irradiance was adjusted at each wavelength using neutral density filters to obtain equal photon irradiance levels at each wavelength. Even so, there were slight vari- ations in stimulus irradiance levels, which ranged from 1.16 X 1013 photons nT2s~' at 400 nm to 2.52 X 1013 photons irT2s~' at 620 nm. Bioluminescence data were subsequently corrected as detailed below to reflect a stan- dard irradiance of 1.20 X 10 photons m Mean bioluminescence was based on the last 20 s of each 60-s test stimulus, and the last 4 min for the standard 5-10-min illumination. Data correction. Because the test stimuli in the spectral sensitivity experiments varied slightly in intensity, all data were corrected to reflect the intensity of bioluminescence at each wavelength based on a stimulus irradiance of 1.20 X 10° photons m"2 s~'. Data collected in the quantum sen- sitivity experiments were used to model the correlation between bioluminescence intensity and stimulus intensity at 490 nm. One of two regression equations (for either San Diego or Santa Barbara collected specimens) was used to calculate (1) the predicted bioluminescence at 1.20 X 1013 photons m~2 s~', and (2) the predicted bioluminescence at the irradiance level measured for each test wavelength stim- ulus. Dividing (1) by (2) gave a proportional correction factor that was multiplied by the bioluminescence value at each test wavelength. Because not enough specimens were available to empirically derive the relationship between stimulus intensity and bioluminescence intensity at every wavelength tested, the assumption was made that quantum sensitivity did not change with stimulus wavelength, so that the relationship observed at 490 nm holds for the other wavelengths. This assumption of univariance is supported by the visual sensitivity and MSP experiments which dem- onstrated that only a single visual pigment is present. The correction for variable stimulus intensity also as- sumes that the observed relationship between biolumines- cence intensity and stimulus intensity holds at all levels of light adaptation or for changes in specimen responsiveness due to fatigue. This is an important assumption because in 4 of 9 spectral efficiency experiments, bioluminescence intensity showed slight but significant decreases over the course of the experiment. Nevertheless, in all cases the bioluminescent responses to test stimuli showed the char- acteristic fast kinetics indicative of the induced counterillu- mination condition (Latz and Case, 1992). In those 4 spec- imens demonstrating a decrease in the standard response at 490 nm. a correction factor for each data point for each of these individuals was determined based on a linear regres- sion describing the intensity of bioluminescence in response to the standard stimuli as a function of time. The effects of these corrections are shown for a single individual (Fig. 1). Responses were corrected for a standard stimulus irradiance of 1.20 X 1013 photons m~2s~' as shown in the following example. For this individual, the relationship between bioluminescence irradiance (in origi- nal units of volts s~') and stimulus irradiance at490nm (the standard stimulus wavelength) is best described by the equation: bioluminescence = 3.66 X 10~h * irradiance0'3 (r- = 0.40, F = 20.03, d.f. = 1.31, P = 0.0001). Using this regression equation, the predicted bioluminescence at an irradiance of 1.20 X 1013 photons m"2 s"1 is 0.4805 volts s 1. For a test wavelength of 400 nm, the measured stimulus irradiance was 1.28 X 1013 photons m"2 s 1. The predicted bioluminescence (using the regression equation) for a 490 nm stimulus at this irradiance level is 0.4924 volts s"', giving a correction factor of 0.976 (0.4805/0.4924). Thus, for a 490-nm stimulus, bioluminescence intensity at 1.20 X 1013 photons m~2s~' is 0.976 times that at 1.28 X 1013 photons m~2s"1. Making the important assumption that quantum sensitivity does not change with stimulus wave- length, the bioluminescence value measured at 400 nm is multiplied by 0.976 to reflect the response to a "standard" " 20-, E 18-1 § <6- 1 14- d ™ 12- 380 420 460 500 540 580 620 660 Wavelength (nm) Figure 1. Correction of spectral efficiency data for a single specimen ol Si'rxextex ximilis. Uncorrected data (solid circles) were corrected for variations in stimulus intensity (open circles) and for both stimulus inten- sity and temporal decrease in responsiveness (closed triangles). Reter to Materials and Methods for details on data correction. VISION AND BIOLUMINESCENCE IN SERGESTES SIMILIS 353 stimulus of 1.20 X 1013 photons m 2 s '. This correction was made in turn for each stimulus wavelength. Data from this individual were also corrected for a de- crease in responsiveness, because the least-squares linear regression between bioluminescence intensity (in volts s ) and the order of presentation of the 490 nm standard stim- ulus showed a slight but significant decay in response ac- cording to the following equation: bioluminescence = -0.022 * (order of presentation) + 0.887 (r = 0.72, F = 36.35, d.f. = 1,15, P < 0.0001). Because the response to 490 nm standard stimuli was somewhat variable over time, this regression equation is the best description of the general decay in response. The decay correction factor was calcu- lated based on time of stimulus presentation by first calcu- lating the predicted bioluminescence at a given stimulus time using the previous equation, and then dividing that result by the bioluminescence measured at the first 490 nm standard stimulus. For example, bioluminescence in re- sponse to the fifth 490 nm standard stimulus was calculated to be 0.7771 using this regression equation. Dividing this value by the value for the first standard stimulus (0.7857) gives a decay correction factor of 0.989. Making similar calculations for the order of presentation of each test wave- length stimulus, the bioluminescence value for each test stimulus was divided by the appropriate decay correction factor. The decay correction assumes that the effects were equivalent at all wavelengths tested. Following corrections for stimulus intensity and response decay, all biolumines- cence data were converted from units of volts s~' to pho- tons m~2 s"1 as previously described, based on the photo- metric calibration of the photomultiplier and the cross- sectional area of the ventral surface of the organs of Pesta for an adult shrimp of size 14 mm carapace length (Latz, 1983). To directly compare visual and behavioral spectral sensitivity, irradiance values from the electrophysiological experiments and bioluminescence values from the behav- ioral experiments were normalized for each individual. Results Microspectrophotometry (MSP) Spectral absorbance, based on MSP measurements of five sections of rhabdomeric tissue from a single individual, was unimodal with maximum absorbance in the blue-green (Fig. 2 A). Assuming that the initial scans were uncontaminated with metarhodopsin (M) pigment, template fitting to the difference spectra (Fig. 2B) indicated a rhodopsin (R) pig- ment with a Amax of 492 nm. The template best-fitting the R/M mixture after red light exposure yields a Amax of 485 nm. This value was corrected to allow for the residue of R in the R/M mixture to give a best estimate of the M pigment Amax of 484 nm, and an M/R extinction ratio (at the respec- tive Amax values) of 1.333. Iterative template-fitting methods (Kent, 1997) suggest, however, that the fraction of M in the 0.2-i 0.15- 0.1- 1 0.05- -0.05 300 400 500 600 Wavelength (nm) 700 I 0.6 0.2- -0.2 300 400 500 600 Wavelength (nm) 700 Figure 2. Spectral absorbance based on microspectrophotometry of five sections of rhabdomeric tissue from the retina of Sergesres similis. (A) Averaged absorbance spectra; bold trace shows the initial absorbance, light trace shows the absorbance following saturating red light illumination, dashed trace shows the absorbance following photobleaching with bright white light. For display, spectra have been standardized to an absorbance of zero at 730 nm. the limit of the spectral scan. (B) Averaged difference spectra for photobleaching of the rhabdom from its initial state [bold; derived from bold trace minus dotted trace in ( Al] and for the photobleach- ing of the rhabdom from its steady state R/M mixture following saturating red light [light; derived from light trace minus dotted trace in (A)]. Specific absorbances at the Amax for these absorbance spectra were 0.0078 jum~' and 0.0094 /M,m~', respectively. Smooth solid traces are best-fit templates (Stavenga et al.. 1993) with Amax values of 492 nm and 485 nm. respec- tively. The dashed line is the estimated metarhodopsin absorbance spec- trum (Amax = 484 nm) resulting from the correction for the residual M in the R/M mixture difference spectrum (light trace), and the absorbance spectrum of the rhodopsin (dotted; Amil, = 495 nm), after correcting for contaminating M in the initial scan (bold). initial measurements may have been as high as 15% and, after correction for this contamination, the R and M Amax values can be revised to 495 nm and 484 nm, respectively, with a M/R extinction ratio of 1 .406. Visual sensitivity of the eye (ERG) The electrophysiologically determined visual spectral sensitivity of dark-adapted specimens of Sergestes similis indicated that the sensitivity maximum was centered at approximately 500 nm in the blue-green region of the spec- trum (Fig. 3). Both blue (480 nm) and near-UV (400 nm) chromatic adaptation uniformly depressed the sensitivity 354 S. M. LINDSAY ET AL. 0.8- 5 0.6- 0.2- -0.6-1 380 420 460 500 540 580 620 Wavelength (nm) Figure 3. Visual spectral sensitivity of Sergestes similis based on electroretinogram (ERG) measurements. Sensitivity, based on the inverse of the irradiance required to elicit a !()() or 200 /u.V response, showed a broad maximum centered around 500 nm. Symbols represent means ± SE for 6 specimens. curve across the spectrum (Fig. 4), and had no effect on ERG waveform (Fig. 5). These results are consistent with those of the MSP study indicating that only a single visual pigment is present. Behavioral sensitivity The magnitude of luminescent countershading by 5. si- milis depended on the level of stimulus irradiance (Fig. 6). -8.0-, -9.0- y> 0-10.0- o jz Q. ra-11.0- o -12.0 dark adapted 480 nm adapted 380 420 460 500 540 580 620 -8.0-, -9.0- B dark adapted 400 nm adapted \ 0-10.0- • o £. Q. ra-11 0- 3 -19 r>- ^a********* 380 420 460 500 540 580 620 Wavelength (nm) Figure 4. fitted »t chromatic adaptation on the spectral sensitivity of Sergestes similis. Illumination of the eyes with (A) 480 nm, and (B) 400 nm light uniformly depressed spectral sensitivity across the spectrum compared to the response of dark-adapted eyes. A ]B 1C D -\N%*- .*nr**" .*\P"** .*\r»**' 0 0.5 1 1.5 0 0.5 1 1.5 0 0.5 1 1.5 0 0.5 1 1.5 G H O> cr 0 0.5 1 1.5 0 0.5 1 1.5 0 0.5 1 1.5 0 0.5 1 1.5 Time (s) Figure 5. Effect of chromatic adaptation on the ERG waveform of Sergestes similis. Dark bars indicate light stimulus off; white bars indicate light stimulus on. (A-D) 200 /xV responses measured in one specimen. (E-H) 100 /J.V responses measured in another specimen. Response to a 430 nm stimulus for the (A) dark-adapted condition and (B) after 400 nm light adaptation. Response to a 530 nm stimulus for the (C) dark-adapted condition and (D) after 400 nm light adaptation. Response to a 430 nm stimulus for the (E) dark-adapted condition and (F) after 480 nm light adaptation. Response to a 530 nm stimulus for the (G) dark-adapted eye and (H) after 480 nm light adaptation. A behavioral threshold occurred at approximately 2-3 X 1C)'2 photons m^s"1 (Fig. 7). At lower stimulus irradi- ances, light levels were near background as measured in an empty chamber. At higher stimulus irradiances. biolumines- cence increased as the 0.35 power of irradiance according to a power law (log-log) regression (r = 0.61). Thus the increase in bioluminescence by S. similis did not match the increase in stimulus irradiance. The magnitude of bioluminescence varied according to stimulus wavelength (Fig. 8). After correcting the data to reflect a standard irradiance of 1.20 X H)1' photons m~2s~', and when necessary, for temporal decreases in responsiveness, the mean spectral efficiency curve for lu- minescent countershading showed a broad peak between 480 and 540 nm (Fig. 9) in the blue-green region of the visible spectrum. The wavelength dependence of the spec- tral efficiency was not symmetrical. Bioluminescence de- creased dramatically approaching the red wavelengths and was negligible above 600 nm, while an intermediate re- sponse still occurred in the near-UV at 400 nm. A compar- ison of the mean data for all 9 specimens tested showed that the corrected data fell within the 95% confidence limits of the original data. Discussion In the midwater shrimp. Sergestes similis. there is a close similarity between visual and behavioral photon and spec- tral sensitivities. The microspectrophotometry results indi- VISION AND BIOLUMINESCENCE IN SERGESTES SIM1L1S 355 v, 4.5 6.4x1011 3.9x10 photons m s" 12 10x1013 2.2x101 0 50 Figure 6. Representative bioluminescent responses by a single specimen of Sergestes similis to increasing stimulus irradiance. Bioluminescence was first induced using a 490 nm stimulus of intermediate intensity, then the specimen was presented with 60-s stimuli from a range of intensities, all at 490 nm. The line represents the level of bioluminescence versus time. Dark bars indicate stimulus light off; white bars indicate stimulus light on. Values above graphs are stimulus intensities. cated that S. similis possesses a single visual pigment with maximum absorbance around 495 nm. Such data can be used to calculate the spectral sensitivity of a photoreceptor containing this visual pigment by first calculating the spec- tral absorptance of an axially illuminated rhabdom. To do this, two additional pieces of information are required: rhabdom length and the specific absorbance (i.e.. absor- bance jumT1) of the visual pigment in the photoreceptor. Rhabdom lengths vary somewhat with eye size, but by sectioning the aldehyde preserved eyes of 5. similis, rhab- dom lengths were found to range from 128 to 161 /urn for shrimps with carapace lengths of 1 1.8 to 14.2 mm (T. Frank, unpublished data). For the specimens used in the ERG 1X101 1X101 1x101 5 1x10" ® i 5 1x103 1x10 1x1012 1x101 1X101 Irradiance (photons m'J s ) Figure 7. Effect of light intensity on average maximum biolumines- cence produced by Sergestes similis. All stimuli were at a wavelength of 490 nm. The magnitude of bioluminescence measured during the last 20 s of each 60-s test stimulus was averaged for each individual. Ten specimens were tested; means ± SE are shown. Responses for stimulus irradiance > 2 X 1012 photons m~2 s~' were best described by the power law (log-log) equation y = (1.87 X lO^tx"15 (r = 0.61). At lower stimulus irradiance values, light levels were near background. Note the separate scale for above-threshold bioluminescence. experiments, which ranged in size from 10.3 to 13.5 mm carapace length, the estimated upper and lower bounds for rhabdom lengths in these animals were approximately 120 to 150 /xm, with a mean of approximately 135 ju,m. This value is similar to that reported by Hiller-Adams et al. (1988) for the sergestid Sergia tenuiremis. MSP measure- ments of specific absorbance for S. similis suggest a specific absorbance of 0.0074 /uirT1 (Kent, 1997) although this is significantly lower that the value of 0.01 jam"1 reported by Cronin and Frank (1996) in Systellaspis debilis, but only slightly lower than the value of 0.008 ju.m~' reported as being 'typical' of crustacean photoreceptors (e.g., Cronin and Goldsmith, 1982). At wavelengths greater than the peak absorbance (495 nm). the spectral absorptance closely matches spectral sensitivity data from ERG measurements and the spectral efficiency curve of luminescent counter- shading (Fig. 10). Using maximum or minimum values instead of mean rhabdom length has little effect on spectral absorptance, while increasing specific absorbance to levels more typical of crustacean photoreceptors leads to a better fit between the different data sets at long wavelengths. At short wavelengths, however, there is significant divergence which probably cannot be attributed to the photosensitivity spectrum of the rhodopsin departing from the absorptance spectrum at short wavelengths, although data on this subject are limited (Dartnall, 1972). It is more likely that the effec- tive spectral sensitivity of the eye is affected by intraocular, pre-retinal filters which selectively filter short wavelength light (Goldsmith, 1978; reviewed by Fein and Szuts, 1982). Electrophysiological measurements using the electroreti- nogram (ERG), corresponding to the summed mass re- sponse of a large number of photoreceptor cells to a light stimulus, were performed to determine the behaviorally relevant spectral sensitivity of S. .similis. While this tech- nique provides a more comprehensive assessment of the visual spectral sensitivity of an organism than do measure- 356 S. M. LINDSAY ET AL. 420 nm 500 nm 580 nm 600 nm 50 Figure 8. Representative bioluminescent responses by a single specimen of Sergestes similis to different wavelengths of light. Bioluminescence was first induced using a 490 nm stimulus of intermediate intensity, then the specimen was presented with 60 s stimuli at various wavelengths, at approximately equal irradiance of 1.5 x 10'-' photons m~- s '. Bars as for Figure 5. ments from single photoreceptors (reviewed by Goldsmith. 1986). ERG results do not reflect the amount of higher order processing of visual input, nor the behavioral response to visual stimuli. Thus the behavioral studies of luminescent countershading extend the physiological assessment, result- ing in a comprehensive description of the organism's sen- sory and behavioral response to ecologically relevant light stimulation. Luminescent countershading by S. xiniilis occurred over a relatively narrow range of irradiance. A behavioral thresh- old occurred at approximately 3 X 10|: photons m~2 s~', as lower irradiance levels resulted in minimal levels of biolu- minescence which were not significantly different from background. This illumination level may represent the min- imum irradiance causing light adaptation of the eye. which appears to be required for luminescent countershading (Latz and Case. 1992). Under ideal conditions, bioluminescence should exactly match stimulus irradiance. As discussed by Young et al. (1980), differences in geometry between stim- ulus and response as well as calibration assumptions make direct comparisons difficult, although relative changes should still be valid. In the present study, the range of stimulus irradiance tested was less than two orders of mag- nitude. Within this range, bioluminescence increased with stimulus irradiance according to a power law (log-log) regression as found by Young et al. (1980) for midwater squid and fish. However, the magnitude of the increase in bioluminescence did not match the magnitude of the in- crease in stimulus irradiance. Other counterilluminating an- imals may not precisely match changes in the stimulus irradiance (Young et al., 1980). yet they exhibit a better match over a larger dynamic illumination range for lumi- nescent countershading than did 5. similis in the present study. In Sergestes similis, behavioral spectral efficiency was similar to visual spectral sensitivity. A survey of species for which behavioral and physiological spectral sensitivity data are available (Table I) suggests that behavioral spectral I ,,H "° 09- 3 380 420 460 500 540 580 620 660 Wavelength (nm) Figure 9. Behavioral spectral sensitivity of Sergestes similis based on the bioluminescence spectral efficiency, corrected for a stimulus irradiance of 1. 2 X 10" photons m~~ s"1 (see text). Symbols represent means ± SE for 9 specimens; the curve is a fourth degree polynomial function fitted to the data, where y = 6.1 X 10V - 1.2 X 10V + 9.2 x 10'V - 3.0 x 10"x + 3.6 x 1015. 380 420 460 500 540 580 620 660 Wavelength (nm) Figure 10. Comparison of relative visual and behavioral spectral sen- sitivity of Sergesres similis. Symbols represent means ± SE. Both the normalized ERG sensitivity (solid circles) and normalized biolumines- cence spectral efficiency (open circles) coincide well at long wavelengths with the calculated spectral absorbance (solid line) of a rhodopsin with peak absorbance at 495 nm. a rhabdom axial length of 135 /urn and .1 specific absorbance of 0.0074 /xn-T1. VISION AND BIOLUMINESCENCE IN SERGESTES SIMII.IS 357 ri "o II oo § ~ 5 C oo o-. 00 ON is ON c a oo S "O r- DO £ ~J ~J 0. —' ^£ o 1- — ON ed '•:: - ON •s. c TD u i— 1 -o o. P-, ^O 3 "O ^_ u< • ra yi TD c ~^; ir, « 3 "3 CX3 o> i o U •a -J i .s O *~ ^ US ij vi — ^ JJ 4J "J a S >- 2 — S c1 c g SJ . Q Lj_ 0> 3 X ca c •i o "id Oi) C 1 J 3 CU 5 CQ * 12 c v || ^ u Cu c n 1 S ^ .ti 3 at> c ca — > >. JCj § d in .£ "3 'S '-S E — . D "> •i ^ 5 •p o " J3 H ^ ^ 2 rN § £ 3 0 ^ .C X *- U "rt c " C £ TD ^> ^_ is Is "3 C •p "I .E "E. 3 rt **^ o. i/-, flj O 1J O "^ ^d in '7 m ^ o o^ g < < C3 1 •o o P D P' z s £ c m ^ a "* ""' ir< "^ 2 5 -o c X o in 3 i OJJ S X 1 - a rt D- t o o — O 1 £r> u" O" O\ ^ p- OJ -b f 1 O tr O UJ TD Ot) c i |£ 8. O O S! u = 0. TD -o K & S c o ^ 2 g S 8 « s PQ CQ -t -t c S "§ 3 ^ d •3 -O ^ £ So a — |*V^ .y — , T^ 'MI ^ 1 2 pj 1J ix ^ 1 £ = y5 ( = -C 1 2 1 _3 ii ft D O " § «3* *O z s 1 ^ s; — Si '-* ^ "O 1-1 ^ ca i minesci pompi If "•3 -C: | 4 2 I 1 'o. "§ p c X ^ " -5 E „ a j o -< c — s = 1 5 '1 il £' cu 3 3 « s" ^J '^- 3 C "3 ^ 3 = j-, D- ~ | £ •S q .2 5 - >' •y ^ ^ ti 0 C c ^ _ "^J -r: rt OJ 3 1 > 1 s £ ^ >5 c ;? o O O £ •^ ftl « * — s u 358 S. M. LINDSAY ET AL Table II Relative behavioral and visual sensitivities to near-UV versus blue-green light for bioluminescent deep-sea crustaceans possessing a single visual pigment Species Visual Spectral Relative Visual Relative Sensitivity Sensitivity Behavioral Maximum (nm) (400 nm/500 nm) Sensitivity (400 nm/500 nm) Acanthephyra 510 0.24 0.18 curtirostris Acanthephyra 510 0.30 0.16 smithi Notostomus 490 0.24 0.10 gibbosus Sergestes 500 0.27 0.48 similis For species except Sergestes simi/is. behavioral sensitivity was defined as the reciprocal of the irradiance required to elicit a simple movement behavior for 400 nm or 500 nm stimuli (Frank and Widder. 1996). For S. similis. behavioral sensitivity was based on the standardized magnitude of bioluminescence. For all species, visual sensitivity was based on electro- retinogram (ERG) measurements (data from present study and Frank and Case. 1988). sensitivity tends to be somewhat broader than that measured electrophysiologically, though the behavioral maxima are similar to maxima in visual sensitivity and photopigment absorption. Douglas and Hawryshyn (1990) noted that be- havioral measurements of spectral sensitivity in fish varied with method, with some behaviors, such as the tail-flip response, being activated only by certain wavelength stim- uli. Behavioral sensitivity also may depend on the type of stimulus applied. For example, the initiation of biolumines- cent flashing by the firefly. Plwtinus scintillnns. has differ- ent spectral sensitivity maxima for horizontal and vertical light stimuli (Table I; Lall. 1993). These different sensitiv- ities appear to be associated with different classes of pho- toreceptors. Similarly, multiple peaks in behavioral sensi- tivity in the tree frog Hyla cinerea (King et al.. 1993) and the tick Hyalommct ilromeilarii (Kaltenrieder et cil., 1989) correspond to peak sensitivities of different classes of pho- toreceptors in these animals (Table I). Sergestes similis shows somewhat greater behavioral sen- sitivity in the near-UV compared to several other species of deep-sea decapods that have single visual pigments and similar ERG-measured visual sensitivities (Table II; Frank and Widder. 1996). This difference may be a reflection of the different behaviors assayed (i.e.. luminescent counter- shading versus movement behaviors) and their ecological context The ecological significance of sensitivity to near-UV and UV light remains unknown. The intensity of downwelling irradiance at mesopelagic depths, calculated utilizing attenuation coefficients measured in the epipelagic zone, may be sufficient to be visually detected (Frank and Widder, 1996). High UV visual sensitivity may aid in luminescent countershading where the strategy is to match the downwelling irradiance field. Alternatively, UV sensi- tivity may play a role in regulating the diurnal vertical migrations of S. similis and other vertically migrating crus- taceans (Forward, 1988). The present study is the first to measure the complete spectral efficiency of behavior in a deep-sea animal using ecologically relevant light stimuli. Previously, Kay (1965) measured peak behavioral spectral sensitivity of 470-490 nm in the euphausiid, Meganyctiphanes non'egica, based on the number of individuals responding to bright photoflash stimulation. The peak in spectral efficiency corresponds to the visual pigment absorbance maximum at 488 nm deter- mined by MSP (Denys and Brown, 1982) and to the peak visual sensitivity of 490 nm measured by electroretinogram (Frank and Widder, in press). As with 5. similis. both visual and behavioral peaks for M. non'egica lie in the blue-green region of the visible spectrum. Even though it has long been suggested that bioluminescence by euphausiid crustaceans may serve as camouflage (Herring and Locket. 