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 .... R 1 THE BIOLOGICAL BULLETIN THE BIOLOGICAL BULLETIN is published six limes 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 tor libraries; $95 for individuals. 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Physio/., not J. Cell. Comp. Physiol.} E. Unusual words in journal titles should be spelled out in full, rather than employing new abbreviations invented by the author. For example, use Rit Visindafjelags Islendinga without abbreviation. F. All single word journal titles in full (e.g., Veliger, Ecol- ogy. Brain). G. The order of abbreviated components should be the same as the word order of the complete title (i.e., Proc. and Trans. placed where they appear, not transposed as in some BIOLOGICAL ABSTRACTS listings). H. A few well-known international journals in their preferred forms rather than WORLD LIST or USASI usage (e.g.. Nature. Science, Evolution NOT Nature. Land., Science, N.Y.; Evolution, Lancaster. Pa.) 6. Reprints, page proofs, and charges. 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. PIERCE 1 *, TIMOTHY K. MAUGEL 1 . MARY E. RUMPHO 2 . JEFFREY J. HANTEN 1 , AND WILLIAM L. MONDY 1 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 I4 CO : , and the I4 C 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 (2-5C), 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 (4C), and the supernatant was centrifuged at 20,000 X g for 30 min (4C). The super- natant from the second spin was layered over a 20% sucrose cushion and centrifuged at 180,000 X g for 2 h (4C) 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 (4C). 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 MgCl 2 , 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 32 P-TTP (ICN, 10 /xCi//xl). The final volume was adjusted to 100 /xl with buffer, as neces- sary, to compensate for 12 P half life. This solution was mixed and incubated at 37C 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% OsO 4 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 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 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 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 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 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 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 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 R 2 = 0.84 2 4 6 8 10 12 14 16 18 U T 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 R 2 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 - 0.44x 2 . (B) Cilium length (n = 22 larvae); cilium length = 18.67 + 2.16x - 0.07x : . (C) Larval volume (n = 36 larvae); larval volume = 10 5 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 10 20 30 40 50 60 70 C 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 (A m . 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 8C 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 (A max = 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. BUNGE 1 *, 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 m 2 ; 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 13C. 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 0.25 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 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 D 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 (x 2 = 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 (x 2 = 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; x 2 = 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 (x 2 = 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 (x 2 = 31.36, 100 , 80 60 40 20 100 80 60 40 20 | g 100 8 80 60 40 20 X o z 1 I 80 g 60 1 "0 20 j/i 2 100 >- 80 60 40 20 100 80 60 40 20 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 20C and 13C for 21 days, both algae fixed and translocated carbon at greater rates at 20C (trans- location rates: 0.38 pg C /zoochlorella/h; 1.12 pg C /zoo- xanthella/h) than at 13C (translocation rates: 0.06 pg C /zoochlorella/h; 0.37 pg C /zooxanthella/h). However, zoochlorellate anemones received 3.5 times less carbon at 20C than at 13C 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 I4 C, 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 I4 C- 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 11C. 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 14 C 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 20C; for the second experiment the anemones were incu- bated at 13C. 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 14 C 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 I4 C method (O'Brien, 1980; Battey and Patton, 1987), with some modifications. One hour prior to the I4 C 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 I4 C 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 14 C-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 I4 C 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 14 C 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 I4 C label. Assay of homogenate was used to determine the amount of I4 C fixed by the whole anemone. The algae were separated from the host fraction to mea- sure the distribution of I4 C 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 14 C by freshly collected field anemones to the anemones in the experimental treatments, anemones gathered from the field were subjected to I4 C 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 1C) until I4 C 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 I4 C 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 I4 C 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 I4 C added and the hours of incubation with I4 C. 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 I4 C 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 20C than at 13C (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 v v 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 11C 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 - r 1 ] 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 13C and 20C 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 13C 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 13C than at 20C (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 20C than at I3C (Fig. 4a). At 13C. 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 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 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 12 b 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 13C and 20C; however, while zooxanthellae translocated approximately 2.5 times less carbon at 13C as at 20C, zoochlorellae translocated almost 4 times less carbon at 13C as at 20C (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 20C contained similar densities of algae; however, at 13C 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 20C (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 20C than at 13C (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 I4 C 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 I4 C. 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 14 C 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 14 C 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 I4 C 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) I4 C 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 20C. 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 20C than at 13C (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 20C 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 13C under high light receives 0.048 /xg C/mg protein/h from its algae (Fig. 4a). To maintain this rate of carbon translocation at 20C. the anemone would require an algal density of only 9.6 X 10 4 algae/mg protein because indi- vidual zoochlorellae translocate 2.5 times more at the higher temperature. However, the density of zoochlorellae at 20C 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 10 4 algae/mg protein at 20C to maintain a translocation rate equivalent to that obtained at 13C. However, zooxanthellate anemones held at 20C contained 3.5 X 10 4 algae/mg protein (Fig. 5), about 18% more than required to maintain the translocation rate obtained at 13C. This slightly elevated density of zooxanthellae was not sufficient to yield any significant difference in translocation rate (Fig. 4a). Using carbon translocation at 13C as the basis of comparison, zoochlo- rellate anemones lost more algae than they should have at 20C, 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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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. LACY 3 ' 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/cm 2 ). 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/cm 2 ) 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 CO 2 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 4C 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 56C. 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 M 111 on an IEC CTF cryostat (International Equipment Company). Sections were floated on Hansson's medium (1.86 mM CoSO 4 . 55.9 mM H 2 SO 4 , 3.73 mM KH,PO 4 , 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) f or 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 5C, 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; CaCl 2 . 3.8 mM; NaHCO v 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% CO 2 , 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/cm 2 , 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/HCO 3 -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 Fe f * 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 cm 2 , 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/HCO 3 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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KRUG 1 * AND ADRIAN A E. MANZI 2 ' Department of Biology, University 1 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 cm 2 ) 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 -20C 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 cm 2 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 4C. 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 (V ) 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, 75C), cold water (50 ml, 4 d, 20C), hot water (50 ml, 24 h, 65C), and 4% sodium hydroxide (50 ml. 24 h, 20C) (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 4C 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 4C 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 50C for 24 h. The proteinase was then inactivated by heating at 100C for 15 min. A control was done by adding proteinase to BVE immediately prior to heating at 100C 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 100C 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 100C 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 . r 1 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. ln<;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 - 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 (V ), 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