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. Subscription per volume (three issues): $102.50 for libraries; $47.50 for individuals. Back and single issues (subject to availability): $40 for libranes; $20 for individuals. Communications relative to manuscripts should be sent to Michael J. Greenberg, Editor-in-Chief, or Pamela Clapp Hinkle. Managing Editor, at the Marine Biological Laboratory, 7 MBL Street. Woods Hole. Massachusetts 02543. Telephone: (508) 289-7428. FAX: 508-457-1924. E-mail: pclapp@mbl.edu. http://www.mbl.edu/BiologicalBulletin/ The home page for the electronic companion to THE BIOLOGICAL BULLETIN the Marine Models Electronic Record and other BIOLOGICAL BULLETIN publications is available on the World Wide Web at the address shown above. THE BIOLOGICAL BULLETIN is indexed in bibliographic services including Index Medicus and MEDL1NE, Chemical Abstracts, Current Contemn, Elsevier BIOBASE/Current Awareness in Biological Sciences, and Geo Abstracts. Printed on acid free paper, effective with Volume ISO, Issue 1, 1991. POSTMASTER: Send address changes to THE BIOLOGICAL BULLETIN, Marine Biological Laboratory, 7 MBL Street, Woods Hole. MA 02543. Copyright 1999. by the Marine Biological Laboratory Periodicals postage paid at Woods Hole. MA, and additional mailing offices. ISSN 0006-3185 INSTRUCTIONS TO AUTHORS The Biological Bulletin accepts outstanding original research reports of general interest to biologists throughout the world. Papers are usually of intermediate length (10-40 manuscript pages). A limited number of solicited review papers may be accepted after formal review. A paper will usually appear within four months after its acceptance. Very short, especially topical papers (less than 9 manuscript pages including tables, figures, and bibliography) will be published in a separate section entitled "Research Notes." A Research Note in The Biological Bulletin follows the format of similar notes in Nature. It should open with a summary paragraph of 150 to 200 words comprising the introduction and the conclusions. The rest of the text should continue on without subheadings, and there should be no more than 30 references. References should be referred to in the text by number, and listed in the Literature Cited section in the order that they appear in the text. Unlike references in Nature, references in the Research Notes section should conform in punctuation and arrangement to the style of recent issues of The Biological Bulletin. Materials and Methods should be incorpo- rated into appropriate figure legends. See the article by Loh- iTumn et al. (October 1990. Vol. 179: 2I4-2IX) for sample style. A Research Note will usually appear within two months after its acceptance. The Editorial Board requests that regular manuscripts con- form to the requirements set below; those manuscripts that do not conform will be returned to authors for correction before review. 1. Manuscripts. Manuscripts, including figures, should be submitted in quadruplicate, with the originals clearly marked. (Xerox copies of photographs are not acceptable for review pur- poses.) The submission letter accompanying the manuscript should include a telephone number, a FAX number, and (if possible) an E-mail address for the corresponding author. The original manu- script must be typed in no smaller than 12 pitch or 10 point, using double spacing (including figure legends, footnotes, bibliography, etc.) on one side of 16- or 20-lb. bond paper, 8 by 1 1 inches. Please, no right justification. Manuscripts should be proofread carefully and errors corrected legibly in black ink. Pages should be numbered consecutively. Margins on all sides should be at least 1 inch (2.5 cm). Manuscripts should conform to the Council of Biology Editors Style Manual. 