1978), to date there has been no direct demonstration that dim biolu- minescence appropriate for luminescent countershading is produced by euphausiids in response to environmentally relevant light cues. The response thresholds for light-induced behaviors of three species of deep-sea caridean shrimp with single visual pigments occurs at approximately 0.4-4 X 10" photons irT2 s~ ' at 500 nm (Frank and Widder, 1994b). Because the eyes of the caridean shrimp were in the dark-adapted state, it is expected that the response threshold for S. similis bioluminescence would occur at higher illumination levels because of the apparent need for the eyes to be light-adapted for the initiation of luminescent countershading (Latz and Case, 1992). The 5. similis response threshold of 2-3 X 1012 photons m~2 s~' at 490 nm for the light-adapted eye suggests that the absolute visual sensitivity of S. similis is similar to that for the caridean shrimp. Sergestes similis and other midwater animals inhabit day- time depths where dim downwelling light is sufficient to silhouette their opaque body structures, making them po- tentially more detectable by predators. Previous studies have demonstrated that the spectral emission (Widder et al., 1983). angular distribution (Latz and Case, 1982) and irra- diance of bioluminescence (Warner et al.. 1979) of S. similis are consistent with a camouflage function. The results of the present study show that the behavioral sensitivity of biolu- minescence is also appropriate for camouflage, based on the daytime optical environment encountered by this species. In the northeast Pacific, 5. similis inhabits daytime depths of approximately 200-700 m (e.g., Clarke, 1966; Pearcy et al.. 1977). The apparent behavioral threshold for luminescent countershading at approximately 3 X 10|; photons m'2 s~' (for a 490 nm light stimulus) would be reached at a daytime VISION AND BIOLUMINESCENCE IN SERGESTES SIMILIS 359 depth of approximately 350 m in the coastal waters off San Diego (Kampa, 1960). Therefore, ambient light levels at the depths inhabited by 5. siniilis would be sufficient to induce and maintain luminescent countershading. The control of luminescent countershading in S. siniilis represents a simple case, where the visual system, biolumi- nescence emission, and oceanic downwelling light all op- erate in the same spectral range. There are other cases where these simple conditions do not hold. All vertically migrating animals including 5. similis experience diel changes in their optical environment. However, animals such as the squid Abralia. which is able to modify the spectral emission of bioluminescence (Young and Mencher, 1980), must coor- dinate the adjustable spectral emission of its biolumines- cence with diel changes in the spectral distribution of down- welling light in order for luminescent countershading to be effective. Animals with multiple visual pigments, such as the oplophorid shrimp Systellaspis debilis (Frank and Case, 1988). may use only one of their photoreceptor classes to drive luminescent countershading. In these more complex cases, the relationship between visual sensitivity and behav- ioral spectral efficiency promises to offer an intriguing insight into the coordination of luminescent camouflage behavior in midwater animals. 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Action spectra of the biolumi- nescence response in firefly Photinus pyralis females (Coleoptera. Lampridae). Soc. Neurosci. Abstr. 22(1-3): 1145. Young, R. E. 1983. Oceanic bioluminescence: an overview of general functions. Bull. Mar. Sa. 33: 829-845. Young. R. E., and F. M. Mencher. 1980. Bioluminescence in mesope- lagic squid: diel color change during countenllumination. Science 208: 1286-1288. Young, R. E., E. M. Kampa. S. D. Maynard, F. M. Mencher, and C. F. E. Roper. 1980. Counterillumination and the upper depth limits of midwater animals. Deep-Sea Res. 27A: 671-691. Reference: Bio/. Bull. 197: 361-367. (December Enhancement of the Response of Rock Crabs, Cancer irroratuSy to Prey Odors following Feeding Experience ANDREW RISTVEY1 AND STEVE REBACH* Department of Natural Sciences. University of Man-land Eastern Shore, Princess Anne, Man-land 21853 Abstract. The rock crab. Cancer irroratus Say, uses chemically mediated learning in the search for food. Rock crabs are opportunistic benthic predators and scavengers. Observations indicate that although they eat a variety of items, they are more sensitive to, and prefer, odors of food items that they have been eating. We found that C. irroratus is more responsive to a familiar food source than to an unfamiliar one and can distinguish between the odors of two different prey after being fed one species for an extended time. Initial preferences for two mytilid bivalves, Mytiliis edulis and Geukensia demissa, were determined in a Y- maze. Crabs were then fed only one of the mussel species for 28 days and retested, using sequential and simultaneous presentations, for their responses to familiar and unfamiliar prey odors. Crabs increased their responses to familiar prey odors, but not to unfamiliar odors. In foraging tests, crabs ate M. edulis more often regardless of the species to which they had been familiarized. Introduction A search image can be defined as a perceptual filtering mechanism learned from experience. It may be only a transitory improvement in perceptual ability, but this selec- tive attention can increase the possibility of stimulus detec- tion (Bond and Riley, 1991 ). In foraging behavior selective attention results in certain prey characteristics being dis- criminated by a predator, facilitating more efficient forag- ing. Search images increase accuracy and decrease response time because the predator requires less information about the prey and becomes more efficient in locating it (Law- rence, 1985a). Received 19 August 1998; accepted 2 September 1999. 1 Present address: 10470 Longwoods Rd., Easton. Maryland 21601. * Author to whom correspondence should be addressed. E-mail: srebach@umes-bird.umd.edu. Search images, originally postulated for visual stimuli (Croze, 1970; Pietrewicz and Kamil, 1979; Lawrence, 1985a, b) could also be associated with chemical stimuli (Atema et ai, 1980; Aterna and Derby, 1981; Derby and Atema, 1981). In the aquatic environment, especially in the absence of light, chemical signals may be the best cues for information about the surrounding environment. Organic molecules are part of the ambient milieu, and an organism must sift out extraneous "noise" to find the information needed for foraging, predator avoidance, and mating (Zim- mer-Faust, 1991). Chemosensory cues play a major role in agonistic (Kara- vanich and Atema, 1998), sexual (Gleeson, 1980), host- finding (Atema and Derby, 1981), and foraging (Pearson and Olla, 1977) behaviors in crustaceans. Experience influ- ences an animal's response to those cues. Derby and Atema ( 1981 ) demonstrated that after lobsters (Homarus america- nus) fed on a specific prey, their sensitivity to that prey odor increased, and they developed a preference for that partic- ular prey. The rocky shore gastropod Nucella lamellosa can discriminate between predatory and nonpredatory crab ef- fluents (Marko and Palmer, 1991), and the nudibranch Aeo- lidia papillosa can distinguish between odors from a learned prey anemone and five other possible prey anemones (Hall et nl., 1982). Yellowfin tuna (Thunnus albacares) became more sensitive to specific fish odors after feeding on that prey for a period of time, but lost their sensitivity after a few weeks without reinforcement (Atema et ai. 1980). In preda- torily naive postlarval lobsters, responses to metabolites of Cancer irroratus and Mytiliis edulis (normal lobster prey) were lower than in field-collected adult lobsters that may have had experience with those prey (Daniel and Bayer. 1987a). When naive lobsters were fed amphipods or clams, those fed amphipods developed stronger responses to am- phipod and not to clam metabolites, but those fed clams did not develop strong responses to either prey (Daniel and 361 362 A. RISTVEY AND S. REBACH Bayer, 1987b). In theory, search images need to be rein- forced (Atema et ai, 1980; Atema and Derby, 1981). and their strength may vary with experience and with the avail- ability and palatability of the food (Gendron, 1986). Many studies have tested the role of chemoreception in foraging, but few have centered on search images. We examined search images and foraging behavior in the rock crab C. irroratus preying on the bivalve mussels Geukensia and Mvtihts. These two groups of mussels occur in the range of C irroratus. are frequently taken as prey items (Stehlik. 1993), and have similar physical characteristics. Mytilus is found on hard substrates in the tidal zone, and individuals located close to shore are known to be eaten by rock crabs (Drummond-Davis etui., 1982; Stehlik, 1993). Geukensia is found in soft substrates in tight clumps attached to marsh grasses, and is less likely to be encountered by a crab. No data are available on innate prey preferences in C. irroratus. Using effluents from these two species, we examined changes in the responsiveness and sensitivity of crabs to prey. Chemoreceptors on various body parts appear to in- fluence behaviors such as walking, searching, and dactyl grasping, and these actions are dependent upon the concen- tration of the stimulus (Derby and Atema, 1982). The po- sition and movements of these structures are good indicators of the sensitivity to food odors (Derby and Atema, 1981). We determined a baseline response for rock crabs, famil- iarized them with a single prey, and retested them to deter- mine if there were acquired or changed responses to familiar and unfamiliar odors. Materials and Methods Rock crabs were collected by local watermen using traps off the coast of Delaware and Maryland and by the inves- tigators at Chincoteague on Assateague Island, Virginia. Ribbed mussels. Geukensia demissa, between 2 and 6 cm. were collected from salt marsh environments in Girdletree. Maryland, and Chincoteague, Virginia. Blue mussels, Myti- lus edulis. between 2 and 6 cm, were collected on the rock jetty at the Ocean City, Maryland inlet. Maintenance conditions A recirculating, biologically filtered saltwater system was used for tests. The water temperature was 1 1° ± 3°C, the salinity was 32 to 35 ppt, and the photoperiod was 12 h light: 12 h dark. Crabs were kept in 40-1 tanks (50 X 25 cm), two per tank, with an acrylic plastic divider separating them to prevent aggression and to enable staggered feeding. They were acclimated to laboratory conditions for 1-2 weeks and fed a diet of squid (Loli^o sp.) every other day. Mussels were housed in an isolated 80-1 tank equipped with a power filter. Fresh mussels were collected every week. The water in the mussel tank was kept at 15" ± 2"C and the salinity at 33 to 34 ppt. The Y-maze Crabs were tested in a 92 X 33 X 20 cm acrylic plastic Y-maze containing 55 to 60 1 of salt water. A piece of acrylic divided the top 50 cm of the maze into two arms (see Fig. 1, Rebach. 1996). A dual-head MasterFlex peristaltic pump (Cole-Parmer #7553.20) delivered liquids through plastic tubing to a 2.5-cm hole in either arm of the Y-maze at the rate of about 0.33 1 min~'. A concentration gradient was established, with the odor becoming more dilute at the drain located at the base of the maze. In dye trials using methylene blue, the average dilution at the base of the maze was determined by spectrophotometer to be 13.6% that of the original concentration. These trials indicated that odor reached the crab within 3 min, with little mixing. In pre- liminary trials, crabs responded to odor within a few min- utes of its reaching them. Each test lasted 10 min during which the observer recorded, from a blind, crab location in the maze and behaviors exhibited. Mussel effluent Mussel effluent was produced daily by placing live mus- sels (equivalent to 10 g soft tissue 1~') in seawater for 10 h (Derby and Atema, 1981). Mussels were checked weekly for reproductive condition to ensure that effluents would be consistent in character. Behaviors We adapted methods used by Derby and Atema (1981) for tests of chemoreceptive sensitivity and measured changes in behavior in the Y-maze that reflected changes in sensitivity. An approach to the source of the effluent was defined as a high-sensitivity behavior. Low sensitivity was characterized by the following behaviors: I Chela raise — claws lifted beyond normal position. 2. Antennule burst — flicking rate increased suddenly. 3. Antennule wipe — antennules groomed, usually with third maxillipeds. May occur in bouts. Wipes occur- ring within 5 s of each other were considered to be one wipe. 4. Maxilliped wave — third maxillipeds moved slowly back and forth without touching one another. 5. Maxilliped wipe — third maxillipeds rubbed against each other within a 5-s period. 6. Shift — body position changed. 7. Body raise — body raised up on dactyls. 8. Fanning — rapid movement of second maxillipeds along with third maxillipeds opened widely to ex- pose mouth parts. An approach was scored when a crab crossed a line 8 cm from the inlet flow at the end of an arm of the Y-maze before a 10-min run was completed. Crabs began the ex- EXPERIENCE AND RESPONSES TO PREY ODORS 363 periment at the base of the maze. If an approach did not occur during a test run. the low-sensitivity behaviors were used for scoring. In every run, each occurrence of a behav- ior other than an approach was counted as one unit (Derby and Atema. 1981). Totals for each crab were then averaged to determine a mean frequency. The higher the value, the more sensitive the crab was, or had become, to the prey odor. Scores were obtained for each crab tested before and after training was complete. Initial response to mussel effluent Sixteen crabs were fasted for 24 h and then tested. In control tests, seawater was used on both sides of the Y- maze. Each crab was allowed to acclimate in the maze for 8-12 h and then tested with effluent and a seawater control. This was repeated for the other mussel species about 10 h later. Initial response tests were completed within 24 days. Familiarization with a specific mussel odor After testing crabs for their initial response, training began. Eight crabs were fed Geukensia and eight M\tilus for a period of 28 days. Crabs were fed whole mussels ad libitum during the training period. The average number of mussels eaten each day was recorded. Response to odors in sequential presentation after familiarization Familiarized crabs were retested as in the first experi- ment. The tests began with a post-familiarization seawater control using seawater in both arms of the Y-maze. Re- sponses to the two prey effluents (familiar and unfamiliar) were recorded based on sequential presentation: each prey odor was tested against a seawater control. Approaches were recorded when they occurred; if no approach occurred, low-sensitivity behaviors were scored. Response to odors in simultaneous presentation after familiarization Familiarized crabs were retested for preference between the two effluents. Odors were presented simultaneously without a seawater control. Distinguishing which odor elic- ited heightened behavioral responses was not possible in this test, so only approaches were scored. Influence of experience on mussel selection A foraging test was performed using live prey in 40-1 tanks. Five equal-sized mussels of each species were ran- domly positioned in the tank and buried to about 667r of their length in a calcite substrate to make them factually cryptic. Crabs were allowed to forage for 12 h, and each test was videotaped. The total number of mussels handled and the species handled first, eaten first, or rejected after being handled were recorded. Mussel shells were marked with small spots of white epoxy to make them easier to see during videotape analysis. Analysis of data Initial scores for responses to seawater, the Mytilus and Geukensia effluents, and the sequential presentation test results were compared using the Friedman test (Systat 8.0, SPSS Inc., Chicago, Illinois). Responses to familiar and unfamiliar odors for each familiarization group were com- pared with the Wilcoxon signed rank tests. (Systat 8.0). Differences between means were determined with Bonfer- roni post hoc analysis (Systat 8.0). Mussel selection data from the foraging test was analyzed using a Wilcoxon signed rank test, a Mann- Whitney test with tied ranks, and a chi-square 2x2 contingency table (Zar, 1984). Results Behavioral obsen'ations Crabs responded to effluents within 2 to 3 min. Those that did not approach responded by displaying lower sensitivity behaviors. Typically, crabs flicked their antennules slowly or intermittently, with occasional bursts, before odors reached them. A burst, with maxilliped or antennule wipes. occurred when the effluent reached the crab. Within 5 min, chela waves and raises occurred, and crabs began to move. Antennule flicks pointed in the direction of movement. These behaviors continued until tests were concluded. In a typical approach, the initial behavior was similar to that of a non-approach. At about 5 min, crabs began walking towards the effluent. Upon reaching the end of the maze, they often grabbed the inflow hole with their chelae. In some simultaneous presentation tests, crabs entered one arm of the maze, turned back, and then proceeded down the other side, through which the familiar effluent flowed. Initial response to mussel effluent Figure 1 shows the responses of 16 crabs to mussel effluent before the crabs were familiarized with other mus- sel species. The Friedman test revealed no differences be- tween the responses to the seawater control and to the Mytilus or Geukensia effluents (P > 0.05, Table I). Familiarization with mussel odor Familiarization periods began after initial responses were obtained. Crabs were fed their assigned species of mussel ad libitum. At first, crabs consumed 6 to 8 mussels a day, although within 4 weeks this decreased to 2 to 3 mussels a day, especially those fed Geukensia. During familiarization, crabs exhibited periods of increased activity. Usual behav- 364 A. RISTVEY AND S. REBACH 3.5 3.0 • 2.5 ° 2.0 H s 1-5H 0.05). Response to odors in simultaneous presentation after familiarization Of the 8 crabs familiarized with Mytilus, 4 approached familiar prey effluent; of the 8 familiarized with Geukensia, Table I Responses to effluents Odors compared Method ot comparison P value Initial odors Pre-familiarization and post-familiarization seawater, familiar and unfamiliar mussel effluents familiar vs. unfamiliar effluents familiar effluent vs. post-familiarization seawater familiar effluent r.v. pre-familiarization seawater unfamiliar effluent vs. pre-familiarization seawater unfamiliar effluent vs. post-familiarization seawater Familiar and unfamiliar odors-familiarized w/ Mvtiltis Familiar and unfamiliar odors-familiarized w/Geitkcnsta familiar r.v. unfamiliar odors-familiarized w/ Mvtilus. familiar vs. unfamiliar odors-familiarized vt/Gettki'iixia unfamiliar odor-familiarized vj/Gi'iikensia vs. unfamiliar odor-familiarized w/ Mytilus familiar odor-familiarized w/Geukensia r.v. familiar odor-familiarized w/ Mytilus Mylilns eaten vs. Geukensia eaten Mytilus rejected vs. Geukensia rejected No. mussels eaten-familiarized on Mytilus or Geukensia No. mussels rejected-familiarized on Myiilitx or Geukcnsiu Species eaten vs. familiarization species Species rejected r.v. familiarization species Friedman statistic (Fig. 1) 16.16.16 >0.05 Friedman statistic (Fig. 2) 12, 12, 12. 12 <0.01 Bonferroni test (Fig. 2) 2. 12 <0.01 Bonferroni test (Fig. 2) 2. 12 <0.01 Bonferroni test (Fig. 2) 2, 12 <0.01 Bonferroni test (Fig. 2) 2. 12 >0.05 Bonferroni test (Fig. 2) 2. 12 >0.05 Wilcoxon signed ranks test (Fig. 3) 5 <0.05 Wilcoxon signed ranks test (Fig. 3) 7 <0.05 Bonferroni test (Fig. 3) 5, 5 0.05 Bonferroni test (Fig. 3) 7. 5 >0.05 Wilcoxon signed ranks test 14 <0.05 Wilcoxon signed ranks test 9 >0.05 Mann-Whitney tied ranks (Fig. 4) 7. 7 >0.05 Mann-Whitney tied ranks (Fig. 4) 7. 7 >0.05 2X2 chi-square (Fig. 4) 14 >0.05 2X2 chi-square (Fig. 4) 14 >0.05 EXPERIENCE AND RESPONSES TO PREY ODORS 365 14 T 8 • • H Initial Post Seawater Seawater Control Control Unfamiliar Familiar Odor Odor Figure 2. Comparison of mean (±SEM) frequenaes of initial and post-familiarization control tests, and tests of familiar and unfamiliar odors after familiarization. No differences were found between responses to seawater controls and unfamiliar odors, but responses to familiar odors were significantly greater than to unfamiliar odors and controls (as indi- cated by an asterisk). 1 approached unfamiliar effluent. These tests failed to yield significant responses because only approaches were scored. The 1 1 crabs that did not approach during the simultaneous presentation exhibited increased sensitivity, as in the se- quential tests. These crabs began to search soon after the odor reached them; they raised chela and walked upcurrent and downcurrent, but they failed to make an approach. 18 -• 16 • • 14 • • 12 • 10 • • 8- 6 • • 4 . . 2 • D Fam w/ Mytilus H Fam w/ Geukensia Unfamiliar Odor Familiar Odor Figure 3. Comparison of mean (±SEM) frequencies between famil- iarization groups. No differences were found in unfamiliar odor responses between familiarization groups. Differences were found in familiar odor responses. Abbreviations: Fam w/ Mytilus = Familiarized with M. edulis; Fam w/ Geukensia = Familiarized with G. demissa. Low-sensitivity scoring could not be used because it would not have been possible to determine which odor was influ- encing the behavior. Influence of experience on prey selection Within 15 min of being placed in the tanks, crabs probed the calcite substrate with their dactyls, attempted to climb tank walls, or walked around. During this period, all crabs touched and moved both species of mussels. More Mytilus (45) were eaten than Geukensia (19), but about the same number of mussels of both species were rejected (Mytilus. 13; Geukensia, 14). A Wilcoxon signed ranks test determined that, overall — regardless of familiar- ization group — significantly more Mytilus were eaten than Geukensia (P < 0.05), but there was no difference in numbers of Mytilus or Geukensia rejected (P > 0.05) (Ta- ble I). Figure 4 compares the species of prey eaten and rejected by crabs in the two familiarization groups. Crabs familiar with Geukensia cumulatively ate 21 Mytilus and 5 Geuken- sia, and rejected 1 1 Mytilus and 1 1 Geukensia. Crabs fa- miliar with Mytilus ate 24 Mytilus and 14 Geukensia. and rejected 2 Mytilus and 3 Geukensia. In all cases, Mytilus was handled first and eaten first. Both groups of crabs handled about the same number of mussels (43 for Mytilus- familiarized crabs and 48 for Geukensia-famiYiarized crabs). A Mann-Whitney test with tied ranks determined that there was no significant difference in total mussels, regard- less of species, eaten (P > 0.05) or rejected (P > 0.05), between crabs familiarized with Mytilus and crabs familiar- ized with Geukensia (Table I). 30 i 25 20- 15 10 • • 5 • • QFam w/ Mytilus HFamw/ Geukensia Mytilus Eaten Geukensia Eaten Mytilus Rejected Geukensia Rejected Figure 4. Comparison of lotal number of prey eaten and rejected by crabs familiarized wilh Myiilus and Geukensia. No differences were found in ratios. Abbreviations: Same as in Figure 3. 366 A RISTVEY AND S. REBACH A chi-square analysis of a 2 X 2 contingency table determined that the ratio of Mytilus to Geukensia eaten was not different (P > 0.05), nor was there a difference between the ratio of mussel species rejected (P > 0.05) for each of the familiarization groups (Table I). Regardless of the mus- sel species that the crabs were familiar with, they ate and rejected the same proportion of mussel species. Discussion Our results show that, in the absence of recent experi- ence, Cancer irroratus did not strongly respond to the effluent of either Mytilus editlis or Geukensia demissa. Exposure to one prey type increased the sensitivity of the crabs to that prey's odor, and responsiveness increased with experience. The results of the sequential presentation tests indicated a significantly increased sensitivity towards famil- iarized prey. Scores of low-sensitivity behaviors were higher for familiar effluents than for unfamiliar effluents. However, the simultaneous presentations could not distin- guish the responses to the two odors because it was not possible to determine which odor was eliciting the height- ened behaviors. Crabs did not often approach the effluent source in either the sequential or simultaneous tests. These results may be misleading since crabs did react to the odors. The use of prey effluents instead of live prey can influence observed behaviors, because the lack of reinforcement with actual prey may have been responsible for the observed decreases in response. It is also possible that the concentrations of stimulatory compounds may have been below the thresholds necessary to initiate search or approach. The amino acids glycine, taurine, glutamate, serine, and threonine have been found to be the most stimulatory in feeding assays in several species of Cancer (Case, 1964; Allen et at., 1975). These amino acids may have been present at low concentrations in test- mussel metabolites. Palaemonetes pugio. a grass shrimp, specifically recognizes various foods by qualitative and quantitative differences in combinations of low molecular weight substances intrinsic to those foods (Carr, 1978). Concentrations that elicited antennular responses may have been too low for activation of approaches and feeding behaviors. Rebach et at. ( 1990) found antennular sensitivity for mussel extract in C. irroratus to be as low as 10""' g I '. Pearson et at. (1979) found similar sensitivities for littleneck clam extract in C. magister. Both of those studies used tissue extracts, whereas this study used prey rinse (body odor) from intact animals. The threshold to elicit feeding is 10s higher than the arousal threshold in rock crabs (Rebach et at.. 1990) and H)1" to l()17 times higher in blue crab (Cullinectes sapidus; Ache, 1982). Arousal thresholds are found at concentrations of picograms ( 10" i: g) per liter, search behavior thresholds at micrograms (10~6 g) per liter, and handling and ingestion of food at milligrams (10"' g) per liter (McLeese, 1973; Mackie, 1973; Pearson and Olla, 1977; Ache, 1982). The effluents used in this study did not often direct the crabs' responses towards familiar effluents, but did arouse them. We may therefore infer that these effluents had concentrations between 10~' and 10"' g I"1. The stomach contents of rock crabs indicate that they are opportunistic feeders (Drummond-Davis et ai, 1982). An assortment of algae, polychaetes, gastropods, mussels, and bits of hermit crabs and other crustaceans are typically consumed. It is possible that nutritional needs were not being met by a diet restricted to a single food for an extended time, and the crabs may have lost interest in familiarized prey. Again, feeding reinforcements were ab- sent and may have counteracted the effects of training. In the foraging tests, tactile and visual cues were intro- duced by the use of living prey rather than effluents. Both groups of familiarized crabs ate and rejected similar num- bers of mussels, and there was no difference in the ratios of Mytilus to Geukensia eaten and rejected. However, even though crabs encountered both species of mussels, they handled Mvtilns first, ate Mytilus first, and consumed more Mytilus than Geukensia regardless of familiarization group. Since crabs walked over both species before selecting any prey, both species should have had an equal chance to be handled. Geukensia has a heavier shell than Mytilus. possi- bly making it more difficult to open, but this did not account for the crabs' preference, because equal numbers of both species were rejected. Metabolites from both species were present in the test tank. As soon as a mussel was eaten, freshly killed prey odors would have filled the tank, possibly decreasing the importance of odor in choosing the next prey, and other cues may have become more important. If crabs use more than one sensory cue in prey choice, a hierarchy may exist for all sensory functions in determining prey selection. Maynard and Sallee (1970) found that che- motactile stimulation of lobster dactyls overrode antennular stimulation. Our tests were run in the light to permit video- taping, so visual cues might have played a role in prey selection. Geukensia was difficult to see against the mottled calcite, whereas the blue-black colored Mytilus contrasted well with the background. Arthropod compound eyes are adept at discerning contrasts (Evans. 1984). Mytilus may have been visually less cryptic and thus more susceptible to predation. The crabs were more likely to have been in contact with Mytilus than with Geukensia before they were caught, and might have retained their sensitivity for that species. Alter- natively, Mvtilim may have been easier to open, or may have simply tasted better than Geukensia. Lobsters are also able to detect differences between two closely related mus- sels— in this case Mytilus and Modiolus — and showed in- creased sensitivity with experience and training (Derby and EXPERIENCE AND RESPONSES TO PREY ODORS 367 Aterna, 1981). Atema et al. (1980) found qualitative differ- ences in the amino acid content of live prey rinses. The foraging study supported the differences found in sensitiv- ities between crabs familiarized with Mvtilus and crabs familiarized with Geukensia. However, M\tilus appeared to be more attractive than Geukensia when crabs were given a choice between live prey. The responses of crabs to mussel odors before and after experience with those mussels indicated that familiarization increased sensitivity towards a prey item. Whether this resulted in the formation of a chemosensory search image or a species-specific preference is not clear. However, recog- nition and remembrance of familiar prey odors facilitates the location of suitable prey in a benthic habitat where few other cues are available. Acknowledgments We thank D. French. C. Loshon, and V. Kennedy for their advice and assistance, E. Layman, M. Ailes, and D. Birkett for their help and support. We also thank two anonymous reviewers for their helpful suggestions and thoughtful com- mentary. AR submitted parts of this manuscript in partial fulfillment of the MS degree at UMES. This research was partially supported by NSF Grant # RII-8704054. This is Contribution No. 30 from the Crustacean Research in Ecol- ogy and Mariculture (CREAM) Institute of the University of Maryland Eastern Shore. Literature Cited Ache, B. 1982. Thermoreception and chemoreception. Pages 369-398 in The Biology of Crustacea. Vol. 3. H. L. Atwood and D. C. Sandeman. eds. Academic Press. New York. Allen, W., E. Frederick, and R. Wong. 1975. 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K. 1991. Chemical signal-to-noise detection by spiny lobsters. Biol. Bull. 181: 419-426. Reference: Biol. Bull. 197: 368-376. (December 1999) The Role of Latero-Frontal Cirri in Particle Capture by the Gills of Mytilus edulis HAROLD SILVERMAN1, JOHN W. LYNN, PETER G. BENINGER*. AND THOMAS H. DIETZ Department of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana 70803; and * Departement de Biohgie, Faculte des Sciences, Universite de Nantes, 2 rue de la Houssiniere, 44322 Nantes Cedex, France Abstract. In this study we examined the mechanism of particle capture in Mytilus edulis, using radioactive-label clearance studies, progressive fixation, and scanning elec- tron microscopy to visualize in detail the cirri and their range of motion. Confocal laser scanning microscopy was used to observe the interaction of cirri with 1 /xm fluores- cent latex particles on live strips of control and serotonin- treated isolated gill tissue. The gills of M. edulis possess large, complex latero- frontal cirri composed of 18-26 pairs of cilia. Particles that were intercepted by the cirri were transferred to the water current on the frontal surface of the filament where they were propelled toward the ventral par- ticle groove. Clearance studies demonstrated that M. edulis removed Escherichia coli from 5°C seawater bathing me- dium at 4.9 ml g~' dry tissue min~'. When the gills were exposed to 10 3 M serotonin, the latero-frontal cirri stopped moving and became fixed in a flexed position that partially blocked the frontal surface of the filament. Clearance stud- ies demonstrated that removal of E. coli from the seawater bathing medium was reduced 90% to 0.5 ml g~' dry tissue min"1 when 10~3 M serotonin was present. These data demonstrated that for small particles (< 2 ;um) in the near field, movement of cirri was essential for successful capture either by direct contact or with water acting as a hydrome- chanical coupler. Introduction Considerable progress in understanding particle process- ing mechanisms in suspension-feeding bivalves has been made in recent years (Beninger et ai, 1993, 1997; J0r- gensen, 1990; Ward et ai, 1998b). Investigations have focused on various components of the feeding sequence: Received 3 June 1999; accepted 5 October 1999. 1 To whom correspondence should be addressed. E-mail: cxsilv@lsu.edu capture-transport-selection-ingestion. However, the actual mechanism(s) of particle capture remains difficult to deter- mine. A number of components are important in understanding the mechanism of particle capture. Fluid mechanics has been used to describe both the delivery of particles to the gill filaments and their movement on the gill (J0rgensen, 1983; Nielsen et ai, 1993; Ward et ai. 1998a; Riisgard and Larsen, 2000). The potential interaction of particles with a structure on the gill would be an equally important compo- nent to understanding the mechanisms leading to particle capture events. Generally, suspension feeding bivalves can capture particles over a wide range of sizes from 10"' /urn to 102 pun, with most showing efficient clearance of parti- cles in the range of a few to tens of /xm (M0hlenberg and Riisgard, 1978; Ten Winkel and Davids, 1982; Sprung and Rose, 1988). The mechanisms used to capture particles at the extremes of the size range may differ, because the behavior in fluid would change depending on particle den- sity and geometry. To observe the actual particle capture event, one needs to be able to resolve both the particle and the cellular or- ganelles that are involved with the capture. To date, Riis- gard and his colleagues have used isolated preparations and constructed half filament models to analyze particle move- ments close to the filaments of Mvtilus edulis (Nielsen et ai, 1993; Riisgard et ai, 1996). Their observations indicate that particles can be directed to the frontal surface of gill fila- ments by direct interception of particles by latero-frontal cirri. They have extended these observations by determining the path as particles approach the gill filaments, in vivo, using a side-mounted microscopic preparation (Riisgard and Larsen, 2000). Silverman et ai. ( 1996, 2000) and Beninger et ai. (1997) have examined gill strips using confocal laser scanning 368 CIRRI AND PARTICLE CAPTURE 369 microscopy (CLSM). The disadvantages of this system are that the observations are made on gill strips that are isolated: normal fluid flow is altered; and normal neural or hormonal cues are modified or missing. However, the advantages ot this system include magnification, resolution, tunable depth of focus, and preselection of a gill strip where all cilia are in motion as confirmed by observation before adding particles. The near-field events of particle interaction with cirri are not altered by isolation procedures. The only bivalve investi- gated to date using this technique is Dreissena polymorpha, a species with a homorhabdic eulamellibranch gill type (Silverman et 30 mm anterior-posterior shell length), the posterior extremity of the gill was used for CLSM preparations, because the demibranchs are thinnest and shortest in this region, while still presenting the typical gill architecture. This preparation facilitated the observation of 4-5 mm wide strips of gill extending from the gill arch to the ventral particle groove. Strips from the posterior extremity of each demibranch were removed using micro- surgical instruments, and placed in isosmotic (1060 mosm) seawater on a microscope slide lined with Nitex screen (125 /urn mesh). This screen allowed cilia-generated water cur- rents to flow on both sides of the demibranch. and mechan- ical disturbance of the gill was avoided by supporting the coverslip above the preparation with silicone vacuum lubri- cant posts at each corner. For larger animals with thicker gills, we also observed single isolated filaments. For spec- imens < 30 mm. the gill was sufficiently small and thin to allow any region to be used. We observed the gills of more than 40 specimens (range of shell length = 18-81 mm) during this study. The preparations were observed using the CLSM tech- nique previously described (Silverman et al.. 1996). The gills were examined with a Noran Instruments confocal system attached to a Nikon Optiphot microscope with a 40X fluor lens (NA 1.3). The laser wavelength was set at 529 nm with an FITC barrier filter in the return image path. Reflected channel images were acquired (laser scan and image digitization < 1 /us/frame) and sequentially stored at 120 frames s~' (~ 8 ms between frames). Images were captured and analyzed using the Odyssey InterVision (No- ran) software on a Silicon Graphics Indy computer. Images were not altered except to adjust the contrast. Fluorescent beads of 0.7 ju,m or 1 .0 jnm diameter were added to the preparations to track particle motion in cilia- aenerated water currents and particle transport on the cili- ated epithelia. Gill preparations were oriented such that beads could be added under the coverslip in the dorsal resjion of the strip and subsequently moved from dorsal to ventral. Particle velocities were determined after observing the movement of individual fluorescent beads over known distances and times. Scanning electron microscopy To examine the spatial relationships of cirri and cilia, gill tissue was prepared for scanning electron microscopy using a progressive fixation technique that allowed the arrest of cilia at all stages of their beat cycles. Small mussels were placed in containers with enough seawater (~ 20-30 ml) to cover the shells. They were left undisturbed until their 370 SILVERMAN ET AL. siphons were clearly visible. Drops of 2% OsO4 (1-2 mil were slowly added near the edge of the container; the specimen was left intact for 20 min. The posterior adductor muscle was then cut and the animal exposed to fixative for another 40 min. In a few cases, the adductor muscle was carefully cut before OsO4, was added, with total exposure to osmium in the bath being 30 min. The mussels were then rinsed in seawater and fixed for an hour in 2% glutaralde- hyde in seawater. After fixation, the gill was removed, dehydrated in a graded ethanol series, critical-point dried, and sputter-coated with gold-palladium. Specimens were mounted on stubs and visualized with a Cambridge Scan- ning Electron Microscope. Some gills were removed and photographed before and after fixation, and again after critical-point drying to monitor tissue shrinkage. Bacterial clearance Clearance of "S-labeled Excherichia coli from the bath- ing medium was quantified with an adaptation of the method of Riisgard ( 1988) as described previously (Silver- man et al, 1995, 1997). Mussels (< 30 mm shell length) were placed in individual test tubes containing 20 ml aer- ated artificial seawater (5°C) with or without 10~3 M sero- tonin for 15 min. Radiolabeled bacteria were added to each tube after the mussels opened their siphons, and after 1 min equilibration, four samples (100 /Ltl) were collected at 10- min intervals. The reduction of bathing medium radioactiv- ity followed first order kinetics, and the clearance was expressed as ml g~ ' dry tissue min" ' . Data are expressed as the mean ± one standard error with the number of animals in parentheses. Results Cimil structure Progressive fixation stopped the cirri and cilia in various stages of their beat cycle. The cirri position varied from being arched over the frontal surface of a gill filament to extending into the interfilument space (Fig. 1). Adjacent cirri on a filament were arrested during fixation in a stag- gered pattern, such that neighboring cirri were not in the same position relative to the frontal surface (Fig. 1 ). The CLSM time series micrographs (Figs. 3-6) are of filaments in the same orientation as in Figure 1. Confocal images of a cirrus beat cycle indicated that during the power stroke the cirrus bent over the frontal surface into a flexed position (Fig. 2D). On recovery it returned to an extended position perpendicular to the apical surface of the latero-frontal cirrus cell (Fig. 2 A, I). The body of the cirrus beat as a single unit; a complete beat required the cirral body to pivot at its base in addition to moving through the ciliary wave- form (Fig. 2). The cirrus moved out of the plane of focus during the flexion, but as the free ciliary tips were moving out of view they were clearly resolvable (Fig. 2E). The high Figure 1. Scanning electron micrograph of gill filaments from A/V///II.V etlnlis treated to allow fixation of cirri during the beat cycle. Cirri flex during their power stroke moving over the frontal surface (f) of the filament. Cirri are in the extended position (arrowhead) in the interfilament space, and in the flexed position (c) with their free tips bent over the frontal surface. Adjacent cirri on a filament were stopped at different stages of a heat cycle (arrow). The inset of a higher magnification indicates that the tree ciliary tips associated with the two ciliary plates of a cirrus angle away from the body of the cirrus to give a 'V like appearance ( *). Bar = 25 ;u,m; inset bar = ? /uni- rate of capture of each image (< 1 /x.s) minimized blurring due to the movement of the cirrus (Fig. 2E). The cirral beat averaged 7.9 ± 0.4 Hz (n = 6). Cirral interaction with particles Particles interacted with moving cirri in several different ways but usually were (i) swept onto the frontal surface of the filaments and entrained into the frontal water current (Fig. 3). Fluorescent particles (1 /urn) entering the interfila- ment space were captured within the 2.2-2.8 /urn wide "V" of the free ciliary tips of the cirrus and moved toward the frontal surface as the cirrus beat from the extended into the flexed position. In most cases, particles interacting with a cirrus were moved directly into the frontal current and transported at 307 ± 36 /am s l, n = 4 (Figs. 3, 4) toward the ventral particle groove. Three additional types of particle interaction with a beat- ing cirrus were observed, (ii) During flexion of the cirrus the particle may be deposited at the edge of the frontal surface without being immediately transported. Subsequent cirral movement positioned the particle into the frontal surface current (Fig. 4). (iii) Some particles remained between the cirral plates and moved with the cirrus for multiple beat cycles (Fig. 5). These particles were finally incorporated into the frontal flow after several cirral beat cycles. The particles that moved with the cirral extension away from the CIRRI AND PARTICLE CAPTURE 371 Figure 2. A series of confocal video images ( 1 20 frames s ' ) of a single cirrus from an in vitro gill strip of M\tilus etlulix. The time between each frame shown in this series is 25 ms. The focal plane of the video is slightly off the perpendicular to the dorso-ventral axis of the filament. The parallel bright lines flank the lateral surface of the filament. Thus, the beat of the cirral tip is toward the frontal surface of the filament that lies to the right and into the background, at a right angle to the visible lateral surface of the filament. The resolution of individual ciliary tips (arrowhead) on one side of the cirrus (one cirral plate) is clear. The bending (power stroke) in A-D moves the plane of the ciliary tips toward the frontal surface of the filament. Images in frames E-I represent the recovery stroke. The total time taken for this beat cycle was —200 ms (beat frequency ~ 5 Hz). The arrow identifies the midpoint of the cirrus. Bar = 4 /j,m. frontal surface suggest that there was intimate contact, ei- ther directly or within a few tenths of a /xm. from a cirrus (Fig. 5). (iv) Finally, on rare occasions the particle did not reappear, indicating it became dislodged from the cirrus in the interfilament space (not shown). Particle entrainment in the frontal surface current of the gill was observed both in association with mucus rafts (Beninger et at., 1993, 1997) and in the absence of any visible mucus rafts (Figs. 3, 4). Indeed, we have observed filaments where two particles were being transported, but at different velocities. One moving particle can overtake another particle on the frontal surface (Fig. 6). Such events indicate independent trans- port of the two particles along the frontal surface, and would suggest that they were not moving in a single mucus raft, or not at the same height above the epithelium. Quick-time videos used to construct Figures 3-6 are archived in the electronic data base of The Biological Bulletin at www.mbl.edu/litml/BB/ VIDEO/BB.video.html. Serotonin effects on cirral position and clearance of Escherichia coli Progressive fixation (as described above) revealed that the cirri in the serotonin-treated animals were arrested in the flexed position, occluding most of the frontal surfaces of the gill filament (Fig. 7). The amount of frontal surface covered by the cirri is somewhat exaggerated in this figure because the fixation caused 44 ± 3% (;; = 4) shrinkage. From our CLSM observations in some sections of the gill, both frontal and lateral cilia continued to beat in the presence of 10 M 372 SILVERMAN ET AL Figure 3. A confocal microscopy time series or the frontal surface of an in vitn> gill filament from Myiilus ednlis. Dorsal is at the top of each image and ventral toward the bottom. The time elapsed between each successive frame is 33 ms. Cirral tips are visible as 'V structures (arrows). The cirri beat in and out of the plane of the image and the 'V's are the tips of the cirri as they flex over the frontal surface on each side of the filament. One cirrus directed a I-/LUTI fluorescent latex particle onto the edge of the filament from the right interhlament space (A: bright spot at top right). The particle was entrained in the frontal water current and moved ventrally down the frontal surface (B). The particle was transported on the frontal surface of the filament (C and D) moving 20 ju.nl in 100 ms by frame D. Bar = 10 |um. serotonin. This was also evident in some areas of gill that were fixed for scanning microscopy. This motion is inferred in the wave-like pattern formed by the tips of lateral cilia (Fig. 7). Although the fixation was not instantaneous, the positions of the lateral cilia tips are suggestive of a syn- chronized or metachronal motion (Fig. 7). To assess particle capture in the absence of movement by latero-frontal cirri, cirral motion was arrested by adding 10 M serotonin to the seawater bathing medium (Jor- gensen, 1983; Ward el ai. 1998a). Clearance (ml g ' dry tissue min ') of 35S-labeled E. coti by M. edulis in 5°C seawater was 4.92 ± 0.43 (n = 3) for controls (130 ± 12 mg dry tissue) compared to 0.51 ± 0.18 (n = 3) for treated animals (142 ± 26 mg dry tissue). Clearance of bacteria from control mussels displayed first order kinetics with 21 nun required to remove 5Q7c of the label (Fig. 8). The serotonin-treated mussels also displayed first order kinetics, but experienced an 89.6% reduction in the rate of removal of bacteria from the seawater. Discussion The movement of an individual cirrus on a living strip of Mytilits i'tlulis gill can be observed at high resolution using confocal laser scanning microscopy (CLSM). The beat cy- cle is similar to that described for the cirri of Drcissena IHilyniorplui (Silverman et ai, 1996). In the flexed position the tips of the cirri are located over the frontal surface of the gill filament, whereas in the extended position they project into the interfilament space at the level of the latero-frontal cells from which they originate. Confocal microscopy permits resolution of individual cirri and. depending on reflectance and light scattering, includes their individual ciliary tips. The lateral (x-y plane) resolution (about 0.2 /xrn) of CLSM, the enhanced rate of sequential image acquisition (~ 8 ms/frame), and high speed of capture of individual images (< I /xs) enabled observation of the interaction of l-/xm particles with latero- frontal cirri. Although particles were seen to be directed Figure 4. A cnnlocul microscopy time series of an in run* Myiiltis cJnli.\ gill filament. A l-/j,m fluorescent particle (bright spot under the letter A) interacted with a cirrus and was moved toward, but not onto, the frontal surface of the filament (A-C). On the next contact with a cirrus, the particle was moved into the frontal surface water current and transported rapidly (D-F). The reference line marks the initial location of the particle in all ol the images. The elapsed time ol the series was 125 ms. Bar = 10 /urn CIRRI AND PARTICLE CAPTURE 373 Figure 5. A confocal microscopy time series of an in vitro Mytilus eiliilis gill tilumenl. A fluorescent particle moved rapidly down the center of the filament ( A-F) in 1 25 nis. A second particle was associated with a cirrus (A; upper left) whose ciliary tips were partially out of the plane of focus. As the cirrus continued to move hack toward the extended position in the left interfilament space, the particle was drawn back with the cirrus (B-C) until it moved out of the plane of focus in D. It reappeared in E-F as the cirrus moved into its flexed position over the frontal surface of the filament. Bar = 10 fim. onto the frontal surface during the power stroke of a cirrus, we could not determine whether the interaction was direct or if water acted as a mechanical coupler between the cirrus and particle (Riisgard ct til.. 1996). However, the close interaction (herein defined as within a few tenths of /urn) of the beating cirrus with the particle is responsible for the movement of many particles to the frontal surface of the filament where particles subsequently move in the frontal water flow. Under the experimental conditions described here for M. edulis. the particles can be moved to the frontal Figure 6. A confocal microscopy time series (25 ms between each image) of an HI vitro Mytilus ediilis gill filament showing that transport of particles along a filament can occur at different rates. In these images two particles moved along the frontal surface of the filament. The larger particle (arrowhead) moved at 750 jum s~'. while the velocity of the smaller particle (arrow) was 350 /j.m s '. Bar = 10 jim. surface of the filaments even when no mucus is visible or onto visible mucus rafts that are observed to be moving along the frontal surface (Beninger el at, 1997). In both cases, transport of panicles along the frontal surface aver- aged about 307 ^im & l, and was similar to previously reported rates (Jones et at. 1990; Ward et at, 1991. 1993; Nielsen et at. 1993). Figure 7. A scanning electron micrograph of two filaments from a Mytilus eilnlis gill treated with 10 ' M serotonin. The space between these fixed filaments \\ as larger than that observed in Figure I (20 /im. versus the 15 jj.m). Visible in the enlarged interfilament space are the tips ot the underlying lateral cilia (Ic). whose origins from two adjacent filaments are easily discernible. The arrows indicate areas where the lateral cilia were fixed in the \arious phases of beat associated with the metachronal wave form unset, lower magnification). The cirri (c) in these preparations are stopped over the frontal surface of the filament, occluding much of the frontal surface. Each cirrus is composed of pairs (arrowheads) of cirral plates containing IS -26 cilia. Bar = 10 /im. inset bar = 20 /j.m. 374 SILVERMAN ET AL 12.5 12.2- E 12.0- E Q. 73 •11.8- 1 1.5- 1 1.2 10 20 30 time (min) Figure 8. Time-dependent removal of Escherichia coli from 5°C sea- water by Mytilin ctlnlis controls (open square) or treated with 10"' M serotonin (filled square). Each point represents the mean ± SEM for 3 animals (SEM smaller than the symbols are not visible). The regression equation for the control mussels was: Y = 0.033X + 12.365. r = 0.994. and treated: Y = -0.003X + 12.376. r = 0.908. The role of these ciliary organelles in particle capture associated with suspension feeding has been controversial. Early workers including Atkins (1937) and Oral (1967) suggested that cirri were important as "mechanical" filters or traps that moved particles onto the frontal surface of the gill filament for subsequent transport. Support for this view is gained from the differential particle capture in bivalve species with cirri of different sizes (Owen and McCrae, 1976; M0hlenberg and Riisgard, 1978; J0rgensen et ai, 1984). McHenery and Birkbeck ( 1985) suggested that Esch- erichia coli were captured more effectively by marine bi- valves with larger cirri than by those with small or no latero-frontal cirri. A similar relationship between cirral size and particle capture has been reported for several freshwater bivalves (Silverman ft cil.. 1995. 