5th Edition (Council of Biology Editors, 1983) and to American spelling. Unusual abbreviations should be kept to a minimum and should be spelled out on first reference as well as defined in a footnote on the title page. Manuscripts should be divided into the following components: Title page. Abstract (of no more than 200 words), Introduction, Materials and Methods, Results, Discussion, Acknowledgments, Literature Cited, Tables, and Figure Legends. In addition, authors should supply a list of words and phrases under which the article should be indexed. 2. Title page. The title page consists of a condensed title or running head of no more than 35 letters and spaces, the manuscript title, authors' names and appropriate addresses, and footnotes listing present addresses, acknowledgments or contribution num- bers, and explanation of unusual abbreviations. 3. Figures. The dimensions of the printed page. 7 by 9 inches, should be kept in mind in preparing figures for publication. We recommend that figures be about 1 times the linear dimensions of the final printing desired, and that the ratio of the largest to the smallest letter or number and of the thickest to the thinnest line not exceed 1:1.5. Explanatory matter generally should be included in legends, although axes should always be identified on the illustra- tion itself. Figures should be prepared for reproduction as either line cuts or halftones. Figures to be reproduced as line cuts should be unmounted glossy photographic reproductions or drawn in black ink on white paper, good-quality tracing cloth or plastic, or blue-lined coordinate paper. Those to be reproduced as halftones should be mounted on board, with both designating numbers or letters and scale bars affixed directly to the figures. All figures should be numbered in consecutive order, with no distinction between text and plate figures and cited, in order, in the text. The author's name and an arrow indicating orientation should appear on the reverse side of all figures. Color: The Biological Bulletin will publish color figures and plates, but must bill authors for the actual additional cost of printing in color. The process is expensive, so authors with more than one color image should consistent with editorial concerns, especially citation of figures in order combine them into a single plate to reduce the expense. On request, when supplied with a copy of a color illustration, the editorial staff will provide a pre-publi- cation estimate of the printing cost. 4. Tables, footnotes, figure legends, etc. Authors should follow the style in a recent issue of The Biological Bulletin in preparing table headings, figure legends, and the like. Because of the high cost of setting tabular material in type, authors are asked to limit such material as much as possible. Tables, with their headings and footnotes, should be typed on separate sheets, num- bered with consecutive Roman numerals, and placed after the Literature Cited. Figure legends should contain enough informa- tion to make the figure intelligible separate from the text. Legends should be typed double spaced, with consecutive Arabic numbers, on a separate sheet at the end of the paper. Footnotes should be limited to authors' current addresses, acknowledgments or contri- bution numbers, and explanation of unusual abbreviations. All such footnotes should appear on the title page. Footnotes are not normally permitted in the body of the text. 5. Literature cited. In the text, literature should be cited by the Harvard system, with papers by more than two authors cited as Jones et al.. 1980. Personal communications and material in prep- aration or in press should be cited in the text only, with author's initials and institutions, unless the material has been formally accepted and a volume number can be supplied. The list of references following the text should be headed Literature Cited, and must be typed double spaced on separate pages, conforming in punctuation and arrangement to the style of recent issues of The Biological Bulletin. Citations should include complete titles and inclusive pagination. Journal abbreviations should normally follow those of the U. S. A. Standards Institute (USASII. as