1997; Tankersley. 1996). The observations of particle/cirral interactions described here extend the observations of Riisgard et a/.. (1996). These earlier observations were based on traditional light microscopic lenses immersed in water (theoretical maxi- mum resolution approximately 0.5 jam) to observe particle interaction with cirri in isolated M. cdulis gills. Their iso- lated gill preparation contained 10 7 M serotonin to stim- ulate ciliary activity and were positioned to maintain the normal interfilament gap (40 jiim) at the level of the lateral ciliated cells. The greater resolution of CLSM provides more structural detail, and the high image capture speed (< 1 jas) and recording speed (120 frames s~') allow better visualization of particle interaction with the cirri. The con- focal images indicate that the cirrus was responsible for the movement of 1 jam particles onto the frontal surface of the gill filament. The similarity of the results between these two studies, where different methods were used, supports the accuracy of information gained from both studies. While observation of cirral/particle interactions with the CLSM provides evidence for (he role of cirri in the particle capture process, it does not provide the information under the normal water flow conditions expected in vivo. Ward and his colleagues have provided endoscopic videos ( 1998a, b) that permit observation of dye streams and the motion of relatively large particles (7 jam) as they move within the pallial cavity toward the gill of a living M. edulis. Ward et ul. ( 1998a) indicate that particles approach the gill at a low angle (less than 30°) and are either captured by the filament or bounce until they are subsequently captured. Observing these endoscopic videos, it appears that there is a non- random location of the particles interacting at or near the edge of the filament. These near-field interactions probably represent interception by the cirri, rather than particle cap- ture by the frontal water flow (Ward et ul.. 1998a). The endoscopic observations are valuable to describe flows and gross movements of particle approach to the filaments. The variable resolution and relatively low magnification limit the observations that are necessary to identify precise mech- anism! s) of interaction between filament organelles and individual particles. Thus, it is not possible to determine the distances separating particles from the structures on the gill filament. Despite the particular limitations of both the con- focal and the endoscopic observational approaches, the data obtained from each appear to be complementary and sug- gest the importance of both cirral interaction (near-field) and water flow (far-field) for particle capture in this species. These data also are complementary to those obtained by Riisgard and his colleagues (1996, 2000) demonstrating cirral interaction with particles in various isolated prepara- tions and also in young undisturbed mussels. Although most reports indicate that isolated M. edulis gills must be stimulated by 10 7 M serotonin to maintain ciliary activities, we have found that sections of isolated gills commonly have beating cilia. We selected preparations where latero-frontal cirri, and frontal and lateral cilia were all beating. The beat frequency of the cirri (8 Hz) and the patterns of motion were similar to the 4-9 Hz observed by Oral (1967) in young mussels in vivo. He noted that intact M. edulis displayed a diverse repertoire of ciliary activity, including spontaneous arrest. While Oral (1967) highlights a beat cycle that places adjacent cirri offset by Vi beat cycle, he points out that there are many other relationships be- tween adjacent cirri that would not be detected with the methods used. The data presented in this study also are consistent with the recent in vivo observations of Riisgard and Larsen (2000) and with the modeling done by Riisgard and colleagues based on isolated filament preparations (Nielsen et ul.. 1993: Riisgard et a!., 1996). Indeed, the observations made here are consistent with the calculations of Silvester and Sleigh (1984) who suggest that latero- frontal cirri can act as "sieves." although perhaps more water moves around rather than through the cirrus (Riisgard et ul.. 1996). The finding of an 83% reduction in particle capture in Mvtilus edulis after serotonin is used to block cirral move- CIRRI AND PARTICLE CAPTURE 375 merit (Ward et til., 1998a) also is consistent with the previ- ous results of Jorgensen (1983), who showed that cirral movement was critical to particle capture. The reduction in particle capture efficiency after serotonin administration has been ascribed to a breakdown in cirral-generated water currents (Ward et ai, 1998a). However, the data of the present study provide an alternate explanation. The obser- vations of the present study confirm that the cirri of treated specimens are arrested in the flexed position (Jorgensen, 1975, 1983). eliminating direct cirri particle interaction while the frontal and lateral cilia continue to beat. Further- more, the positions of the arrested cirri physically block particles from approaching the frontal surface following serotonin treatment. Bacterial clearance was reduced 90% under these conditions. On occasion we have noted that cirral beating may be spontaneously arrested. Oral (1967) also noted that ciliary organelles on /;; vivo mussel gills would spontaneously become motionless. Fluorescent par- ticles moving in the frontal water current were observed to "fall" occasionally or be deflected from the frontal surface (Nielsen et ai, 1993; unpub. obs. in D. polymorpha). Thus, cirral activity is important for both particle capture and. perhaps, keeping particles entrained in the frontal water current. In addition to the cirral arrest, serotonin also caused gill musculature to relax in preparations (J0rgensen, 1983; Me- dler and Silverman. 1997). which can increase the interfila- ment distance. In modeling particle-capture mechanisms, the dimensions of gill structures (ostial size, intertilament space, filament size, cirral size), rate of ciliary beat, and ambient hydrostatic pressure are all critical components (Silvester and Sleigh, 1984; Famme et ai. 1986; Griinbaum ft ul.. 1998). Riisgard and Larsen (2000) point out that the calculations based on /';; vitro gill dimensions must at least match intact clearance values, microscopic measurements on intact tissues, and particle transport rates, if such calcu- lations are to accurately predict mechanism. Many of these components are neither fixed nor necessarily uniform across a gill at any particular time (Medler and Silverman. 1997; Medler f/ «/., 1999). We cannot determine whether the particle-delivery mech- anism involves direct particle contact with the cirri. How- ever, at the observed proximity of the particle with the cirrus (< 1 ju,m), the question of 'hydromechanical' versus 'me- chanical' becomes irrelevant, as any intervening water is essentially a mechanical coupler. Evidence for this reason- ing comes from particles that were not successfully moved to the frontal surface on the first cirral interaction. Many of these particles were drawn back with the cirrus as it moved toward the extended position (Figs. 4. 5). These particles appeared to reside within the space (2.2-2.8 /urn) between the free tips of the two ciliary plates making up the cirrus. The particle ( 1 ^im in diameter) could be physically wedged between the plates, moving within the water being drawn back by the cirrus during its extension, or by cohesive contact with the plates. The amount of water that might be moving with the cirri is small according to the model of Riisgard et ul. ( 1996). In addition, their model indicates that particles can make contact with the cirral plate. The mech- anism for particle capture is close interaction with the cirrus, and at low Reynolds numbers, hydromechanical and me- chanical are essentially the same, and consistent with the confocal observations made in this study. Both the eulamellibranch. Dreissena polymorpha and the filibranch Mytilus edulis species have homorhabdic gills and complex cirri, and capture small particles in the near-field when cirral movements cause particles to be deflected onto the frontal surface. The cirri stop the particles and then transfer them to the frontal flow (Riisgard et ai. 1996; Beninger et ai. 1997; Riisgard and Larsen. 2000; this study). The environment of the transfer is non-steady state. low Reynolds number (Griinbaum et ai. 1998). Predicting exact particle movements without actually observing them is difficult (Shimeta and Jumars, 1991). Far-field observa- tions are possible using endoscopy that situate particle cap- ture in intact specimens near the latero-frontal cirri (Ward et ul.. 1998a). Near-field interactions are visible with CLSM (Silverman et ai. 1996; this study). These observations, together with microscopic observations on single filaments or isolated gills (Nielsen et ai. 1993; Riisgard et ai. 1996), and studies using in vivo preparations (Oral, 1967; Riisgard and Larsen, 2000) all demonstrate the importance of cirri and their direct interaction with particles during the capture process. Acknowledgments This project was supported by Louisiana State University Sea Grant Program NA46RG0096 R/ZMM-5. We thank Julie Cherry, Ron Bouchard, Paul Bruce, and Chris Thi- bodaux for technical assistance. This research was aided by the M. D. Socolofsky Microscopy Facility. Additional video images may be viewed at http://www.biology.lsu.edu/re- search. Literature Cited Atkins, D. 1937. On the ciliary mechanisms and interrelationships of lamellibranchs. II. Sorting devices on the gills. Q. J. Microsc. Set. 79: 339-373. Beninger, P. G., S. St-.|ean, Y. Poussart, and J. E. Ward. 1993. Gill function and mucocyte distribution in Placopecten magellanicus and M\tilus eilulis (Mollusca: Bivalvia): the role of mucus in particle transport. Mm: /-..,>/. Prog. Ser. 98: 275-282. Beninger, P. G., J. VV. Lynn, T. H. Dietz, and H. Silverman. 1997. Mucociliary transport in living tissue: the two-layer model confirmed in the mussel Myrilm. edulis L. Biol. Bull. 193: 4-7. Chambers, E. 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Mohlenberg, F., and H. U. Riisgard. 1978. Efficiency of particle re- tention in 13 species of suspension feeding bivalves. Ophelia 17: 239-246. Moore, H. J. 1971. The structure of the latero-frontal cirri on the gills of certain lamellibranch molluscs and their role in suspension feeding. Mar. Biol. 11: 23-27. Nielsen. N. F., P. S. Larsen, H. U. Riisgard. C. B. Jorgensen. 1993. Fluid motion and particle retention in the gill of Mytilus ediilis: video recordings and numerical modelling. Mar. Biol. 116: 61-71. Owen. G. 1974. Studies on the gill of Mytilus ediilis: the eu-latero- frontal cirri. Proc. R. Soc. Loiul. B. 187: 83-91. Owen, G., and J. M. McCrae. 1976. Further studies on the latero-frontal tracts of bivalves. Proc. R. Soc. Lund. B. 194: 527-544. Riisgard. H. U. 1988. Efficiency of particle retention and filtration rate in 6 species of Northeast American bivalves. Mar. Ecol. Prog. Ser. 45: 217-223. Riisgard, H. U., and P. S. Larsen. 2000. A comment on experimental techniques for studying particle capture in filter-feeding bivalves. Lim- nol. Oceanogr. (In press). Riisgard. H. U., P. S. Larsen, and N. F. Nielsen. 1996. Particle capture in the mussel Mytilus ediilis: the role of latero-frontal cirri. Mar. Biol. 127: 259-266. Shimeta. J.. and P. A. Jumars. 1991. Physical mechanisms and rates of particle capture by suspension-feeders. Oceanogr. Mar. Biol. Annn. Rc\: 29: 191-257. Silverman, H., E. C. Achberger, J. W. Lynn, and T. H. Dielz. 1995. Filtration and utilization of laboratory-cultured bacteria by Dreissena polymorpha, Corhicula fiuminea, and Caninciilina te.\asensis. Biol. Bull. 189: 308-319. Silverman, H., J. W. Lynn, and T. H. Dietz. 1996. Particle capture by the gills of Dreissena polymorpha: structure and function of latero- frontal cirri. Biol. Bull. 191: 42-54. Silverman, H., S. J. Nichols, J. S. Cherry, E. Achberger, J. VV. Lynn. and T. H. Dietz. 1997. Clearance of laboratory-cultured bacteria by freshwater bivalves: differences between lentic and lotic unionids. Can. J. Zool. 75: 1857-1866. Silverman, H.. J. VV. Lynn, and T. H. Dietz. 2000. In vitro studies of particle capture and transport in suspension-feeding bivalves. Liinnol. Oceanogr. (In press). Silvester. N. R., and M. A. Sleigh. 1984. Hydrodynamic aspects of particle capture by Mytilus. J. Mar. Biol. Assoc. UK 64: 859-879. Sprung, M., and V. Rose. 1988. Influence of food size and food quantity on the feeding of the mussel Dreissena polymorpha. Oeco/ogia 77: 526-532. Tankersley, R. A. 1996. Multipurpose gills: effect of larval brooding on the feeding physiology of freshwater unionid mussels. Invertebr. Biol. 115: 243-255. Ten Winkel, E. H., and C. Davids. 1982. Food selection by Dreissena polymorpha Pallas (Mollusca: Bivalvia). Freshwater Biol. 12: 553- 558. Ward, J. E., P. G. Beninger, B. A. MacDonald, and R. J. Thompson. 1991. Direct observations of feeding structures and mechanisms in bivalve molluscs using endoscopic examination and video image anal- ysis. Mai: Biol. Ill: 287-291. Ward, J. E., B. A. MacDonald. R. J. Thompson, and P. G. Beninger. 1993. Mechanisms of suspension feeding in bivalves: resolution of current controversies by means of endoscopy. Liinnol. Oceanogr. 38: 265-272. Ward. J. E., L. P. Sanford. R. I. E. Newell, and B. A. MacDonald. I998a. An new explanation of particle capture in suspension-feeding bivalve molluscs. Liinnol. Oceanogr. 43: 741-752. Ward, J. E.. J. S. Levinton, S. E. Shumway, and T. Cucci. 1998b. Particle sorting in bivalves: in vivo determination of the pallial organs of selection. Mar. Biol. 131: 283-292. Reference: Bio/. Bull. 197: 377-387. (December 1999) The Physiological Basis for Faster Growth in the Sydney Rock Oyster, Saccostrea commercials BRIAN L. BAYNE1'*, SUSANNE SVENSSON1'2 AND JOHN A. NELL3 1 Centre for Research on Ecological Impacts of Coastal Cities, Marine Ecology Laboratories All, University of Sydney, NSW 2006, Australia; 2 Department of Zoophysiology, University of Goteborg. Medicinaregatan 18, 413 90 Goteborg, Sweden; and 3 NSW Fisheries, Port Stephens Research Centre, Tavlors Beach, NSW 2316, Australia Abstract. Sydney rock oysters were sampled from a mass selection experiment for growth (the "selected" category) and from a control ("not selected") population and held in the laboratory at three ration levels. We evaluated three models to explain faster rates of growth by selected oysters. Selection resulted in oysters feeding at up to twice the rate and with greater metabolic efficiency than controls. A field experiment confirmed that selection leads to faster rates of feeding across a wide range of food concentrations. Selected oysters also grew more efficiently, at a smaller cost of growth (Cj: mean values for Cg were 0.43 J • J ' in selected individuals and 0.81 J • J~' in the controls. In contrast, oysters in both categories showed similar meta- bolic rates at maintenance, i.e., at a ration supporting zero growth. There was no evidence that differential energy allocation affected the balance between total metabolic re- quirements above and below zero net energy balance. By experimenting with selected and control oysters of different sizes and ages, then standardizing the data for size, we found no effects of age on the differences due to selection. Faster-growing oysters feed more rapidly; invest more en- ergy per joule ingested; show a higher net growth effi- ciency: and are able to allocate less energy per unit of tissue growth, than slower-growing individuals. Introduction The physiological processes that constitute growth are of fundamental interest. A striking feature of growth in nature is its variability amongst individuals, which is a result of the Received 16 December 1998; accepted 29 September 1999. * To whom correspondence should be addressed. E-mail: blb@bio. usvd.edu.au effects of exogenous and endogenous factors. Of the various endogenous factors involved, genotypic composition may play a significant part. Genetic properties may affect growth in various ways (Koehn, 1991), including correlations between growth rate and genetic heterozygosity (Mitton and Grant, 1984; Zouros el al., 1988; Britten, 1996). For marine bivalve molluscs in particular, presumed interactions between genotype and growth are of particular interest in aquaculture (Newkirk, 1980), and the selective breeding of oysters has often suc- ceeded in increasing average rates of growth (see review by Sheridan, 1997). Analysis of the bioenergetics of growth is useful in stud- ies seeking to link phenotypic variability in growth to ge- netic causes. This approach involves the dissection of growth into its component processes, as represented by the "balanced energy equation" of Winberg (1956; see review by Wieser, 1994). For example, Present and Conover (1992) have described how genetically based latitudinal differences in the growth rate of the fish Menidia menidia were due to differences in both food consumption rates and somatic growth efficiency. In this present study, we set out to identify physiological mechanisms to explain observed vari- ability in growth in oysters. We postulated three ways by which an individual animal may increase its rate of growth above that of other individuals, when held in the same environmental conditions. Though not fully independent nor mutually exclusive, these are sufficiently different in both underlying mechanisms and likely ecological conse- quences to act as useful alternative models to explain vari- ability in growth rate among individuals. We then evaluated these models by comparing oysters artificially selected for faster growth with control, "not-selected," oysters. 377 378 B. L. BAYNE ET AL First, in the increased acquisition model, an individual may obtain more food per unit time by feeding more rapidly than others, so increasing its metabolizable energy intake (Present and Conover, 1992; Rist et al., 1997; De Moed el til.. 1998). This may be evaluated by comparing a variety of traits of feeding behavior (Bayne et al.. 1999) and relating the results to rates of growth. We tested the hypothesis that, under similar conditions of ration (quantity and quality), oysters selected for faster growth will have faster rates of ingestion than do control (not-selected) oysters. Second, in the modified allocation model, faster growth may be the result of greater proportional allocation of en- ergy to growth at the expense of other energy-demanding processes, such as body maintenance (Wieser, 1989). We evaluated one aspect of this model by estimating the meta- bolic rate of selected and not-selected oysters at mainte- nance, when growth is neither negative nor positive, to test the hypothesis that selected oysters would show reduced maintenance rates. Finally, in the metabolic efficiency model, faster growth may result from a higher growth efficiency (Present and Conover. 1992) from reduced metabolic costs of growth (Wieser, 1994). or from a combination of the two. This model was evaluated in two ways. Firstly, net somatic growth efficiency, defined as the proportion of metaboliz- able energy intake allocated to growth, was determined. Secondly, by measuring metabolic rates at different rates of growth, we tested the hypothesis that reduced costs of growth correlate, amongst individuals, with increased growth rate. We used the Sydney rock oyster, Saccostrea conunercia- lis (Iredale and Roughley). The New South Wales Fisheries Research Centre at Port Stephens. Australia, established a mass selection program for these oysters in 1990 (Nell el al.. 1996). Four selected lines were established for faster growth, and these have been bred in alternate years since. After one generation of selection, Nell et at.. ( 1996) found that oysters from two of the lines were heavier than oysters from two control lines. After two generations, their weight was 18% greater than controls (Nell et a/.. 1998). Oysters from the third generation of selection (referred to in Nell et til.. 1998, as the "loose 2" selection line) were used in the present study and compared with control oysters. The ex- periment was designed to test hypotheses derived from the three models discussed above and to identify the physiolog- ical characteristics that may explain enhanced growth in the selected lines. Materials and Methods Material and general procedures Oysters were provided by the NSW Fisheries Laboratory at Port Stephens. The selected oysters were as described above. Controls were from a commercial oyster farm and Table 1 Mean (±SD: n = 12} shell heights (cm) and whole weights {shell plus flesh, grams} of oysters in the four experimental categories Category Shell height Whole weight Selected. Large Not selected. Large Selected. Small Not selected. Small 7.93 ± 0.33a 7.89 ± 0.47" 6.37 ± 0.32" 6.54 ± 0.36b 52.95 ± 1.05° 49.66 ± 2.40C 38.40 ± 3.87d 36.96 ± 2.84d Values sharing superscripted letters are not significantly different. are referred to as "not-selected." These oysters were grown under identical conditions to the selected oysters, within Nelson Bay, New South Wales, though they were from a natural larval settlement and not cultured as larvae within the hatchery, as were the selected individuals. Within each category, we distinguished one group of "small" and one group of "large" oysters (Table I). The selected oysters were 23 months old. The ages of the not-selected oysters were not known with certainty, but the "not-selected, small (NSS)" individuals are considered to be of similar age to the "se- lected, large (SL)" individuals, though of smaller size, and of similar age and size to the "selected, small (SS)" oysters. The "not-selected, large (NSL)" oysters are thought to be about 6 months older than the selected, large oysters. Twelve oysters from each experimental category (Table I) were tagged for individual identification and held in the water-table of a research aquarium of recirculating seawater in Sydney. The aquarium contained 600 1 of water, of which 33% was replaced every 7 days. Water temperature was controlled at 20 ± 5°C and salinity at 33 ± 1.5%c. The laboratory experiment was as follows: 20 January to 8 Fehnuiry 1998 (20 days). No supple- mentary food added; physiological measurements made from 28 January to 5 February and labeled the "field" condition. 9 February to 4 March (24 days). Food added to make up the "middle ration" condition; measurements made from 23 February to 2 March. 5 March to 18 March (14 days). Food added to comprise the "high ration" condition; measurements made 12 to 18 March. 