adopted by BIOLOGICAL ABSTRACTS and CHEMICAL ABSTRACTS, with the minor differences set out below. The most generally useful list of bio- logical journal titles is that published each year by BIOLOGICAL ABSTRACTS (BIOSIS List of Serials; the most recent issue). Foreign authors, and others who are accustomed to using THE WORLD LIST OF SCIENTIFIC PERIODICALS, may find a booklet published by the Biological Council of the U.K. (obtainable from the Institute of Biology, 41 Queen's Gate, London. S.W.7. England, U.K.) useful, since it sets out the WORLD LIST abbreviations for most biological journals with notes of the USASI abbreviations where these differ. CHEMICAL ABSTRACTS publishes quarterly supplements of addi- tional abbreviations. The following points of reference style for THE BIOLOGICAL BULLETIN differ from USASI (or modified WORLD LIST) usage: A. Journal abbreviations, and book titles, all underlined (for italics) B. All components of abbreviations with initial capitals (not as European usage in WORLD LIST e.g.. J. Cell. Comp. Physiol. NOT J. cell. comp. Physiol. ) C. All abbreviated components must be followed by a period, whole word components must not (i.e.. J. Cancer Res.) D. Space between all components (e.g.. J. Cell. Comp. 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. Staude advised on analysis of video- tapes of U. caupo. We thank J. Marcus for help in printing photographs, and J. Hoffman and two anonymous reviewers for useful comments on the manuscript. Literature Cited Bhuud, M., and C. Cazaux. 1987. Description and identification of polychaete larvae; their implications in current biological problems. Oceania 13: 595-753. Emlet, R. B. 1990. Flow fields around ciliated larvae: the effects of natural and artificial tethers. Mar. Ecol. Prog. Ser. 63: 211-225. Emlet, R. B., and R. R. Strathmann. 1994. Functional consequences of simple cilia in the mitrariu of owcniids (an anomalous larva of an anomalous polychaete) and comparisons with other larvae. Pp. 143- 157 in Reproduction and Development oj Marine Invertebrates, W. H. Wilson, Jr., S. A. Strieker, and G. L. Shinn, eds. Johns Hopkins University Press, Baltimore. Gould, M. 1967. Echiund worms: Urechis. Pp. 163-171 in Methods in Developmental Biology. F. H. Wilt and N. Wessels. eds. Crowell, New York. Hatschek. B. 1880. Oner die Entwicklungsgeschichte von Echiurus und die systematische Stellung der Echiuridae. Arbeiten Zool. lust. Wien 3: 45-78, plates 1-6. Hermans, C. O. 1964. The Reproductive and Developmental Biology of lite Opheliid Polychaete, Armandia brevis. M. S. Thesis, University of Washington. 131 pp Hermans, C. O. 1978. Metamorphosis in the opheliid polychaete Ar- mandia brevis. Pp. 1 13-126 in Settlement and Metamorphosis of Ma- rine Invertebrate Larvae. F.-S. Chia and M. E. R. Rice. eds. Elsevier, New York. Hoegh-Guldberg, O., and D. T. Manahan. 1995. Coulometric mea- surement of oxygen consumption during development of marine inver- tebrate embryos and larvae. / E\p. Biol. 198: 19-30. Jagersten, G. 1972. Evolution of the Metazoan Life Cycle. Academic Press. New York. 282 pp. McEdward, L. R. 1984. Morphometric and metabolic analyses of the growth and form of an echmopluteus. J. Exp. Mar. Biol. Ecol. 82: 259-287. McEdward, L. R., and R. R. Strathmann. 1987. The body plan of the cyphonautes larva of bryo/.oans prevents high clearance rates: compar- isons with the pluteus and a growth model. Biol. Bull. 172: 30-45. McHugh. I). 1997. Molecular evidence that echiurans and pogono- phorans are derived annelids. Proc. Nail. Acud. Sci. USA 94: 8006- 8009. McHugh, I)., and G. VV. Rouse. 1998. Life history evolution of marine invertebrates: new views from phylogenetic systematics. Trends Ecol. Evol. 13: 1X2-1X0. Newby, W. W. 1940. The Embryology of the Ecliiuroid Worm Urechis caupo. American Philosophical Society. Philadelphia. 219 pp. Nielsen. C. 1995. Animal Evolution. Oxford University Press, Oxford. 467 pp. Nielsen, C. 1998. Origin and evolution of animal life cycles. Biol. Rev. 73: 125-155. Phillips. N. E., and B. Fernet. 1996. Capture of large particles by suspension-feeding scaleworm larvae (Polychaeta: Polynoidae). Biol. Bull. 191: 199-208. Richter, G., and G. Thorson. 1975. Pelagische Prosobranchier-Larven des Golfes von Neapel. Ophelia 13: 109-185. Rouse, G. W., and K. Fauchald. 1997. Cladistics and polychaetes. Zool. Scr. 26: 139-204. Salensky, W. 1876. Uber die Metamorphose des Echiurus. Morpholo- gisches Jahrhiich 2: 319-327. Sleigh, M. A. 1984. The integrated activity of cilia: function and coor- dination. J. Proto-ool. 31: 16-21. Strathmann, R. R. 1987. Larval feeding. Pp. 465-550 in Reproduction of Marine Invertebrates. Vol. 9. General Aspects: Seeking Uniiv in Diversity, A. C. Giese, J. S. Pearse, and V. B. Pearse, eds. Blackwell, Palo Alto, CA. Strathmann, R. R. 1993. Hypotheses on the origins of marine larvae. Ainni. Rev. Ecol. Syst. 24: 89-1 17. Strathmann, R. R., and D. J. Eernisse. 1994. What molecular phylog- enies (ell us about the evolution of larval forms. Am. Zool. 34: 502- 512. Strathmann. R. R., and E. Leise. 1979. On feeding mechanisms and clearance rates of molluscan veligers. Biol. Bull. 157: 524-535. Strathmann, R. R., T. L. Jahn, and J. R. C. Fonseca. 1972. Suspen- sion feeding by marine invertebrate larvae: clearance of particles by ciliated bands of a rotifer, pluteus. and trochophore. Biol. Bull. 142: 505-519. Strathmann. R. R., L. Fenaux, A. T. Scwell, and M. F. Strathmann. 1993. Abundance of food affects relative size of larval and postlarval structures of a molluscan veliger. Biol. Bull. 185: 232-239. Suer, A. 1,. 1982. Larval Settlement, Growth, and Reproduction of the Marine Ecluuran Urechis caupo. Ph.D. Thesis. University of Califor- nia. Davis. 198 pp. Reference: Biul. Bull. 197: 26-39. (August I9Q9| Bioluminescence in the Deep-Sea Cirrate Octopod Stauroteuthis syrtensis Verrill (Mollusca: Cephalopoda) SONKE JOHNSEN 1 , ELIZABETH J. BALSER 2 , ERIN C. FISHER 1 , AND EDITH A. WIDDER 1 ' Marine Science Division, Harhor Branch Oceanographic Institution. Ft. Pierce. Florida: and 2 Department of Biology, Illinois Wesleyan University, Bloomington, Illinois Abstract. 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. This is Harbor Branch Contribution No. 1287 and Contri- bution No. 479 of the Smithsonian Station at Fort Pierce, Florida. Literature Cited Aldred, R. G., M. Nixon, and J. Z. Young. 1978. The blind octopus Cinotlnniimi. Nature 275: 547-549. Aldred, R. G., M. Nixon, and J. Z. Young. 1982. Possible light organs in tinned octopods. J. Mo/luscan Stud. 48: 100-101. Aldred, R. G., M. Nixon, and J. Z. Young. 1983. Cirrothauma murrayi Chun, a tinned octopod. Philos. Trans. R. Soc. Land. B 301: 1-54. Aldred, R. G., M. Nixon, and J. Z. Young. 1984. Ganglia not light organs in the suckers of octopods. J. Molluscan Snul. 50: 67-69. Buck, J. 1978. Functions and evolutions of bioluminescence. Pp. 419- 460 in Bioluminescence in Action, P. J. Herring, ed. Academic Press, London. Budelmann, B. U., R. Schipp, and S. Boletzky. 1997. Cephalopoda. Pp. 119-414 in Microscopic Anatomy of Invertebrates Vol. 6A, F. W. Harrison and A. J. Kohn, eds. Wiley-Liss Publications, New York. Chun, C. 1910. Die Cephalopoden. Wiss. Ergebn. dl. Thiefsee-Exped. "Valdn-ia" 18: 1-552. Chun. C. 1913. Cephalopoda. Pp. 1-21 in Report on the Scientific Results of the "Michael Sars " North Atlantic Deep-Sea Expedition 1910 Vol. III. Part I. J. Murray and J. Hjort. eds. The Trustees of the Bergen Museum. Bergen. Denton, E. J. 1990. Light and vision at depths greater than 200 meters. Pp. 127-148 in Light ami Life in the Sea. P. J. Herring. A. K. Campbell. M. Whittield. and L. Maddock, eds. Cambridge University Press, New York. Frank, T. M., and J. F. Case. 1988. Visual spectral sensitivities of bioluminescent deep-sea crustaceans. Bio/. Bull. 175: 261-273. Hamner, W. M. 1990. Design developments in the pkmktonkreisel, a plankton aquarium for ships at sea. / Plankton Res. 12: 397-402. Hanlon, R. T., and ,1. B. Messenger. 