19 March to 9 April (22 days). No food added; the "low ration" condition; measurements made from 6 to 10 April, after 16-20 days without food. Rations The food was unicelled algae Isochrysis galbana (strain T-ISO) and Chaetoceros gruclUs, supplied as algal pastes by Reed Mariculture Inc.. California. Individual pastes were combined in the proportion 3 parts T-ISO to 7 parts C. PHYSIOLOGY AND GROWTH OF ROCK OYSTERS 379 gracilis, and the cells were suspended in seawater in a feeding reservoir at the desired concentration (Table II). The cells were dosed to the oysters by peristaltic pump, from 0930 to 1530 daily (middle ration) and 0930 to 1630 (high ration). Cell concentrations in the trays were monitored frequently with a particle size analyzer (Coulter Counter model Zl ). Samples of cells from the feeding reservoir were weighed after drying overnight at 80°C, and then measured for nitrogen content (by Leco CHN analyser). Physiological measurements All oysters were measured for clearance rate, absorption efficiency, rate of oxygen consumption, and rate of ammo- nia-nitrogen excretion, at each ration level. Following pre- liminary studies, care was taken not to use the same indi- viduals in any two measurements without at least 24 h of recovery from the stress of handling. Clearance rate (CR). Clearance rate is a measure of the volume of water cleared of algal cells per hour. When pseudofeces (that is, material cleared from suspension but not ingested) are not produced (as in this experiment), the rate of ingestion of food is calculated as CR X [food concentration]. Oysters were placed individually in 1-1 beakers in water from the feeding trays and left undisturbed in a water bath for 30 min. A beaker without an oyster was used as the blank control. The water was gently aerated using Pasteur pipettes coupled to a compressed air supply. About 600 ml of the water was then siphoned off and replaced with seawater containing algal cells at a concentration equivalent to the experimental ration. A 10-ml sample was taken after 10 min and then at 10-min intervals for a further 40 min. Cell concentrations were measured with a particle size analyzer (Coulter Counter model Zl). For data analysis, a check was first made for linearity ( In cell concentration plotted over time), then clearance rate as Table II Maximal concentrations of cells (T-ISO + C. gracilis) and total paniculate matter (TPM) in the oyster trays for each of the experimental rations Maximal TPM: Maximal cell mg • 1~' % body weight Ration conc:103 mP' [ = "Peak rations"] ingested • d~' Low- 2.9 ± 0.9 0.07 ± 0.02 Middle 44.0 ± 1.9 0.59 ± 0.02 0.87 ± 0.37 High 90.9 ± 5.3 1 .07 ± 0.07 2.41 ± 1.58 TPM is based on a conversion from cell numbers (106 cells = 0.013 ± 0.003 mg dry mass). The "% body weight ingested" is calculated from mean ingestion rates across the four experimental categories, as presented later in the Results section. All values are means ± SD. liters per hour was calculated according to Coughlan (1969): CR = ([lnC,,-lnC,]- Vol/M-Blank, where C0 and C, are concentrations of cells at the beginning and end of incubation time /, Vol is the volume of water in the beaker, and Blank is the change in cell concentration in the blank control beaker. Absorption efficiency (AE). Absorption efficiency mea- sures the efficiency with which ingested organic material is absorbed by the animal. When multiplied by the ingestion rate, AE estimates absorption rate (milligrams of organic matter per hour). Samples of feces were collected from the beakers in which the oysters were held for CR measurements. It proved impossible to collect enough material for analysis by indi- vidual; rather, samples were pooled according to category, at each of the middle and high ration levels. Food cells were sampled at the same time. Samples were filtered onto ashed, preweighed, GF/C filters, washed with 0.9% ammonium formate, dried overnight at 80°C, weighed, ashed for 4 h at 450°C, and weighed again. AE was estimated according to Conover (1966): AE= F - £/[(! - £)• F], where F and E are the ratios of ash-free dry weight to dry weight of the food and feces, respectively. Oxygen consumption (VO:). Oxygen consumption is an indirect measure of the metabolic rate, or rate of energy expenditure, by the animal. Oysters were placed individually in airtight flasks of -500 ml volume, on a perforated base that allowed stirring of the water by magnetic stirrers. Each flask was fitted with a Strathkelvin oxygen electrode to record the rate of decline of dissolved oxygen in the flasks. The flasks were also fitted with two 5-ml syringes, one containing algal cells, the other empty as a compensation chamber. After 30 min at constant temperature in a water bath, the algal cells were injected into the flask to achieve a cell concentration equivalent to that measured in the feeding trays (middle and high ration levels only). The rate of oxygen consumption was then recorded for a further 60-90 min. For each set of measure- ments of five oysters, one flask was used as a blank control. The rate of oxygen consumption, as milliliters of oxygen per hour, was calculated as: VO, = ([<9:il - O2l2] • Vol//) - Blank. where 0-,,, and O-,,2 are oxygen concentrations (milliliters per liter) at least 30-min apart; Vol is the volume of water in the flask; / is the time in hours, and Blank is the change of oxygen concentration in the blank control respirometer. Excretion rale (VNH4~N). This is the rate at which nitrogen is excreted as ammonia. Oysters were placed indi- 380 B. L. BAYNE ET AL. vidually in 1 1 of filtered seawater and left undisturbed for 3 h. Beakers without oysters served as blank controls. Con- centrations of ammonia were measured using the phenol- hypochlorite method of Solorzano (1969); a full set of standards was analyzed for each experimental run. Rates of excretion, as milligrams of ammonia-nitrogen per hour, were calculated as: VNH4 - N = (Conccvpll - Conca,nlrill)-Vol//. where Concexptl and Concconlro| are ammonia concentrations in experimental and control beakers, respectively, Vol is the volume of water in the beaker, and / is the incubation time (3 h). Oxygen:nitrogen ratio. The ratio, in molar equivalents, of oxygen consumed to nitrogen excreted serves as an index of catabolic substrate (Bayne and Newell, 1983). and was calculated to evaluate whether the oysters in the different growth and size categories were utilizing different biochem- ical substrates, a difference which might then explain other observed metabolic differences. Growth. The 12 oysters in each experimental category were weighed (shell plus flesh) at the beginning (Table 1) and end of the experiment. Growth was calculated by sub- traction and related to the period spent at high ration (14 days) to convert to a daily rate. At the end of the experi- ment, the oysters were shucked and dry flesh weights de- termined after drying overnight at 80°C. Due to uncoupling in the growth of shell and tissue in bivalves (Hilbish, 1986; Lewis and Cerrato, 1997) conver- sions of total weight to weight of tissue, using a constant conversion factor, must be made with caution. For this study, we derived such conversion factors for each individ- ual at the end of the experiment. Given the relatively short duration of the experiment, we considered it appropriate to use these factors to estimate equivalent dry flesh weight at the start, and so to estimate growth also as milligrams of dry tissue per day. Converting rates to a standard body size This conversion was based on allometric relationships between dry flesh weight and the measured physiological rates, following Bayne and Newell (1983): where V/stanJ and VVsland are the standardized rate and dry flesh weight, respectively; Vmeas and Wmeas are the rate and dry flesh weight as measured; and |3 is the allometric expo- nent in the equation describing physiological rate as a function of body size. Estimates of /3 were derived for clearance and respiration rates across all experimental categories from the "field condition" measurements. The exponent for excretion rate is based on a separate sample of oysters (Svensson, unpub- lished). The exponent for growth was determined from rates of growth calculated (see above) for the high ration condi- tion. The values were as follows: Clearance rate: ft = 0.641 ± 0.1 13(« = 32) Oxygen consumption rate: J3 = 0.536 ± 0.107(>i = 25) Excretion rate: /3 = 0.772 ± 0.156(/( = 50) Growth rate: j3 = 1.96 ± 0.58(/i = 45). As measured over all categories (n = 48), the mean dry flesh weight of the experimental oysters at the end of the experiment was 0.920 ± 0.243 g. Wstand was set at 1.0 g. Field measurements Rates of ingestion were measured in the field on two occasions, as a test of the hypothesis that results of the laboratory experiment on feeding rates, and in the context of a model of energy acquisition, would be repeated under the more natural conditions of food availability. Selected and not-selected oysters of similar size (64.0 ± 3.35 g and 62.7 ± 2.86 g whole weight, respectively) were held over- night in wide-mesh bags at the mouth of the Karuah River as it enters the Port Stephens estuary, on 9-10 September and 3-5 November. 1998. This is an area used for cultivat- ing oysters. The selected oysters were from the same mass selection as those used in the laboratory. During the field measurements, water temperatures were 19.1 ± 1.9°C(over both months) and salinities were 28.2 ± 1.9%o (September) and 32.5 ± 8.3%c (November). Total paniculate matter in suspension was 8.0 ± 2.3 and 29.3 ± 5.8 mg • 1 in September and November, respectively. The oysters were placed individually in specially de- signed trays (36 X 16 X 8 cm. with a sill at one end to reduce turbulent flow) at flow rates of 450 ± 15 ml • min " ' of water pumped directly from the river. After 1 h all biodeposits were removed from the trays and the oysters left undisturbed for a further 30 or 60 min. Feces and pseudo- feces were then collected quantitatively, together with sam- ples of suspended particulate matter, and filtered onto ashed and weighed GF/C filters. The filters were dried overnight at 80°C, weighed, ashed at 450°C for 4 h, and weighed again. The results were used to calculate rates of filtration and ingestion by the "biodeposition" method as described by Iglesias et al. (1998) and Bayne er al. (1999). Statistical anal\sis The results of the laboratory experiment were analyzed in three stages, using SYSTAT 6.0 (Wilkinson, 1996). 1 . For each ration condition the physiological measure- ments, standardized to an animal size of 1 g dry flesh weight, were analyzed as a two-way ANOVA with "selection" (i.e.. selected or not-selected) and "size" PHYSIOLOGY AND GROWTH OF ROCK OYSTERS 381 (large or small) as the main effects. In all cases the "selection X size" interaction was not significant. 2. Given the "repeated measures" nature of the experi- mental design, a different approach was taken to analyze the data across rations. Three groups of four individuals were first selected at random from each category (selection and size). These were then allo- cated, again at random, to one of the three ration levels. A three-way ANOVA was then done, with "selection," "size," and "ration" as the main effects. Where both "selection" and "ration" showed signif- icant effect, a regression analysis was performed, with ration as the independent variable, to compare oysters from the different categories. 3. Finally, data for each individual oyster over the three ration levels were analyzed by linear regression, and comparisons between categories were made on the basis of the average "within category" values for the slope and intercept in the fitted equations. These regressions were for three data points only, per indi- vidual: only those for which the level of significance in the analysis was P < 0.10 were used for compar- isons. The results of the measurements in the field were ana- lyzed by two-sample / test with pooled variance. Results Laboratory experiment Ration levels. The cell concentrations in the feeding trays (Table II) were converted to equivalent dry mass using the constant 0.013 mg per 106 cells, and to nitrogen content using 5.6% N by weight. "Peak rations" are the levels recorded between 1100 and either 1500 (low and middle ration) or 1600 (high ration), and are the concentrations applied during the physiological measurements (except ex- cretion rates, which were measured in filtered seawater). Peak rations were as follows: low ration, 0.074 ± 0.018; middle ration, 0.593 ± 0.024: high ration, 1.071 ± 0.068 mg • 1 ~ ' . Clearance and ingestion rates. These were measured for the middle and high ration levels only (Table III). Differ- ences due to size alone, following standardization to 1 g dry flesh weight, were significant only for ingestion rates at the middle ration. This result indicates that the effects of age on feeding rates were not greatly significant overall. The ef- fects of selection were greater, particularly at high ration, where the selected, large oysters had significantly faster rates of clearance and ingestion than the not-selected, large and not-selected, small oysters. When the original ingestion rates (i.e.. before standard- izing them to 1 g dry flesh weight) were converted to percentages of body weight (as dry mass in milligrams), significant differences among categories were evident (Fig. 1: high ration). Selected oysters at both middle and high ration levels had faster relative ingestion rates than the not-selected oysters, with no significant differences between large and small oysters. For example, at the middle ration. SL oysters ingested 1.14 ± 0.47 %bw • d"1, compared with 0.80 ± 0.22 %bw • d"1 for NSL individuals; the values for SS and NSS oysters were 0.88 ± 0.44 and 0.68 ± 0.33 %bw • d 1, respectively (see Fig. 1 for the high ration data). Absorption efficiency. There were no significant differ- ences due either to selection or to size. Values were all between 0.66 and 0.78 (mean 0.71 ± 0.06), with slightly lower values at high than at middle ration (P < 0.05). Rates of oxygen consumption. Differences among cate- gories (Table IVA) were significant (P < 0.05) only for size effects at the middle ration, where the VO2 was higher for the NSL oysters than for the NSS oysters. Differences between selected, large, and not-selected, large, and between selected, small, and not-selected, small, oysters were not significant at any of the ration levels. The effects of ration on rates of oxygen consumption, however, were highly significant for all categories, with Table III Clearance rates (CR: I • h~'> and ingestion rates (IR: mg • h ' } for oysters in each of four experimental cuti'xurii'x. at twn rution* CR IR Category Middle ration High ration Middle ration High ration Selected. Large 2.53 ± 0.28 3.10 ± 0.37 10.36 ± 1.13 23.59 ± 2.74 Not selected. Large 2.12 ± 0.18 2.05 ± 0.29 8.59 ± 0.73 16.42 ± 2.14 Selected. Small 2.07 ± 0.25 3.00 ± 0.34 8.45 ± 1.04 23.54 ± 2.32 Not selected. Small 1.65 ±0.22 2.01 ± 0.29 6.73 ± 0.88 15.09 ± 2.16 P for selection <0.0] <0.01 ns <0.005 P for size ns ns <0.05 ns Values are means ± SE, for weight-standardized data; n = 1 2 per category. The results of an analysis of variance are shown as the relevant probability, P, comparing selected with not-selected, and large with small oysters; ns signifies P > 0.05. 382 B. L. BAYNE ET AL TO • cu Q. T3 O LU CO LU O ? 0 SL NSL SS NSS CATEGORY Figure 1. Rates of ingestion (not standardized for differences in body weight), as percent of dry body weight per day, by oysters in the tour experimental categories at high ration; means ± SD. The categories are SL. Selected, large; NSL, Not-selected, large; SS. Selected, small; NSS. Not- selected, small. respiration rate increasing as ingested ration increased. Ox- ygen consumption rates were converted to energy equiva- lents as 20.1 J • ml O: ' (Gnaiger, 1983), and ingestion rates converted as 26.5 J • mg ' (Widdows and Hawkins, 1989). The energy ingested per unit of energy respired was then calculated. At the middle ration, differences in this efficiency mea- sure between categories were not significant; the overall mean value was 0.83 ± 0.38 joule ingested per joule re- spired, which indicates a ration level below the maintenance requirement. At the high ration, average energy ingested per unit respiration was higher (1.53 ± 0.55 J • J"'), with significant differences due to selection (P < 0.01 ). Selected oysters (large and small: 1.86 ± 0.78 J • J"1) were more efficient in this respect than the not-selected oysters ( 1 .20 ± 0.55 J • J"1). This conclusion was confirmed by the analysis of data for individuals. For each oyster, a regression analysis was made of respiratory energy loss. R (J • d '), as a function of ingested ration, IR (J • d"1). Figure 2 shows the means and standard deviations of the fitted slopes, grouped by cate- gory. Categories SL and NSL (P < 0.02), and SS and NSS (P < 0.001) were significantly different. Differences be- tween size categories (SL vs. SS and NSL vs. NSS). how- ever, were not significant. On average, selected oysters respired 0.24 J for every joule ingested, across ration levels, compared with 0.45 J by the not-selected oysters. Excretion rates. At all ration levels, selected oysters excreted more ammonia than the not-selected oysters (Table Table IV Metabolic measurements for oysters in four experimental categories at low, middle, ami high ration levels Ration Category Low ration Middle ration High ration A. Oxygen consumption rate (VO2): ml O, • h~': Selected, Large 0.408 ± 0.080 0.591 ± 0.138 0.738 ± 0.218 Not selected. Large 0.391 ± 0.105 0.662 ±0.148 0.701 ± 0.180 Selected. Small 0.363 ± 0.074 0.538 ±0.126 0.614 ± 0.150 Not selected, small 0.333 ± 0.061 0.514 ± 0.096 0.631 ±0.110 P for selection ns ns ns P for size ns <0.05 ns B. Excretion rate (VNH4 • N): /ag NH4 • h~'* Selected, Large 25.0 ± 4.6 28.7 ±4.1 27,4 ± 3.4 Not selected. Large 18.5 ± 2.8 19.9 ± 2.7 17.1 ± 2.5 Selected. Small 22.9 ± 4.3 25.4 ± 3.5 31.9 ± 4.6 Not selected, small 8.9 ± 1.2 18.1 ± 2.5 25.2 ± 4.5 P for selection <0.05 <0.05 <0.05 P for size ns ns ns C. Scope for growth (SFG): J • g~' • d~' Selected. Large -212 ± 11 -76 ± 23 87 ± 29 Not selected. Large -199 ± 15 -154 ± 17 -33 ± 31 Selected, Small -175 ± 10 -122 ± 28 154 ± 47 Not selected, small -160 ± 8 -106 ± 25 -33 ± 26 P for selection ns ns <0.001 P for size 11 S ns ns Values are means ± SE; n = 12 per category. The results of an analysis of variance arc shown as the relevant probability. P, comparing selected with not-selected, and large with small oysters; ns signifies P > 0.05. * Values represent weight-standardized data. PHYSIOLOGY AND GROWTH OF ROCK OYSTERS 383 u.o T3 % 0.7 Q) O) •E 0.6 _ - o 0.5 1 0.4 1 °3 ! °2 - - - 1 0.1 0.0 - •" SL NSL SS NSS CATEGORY Figure 2. Energy respired (J • d ') per unit of ingested energy (J -d ') by oysters in the four experimental categories; means ± SD. The tour categories are SL, Selected, large; NSL. Not-selected, large; SS. Selected, small; NSS. Not-selected, small. IVB). Size (age) effects were not significant. Only for the small oysters was there a suggestion of excretion rates increasing with increased rates of ingestion. At the middle ration, excretion rates were fast relative to ingested nitrogen (1.50 ± 1.13 mg excreted • mg~' in- gested, across all categories), indicating that this ration was well below the maintenance requirement for nitrogen, as it was also for energy (see above). At high ration, on average, 56% of ingested nitrogen was lost in excretion. Oxygen:nitrogen ratio. Analysis of variance indicated no significant effects of either growth category or ration on the O:N ratio. Mean ratios for selected and not-selected indi- viduals were 59.8 ± 7.0 and 66.6 ± 7.3, respectively. Scope for growth {SFG) and maintenance metabolic rate. SFG was calculated as the difference between metaboliz- able energy intake (ingested ration X absorption efficiency) and the sum of respiratory and excretory energy losses. At low ration, SFG is assumed equal to the summed energy losses (i.e., there was no significant energy intake). There were no significant differences between either selection or size categories at this ration. Similarly, at the middle ration (Table IVC), where the SFG was negative in all cases (i.e., metabolizable energy intake was below the maintenance requirement), there was no significant effect of selection or size overall. At high ration, however, the SFG was high and positive for selected oysters (both large and small) and negative for not-selected oysters, a highly significant difference (Table IVC; P < 0.001). Differences due to size (age) in these weight-standardized data were not statistically significant. The maintenance metabolic rate (/Jmaim; joules per day) is the rate expressed when growth is neither positive nor negative. This was estimated by plotting R as a function of the SFG (Fig. 3): the intercept at zero growth indicates the metabolic rate at maintenance. Linear regression analysis was applied to all individuals, and the slopes and intercepts were compared by analysis of variance (Table V). Data from eight individuals (2. 3. 2. and 1 in the SL, NSL, SS, and NSS categories, respectively) were rejected as not meet- ing the chosen level of significance (P < 0.10). There was no significant effect of selection on the esti- mated maintenance metabolic rate (mean = 308 ± 19 J • d~'). Size, however, did have a significant effect; mainte- nance rate was 337 ± 19 J • d ! for large oysters and 278 ± 19 J • d"1 for small oysters. Regression analysis demonstrated a significant linear re- lation, over all individual oysters, between intercepts and slopes from the individual regressions of R vs. SFG. There- fore, to confirm the absence of any significant differences in estimated Rmainf due to selection, a separate statistical test was performed. Individual oysters were ranked for Rmamt, and the two selection categories were compared by the Mann-Whitney U test. The result was not statistically sig- nificant, 0.10 > P > 0.05, over 34 cases. Growth, the costs of growth, and growth efficiency. Rates of growth, standardized to 1 g dry flesh weight, were de- rived for the 14 days spent at high ration, from measures of whole weight (shell plus flesh). Growth in selected oysters was faster than in the not-selected oysters (Table V). The data for growth in whole weight were converted to equivalents in growth of dry tissue weight using total/dry ro T3 I co CO CO O rr O £ cr Q. CO HI 500 400- 300- "-200 -100 100 200 300 SCOPE FOR GROWTH: Joules per day Figure 3. Respiratory energy loss (J • d ' ) at different levels of scope for growth (J • d ' ) in three oysters from each of two categories; selected, large (circles) and not-selected, large (triangles). Fitted regression lines are shown. The intercepts at zero scope for growth represent energy losses at maintenance (R, „.„„,). 384 B. L. BAYNE ET AL. Table V Rates of growth and growth efficiency tit hi^li ration, the estimated costs of growth and maintenance metabolic rate (Rmjln[), /or oysters in each of four experimental categories Category Growth at high ration: mg total weight • d~' Growth efficiency Cost of growth: j-r1 Maintenance metabolic rate: J • d~' Selected, Large 71.4 ± 4.6 0.26 ± 0.03 0.48 ± 0.07 313.1 ± 23.2 Not selected. Large 58.8 ± 6.2 0.16 ± 0.02 0.75 ± 0.14 361.6 ± 25.8 Selected, Small 63.9 ± 5.0 0.29 ± 0.03 0.35 ± 0.07 248.4 ± 16.4 Not selected. Small 52.0 ± 3.7 0.25 ± 0.03 0.85 ±0.15 308.9 ± 32.2 P for selection <0.01 <0.05 <0.05 ns P for size ns <0.05 ns <0.05 Values are means ± SE for n = 12 per category (growth and growth efficiency) and n = 10, 9, 10. and 1 1 for categories SL, NSL, SS, and NSS, respectively (costs of growth and /?„,.„„,). Growth values are for shell + flesh. Growth efficiency is for tissue growth as a proportion of metabolizable energy intake. The costs of growth and maintenance metabolic rate are estimated as described in the text. The results of an analysis of variance are shown as the relevant probability, P. comparing selected with not-selected, and large with small oysters; ns signifies P > 0.05. tissue conversion factors tor each individual. When values for metabolic rate associated with growth (/?grow. in units of joules per day), calculated as R - /?maim. are plotted against these estimates of tissue growth in energy units, the slope of the regression provides an estimate of the cost of growth, i.e., /vgrovv per unit tissue growth (joules per joule). This analysis yields (see Table V) 0.43 ± 0.19 J • J"1 for the selected oysters (categories 1 and 3 together) and 0.81 ± 0.26 J • J" ' for the not-selected oysters (categories 2 and 4). These estimates are significantly different (P < 0.01 ). Growth efficiency was calculated as metabolizable en- ergy intake/tissue growth, both in units of joules per day. for high ration (Table V). The effects of both selection {P < 0.05) and size (P < 0.05) were significant. Over all cate- gories of selection and size, growth efficiency was low. 0.24 ± 0.04. Field measurements Selected oysters (n = 17) had significantly faster rates of both filtration and ingestion than not-selected oysters (n = 21 ) on both occasions in the field (September and Novem- ber; Table VI). Rates were faster in November, when con- centrations of suspended paniculate material were higher (TPM, 29.3 ± 5.8 mg • T1 compared to 8.0 ± 2.3 in September: particulate organic matter. POM. 4.0 ± 1.5 mg • 1~' compared to 1.6 ± 0.4 in September). The ratios of ingestion rates for selectedmot-selected oysters were 2.70 for September and 2.06 for November. Discussion The rock oysters (Saccostrea commercialis) used in this experiment were taken from the third generation of a mass selection program (the "selected" categories) and from a control ("not-selected") population from the same location in the Port Stephens estuary, Australia. At a ration level that peaked daily at 1.1 mg total particulate matter per liter (the "high ration" level), the selected oysters grew, on average, 22% faster than the not-selected oysters. This accords with Newkirk and Haley (1982) for selection for growth in Ostrea edulis (23% gain over controls), Paynter and Dim- ichele (1990) for Crassostrea virginica (24%-28% gain), and Toro et al. ( 1994) for Ostrea chilensis ( 13%-33% gain). It also accords with assessment of the Port Stephens selec- tion study itself, where an 18% improvement in growth rate was recorded after two generations (Nell et al.. 1998). The molar ratio of oxygen consumed to nitrogen excreted was calculated to evaluate whether the two groups of se- lected and not-selected (=control) oysters were catabolizing Table VI Filtration and ingestion rates of oysters measured on Mo occasions in the field at Karuah. Ne»: South Wales Filtration: mg Ingestion: mg • h ' Category September November September November Selected Not selected P comparing categories. 