1996. Cephalopod Behaviour. Cambridge University Press, Cambridge. Harvey, E. N. 1952. Bioluminescence. Academic Press, New York. Herring, P. J. 1983. The spectral characteristics of luminous marine organisms. Pruc. R. Soc. Loud. B 220: 183-217. Herring, P. .1. 1988. Luminescent organs. Pp. 449-489 in The Molluxca Vol. II. E. R Tru<;man and M. R. Clarke, eds. Academic Press. New York. Herring, P. J., P. N. Dilly. and C. Cope. 1987. The morphology of the bioluminescent tissue of the cephalopod Japetella diaphana (Cepha- lopoda: Bolitaemdael. / Zoo/. Lund. 212: 245-254. Herring, P. J., P. N. Dilly, and C. Cope. 1992. Different types of photophore in the oceanic squids, Octopoteuthis and Tuningiti (Cepha- lopoda: Octopoteuthidae). J. Zoo/. Loud. 227: 479-491. Herring, P. J., P. N. Dilly, and C. Cope. 1994. The bioluminescent BIOLUMINESCENCE IN A DEEP-SEA OCTOPOD 39 organs of the deep-sea cephalopod Vampyroteuthis infemalis (Cepha- lopoda: Vampyromorpha). J. Zool. Land. 233: 45-55. Jerlov, N. G. 1976. Marine Optics. Elsevier Scientific Publishing, New York. Johnsen, S., E. J. Balser, and E. A. Widder. 1999. Light-emitting suckers in an octopus. Nature 398: 1 13-1 14. Johnston, I. A., and P. J. Herring. 1985. The transformation of muscle into bioluminescent tissue in the fish Benlhalbella infans Zugmayer. Proc. R. Soc. Land. B 225: 213-218. Kier, W. M., and A. M. Smith. 1990. The morphology and mechanics of octopus suckers. Bio/. Bull. 178: 126-136. Kirk. J. T. O. 1983. Light and Photosynthesis in Aquatic Ecosystems. Cambridge University Press, Cambridge. Mertens, L. E. 1970. In-water Photography: Theory and Practice. Wiley Interscience, New York. Morin, J. G., A. Harrington, K. Ncalson, N. Krieger, T. O. Baldwin, and J. W. Hastings. 1975. Light for all reasons: versatility in the behavioral repertoire of the flashlight fish. Science 190: 74-76. Nixon, M., and P. N. Dilly. 1977. Sucker surfaces and prey capture. Symp. Zool. Soc. Land. 38: 447-5 1 1 . Packard, A. 1961. Sucker display of Octopus vulgaris. Nature 190: 736-737. Partridge, J. C., S. N. Archer, and J. Van Oostrum. 1992. Single and multiple visual pigments in deep-sea fishes. J. Mar. Bio/. Assoc. U.K. 72: 113-130. Pereyra, W. T. 1965. New records and observations on the flapjack devilfish Opistoteuthis cali/orniana Berry. Pac. Sci. 19: 427-441. Robison, B. H., and R. E. Young. 1981. Bioluminescence in pelagic octopods. Pac. Sci. 35: 39-44. Roper, C. F. E., and W. L. Brundage. 1972. Citrate octopods with associated deep-sea organisms: new biological data based on deep benthic photographs (Cephalopoda). Smithson. Cnntrib. Zool. 121: 1-45. Vecchione, M. 1987. A multispecies aggregation of curate octopods trawled from north of the Bahamas. Bull. Mar. Sci. 40: 78-84. Vecchione, M., and R. E. Young. 1997. Aspects of the functional morphology of cirrate octopods: locomotion and feeding. Vie Mi/ic'ii 47: 101 -1 10 Villaneuva, R.. and A. Guerra. 1991. Food and prey detection in two deep-sea cephalopods: Opistoteuthis aga.isizi and O. vossi. Bull. Mar. Sci. 49: 288-299. Villaneuva, R., M. Segonzac, and A. Guerra. 1997. Locomotion modes of deep-sea cirrate octopods (Cephalopoda) based on observations from video recordings on the Mid-Atlantic Ridge. Mar. Bio/. 129: 1 13-122. Voight, J. R. 1997. Cladistic analysis of the octopods based on anatom- ical characters. J. Molluscan Stud. 63: 311-325. Voss, G. L. 1967. The biology and bathymetric distribution of deep-sea cephalopods. Stud. Trop. Oceanogr. 5: 51 1-535. Voss, G. L. 1988. The hiogeography of the deep-sea octopoda. Malaco- logia 29: 295-307. Voss, G. L., and W. G. Pearcy. 1990. Deep-water octopods (Mollusca: Cephalopoda) of the northeastern Pacific. Proc. Calif. Acad. Sci. 47: 47-94. Widder, E. A. 1999. Bioluminescence. Pp. 555-581 in Adaptive Mech- anisms in the Ecology of Vision. S. N. Archer, M. B. A. Djamgoz. E. Loew, J. C. Partridge, and S. Vallerga, eds. Kluwer Academic Pub- lishers. Dordrecht, The Netherlands. Widder, E. A., M. I. Latz, and J. F. Case. 1983. 