20.2 ± 1.6 13.3 ± 1.4 <0.005 70.3 ± 17.7 22.3 ± 5.4 <0.05 2.7 ± 0.3 1.0 ± 0.2 <0.001 16.1 ± 3.0 7.8 ± 1.4 <0.01 Values are means ± SE for n = 16 (September) and n = 22 (November). P values are the result of / tests comparing categories. PHYSIOLOGY AND GROWTH OF ROCK OYSTERS 385 different energy substrates at the time of the experiment. Such differences might indicate different physiological states in the two growth categories, which would render more detailed metabolic comparisons complex. There was, however, no significant difference in the O:N ratios (P > 0.05), in spite of differences in rates of excretion, suggesting that selection for growth did not shift the normal seasonal pattern of metabolism significantly in these oysters. Until we know more about nitrogen metabolism in this species, we cannot fully interpret the observed differences in excretion rates. However, the selected oysters both fed more quickly and excreted nitrogen at a higher rate than the control individuals. This is not unexpected, but more infor- mation on the relationship between ingested and excreted nitrogen is needed before these observations can be set in context with selection for growth. We proposed three models to explain the observed dif- ferences in rates of growth: faster growing individuals may feed more rapidly; they may reduce their maintenance en- ergy requirement; or they may grow more efficiently than slower growing individuals. The results of the laboratory experiment supported the first and third, but not the second, of these models. Further, by experimenting with different sizes (and therefore ages) of oysters, but correcting the measurements according to observed size/rate relationships, we demonstrated that age was not a significant factor in explaining most of the observed differences between indi- viduals. The exceptions were estimated maintenance meta- bolic rate, which increased with age. and growth efficiency, which declined with age. Oysters from the selected line had faster clearance rates (volume of water cleared of food cells per hour) than control, not-selected oysters. This was reflected in a 44% increase in ingestion rates at the greatest ration. Because of a similarity across experimental categories in the efficiency with which these cells were absorbed in the gut, an identical difference in metabolizable energy intake was observed. In experiments with hybrid and inbred lines of Pacific oysters (Crassostrea gigeis), Bayne et al. (1999) recorded faster clearance rates by hybrids in three out of four comparisons, consistent with observed differences in growth rate. Genet- ically based differences in feeding behavior are an impor- tant component of differences in rates of growth among individual oysters. A similar difference between selected and not-selected oysters was observed in the field experiment, in which a different technique for measuring feeding behavior was used (the biodeposition method in the field, in contrast to direct cell counts in the laboratory), and the concentration of food was significantly greater (8.00 ± 2.28 and 29.3 ± 5.8 in the field, compared with a maximum of 1 .07 ± 0.07 mg • 1~' in the laboratory). Under these conditions the differ- ences between the two experimental categories of oyster were actually more marked than in the laboratory. This finding lends general support to the "energy acquisition" model and demonstrates that the inferred genetic component of variability in feeding behavior supports faster growth, both under natural circumstances in the field and in the laboratory. Feeding rates were not only faster in selected oysters, they were also more metabolically efficient. Feeding is not itself energetically expensive in bivalve molluscs, although the total costs of feeding and digestion plus absorption may account for up to 20% of total metabolic rate (Hawkins and Bayne, 1992). Feeding rates may increase significantly without seriously compromising net energy yield. The rock oysters selected for growth achieved a greater gain of en- ergy per unit of energy lost in metabolism than did control oysters. The actual mechanisms of feeding that are respon- sible for these differences remain unknown. We presume, however, that increasing rate of ingestion as a mechanism for increasing gross energy yield will be limited eventually by decreased gut passage time, which ultimately limits maximum absorption efficiency (Bayne et al., 1989). Our second model, which we call the energy allocation model, was not supported by the results of the experiment. The estimated rate of metabolism at maintenance varied between 250 and 360 joules per day, standardized for oys- ters of 1 g dry flesh weight, and it increased with age but not with selection. An average of 308 J • d~' is equivalent to a maintenance requirement for metabolizable energy intake of — 1.4% of body weight per day. This is similar to published values for other bivalves of similar size (reviewed by Bayne and Newell, 1983) and accords with our conclusion that the middle ration level in the laboratory was insufficient to meet the requirements for maintenance of these oysters. Increased maintenance costs with age have commonly been reported for other bivalves (review by Griffiths and Griffiths, 1987). Studies with blue mussels, Mytilus edulis, by Hawkins et ul. (1986) and Bayne and Hawkins (1997), and with rain- bow trout, Oncorhvnchus mykiss, by McCarthy et al. (1994), have demonstrated how reduced rates of protein turnover contribute to reduced metabolic costs and higher rates of growth. These processes appear to be genotype dependent, and they support the concept of differential energy allocation (Wieser, 1989) as a means of increasing growth. In our experiments, differences in maintenance metabolic rate between fast- and slow-growing oysters were not statistically significant. This result merits further re- search, however. For example, the data (Table V) show a tendency towards higher maintenance metabolic rates in the not-selected oysters, particularly among the smaller size categories, but with high variance. In similar experiments with Pacific oysters, Crassostrea gigas (Bayne, in press), we have observed significant differences in flmaim among individuals, which correlated with differences in growth rate. The energy allocation model remains a possibility in 386 B. L. BAYNE ET AL Metabolic energy loss *Snaint MEI select MEI control v G control ' select [-ve] zero [+ve] Growth Figure 4. A qualitative illustration of the main findings of this study. Metabolic energy loss is plotted as a function of growth for oysters selected for fast growth, and for control oysters. Below the maintenance requirement, where growth is negative, there is no difference in metabolic expenditure due to selection; the maintenance metabolic demand (ftma,nl) is the same for both experimental categories. However, selected oysters achieve a higher metabolizable energy intake (MEI) than the controls and express a lower cost of growth. The net result is an increased growth rate and a higher growth efficiency (Gxka vs. Gcomro|). Differences due to selection have been exaggerated for illustration purposes. the general case, therefore, although not supported directly by these data on Saccostreu. Our third model concerned growth and metabolic effi- ciency. This was evaluated by estimating both the costs of growth and net growth efficiency in selected and not-se- lected oysters. The results supported the hypothesis that selected oysters would show a lower cost of growth (0.43 J • J ') than control oysters (0.81 J • J~'). Both values are high compared with published values (Wieser, 1994; average for "ectothermic metazoans" of 0.30 J • J~ ' ), possibly reflecting a relatively poor-quality diet and slow overall rates of growth. Nevertheless, the differences due to selection, and the lack of significant differences due to age, are evident. Clearly, selection for growth in this species, as in others (Bayne and Hawkins, 1997), involves selection for reduced costs of growth. Selected and not-selected oysters also differed in growth efficiency measured as the proportion of metabolizable en- ergy intake utilized in tissue growth. This efficiency was low in all cases— for example, 0.28 ± 0.09 and 0.21 ± 0.08 for selected and not-selected oysters, respectively. The re- sults do, however, support the hypothesis that selected oys- ters utilize a higher proportion of absorbed ration for growth, and do so at a reduced cost of growth relative to the controls. In summary (Fig. 4), our experiments indicate that mass selection for growth in the rock oyster resulted in individ- uals that had a greater intake of metabolizable energy by virtue of faster (and more metabolically efficient) feeding, and were able to use this intake more efficiently for growth. Selected and control oysters did not differ in their energetic costs at maintenance. The field experiment confirmed that selected oysters fed more rapidly than the controls. The challenge now is to analyze in more detail the feeding behavior and the metabolic processes that contribute to the costs of growth and to link these processes more directly to observed individual differences in genotype. Acknowledgments We are grateful to Shannon Long, who helped with many aspects of this work, and to Shannon and Alison Phillips for their work in the field. Graham Housefield provided invalu- able support throughout the project. The research was sup- ported by a grant from the Australian Research Council to the Special Research Centre on Ecological Impacts of Coastal Cities. Susanne Svensson was supported by the Foundation for Strategic Environmental Research, Sweden (Sucozoma Project. DNR 95005). The manuscript benefited significantly from the comments of Gee Chapman and Tony Underwood. 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Heterozygosity and growth in marine bivalves: further data and possible explanations. Evolution 42: 1332-1341. Reference: Bial. Bull. 197: 388-394. (December 1999) Hydrostatic Pressure Alters the Time Course of GTP[S] Binding to G Proteins in Brain Membranes from Two Congeneric Marine Fishes JOSEPH F. SIEBENALLER1 * AND THOMAS F. MURRAY2 1 Department of Biological Sciences, Louisiana State University, Baton Rouge. Louisiana 70803; and ' Department of Physiology and Pharmacology, University of Georgia College of Veterinary Medicine, Athens, Georgia 30602 Abstract. The effects of hydrostatic pressure on the re- ceptor-stimulated exchange of guanosine triphosphate (GTP) for guanosine diphosphate (GDP) on the a subunit of G proteins were studied in two congeneric marine teleost fishes that differ in their depths of distribution. The poorly hydrolyzable GTP analog [35S]guanosine 5'-[y-thio]tri- phosphate ([35S]GTP[S]) was used to monitor the modula- tion of signal transduction by the A, adenosine receptor agonist Nh-R-(phenylisopropyl)adenosine (R-PIA) in brain membranes of the scorpaenids Sebastolohux ulascunus and S. altivelis. The maximal binding (BmaJ and dissociation constant (Kd) values, determined from equilibrium binding isotherms at atmospheric pressure (5°C), were similar in the two species. The fim.lx values for these species are much lower than literature values for mammalian brain tissue (25°C); however, the Kd values of the teleost and mamma- lian G proteins are similar. The EC5(, values for the A, adenosine receptor agonist R-PIA were similar in the two species. Hydrostatic pressure of 204 atm altered the binding of ["S]GTP|S]; basal [XSS]GTP[S] binding decreased 25%. The A, adenosine receptor agonist R-PIA and the musca- rinic cholinergic receptor agonist carbamyl choline stimu- lated [ '5S]GTP|S] binding at 1 and 204 atm. At atmospheric pressure the halt-time (/,,,) of [35S]GTP[S] binding differed between the two species. The GTP[S] on rate (knn) is larger in the shallower-living S. alascanus. Increased hydrostatic pressure altered the time course, decreasing the ;,/: in both species. The pressures that elicit this change in the time course differ between the species. However, interpolating over the ra uze of in situ pressures the species experience. Received 16 June 1998; accepted 8 September 1999. * To whom correspondence should be addressed. E-mail: zojose @ unixl.sncc.lsu.edu the values are similar in the two species. The guanyl nucle- otide binding properties of the G protein « subunits appear to be conserved at the environmental temperatures and pressures the species experience. Introduction The high hydrostatic pressures characteristic of the deep ocean significantly influence guanine-nucleotide-binding protein (G protein (-coupled signal transduction systems (Siebenaller and Murray, 1995). Because of the large num- ber and ubiquity of G protein-coupled signaling complexes, the effects of pressure on such systems will have an impor- tant role in shaping the evolution of signal transduction systems in marine species and may play a part in determin- ing the bathymetric distribution of species. G proteins couple a diverse superfamily of cell surface receptor proteins, characterized by seven membrane-span- ning regions, to a variety of effector elements, such as adenylyl cyclase, ion channels, and phospholipases (Spiegel ct til., 1994). The subunits of the heterotrimeric G proteins are designated a, /3, and y. The classes of G proteins, such as G, and Gs, which inhibit and stimulate adenylyl cyclase, respectively, and G0. a common G protein in brain tissue that may be coupled to Ca+ f channels and phospholipase C, are defined by the « subunit type (Oilman, 1994). Receptors with bound agonist interact with heterotrimeric G proteins, promoting the binding of GTP in exchange for GDP on the a subunit. The binding of GTP evokes a conformational change that results in the dissociation of the G protein into a • GTP and a /3y dimer (Coleman ct 0.05), based on a partial F test (Hoyer et ul., 1984). PRESSURE ALTERS GTP[S] BINDING 391 Results High-affinity binding sites for [3?S]GTP[S] were charac- terized in equilibrium saturation experiments at 5°C and atmospheric pressure, using concentrations of [35S]GTP[S] up to 13.5 nM. Incubations were carried out for 3 h. The binding capacities and binding affinities of brain membrane preparations of the two species were identical (Bmav P = 0.27; Kd, P = 0.88: Table I). The 1C (inhibitory concentration )50 values for GDP were determined at atmospheric pressure using 0.3 nM [35S]GTP[S]. The values were 20.66 ± 19.23 juM for S. alascamts and 27.60 ± 26.24 juM for S. altivelis (mean ± SE, three independent determinations for each species). The IC50 values do not differ between the two species (P > 0.05). The EC (effective concentration )50 values for R-PIA stimulation of [^SJGTPJS] binding at atmospheric pressure were determined using 0.3 nM [35S]GTP[S] in the standard assay mixture. The values were 40.92 ± 17.45 nM for 5. alascamts and 67.04 ± 47.70 nM for S. altivelis (mean ± SE, three independent determinations for each species). The values do not differ between the species (P = 0.63). [35S]GTP[S] binding was maximally stimulated by 3 JJ.M R-PIA. Increased concentrations of R-PIA ( 10 \iM and 100 juM) did not increase the maximal binding of [35S]GTP[S]. For assays of agonist-stimulated [35S]GTP[S] binding, 10 H.M R-PIA was used. The effects of 204 atm pressure on the stimulation of [35S]GTP[S] binding by the A, adenosine receptor agonist R-PIA and the muscarinic cholinergic receptor agonist car- bamyl choline (carbachol) were examined in 3-h incu- bations (Fig. 1). Increased pressure inhibited basal [35S]GTP[S] binding about 25% in both species. At 204 atm, both 10 juM RPIA and 100 p.M carbachol increased [35S]GTP[S] binding over the basal binding at 204 atm. For these agonists, the percent stimulation was similar in both species. The time course of the association of [35S]GTP[S] with G proteins was determined at atmospheric pressure (Fig. 2). The time course differed between the Sebastolobus species. At atmospheric pressure, brain membrane preparations from S. altivelis had tu2 values for binding of [35S]GTP[S] that were significantly higher than for S. alascanus. The values Table I BmajL and Kd determinations from ['fS]GTP/Sl equilibrium binding isotherms at 5°C and atmospheric pressure in the absence of other added giianyl nucleotides S. alascanus Species Bmax (pmol/mg) K,, (n/W) 5. a!ascam/s S. altivelis 1.49 ±0.25 1.15 ±0.10 0,41 ±0.10 0.43 ± 0.07 Mean ± SE of three independent determinations for each species. The values were identical in the two species: Bmax. P = 0.27; Kd, P = 0.88. l I Control 11 o c 0.05). For S. ultivi'lix the i,,, value at I atm differs from those at 204 atm (P < 0.05) and 40H atm (P < 0.05); the values at 204 aim and 40S aim do not differ (P > 0.05). PRESSURE ALTERS GTP[S| BINDING 393 ulation of G proteins appears to be largely GDP-liganded (Murray and Siebenaller, 1993). The bulk phospholipid and fatty acid compositions of the brain membranes of the Sebastolobus species are the same (Siebenaller et ai, 1991). The ordering effects of increased pressure on membrane acyl chain organization (Cossins and Macdonald, 1989) would be expected to also be the same. The difference in coupling efficiency at atmospheric pres- sure between the species may reflect the need for confor- mational flexibility and mobility in the membranes at the environmental pressures the species experience (Murray and Siebenaller. 1993). The coupling of receptors to G proteins depends on membrane fluidity (Houslay et cil., 1980, 1981; Casado et ai. 1992). The effect of pressure on the time course of GTP binding in the Sebastolobus species supports this interpretation. Increased pressure increases the observed binding con- stant (decreased ?,/2) of [35S]GTP[S], and the pressure sen- sitivities of the observed binding rate constants (k) of the two species differ (Table I). In S. altivelis. the heightened rate of GTP binding may result from increased pres- sure raising the proportion of G proteins accessible to [35S]GTP[S] binding. Alteration of the localization of signal transduction complexes in the plasma membrane (Huang et al, 1997) or alteration of the interacting pool of subunits (Figler et ai, 1997) may contribute to the change in the rate of guanine nucleotide exchange. Because the ?,/-, and observed k values depend on the concentration of GTP, values in vivo will differ from those reported here. However, it is clear that the pressure effects on the observed binding constant, k (Table II), reflect changes in the binding constants, kon and kntl, that are independent of the GTP concentration. Of note is the ob- servation that the k values of the species are similar over the range of /'/; situ pressures that the species experience. At the typical habitat pressures of S. alascanns ( 10 to 85 atm), the k value, calculated from the mean of the 1 and 204 atm values, which do not differ, is approximately 0.0171. Over the range of 30.5 to 177.5 atm, the k values for S. altivelis, obtained by interpolation, are 0.0128 to 0.0180. These k values were calculated from a plot of the replicates of In k versus 1 and 204 atm pressure. This assumes that the change in k is due to a pressure-independent volume change. If the pressure effect diminishes at higher pressures, as suggested by the fact that the 204 and 408 atm values are the same in S. altivelis, our interpolation underestimates how close the values are for the two species. Nonetheless, at their respec- tive depths of occurrence, the binding rates are more similar than apparent from a comparison at atmospheric pressure. Thus the guanyl nucleotide binding properties of the a subunits appear to be conserved at their habitat pressures. As seen previously (Siebenaller et ai, 1991; Siebenaller and Murray, 1994a), increased pressure decreases the effi- cacy of agonists (Fig. 1). The maximal binding (Bm.M) values (Table I) for A, adenosine receptor agonist stimu- lated-! S]GTP[S] binding in Sebastolohus brain mem- branes at 5"C are about 70-fold lower than the Bmax value (25°C) in bovine brain membranes (Lorenzen et ai, 1993). However, the Kd values (Table I) of the mammalian and teleost species are similar. This observation is consistent with the conservation of binding parameters, and is similar to the pattern observed for enzymes and receptors at their environmental temperatures (e.g.. Yancey and Soinero, 1978; Yancey and Siebenaller, 1987; Siebenaller and Mur- ray, 1988) and pressures (e.g.. Siebenaller. 1987). 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LEE, TCHAIKO PARRIS, ZOYA KOROLEVA, NISHAL MOHAN, PIERRE NOAILLES, AND WILLIAM D. COHEN* Department of Biological Sciences, Hunter College of CUNY, 695 Park Are., New York, New York 10021 : and Marine Biological Laboratoiy, Woods Hole, Massachusetts 02543 Abstract. The normal shape of the erythrocytes of the bivalves known as blood clams is maintained by a marginal band (MB) of microtubules. When hemolymph (or "blood") is withdrawn from the animal, its erythrocytes change, within minutes, from the normal smooth-surfaced, flattened ellipsoids (N-cells) to spheroids with folded surfaces (X- cells). This alteration can be prevented by rapidly diluting the hemolymph with physiological medium, yielding N- cells for use in studying the transformation to X-cells. Bioassays showed that shape transformation was induced by a hemolymph activity (Hx) and was a function, in part, of cell responsiveness to this activity. Eventually the shape of the cells spontaneously returned to normal, at a rate depen- dent upon the concentration of the cells and of Hx; recovery was correlated with loss of Hx. The X-cells contained an intact but highly deformed MB, but this was not the effector of the transformation. Erythrocytes made to lack MBs still changed shape, although they did not recover as completely as did the MB-containing controls. When clams were cooled before hemolymph was withdrawn, the concentration of Hx was reduced. Hx was retained after dialysis of hemolymph, and initial filtration and chromatography indicated that its Mr was greater than 500.000. Shape transformation was blocked by EGTA, by serine protease inhibitors, and by sodium azide; the last indicates ATP-dependence. Although the mechanism responsible for shape transformation re- mains to be determined, the data suggest that the change is triggered by a coagulation-related activity in response to the removal of hemolymph from the animal. Received 23 April 1999; accepted 13 August 1999. * To whom correspondence should be addressed. E-mail: cohen@genectr. hunter.cuny.edu Introduction The nucleated erythrocytes of non-mammalian verte- brates are useful for studying the role of cytoskeletal ele- ments in cellular morphogenesis and cell-shape mainte- nance. When mature, these cells are characteristically flattened ellipsoids or discoids with a nuclear bulge. Their cytoskeletal system contains a marginal band (MB), com- posed principally of microtubules, enclosed within the membrane skeleton (MS) in the plane of flattening, and in at least some species, intermediate filaments link sites on the MS to the MS or nucleus. The morphology of these cells is relatively stable and is maintained by the MB against po- tentially deforming mechanical or osmotic forces (Joseph- Silverstein and Cohen, 1984, 1985; Cohen, 1991). Certain invertebrates also have hemoglobin-bearing nucleated erythrocytes (Ratcliffe and Rowley, 1981; Cohen and Nem- hauser, 1985), and in some cases these cells change mor- phologically when they are removed from the animal (e.g., Terwilliger et al., 1985). Erythrocyte-containing species include bivalves of the genera Anadara and Noetia. The cytoskeletal system of their red cells resembles that of non-mammalian vertebrates but, unlike the latter, it also includes a functional centrosome associated with the MB (Cohen and Nemhauser, 1980: Nemhauser et al., 1983). Most importantly for the present work, the shape of the erythrocytes changes drastically — from normal flattened el- lipsoids ("N-cells") to wrinkled spheroids ("X-cells") — in hemolymph samples from these and related species (Sulli- van. 1961; Cohen et al., 1985). We have found that this transformation is induced by a hemolymph activity, and that, surprisingly, it eventually reverses spontaneously, so that the cells return to a near-normal shape ("R-cells"; 395 396 C. LEMA-FOLEY ET AL. Dadacay et ai, 1996). This is the only natural, reversible morphological change in non-mammalian erythrocytes of which we are aware, and it offers an opportunity to analyze the interplay between extracellular factors and cytoskeletal elements in maintaining cellular shape. We present here experimental work that defines major properties of the nat- ural inducing factors and of the cellular response in this system. Materials and Methods Experimental materit.il Specimens of Noetia ponderosa (the "ponderous ark") were obtained from Terry Bros. Co. (Willis Wharf, VA) and were maintained in a refrigerated seawater tank at about 17°C or in the running natural seawater system at the Marine Biological Laboratory. Blood from the mantle cav- ity or foot muscle was drawn using syringes with 22- or 27-gauge needles, or was obtained by cutting muscle during clam sacrifice. Shape-transforming activity in the whole blood, as measured by the percentage of X-cells present, was recorded 5 or 10 min after blood was obtained, when it was typically maximal. Because we occasionally found clams in which activity was very low, we adopted an activity of 85% or greater as the minimal level for subse- quent experimental use. To obtain N-cells (cells of normal morphology that had not undergone transformation), blood was immediately di- luted a minimum of 1:200 into "physiological medium" consisting of MBL formula artificial seawater (Cohen, 1997) containing 0.5 mM NaH,PO4 at pH 7.2, in which cells retained normal shape. In a few experiments, marine molluscan Ringer's solution (MMo-1: Cohen, 1997), a sim- ilar medium also containing 0.5 mM NaH^PO4 at pH 7.2, was employed with the same results. The resulting suspen- sion was centrifuged (2000 rpm, 3 min, Beckman Accuspin at 17°C), yielding an erythrocyte pellet overlaid by a thin layer of white cells and trapped erythrocytes. This layer was removed by aspiration, and the cells were washed two or three times by centrifugation in about 5 times the original blood volume of physiological medium. Washed erythro- cytes were resuspended to the original blood volume and hematocrit was measured to determine cell concentration by volume. N-cells were ready for use after final adjustment to a concentration of 1% by volume and inspection with phase contrast microscopy to verify normal shape. Bioassay of shape transforming nctiritv Cell-free hemolymph was obtained as the supernate after a 3-min microfuge centrifugation of whole blood. As noted previously (Dadacay et with physiological medium, time spent in the fully transformed state was progressively reduced. A typical time course for shape transformation and recovery in diluted hemolymph is pre- sented in Figure 4. and a comparison of cell response to undiluted and diluted hemolymph is shown in Fig. 5. The rate of shape reversal was positively correlated with eryth- rocyte concentration (Fig. 6). Shape transformation was not triggered by a change in hemolymph pH. nor could it be mimicked by simple 398 C. LEMA-FOLEY ET AL. Figure 2. Survey view of Noe tia ponderosa erythrocytes. (a) Normal cells, flattened and ellipsoidal; (h) shape-transformed erythrocytes; (c) erythrocytes during recovery. Scanning electron microscopy; bar = 10 /urn. changes of pH in the medium. Cells were found to be morphologically stable over a tested pH range of 6.6-7.5 in physiological medium, and the pH of native cell-free he- molymph remained stable at about 6.7 as measured before and after shape transformation. Cell responsiveness The percentage of cells that changed shape was markedly lower in native blood samples from certain clams. To test whether this was due to variable H. or to variable cell Figure 3. Higher magnification view of two cells in Figure 2. plus two additional examples: (a. b) Normal erythrocytes, flattened and ellipsoidal (nb = nuclear bulge); (b) shape-transformed erythrocytes, with "lumpi- ness resolved us extensive surface folding. Scanning electron microscopy; bar = 10 ju,m. ERYTHROCYTE SHAPE TRANSFORMATION 399 100-, 90- N. 80- ^ 70- \ 0) 0 X v£ 60- 50- 40- \ 30-I \ 20-I V 10 I ^\ n J 0 20 40 60 80 time (min) Figure 4. Typical time-course of cell-shape transformation and recov- ery in diluted hemolymph. The percentage of X-cells rose to >90% within 5 min, held at >80% for ~15 min. and declined to near zero by 60 min. responsiveness to Hx, N-cells and hemolymph were pre- pared from clams exhibiting high and low levels of native shape transformation respectively, and cells and hemo- lymph were mixed in all four combinations. The results (Fig. 7) show that Hx was high in all hemolymphs, but that erythrocytes of two different clams can vary greatly in their responsiveness to hemolymph from the same clam. 0 10 20 30 60 time (min) Figure 6. Effect of cell concentration on cell-shape transformation and recovery (undiluted hemolymph). At the standard cell concentration (1%) the percentage of shape transformation was maximal, with only modest subsequent reversal. At higher cell concentrations the peak percentage of X-cells was reduced, and reversal rate was progressively greater. Post-recovery cell responsiveness and depiction of Hx Cells that had recovered normal shape (R-cells) were able to undergo a second transformation to X-cells, comparable to that of controls, when transferred into fresh hemolymph 0 10 20 30 60 90 time (min) Figure 5. Effect of hemolymph dilution on cell-shape transformation and recovery. H = undiluted hemolymph; H/5 and H/10 = hemolymph diluted 1:5 and 1:10 with physiological saline, respectively. Note that <50% of the cells responded in H/10. In experiments such as this using undiluted hemolymph. reversal to <10% X-cells was frequently not ob- served for 6 or more hours. • A cells + HA o Acells + HB • B cells + HA B cells +HB 10 15 20 time (min) 25 30 Figure 7. Dependence of shape transformation on cellular factors. Cells and cell-tree hemolymph (H) were obtained from two clams (A and B), and all four mixtures were made as indicated. Clam A cells yielded >90% .X-cells within 5 min and maintained that activity independent of hemolymph source, whereas clam B cells showed submaximal activity independent of hemolymph source. 400 C. LEMA-FOLEY ET AL. N-cells + H o R-cells + H N-cells + Hp-r =¥=* 10 15 20 time (min) 25 30 Figure 8. Assay for hemolymph activity (H,) following cell-shape transformation and recovery, and test of capacity of recovered cells (CR) to undergo a second transformation. Upon recovery, R-cells reincubated with fresh hemolymph again underwent shape transformation, whereas reincu- bation of fresh N-cells in the "post-reversal" cell-free hemolymph (H^r) showed that it lacked H,. (Fig. 8, open circles). However, the converse was not true: N-cells incubated in hemolymph in which other cells had previously undergone shape transformation and recovery either remained normal or exhibited only partial transfor- mation and very rapid recovery, indicating depletion of Hx (Fig. 8. squares). Cytoskeletal structure and function As revealed by indirect anti-tubulin immunofiuorescence, the MB was still present and continuous in the cytoskeletons of shape-transformed cells, but its shape was highly convo- luted compared to that of normal cells (Fig. 9). To test whether the MB was a primary effector of shape transfor- mation, erythrocytes with and without MBs were prepared by temperature cycling in the presence of inhibitors of disassembly (taxol) or reassembly (nocodazole). Similarly cycled controls (DMSO solvent only) contained completely or partially reassembled MBs. Bioassays in diluted hemo- lymph produced more than 96% X-cells in all three prepa- rations within 10 min, and reversal after 3 h. One major difference was noted in cells lacking MBs, however: many of the major transformation-induced surface indentations were not eliminated during reversal (Fig. 10). Shape transformation inhibitors Heating hemolymph in a boiling water bath, followed by cooling and bioassay. showed Hx to be heat-labile. In three experiments in which controls showed very high initial Figure 9. Marginal bands (MBs) of microtubules as revealed by anti- tubulin immunofiuorescence. (a) N-cell cytoskeleton; (b-d) X-cell cy- toskeletons. N-cell MBs were typically ellipsoids, sometimes twisted into figure-8s. X-cell MBs were essentially intact, but assumed highly convo- luted shapes without apparent breakage. A diagram of the basic MB deformation pattern observed in many X-cells (e.g.. b. c) is given in (e); some twist patterns were more complex, however (e.g.. d). Fluorescence microscopy; bar = 10 ;um. activity (99% at 10 min). samples heated for 20 min exhib- ited initial residual activity of 75%-957c, but recovery of cell shape began after only about 20 min. By 60 min, experimentals had 0%-15% X-cells, whereas controls still Figure 10. Shape reversal in cells containing or lacking MBs. (a) MB-containing cells: only minor post-reversal surface deformations were observed; (b) cells lacking MBs; major surface indentations were retained post-reversal. Video-enhanced phase contrast microscopy; bar = 10 /xm. ERYTHROCYTE SHAPE TRANSFORMATION 401 20 30 40 time (min) 50 60 Figure 11. Effect of sodium azide on shape transformation and recov- ery. N-cells were incubated for 30 or 60 min in 3 mM sodium azide in physiological medium (=expenmentals E30 and E60) or in physiological medium alone ( =controls C30 and C60) prior to assay. Azide pre-incuba- tion markedly reduced the percentage of X-cells and accelerated shape reversal, with greater effect for the 60-min period. Controls maintained >90% X-cells throughout. had 95%-99%. In two additional experiments, controls with 9095-9395- initial activity retained 8095—9095- after 30 min, whereas experimental heated for 10 min had only 595--6% X-cells initially, and less than 295- in 30 min. Bioassays were conducted with various potential bio- chemical inhibitors added to hemolymph prior to the addi- tion of N-cells. Shape transformation was completely inhib- ited by 10 mM (or greater) EGTA, as reported previously (Dadacay et ai. 1996); at a given concentration, EDTA was less effective. The protease inhibitors initially surveyed were antipain, aprotinin, bestatin, chymostatin, leupeptin, aminoethylbenzenesulfonyl fluoride (AEBSF), and phos- phoramidon. As assayed after a 10-min exposure, only chymostatin and AEBSF were effective, at 189f and 79f X-cells, respectively (vs. 91% for controls). AEBSF pro- duced complete inhibition at 3 mg/ml (12 mM) and above, and an excess (5 mg/ml) was typically employed in other experiments. ATP-dependence of shape transformation was tested by pre-incubation of N-cells in physiological medium contain- ing 3 mM sodium azide for either 30 min or 60 min, followed by a wash, prior to mixing cells and cell-free hemolymph. Controls were similarly incubated, except without azide. As shown in Figure 11, the percentage of cells undergoing shape transformation was markedly re- duced and shape reversal accelerated in both cases com- pared to controls, with the longer period of ATP depletion having the greater effect. Activity sequence The inhibitor studies indicated that shape transformation involved a calcium-activated step and a proteolytic activity. but in what sequence did they occur? To answer this ques- tion, N-cells were mixed with hemolymph at t = 0, and inhibitors were added at subsequent time intervals (Fig. 12). EGTA was found to be effective only if present initially (t = 0): in contrast, AEBSF was effective initially and was at least partially effective at later time points. Importantly, when added at / = 5 min and 10 min, AEBSF induced some shape reversal, whereas EGTA did not. Triggering of transformation acthitv Shape transformation was apparently triggered during the removal of blood and thus might be a response to wounding. Three pairs of clams were used to test this hypothesis: physiological responsiveness was reduced by pre-cooling clams to 1 °C. and tissue trauma was minimized by remov- ing blood through a very fine (28-ga) syringe needle. The activity of hemolymph obtained using both variables to- gether (assayed using N-cells from a non-cooled clam) was reduced to about 5095- of that of hemolymph obtained from the same clam after re-warming and sacrifice by slashing muscle (Fig. 13). Activity reduction of lesser extent was also observed when each variable was tested separately (low temp. vs. normal: fine needle vs. sacrifice; not shown). Control BEGTA D AEBSF 100n (0 0 5 10 time of inhibitor addition (min) Figure 12. Effect of addition of EGTA and AEBSF at various times after exposure of N-cells to hemolymph. The percentage of X-cells was determined 5 mm after the addition of inhibitor (final concentrations: 20 mM EGTA. 5 mg/ml AEBSF). EGTA was effective only when present initially, whereas AEBSF was still partially effective when added later. 402 C. LEMA-FOLEY ET AL • B cells + HB o B cells + HA • B cells + HA(1°C) 10 15 20 time (min) 25 30 Figure 13. Tissue trauma as a possible factor in Hx level. Two prep- arations of cell-free hemolymph were made from clam A: HA(1,C, was obtained via syringe after pre-cooling clam A to 1°C, and HA from the same clam was obtained by cutting muscle after 3 h of rewarming. N-cells (CB) and cell-free hemolymph for control bioassay were obtained from clam B. Three mixtures were made as indicated, with HA(I 0 exhibiting less than half the activity of HA. Dialysis and initial fractionation of hemolymph Active hemolymph was dialyzed for 1 2 h against physi- ological medium at 0°C. with a molecular weight cutoff of about 12,000. Control hemolymph was similarly treated except that the dialysis sac was simply kept moist. Bioassay with fresh N-cells showed that both samples exhibited ex- cellent activity, with the experimental comparable to the control except for an extended activation lag (Fig. 14). Centrifugation through filters with different molecular weight cutoffs (Centricon) indicated that the Mr for Hx is greater than 500,000, and chromatogaphy on Sephadex G-200SF confirmed that Hx moved together with the blue dextran marker, indicating the M, to be greater than 250,000. Discussion Shape transformation and recovery The results show that shape transformation occurs in response to an activity (Hx) in the cell-free hemolymph, and that the extent of response is a property of the erythrocyte population. In typical assays, more than 907r of cells were responsive to Hx, but individual cells, as well as cells from certain clams, varied in both responsiveness and rate of recovery. Variability was particularly clear at high hemo- lymph dilutions (e.g.. Fig. 5, H/IO), in which only a fraction nl the erythrocytes responded. This was verified by exper- iments using erythrocytes from clams with high vs. low percentages of transformation, in which the variable re- sponse was attributable to cells rather than hemolymph (Fig. 7). In addition, recovery during a typical time course (e.g., Figs. 4-6) was not synchronous, indicating a range of cell sensitivity to Hx. Cells that had undergone shape transformation and recov- ery (R-cells) retained the capacity to change shape a second time (Fig. 8). In contrast, hemolymph assayed after removal of R-cells was depleted of Hx, and thus the recovery phe- nomenon is correlated with loss of Hv The increased rate of recovery with increasing cell concentrations (Fig. 6) is consistent with increased cell-induced loss of Hv Biochemical activities involved in shape transformation Shape transformation was markedly inhibited by EGTA, with EDTA less effective, indicating that the process is Ca++-activated. Transformation was also greatly reduced by preheating of hemolymph and blocked by the serine protease inhibitors AEBSF and chymostatin. Both EGTA and AEBSF were effective when present at t = 0 (mixing of N-cells and hemolymph), but only AEBSF was effective when added later (Fig. 12). Thus, the initial step is presumed to be Ca++-activated, resulting in a hemolymph proteolytic activity for which Ca++ is no longer required. One relatively simple hypothesis is that a proteolytic product maintains cell shape transformation, and that cells recover when the proteolytic activity ceases and product concentration falls. This is at least partly supported by the observation that addition of AEBSF produces considerable reduction in the percentage of X-cells present (Fig. 12) 'Non-dialyzed o Dialyzed 10 15 20 time (min) 25 30 Figure 14. Activity of cell-free hemolymph after dialysis. Non-dia- lyzed and dialy/ed hemolymph reached comparable X-cell levels, with the latter exhibiting a longer lag period. ERYTHROCYTE SHAPE TRANSFORMATION 403 compared to controls. With time, the X-cells presumably inhibit this proteolytic activity or exhaust its product, ac- counting for the loss of transforming activity in post-recov- ery hemolymph. Mechanism of shape transformation and recovery ATP-depletion by pre-incubating the erythrocytes with azide inhibits shape transformation in most of the cell population, showing that it is an active phenomenon. Three hypotheses for an active shape-transformation mechanism are (a) alteration of MB shape by a microtubule-motor protein system, (b) contraction of a cell-surface-associated actomyosin system, and (c) reduction of cell volume by an osmotic pump system. Each is considered here in relation to the data. (a) Microtubule-motor protein mechanism: MB function. Transformation of cell shape, as well as initiation of shape reversal, takes place in the presence or absence of the MB; thus, the MB is not the primary effector, and this mecha- nism can be ruled out. However, the MB is needed to return to completely normal cell shape (Fig. 10), a finding consis- tent with previous studies showing that the MB is required for the erythrocytes of dogfish and blood clams to resist deformation caused by mechanical and osmotic stress (Joseph-Silverstein and Cohen, 1984, 1985). Whereas normally the MB of the blood clam erythro- cyte appears to deform the MS and maintain its shape (Joseph-Silverstein and Cohen, 1985), the mechanism at work here generates sufficient force to produce substan- tial secondary deformation of the MB (Fig. 9b-d). Such deformation without breakage demonstrates that the MB has remarkable flexibility, as expected for shape-mainte- nance function. The contours assumed by many of these deformed MBs appear to be twisted variations of a "base- ball seam" model, in which a spheroidal shape is accom- modated by an initially planar ellipse folding back on itself (Fig. 9e). The complete cell-shape recovery ob- served in the presence of azide is compatible with non- ATP-requiring, mechanical MB function in response to deformation. (b) Cell-surface-associated actomyosin contraction mechanism. The entire X-cell surface has a folded appear- ance, and thus the underlying membrane skeleton (MS) is a potential cytoskeletal effector. We have observed previ- ously that F-actin is prominently associated with the Noetia erythrocyte MS (Lee et al., 1998). but the presence of myosin has not yet been demonstrated in these cells. Myosin resembling that of platelets has, however, been identified in mammalian erythrocytes (Fowler et al., 1985). It can form bipolar filaments (myosin II class) and is believed to par- ticipate in contractile activity associated with the eryth- rocyte membrane (Fowler, 1986; Pasternack and Racusen, 1989; der Terrossian et al., 1994). Thus there is sufficient precedent for serious consideration of such a mechanism in blood clam erythrocytes. (c) Active reduction of cell volume by osmotic efflux. The appearance of shape-transformed cells (Fig. 3) is compatible with osmotic distortion, and osmotic mechanisms are attrac- tive in being potentially readily reversible. However, if an osmotic mechanism is at work here, it cannot be a simple phenomenon attributable to use of physiological media or other experimental manipulations. First, the native shape ot these red cells is flattened and ellipsoidal, as shown previ- ously by immediate fixation of blood samples (Cohen and Nemhauser, 1986); when the "blood" is diluted immediately and extensively with our physiological media, the cells retain their native shape indefinitely. Second, both the shape change and recovery occur in the clam's own hemolymph without any experimental manipulation whatever. Third, hemolymph diluted tenfold with physiological medium in which N-cells are osmotically stable still induces complete shape change in some cells of the population. Fourth, freshly added N-cells are morphologically stable in hemo- lymph in which cells have undergone the complete cycle of transformation and recovery (Fig. 8). Finally, the effect cannot be mimicked by suspension of N-cells in hyperos- motic media, as reported earlier (Dadacay et al., 1996); N-cells get thinner in such media, but retain their flattened ellipsoidal shape. It is quite possible, however, that the shape transforma- tion involves a more complex osmotic mechanism, such as one in which an external signal triggers hyperactivity of an active efflux system that produces and maintains excessive volume loss until reversal. Amende and Pierce ( 1980a) have shown that hypo-osmotically stressed Noetia red cells re- duce their volume to normal levels by a mechanism involv- ing active efflux of taurine and other amino acids. Like the shape transformation reported here, this osmoregulatory mechanism is activated by Ca++ and inhibited by ATP depletion (Amende and Pierce, 1980b; Pierce and Maugel, 1985; Pierce et al., 1989). Thus it is possible that activation of the same system in N-cells by some other means could further reduce cell volume. We have not reported hematocrit volume measurements in this paper, however, because we believe that differences in the packing of N-cells — smooth- surfaced, flattened ellipsoids — and X-cells — spheroids with highly folded surfaces — make such data highly unreliable. Studies of this mechanism will require more sophisticated volume measurements. Triggering of erythrocyte shape transformation The hemolymph of this and closely related molluscan species (Amukira ovulix, A. tmnsversa) does not undergo complete in vitro clotting in the classic sense, but micros- copy of whole-blood samples shows that white hemocytes begin to aggregate shortly after the blood is obtained. In 404 C. LEMA-FOLEY ET AL. other molluscs (oysters, mussels), hemocytes have been shown to aggregate at wound sites and plug them (Sparks and Morado, 1988), and eventual spontaneous disaggrega- tion has also been observed in oysters under certain condi- tions (Bang, 1961; Feng. 1965, 1988). The act of obtaining hemolymph by syringe or by tissue cutting may initiate similar wound-repair mechanisms in blood clams, with el- evation of Ca++ by entry of seawater a possible trigger. Erythrocyte shape transformation might then be a secondary effect triggered and maintained by a local mechanism of wound repair, with recovery advantageous for affected erythrocytes that move away from the site and enter the general circulation. The data are consistent with the idea that the mecha- nism for triggering shape alteration is analogous to that of vertebrate blood clotting, in which Ca+ + is required for early activation steps, serine protease activity yields fibrin, and generation of plasmin causes clot dissolution (reversal). The analogy is not necessarily superficial, as serine proteases are involved in clotting cascades in certain other invertebrates (e.g.. Limnlus: Bergner et al., 1997). Though not the focus of present work, initial characterization of molecular species participating in Hv showed retention of activity in dialyzed cell-free hemo- lymph, indicating that components of relatively low mo- lecular mass (< 12,000 Da) were not involved. Hx was present in fractions produced by column chromatography and centrifugal filtration that respectively indicate Mr values of more than 250,000 and more than 500,000 for critical components. Their identification should provide further insight into the signals triggering this morphoge- netic alteration, as well as into the effector mechanism. Acknowledgments We thank L. Kerr (MBL) for preparation of critical-point- dried SEM samples, and L. Bonacci, K. Brown, A-V. Da- dacay, F. Harrow, and J. Huerta (Hunter College) for addi- tional technical contributions. Student support from the Hunter College Howard Hughes Undergrad. Biology Program, NIGMS-MBRS CMOS 1 76- 1 8, NIGMS-MARC GM07823-18, and the NSF-REU program, as well as research support from PSC-CUNY 668201 and NSF 9808368, is gratefully acknowledged. Literature Cited Amende, L. M., and S. K. Pierce. 1980a. Cellular volume regulation in salinity stressed mollusks: the response of Nuetia ponderosa (Arcidae) red blood cells to osmotic variation. J. Comp. Physiol. 138: 283-289. Amende, L. M., and S. K. Pierce. 1980b. Free amino acid mediated volume regulation of isolated Nuetia ponderosa red cells: control by Ca2 + and ATP J. Comp. Physiol. 138: 291-298. Bang, F. B. 1961. Reaction to injury in the oyster (Crassostrea vir- ginica). Biol. Bull. 121: 57-68. Bergner A., T. Muta, S. Iwanaga, H. G. Beisel, R. Delotto, and \V. Bode. 1997. Horseshoe crab coagulogen is an invertebrate protein with a nerve growth factor-like domain. Biol. Chem. 378: 283- 287. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254. Cohen, W. D. 1991. The cytoskeletal system of nucleated erythrocytes. Int. Rev. Cytol. 130: 37-83. Cohen, W. I)., ed. 1997. Compendium of Physiological Solutions. Biol. Bull. Comp. (Online] Biological Bulletin Publications. Marine Biolog- ical Laboratory, Woods Hole, MA. Available: http://www.mbl.edu/ html/BB/COMPENDlUM/Comp-TabCont.html. [1999, April 12], Cohen, W. 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INDEX A century of science: The Bii>li>i;inil Bulletin looks back — and forward, 113 A cuticular secretion of the horseshoe crab, Limtiltix polyphemus: a poten- tial anti-fouling agent, 274 A new approach for measuring real-time calcium pumping and SR function in muscle fibers, 227 Acarlia tonsa. reproductive output. 294 Acetylcholine. 267 Acoustic behavior and reproduction in five species of Corycoras cattishes (Callichthyidae). 241 Acoustics, underwater, 241 Active factor. 174 ADAMO, SHELLEY A., see Alison J. King, 256 Aggression, 256 AKANKI, F. R.. see S. J. Zottoli, 239 Alkaline gland. 82 Allelochemicals, 332 Allorejection, 188 Amazonian fish. 241 5-arninolevulinate dehydratase, 283 An endogenous SCP-related peptide modulates ciliary beating in the gills of a venerid clam, Mcrcemiria inerct'naria, 159 An ethogram of body patterning behavior in the biomedically and com- mercially valuable squid Loligo pealei off Cape Cod, Massachusetts, 49 Annelida. 14 Annual Report of the Marine Biological Laboratory, v. 197(1), Rl Annual viral expression in a sea slug population: life cycle control and symbiotic chloroplast maintenance. 1 Antennule use by the American lobster. Homarus americanus, during chemo-orientation in three turbulent odor plumes. 249 Anthiipleiira elegantixximu, 72 Anti-fouling. 274 Apoptosis. 271 ARMSTRONG, PETER B.. see John M. Harrington, 274; Rengasamy Asokan. 275: Chhanda Biswas. 276 Ascidian. 188 ASOKAN, RENGASAMY, AND PETER B. ARMSTRONG, Cellular mechanisms of hemolysis by the protein limulin. a sialic-acid-specific lectin from the plasma of the American horseshoe crab. Liimilus polyphemus, 275 Astrocyte, 1 15 ATEMA, JELLE, see Katrin Mjos, 249; John P. Hanna. 250; Erik Zettler. 252; Cnstin Berkey. 253; Leslie C. McLaughlm. 254 Avian fracture, 1 I B Bacteria, marine, 283 BALSER, ELIZABETH J., see Siinke Johnsen. 26 BARLOW. ROBERT B.. see Vanessa J. Ruta. 233: Justin W. Widener, 300 Barnacle, 144 BAYNE. BRIAN L., SUSANNE SVENSSON. AND JOHN A. NELL. The physiolog- ical basis for faster growth in the Sydney rock oyster. Saccuxtmi commercialis, 377 BEARER, E. I . M. L. SCHLIEF, X. O. BREAKEHELD, D. E. SCHUBACK, T. S. REESE, A,;I> .1. H. L/\VAIL. Squid axoplasm supports the retrograde axonal transj oi herpes simplex virus. 257 Behavior, 49, 225 courtship. 63 feeding, 207, 315. 361, 377 Behavior of hemocytes in the allorejection reaction in two compound ascidians. Botryllus scalaris and Symplegma reptans, 188 Behavioral plasticity, 63 BEN-YAKIR, R., see B. Rinkevich. I I BEN-YAKIR, S., see B. Rinkevich, 1 1 BENINGER, PETER G.. see Harold Silverman, 008 BERKEY, CRISTIN, AND JELLE ATEMA. Individual recognition and memory in Hoimmtx americanus male-female interactions, 253 BILLACK, BLASE, JEFFREY D. LASKIN, MICHAEL A. GALLO, AND DIANE E. HECK. Effects of a-bungarotoxin on development of the sea urchin Arhticid piinctiiUttti. 267 Bioacoustics. 241. 242 Bioenergetics. 377 Bioluminescence. 26. 132, 348 Bioluminescence in the deep-sea cirrate octopod Stauroteuthis syrtensis Verrill (Mollusca: Cephalopoda), 26 Biosynthesis of tyrosine O-sulfate by cell proteoglycan from the marine sponge, Microciona prolifera, 279 BISWAS, CHHANDA, AND PETER B. ARMSTRONG. Identification of a hemo- lytic activity in the plasma of the gastropod Busycon canaliculatum, 276 Bivalve feeding, 368 BLACKBURN, JOHN G., see Gregory M. Grabowski, 82 Blood clam, 395 Body coloration. 49 BOLTON, TOBY F., see Florence I. M. Thomas, 7 Botryllus scalaris, 188 BOWEN, J., see A. Evgenidou, 292 BOYLE. KIM-LAURA, see Roger T. Hanlon. 49 Brain slice. 1 15 BREAKEFIELD, X. O.. see E. L. Bearer, 257 Bringing the script to life: the role of muscle in behavior, 225 Brooding. 104 BROWN. E., see A. Evgenidou, 292 BUNGE, RICHARD P.. see Geoffrey K. Ganter. 40 Buoyancy. 309 BURGER. MAX M.. see Jane C. Kaltenbach. 271; William J. Kuhns. 277; Octavian Popescu. 279 BLISHMANN. PAUL J., Concurrent signals and behavioral plasticity in blue crab (Callinectes sapiitits Rathbun) courtship. 63 BUSKEY. E. J., see D. K. Hartline. 132 BYRNE. MARIA, see Francis Chee, 123 Calcium. 115. 227. 229. 268 Calliactis japonica, 3 1 5 Camouflage. 348 Ctincer in'orutits, 361 CANDELARIO-MARTINEZ. AURORA, see Louis F. Gainey, Jr.. 159 CANFIELD. SUSANNAH. Luc CLAESSENS. CHARLES HOPKINSON JR.. EDWARD RASTETTER, AND JOSEPH VALLINO, Long-term effect of municipal water use on the water budget of the Ipswich River Basin. 295 Carbohydrates. 94 Carbon translocation, 72 Curcinux niecnutx. 297 CEBRIAN, J.. see A. Evgenidou, 292 Cell morphology, 395 Cell shape transformation, 395 Cellular mechanisms of hemolysis by the protein limulin. a sialic-acid- specilic lectin from the plasma of the American horseshoe crab, Liniiiliix piilyphi'iintx, 275 406 INDEX TO VOLUME 197 407 Central nervous system. 240 Centrifuge microscopy, 260 Cephalopod. 26. 256 CHAGA. OLEG, see Steven Q. Irvine. 3 1 9 CHAPPARO. O. R.. R J. THOMPSON. AND C. J. EMERSON. The velar ciliature in the brooded larva of the Chilean oyster Ostrea chilensis (Philippi. 1845). 104 CHEE. FRANCIS, AND MARIA BYRNE, Development of the larval serotonergic nervous system in the sea star Patiriella regularis as revealed by confocal imaging, 123 Chemical cues. 94 Chemo-onentation turbulence. 249. 250 Chemoreceptor cells as concentration slope detectors: preliminary evi- dence from the lobster nose. 252 Chemoreceptor filter properties, 252 Chemoreception. 361 Chemotaxis. 250 CHILD, ALICE, see Aimee Vasse, 281 Cilia bands, 14 beating. 159 in feeding. 14 velar, 104 Circadian rhythms. 233 Cirri, 008 CLAESSENS, Luc, see Katherine M. Pease. 289; Susannah Canfield, 295 Clam, 159 CLAY, JOHN R., AND ALAN M. KUZIRIAN, Fluorescence localization of K+ channels in the membrane of squid giant axons. 231 COHEN. WILLIAM D., see Christine Lema-Foley. 395 Colony specificity. 188 Columba livia. I 1 Communication, 49 Concentration slope detectors. 252 Concepts in Imaging and Microscopy: Exploring biological structure and function with confocal microscopy. 1 15 Concurrent signals and behavioral plasticity in blue crab (Callinectes sapidus Rathbun) courtship. 63 Confocal microscopy. 115. 262. 263 Contact with squid eggs increases agonistic behavior in male squid (Loligo pealei), 256 Copepod. 132 Coral reef. 303 Coral reef fish. 242 Coralline algae, 332 CORCORAN, A., see A. Evgenidou, 292 CORCORAN, D.. see A. Evgenidou. 292 Countershading. 348 Courtship, 63 Courtship sounds of the Pacific damselfish, Abiidefduf sordidus (Pomacen- tridae), 242 Crab. 63. 174. 361 GRAIN. JENNIFER A., Functional morphology of prey mgestion by Placetron wosnessenskii Schalfeew zoeae (Crustacea: Anomura: Lithodidae), 207 Cropping of sea anemone tentacles by a symbiotic barnacle, 315 Crustacea, 144. 348, 361 CUBBAGE, ANDREA, DAVID LAWRENCE, GABRIELLE TOMASKY, AND IVAN VALIELA, Relationship of reproductive output in Acartia tonsa, chlo- rophyll concentration, and land-derived nitrogen loads in estuaries of Waquoit Bay, Massachusetts, 294 Cyclodextrin, 284 Cypnd. 144 Cytochrome P450. 303 D D'AMBROSIO, A., see A. Evgenidou. 292 DAILEY, MICHAEL. GLEN MARRS, JAKOB SATZ. AND MARC WAITE. Concepts in Imaging and Microscopy: Exploring biological structure and func- tion with confocal microscopy. 115 Damselfish. 242. 244 DEARHOLT, C.. see A. Evgenidou, 292 Decline of a horseshoe crab population on Cape Cod, 300 DEEGAN. LINDA, see Talia Young, 297: Sharon Komarow. 299 Deep sea, 26. 348 Defense mechanism. 281 DEGNAN, BERNARD M., AND CRAIG R. JOHNSON. Inhibition of settlement and metamorphosis of the ascidian Henlmaitiu cumitu by non-genic- ulate coralline algae. 332 DEMING, NICOLE M.. see Robert B. Silver. 268 Density. 309 DEPlNA. ANA S.. see Torsten Wollert. 265 DESAI. ARSHAD, see Paul Maddox. 263 Development. 123. 267. 319. 341 Development of the larval serotonergic nervous system in the sea star Patiriella regularis as revealed by confocal imaging. 123 Diatom. 292 Dictyostelium, 260 DIETZ. THOMAS H., see Harold Silverman, 368 Dinoflagellate. 292 Dioxm. 303 Disease, 237 Displays, 49 DNA-PK in development. 341 DOBLE, KAREN E., see Louis F. Gainey, Jr., 159 DODGE, FREDERICK A., see Vanessa J. Ruta, 233 Dorsal cell, 239 Dynamic confocal imaging of interphase and mitotic microtubules in the fission yeast, S. pwnbe. 262 Dynamic confocal imaging of mitochondria in swimming Tetrahymena and of microtubule poleward flux in Xenopus extract spindles. 263 Dynein, 259 E Echinoid, life history. 7 Echiuridae. 14 EDDS-W ALTON. P. L.. see R. R. Fay, 240 EDWARDS. KRISTIN A., see Florence I. M. Thomas. 7 Effects of a-bungarotoxin on development of the sea urchin Arbacia punctulata, 267 Effects of green tea polyphenols on lens photooxidative stress, 285 Effects of increased nitrogen loading on the abundance of diatoms and dinoflagellates in estuarine phytoplanktonic communities, 292 Effects of vanadate on actin-dependent vesicle motility in extracts of clam oocytes. 265 Elasmobranch. 82 Electrophysiology. 82 Electroretinogram, 348 Ellipsosomes. 235 Elysia. 1 EMERSON. C. J.. see Benjamin G. Miner, 14; O. R. Chaparro. 104; Dovi Kelman. 309 EMPSON, RUTH M.. see Jyotshna Kanungo. 341 Endosymbiosis, 1 ENGEBRETSON. HILARY P.. AND GISELE MULLER-PARKER, Translocation of photosynthetic carbon from two algal symbionts to the sea anemone Anthopleura eleRiintissima. 72 Enhancement of the response of rock crabs. Cancer irroratus, to prey odors following feeding experience, 361 Epidermal growth factor. 198 Erythrocyte. 395 Escape behavior kinematics, 132 Ethogram. 49 Eutrophication, 290 Evaluation ol a reporter gene system biomarker for detecting contamina- tion in tropical marine sediments, 303 Evaluation of circadian rhythms in the Liinu/iis eye. 233 EVGENIDOU. A.. A. KONKLE, A. D'AMBROSIO. A. CORCORAN. J. BOWEN. E. BROWN. D. CORCORAN. C. DEARHOLT. S. FERN. A. LAMB, J. MICHA- LOWSKY. I. RUEGG, AND J. CEBRIAN, Effects of increased nitrogen 408 INDEX TO VOLUME 197 loading on the abundance of diatoms and dinotlagellates in estuarine phytoplanktonic communities. 292 Evolution, 26, 198. 319 Extracellular survival of an intracellular parasite (Spraguea lophii. Micro- sporea), 270 FAY. R. R.. AND P. L. EDDS-WALTON. Sharpening of directional auditory input in the descending octaval nucleus of the toadtish, Opsanm tan, 240 Feeding. 207 barnacle. 3 1 5 bivalve. 368 FEINSTEIN, DOUGLAS L., see Robert Gould, 259 FERN, S., see A. Evgenidou. 292 Fertilization. 7 FINDLEY, ANN, see Earl Weidner. 270 Fish Amazonian. 241 Coral reef, 242 FISHER. ERIN C, see Sbnke Johnsen. 26 Fluorescence localization of K+ channels in the membrane of squid giant axons. 231 Fluorescence speckle microscopy. 263 Foraging. 361 FRANK. T. M.. see S. M. Lindsay. 348 FREUND, CONCETTA. see Robert Gould. 259 FUKUI, YOSHIO, TARO Q. P. UVEDA, CHIKAKO KITAYAMA. AND SHINYA INDUE, Migration forces in Dictvoste/iitm measured by centrifuge D1C microscopy, 260 Functional and evolutionary implications of opposed bands, big mouths, and extensive oral dilution in larval opheliids and echiurids (Anne- lida), 14 Functional morphology, 2(17 Functional morphology of prey ingestion by Placetron wosnessenskii Schalfeew zoeae (Crustacea: Anomura: Lithodidae), 207 G proteins, 388 GAINEY. Louis F.. JR., KELLY J. VININO. KAREN E. DOBLE, JENNIFER M. WALDO, AURORA CANDELARIO-MARTINEZ, AND MICHAEL J. GREEN- BERG. An endogenous SCP-related peptide modulates ciliary beating in the gills of a venerid clam. Mercenaria merceiniria. 159 /B-galactosidase, 40 GALLO. MICHAEL A., see Blase Billack. 267 Gamete properties. 7 GAMULIN, VERA, see Alexander Skorokhod. 198 GANTER, GEOFFREY K.. RALF HEINRICH. RICHARD P. BUNGE, AND EDWARD A. KRAVITZ, Long-term culture of lobster central ganglia: expression of foreign genes in identified neurons. 40 GARRITT. ROBERT, see Talia Young, 297; Sharon Komarow, 299 GFP-tubulin. 262 Glial cells. Glutathione S-transferase, 268 GOULD, ROBERT, CONCETTA FREUND, FRANK PALMER, PAMELA E. KNAPP, JEFF HUANG. HILARY MORRISON. AND DOUGLAS L. FEINSTEIN: Mes- senger mRNAs for kinesins and a dynein are located in neural pro- cesses. 259 GRABOWSKI. GREGORY M., JOHN G. BLACKBURN. AND ERIC R. LACY, Morphology and epithelial ion transport of the alkaline gland in the Atlantic stingray (Dasyatis siihiiui). X2 GRASSO, FR-ANK. see Katrin Mjos. 249: John P. Hanna. 250 Green crab, 297 Green tea polyph^n iU. 285 Green-fluorescent protein. 40 GREENBERG. MICHAEL J., A century of science: Th? Biological Bulletin looks back — and forward. I 13 GREENBERG, MICHAEL J., see Louis F. Gainey, Jr., 159 Growth, 377 GUNDACKER. DiETMAR, see Alexander Skorokhod. 198 H HANLEY, JANICE S., NADAV SHASHAR, ROXANNA SMOLOWITZ. WILLIAM MEBANE. AND ROGER T. HANLON, Soft-sided tanks improve long-term health of cultured cuttlefish, 237 HANLON, ROGER T., MICHAEL R. MAXWELL, NADAV SHASHAR. ELLIS R. LOEW, AND KIM-LAURA BOYLE, An ethogram of body patterning behavior in the biomedically and commercially valuable squid Loligo pealei off Cape Cod, Massachusetts, 49 HANLON, ROGER T.. see Janice S. Hanley, 237; Kathleen Q. Tang, 247; Alison J. King, 256 HANNA. JOHN P., FRANK W. GRASSO. AND JELLE ATEMA, Temporal corre- lation between sensor pairs in different plume positions: a study of concentration information available to the American lobster. Homarus amcricanus. during chemotaxis. 250 HANTEN, JEFFREY J., see Sidney K. Pierce, 1 HAROSI. FERENC I., see Inigo Novales Flamarique, 235 HARRINGTON, JOHN M., AND PETER B. ARMSTRONG. A cuticular secretion of the horseshoe crab, Liinuhis polypheinus: a potential anti-touling agent, 274 HARRISON. PAUL J. H.. AND DAVID C. SANDEMAN, Morphology of the nervous system of the barnacle cypris larva (Balamis amphitrite Darwin) revealed by light and electron microscopy, 144 HARTLINE, D. K.. E. J. BUSKEY, AND P. H. LENZ. Rapid jumps and bioluminescence elicited by controlled hydrodynamic stimuli in a mesopelagic copepod. Pleuromamma xiphias. 132 Hatching, crab. 1 74 Hearing, 240 HECK, DIANE E., see Blase Billack, 267 HEINRICH, RALF, see Geoffrey K. Ganter. 40 Hemocyanin. 276 Hemocytes. 188 Hemolysis, 275, 276 Herpes virus. 257 Hippocampus, 1 15 HIROSE, EUICHI, see Maki Shirae, 188 Hi7A. N. A., see S. J. Zottoli, 239 HO-SANG, D. A. JR.. see S. J. Zottoli. 239 Homarus americamis. 249, 252. 253, 254 HOPKINSON, CHARLES JR.. see Katherine M. Pease. 289; Susannah Canh'eld. 295 HOSKIN. FRANCIS C. G., DIANE M. STEEVES. AND JOHN E. WALKER. Sub- stituted cyclodextrin as a model for a squid enzyme that hydrolyzes the nerve gas soman. 284 5-HT neurogenesis. 123 HUANG. JEFF, see Robert Gould. 259 Hyaluronic acid, 277 Hyaluronic acid: a component of the aggregation factor secreted by the marine sponge, Microciona pro/ifera, 277 Hyaluronic acid binding protein, 277 Hydrostatic pressure, 388 Hydrostatic pressure alters the time course of GTP[S] binding to G proteins in brain membranes from two congeneric marine fishes. 388 I Identification of a hemolytic activity in the plasma of the gastropod Bu*\con ciiiiiiliciiliiiiiin. 276 Identified neurons. 40 Immunity. 274 Immunocytochemistry. 231 Increased lability of estuarine dissolved organic nitrogen from urbanized watersheds. 290 Individual recognition, 253, 254 Individual recognition and memory in Homarus americanus male-female interactions, 253 INDEX TO VOLUME 197 409 Influence of marsh flooding on the abundance and growth of Fundiilux heteroclitus in salt marsh creeks, 299 Ingestion. 207 INGOGLIA, MARK J., see Lisa M. Kerr. 303 Inhibition of settlement and metamorphosis of the ascidian Herdmunui curvata by non-geniculate coralline algae, 332 INDUE, SHINYA, see Yoshio Fukui. 260: P. T. Tran, 262; Paul Maddox, 263 Insulin receptor-like tyrosine kinase, 198 Intense concanavalin A staining and apoptosis of peripheral flagellated cells in larvae of the marine sponge Microciona prolifera: significance in relation to morphogenesis, 271 INTERIOR, REY, see Octavian Popescu, 279 Internal fixation, 1 1 Invertebrate reproduction. 7 larvae, 207 Ion channels. 23 1 Ipswich River, 289, 295 Ipswich River nutrient dynamics: preliminary assessment of a simple nitrogen-processing model. 289 IRVINE, STEVEN Q.. OLEG CHAGA. AND MARK Q. MARTINDALE. Larval ontogenetic stages of Chaetnptertis: developmental heterochrony in the evolution of chaetopterid polychaetes, 319 IWASAKI. HIROSHI. see Masayuki Saigusa. 1 74 JOHNSEN, SONKE, ELIZABETH J. BALSER, ERIN C. FlSHER. AND EDITH A. WIDDER, Bioluminescence in the deep-sea cirrate octopod Stauroteu- this syrtensis Verrill (Mollusca: Cephalopoda). 26 JOHNSON. CRAIG R., see Bernard M. Degnan, 332 Johnston atoll. 242 K KAATZ, INGRID M., AND PHILLIP S. LOBEL. Acoustic behavior and repro- duction in five species of Corycoras catfishes (Callichthyidae), 241 KALTENBACH, JANE C.. WILLIAM J. KUHNS, TRACY L. SIMPSON, AND MAX M. BURGER, Intense concanavalin A staining and apoptosis of periph- eral flagellated cells in larvae of the marine sponge Microciona prolifera: significance in relation to morphogenesis, 27 1 KANUNGO. JYOTSHNA. RUTH M. EMPSON, AND HOWARD RASMUSSEN, Mi- croinjection of an antibody to the Ku protein arrests development in sea urchin embryos, 341 KAPOOR, TARUN, see Paul Maddox, 263 KAVSAN, VADIM. see Alexander Skorokhod. 198 KELMAN, Dovi, AND RICHARD B. EMLET. Swimming and buoyancy in ontogenetic stages of the cushion star Pteraster tesselanis (Echino- dermata: Asteroidea) and their implications for distribution and move- ment, 309 KENT, J., see S. M. Lindsay, 348 KERR, LISA M., PHILLIP S. LOBEL, AND J. MARK INGOGLIA, Evaluation of a reporter gene system biomarker for detecting contamination in trop- ical marine sediments, 303 KERR, LISA M.. see Phillip S. Lobel, 242 KILHAM, N., see Katherine M. Pease, 289 Kinesin. 259 KING, ALISON J., SHELLEY A. ADAMO, AND ROGER T. HANLON, Contact with squid eggs increases agonistic behavior in male squid (Lotigo pealei), 256 KITAYAMA. CHIKAKO, see Yoshio Fukui, 260 KLIMOV, ANDREI A., see Lawrence C. Rome, 227 KNAPP, PAMELA E., see Robert Gould, 259 Koleolepas avis. 3 1 5 KOMAROW, SHARON, TALIA YOUNG, LINDA DEEGAN. AND ROBERT GARRITT, Influence of marsh flooding on the abundance and growth of Fundulus heteroclitus in salt marsh creeks, 299 KOMAROW, SHARON, see Talia Young, 297 KONKLE, A., see A. Evgenidou, 292 KOROLEVA, ZOYA, see Christine Lema-Foley, 395 KRAVITZ. EDWARD A., see Geoffrey K. Ganter, 40 KROEGER, KEVIN D., see Felisa L. Wolfe, 290 KRUG. PATRICK J.. AND ADRIANA E. MANZI, Waterborne and surface- associated carbohydrates as settlement cues for larvae of the specialist marine herbivore Alderia modesta, 94 Ku antigen, 341 KUHNS, WILLIAM J.. MAX M. BURGER. AND EVA TURLEY. Hyaluronic acid: a component of the aggregation factor secreted by the marine sponge, Microciona prolifera, 211 KUHNS, WILLIAM J., see Jane C. Kaltenbach, 271; Octavian Popescu. 279 KUZIRIAN, ALAN M., see John R. Clay, 231 Lability, 290 LACY, ERIC R., see Gregory M. Grabowski, 82 LAMB, A., see A. Evgenidou. 292 LANGFORD, GEORGE M.. see Torsten Wollert, 265 Larva, 104, 123, 207, 309 cypris, 144 feeding. 14 nonfeeding, 309 settlement, 94 Larval ecology, 207 Larval ontogenetic stages of Chaetopterus: developmental heterochrony in the evolution of chaetopterid polychaetes. 319 LASKIN, JEFFREY D., see Blase Billack, 267 Lateral inhibition, 233 LATZ . M. I., see S. M. Lindsay. 348 LAVAIL, J. H., see E. L. Bearer, 257 LAWRENCE, DAVID, see Andrea Cubbage, 294 Lead (Pb). 283 Lectin staining, 271 LEE, KYENG G., see Christine Lema-Foley, 395 LEMA-FOLEY. CHRISTINE, KYENG G. LEE, TCHAIKO PARRIS. ZOYA KORO- LEVA, NISHAL MOHAN. PIERRE NOAILLES, AND WILLIAM D. COHEN, Reversible alteration of morphology in an invertebrate erythrocyte: properties of the natural inducer and the cellular response, 395 Lens, 285 LENZ, P. H.. see D. K. Hartlme, 132 Leukotriene B4, 268 Leukotriene B4 as calcium agonist for nuclear envelope breakdown: an enzymological survey of endomembranes of mitotic cells, 268 LEWIS, NATHANIEL, see Seymour Zigman, 285 Life cycle. 1 Life history, 7 Limulin. 275 Limulus. 233. 274, 275, 300 LINDSAY, S. M., T. M. FRANK, J. KENT, J. C. PARTRIDGE, AND M. I. LATZ. Spectral sensitivity of vision and bioluminescence in the midwater shrimp Sergestes similis, 348 LOBEL, PHILLIP S., AND LISA M. KERR, Courtship sounds of the Pacific damselfish, Abudefduf sordidus (Pomacentridae), 242 LOBEL, PHILLIP S.. see Ingrid M. Kaatz. 241; Lisa M. Kerr, 303 Lobster, 40, 249, 252, 253, 254 chemo-orientation, 249 LOEW, ELLIS R., see Roger T. Hanlon, 49 Loligo pealfi, 49 Long-term culture of lobster central ganglia: expression of foreign genes in identified neurons. 40 Long-term effect of municipal water use on the water budget of the Ipswich River Basin. 295 LYNN, JOHN W., see Harold Silverman, 008 M MADDOX. PAUL, ARSHAD DESAI, E. D. SALMON, T. J. MITCHISON, KAREN OOGEMA. TARUN KAPOOR, BRIAN MATSUMOTO, AND SHINYA INOUE, Dynamic confocal imaging of mitochondria in swimming Tetrahy- mena and of microtubule poleward flux in Xenopus extract spindles, 263 410 INDEX TO VOLUME 197 MADDOX, P., see P. T. Tran, 262 MALCHOW, ROBERT PAUL. AND DAVID J. RAMSEY, Responses of retinal Muller cells to neurotransmitter candidates: a comparative study, 229 MANZI, ADRIANA E., see Patrick J. Krug, 94 Marginal band. 395 Mariculture. 237 MARRS. GLEN, see Michael Dailey. 1 15 Marsh flooding, 2w MARTINDALE. MARK Q.. see Steven Q. Irvine. 319 Mating behavior. 300 systems. 256 MATSUMOTO, BRIAN, see Paul Maddox, 263 MAUGEL, TIMOTHY K., see Sidney K. Pierce, 1 MAXWELL. MICHAEL R.. see Roger T. Hanlon. 49 MCLAUGHLIN. LESLIE C., JENNIFER WALTERS, JELLE ATEMA, AND NORMAN WAINWRIGHT. Urinary protein concentration in connection with ago- nistic interactions in Homarus umericiiini*. 254 MEBANE, WILLIAM, see Janice S. Hanley, 237 Mechanical resistance to shear stress: the role of echinoderm egg extra- cellular layers, 7 Mechanosensory thresholds. 132 Memory, 253 MENSINGER, ALLEN F., see Nichole N. Price. 246. Kathleen Q. Tang. 247 Mercenaria mercemiria, 159 Messenger mRNAs for kinesins and a dynein are located in neural pro- cesses. 259 Metabolic efficiency. 377 Metamorphosis. 94 MICHALOWSKY. J.. see A. Evgenidou. 242 Microciona prolifera. 211 , 279 Microglia. 1 15 Microinjection of an antibody to the Ku protein arrests development in sea urchin embryos. 341 Microspectrophotometry. 348 Microsporidian. 270 Microtubule. 262, 263 Migration forces. 260 MINER, BENJAMIN G.. ERIC SANFORD. RICHARD R. STRATHMANN. BRUNO PERNET. AND RICHARD B. EMLET. Functional and evolutionary impli- cations of opposed bands, big mouths, and extensive oral ciliation in larval opheliids and echiurids (Annelida). 14 MISEVIC, GRADIMIR, see Octavian Popescu. 279 MITCHISON, T. J.. see Paul Maddox. 263 MJOS, KATRIN. FRANK GRASSO. AND JELLE ATEMA. Antennule use by the American lobster. Homarus americunus. during chemo-orientation in three turbulent odor plumes. 249 MOHAN, NISHAL. see Christine Lema-Foley. 395 Mollusc, 276 MONDRUP, THOMAS, Salinity effects on nutrient dynamics in estuarme sediments investigated by a plug-flux method, 287 MONDY, WILLIAM L.. see Sidney K. Pierce, 1 Morphology, 82, 144. 319 Morphology and epithelial ion transport of the alkaline gland in the Atlantic stingray (Dasyatis sabina). 82 Morphology of the nervous system of the barnacle cypris larva (Balanus amphitrite Darwin) revealed by light and electron microscopy. 144 MORRISON. HILARY, see Robert Gould. 259 Morula cell. 188 MOTTA. M, see S. J. Zottoli. 239 MULLER. ISABEL M., see Alexander Skorokhod, 198 MULLER. WERNER E. G., see Alexander Skorokhod. 198 MULLER-PARKER, GISELE. see Hilary P. Engebretson, 72 Mummichog, 2l>(> MURRAY. THOM « F., see Joseph F. Siebenaller. 388 Muscle. 225. 22: Mussel. 008 Myosin-dependent vesicle iransport. 265 Mwilus edulis. 008 N Natural population. 300 Necrosis. 332 NELL, JOHN A., see Brian L. Bayne. 377 Nerve gas, 284 Nervous system. 144 Neurogenesis. 123 Neuron. 1 15 identifiable. 239 supramedullary. 239 Neuropeptides, 159 Nitrogen cycling. 287. 289 dissolved organic. 290 land-derived, 294 model, 289 NOAILLES. PIERRE, see Christine Lema-Foley. 395 Noetia ponderosa, 395 NOVALES FLAMARIQUE. INIGO, AND FERENC I. HAROSI, Photoreceptor pig- ments of the blueback herring (A/osa aesteva/is. Clupeidae) and the Atlantic silverside (Meniilia menidia, Atherinidae), 235 Nuclear envelope breakdown, 268 Nutrient loadina, 292 o O'NEILL. MAUREEN D.. see Kathleen Q. Tang. 247 Octopus, 26 Odor, 361 OGUNSEITAN. O. A., S. L. YANG, AND E. SCHEINBACH, The S-aminolevuli- nate dehydratase of marine Vibrio alginolyticus is resistant to lead (Ph). 283 Oligodendrocyte, 259 OLIVER, STEVEN J.. AND ELISE WATSON, Threat-sensitive nest defense in domino damselfish (Dascyllus albise/la), 244 OOGEMA, KAREN, see Paul Maddox. 263 Opheliidae, 14 Opisthobranch. 94 Organ culture. 40 Ongin of insulin receptor-like tyrosine kinases in marine sponges, 198 Oslrea clulensis, 104 Ovigerous-hair stripping substance (OHSS) in an estuarine crab: purifica- tion, preliminary characterization, and appearance of the activity in the developing embryos. 174 Oyster. 104. 377 PALMER. FRANK, see Robert Gould. 259 Parasitism. 315 PARRIS, TCHAIKO, see Christine Lema-Foley, 395 Particle capture. 368 PARTRIDGE. J. C.. see S. M. Lindsay, 348 Patiriella regularis. 123 PAUL, ROBERT, see Robert Paul Malchow, 229 PEASE, KATHERINE M.. L. CLAESSENS, C. HOPKINSON, E. RASTETTER, J. VALLINO, AND N. KILHAM. Ipswich River nutrient dynamics: prelim- inary assessment of a simple nitrogen-processing model. 289 Pentose phosphate pathway. 268 PERNET, BRUNO, see Benjamin G. Miner. 14 Phospholipase A2, 268 Photo-oxidation. 285 Photoreceptor pigments of the blueback herring (A/osa aestevulis. Clupei- dae) and the Atlantic silverside (Menidia menidia, Atherinidae). 235 Physiological characterization of supramedullary/dorsal neurons of the cunner. Tautogoldbrus udspersiis, 239 Physiology, 377 Phytoplankton biomass. 294 PIERCE. SIDNEY K.. TIMOTHY K. MAUGEL. MARY E. RUMPHO. JEFFREY J. HANTEN, AND WILLIAM L. MONDY. Annual viral expression in a sea INDEX TO VOLUME 197 411 slug population: life cycle control and symbiotic chloroplast mainte- nance. 1 Polarization. 49 Polychaete. 14. 3 11) Polyclonal antibody. 174 Pomacentrid. 244 POPESCU. OCTAVIAN, REY INTERIOR, GRADIMIR MlSEVlC. MAX M. BURGER. AND WILLIAM J. KUHNS, Biosynthesis of tyrosine O-sulfate by cell proteoglycan from the marine sponge, Microciona prolifera, 279 Population size and summer home range of the green crab, Cphf>rii jiistilliita. 1 1 Substituted c\cl>>.lextrin as a model for a squid enzyme that hydrolyzes the nerve gas sutnan, 284 Summer home range, 297 Supramedullai) neurons. 239 SVENSSON. S; s\NNE, see Brian L. Bayne. 377 Swimbladder. 225 412 INDEX TO VOLUME 197 Swimming. 309 Swimming and buoyancy in ontogenetic stages of the cushion star Pter- asler tesselatiia (Echinudermata: Asteroidea) and their implications for distribution and movement. 309 Symbiosis. 1, 72. 315 Symplegma t't'phui\. 188 TAN. X., see S. J. Zottoli. 239 TANG. KATHLEEN Q., NICHOLE N. PRICE, MAUREEN D. O'NEILL. ALLEN F. MF.NSINGER, AND ROGER T. HANLON, Temperature effects on first-year growth of cultured oyster toadfish, Opsanns tan. 247 Teleost. 246 Temperature effects on first-year growth of cultured oyster toadfish. Op- \iiintx tan. 247 Temporal correlation between sensor pairs in different plume positions: a study of concentration information available to the American lobster. Homanm iiiiu'rifiiniix. during chemotaxis, 250 Territoriality, interspecific. 244 The 5-aminolevulinate dehydratase of marine Vihrio alginolyticus is resis- tant to lead (Pb), 283 The physiological basis for faster growth in the Sydney rock oyster, Saccostrea commercialis, 377 The role of latero-frontal cirri in particle capture by the gills of Mytilus ciliilis, 368 The velar ciliature in the brooded larva of the Chilean oyster Ostrca chilensis (Philippi. 1845), 104 THOMAS. FLORENCE I. M.. KRISTIN A. EDWARDS, TOBY F. BOLTON, MARY A. SEWELL, AND JILL M. ZANDE, Mechanical resistance to shear stress: the role of echinoderm egg extracellular layers, 7 THOMPSON, R. J., see O. R. Chaparro. 104 Threat-sensitive nest defense in domino damselfish (Diiscvllns albixclhn, 244 Time-lapse, 1 1 5 Toadfish, 225, 227, 240. 246. 247 TOMASKY, GABRIELLE, see Andrea Cubbage, 294 TRAM, P. T., P. MADDOX, AND S. INOUE. Dynamic confocal imaging of interphase and mitotic microtubules in the fission yeast. S. ponihe, 262 Translocation of photosynthetic carbon from two algal symbionts to the sea anemone Anthopleura elegantissima, 72 Trochophore. 14 TURLEY, EVA, see William J. Kuhns, 277 Tyrosine O-sulfate, 279 Tyrosine sulfation substrates, 279 Vanadate. 265 VASSE, AIMEE, ALICE CHILD, AND NORMAN WAINWRIGHT. Prophenoloxi- dase is not activated by microbial signals in Limulus polyphemus, 281 Veliger. 104 Velum. 104 VINING, KELLY J., see Louis F. Gainey, Jr.. 159 Vision. 235 Visual pigment. 235. 348 w WAINWRIGHT, NORMAN, see Leslie C. McLaughlin, 254; Aimee Vasse, 281 WAITE. MARC, see Michael Dailey. I 15 WALDO, JENNIFER M., see Louis F. Gainey. Jr., 159 WALKER, JOHN E., see Francis C. G. Hoskin, 284 WALTERS, JENNIFER, see Leslie C. McLaughlin. 254 Water budget. 295 Waterborne and surface-associated carbohydrates as settlement cues for larvae of the specialist marine herbivore Ahleria mndesta. 94 WATSON, ELISE, see Steven J. Oliver. 244 WATTS. K. M., see S. J. Zottoli, 239 WEIDNER, EARL, AND ANN FINDLEY, The extracellular survival of an intra- cellular parasite (Spraguca lophii, Microsporea). 270 WIDDER. EDITH A., see Sonke Johnsen. 26 WIDENER, JUSTIN W.. AND ROBERT B. BARLOW, Decline of a horseshoe crab population on Cape Cod. 300 WOLFE. FELISA L., KEVIN D. KROEGER, AND IVAN VALIELA. Increased lability of estuarine dissolved organic nitrogen from urbanized water- sheds. 290 WOLLERT, TORSTEN, ANA S. DfiPlNA, AND GEORGE M. LANGFORD. The effects of vanadate on actin-dependent vesicle motility in extracts of clam oocytes. 265 YAMATO, SHIGEYUKI. see Yoichi Yusa. 315 YANG, S. L., see O. A. Ogunseitan, 283 YOUNG, IAIN S., see Lawrence C. Rome. 227 YOUNG. TALIA, Sharon Komarow. Linda Deegan. and Robert Garritt, Population size and summer home range of the green crab, Carcinus IIHICIIUS, in salt marsh tidal creeks, 297 YOUNG, TALIA, see Sharon Komarow. 299 YUSA, YOICHI, AND SHIGEYUKI YAMATO. Cropping of sea anemone tenta- cles by a symbiotic barnacle, 315 u Ultrastructure, 144 Urchin. 267 Urinary protein, 254 Urinary protein concentration in connection with agonistic interactions in Hnimiriix iinwricaniix. 254 UYEDA. TARO Q. P., see Yoshio Fukui. 260 VALIELA, IVAN, see Felisa L. Wolfe. 290; Andrea Cubbage. 294 VALLINO, JOSEPH, see Katherine M. Pease, 289; Susannah Canfield, 295 ZANDE, JILL M.. see Florence I. M. Thomas, 7 ZETTLER, ERIK, AND JELLE ATEMA, Chemoreceptor cells as concentration slope detectors: preliminary evidence from the lobster nose, 252 ZIGMAN, SEYMOUR, NANCY S. RAI-TERTY. KEEN A. RAFFERTY, AND NATHANIEL LEWIS, Effects of green tea polyphenols on lens photooxi- dative stress, 2S5 Zoochlorellae, 72 Zooxanthellae. 72 ZOTTOLI. S. J., F. R. AKANKI, N. A. HI/A. D. A. Ho-SANG, JR.. M. MOTTA, X. TAN. K. M. WATTS, AND E.-A. SEYFARTH. Physiological charac- terisation of supramedullary/dorsal neurons of the cunner, Tautogo- liihru\ adspersuSi 239 .