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. Oxford University Press, New York. Dubas, F., R. T. Hanlon, G. P. Ferguson, and H. M. Pinsker. 1986. Localization and stimulation of chromatophore motoneurons in the brain of the squid. Lolliguncula brevis. J. E.\p. Biol. 121: 1-25. Flamarique, I. N., and C. A. Hawryshyn. 1997. Is the use of under- water polarized light by fish restricted to crepuscular time periods? Vision Res. 37: 975-989. Gilbert, D. L., W. J. Adelman, and J. M. Arnold, eds. 1990. Squid as Experimental Animals. Plenum Press, New York. Griswold, C. A., and J. Prezioso. 1981. In situ observations on repro- ductive behavior of the long-finned squid, Loligo pealei. Fish. Bull. 78: 945-947. Hanlon. R. T. 1982. The functional organization of chromatophores and iridescent cells in the body patterning of Loligo plei (Cephalopoda: Myopsida). Malacologia 23: 89-1 19. Hanlon, R. T. 1988. Behavioral and body patterning characters useful in taxonomy of field identification of cephalopods. Malacologia 29: 247- 264. Hanlon, R. T. 1996. Evolutionary games that squids play: fighting, courting, sneaking, and mating behaviors used for sexual selection in Loligo pealei. Biol. Bull. 191: 309-310. Hanlon. R. T. 1998. Mating systems and sexual selection in the squid Loligo: How might commercial fishing on spawning squids affect them? Calif. Coop. Oceanic Fish Invest. Rep. 39: 92-101. Hanlon, R. T., and J. B. Messenger. 1996. Cephalopod Behaviour. Cambridge University Press, Cambridge. Hanlon, R. T., R. F. Hixon, and W. H. Hulet. 1983. Survival, growth, and behavior of the loliginid squid, Loligo plei, Loligo pealei and Lolliguncula brevis (Mollusca: Cephalopoda) in closed sea water sys- tems. Biol. Bull. 165: 637-685. Hanlon, R. T., K. M. Cooper, B. U. Budelmann, and T. C. Pappas. 1990. Physiological color change in squid iridophores. I. Behavior, morphology and pharmacology in Lolliguncula brevis. Cell Tissue Res. 259: 3-14. Hanlon, R. T.. M. J. Smale, and W. H. H. Sauer. 1994. An ethogram of body patterning behavior in the squid Loligo vulgaris reynaudii on spawning grounds in South Africa. Biol. Bull. 187: 1-10. Hanlon, R. T., M. R. Maxwell, and N. Shashar. 1997. Behavioral dynamics that would lead to multiple paternity within egg capsules of the squid Loligo pealei. Biol. Bull. 193: 212-214. Horvath, G., and D. Varju. 1997. Polarization pattern of freshwater habitats recorded by video polanmetry in red, green and blue spectral ranges and its relevance for water detection by aquatic insects. / Exp. Biol. 200: 1155-1163. Kattawar, G. W. 1994. Polarization of light in the ocean. Pp. 203-225 in Ocean Optics. R. W. Spinrad, K. L. Carder, and M. J. Perry, eds. Oxford University Press, New York. McConathy, D. A., R. T. Hanlon, and R. F. Hixon. 1980. Chromato- phore arrangements of hatchlmg loliginid squids (Cephalopoda. Myop- sida). Malacologia 199: 279-288. McKiernan, D. J., and D. E. Pierce. 1995. Loligo squid fishery in Nantucket and Vineyard Sounds. Mass. Div. Mar. Fish. Publ. No. 17648-75-200: 1-200. NEFSC, Northeast Fisheries Science Center. 1995. Status of fishery resources off the northeastern United States for 1993. NOAA Tech. Mem. NMFS-F/NEC. Woods Hole, Massachusetts. 1-138. Packard, A., and F. G. Hochberg. 1977. Skin patterning in Octopus and other genera. Symp. Zool. Soc. Loiul. 38: 191-231. Porteiro, F. M., H. R. Martins, and R. T. Hanlon. 1990. Some obser- vations on the behaviour of adult squids. Loligo forbesi, in captivity. J. Mar. Biol. Assoc. UK 70: 459-472. Saidel, W. M., J. Y. Lettvin. and E. F. MacNichol. 1983. Processing of polarized light by squid photoreceptors. Nature 304: 534-536. Sauer, W. H. H., M. J. Roberts, M. R. Lipinski, M. J. Smale, R. T. Hanlon, D. M. Webber, and R. K. O'Dor. 1997. Choreography of the squid's "nuptial dance." Biol. Bull. 192: 203-207. Shashar, N., and R. T. Hanlon. 1997. Squids (Loligo pealei and Eu- prymna scolopes) can exhibit polarized light patterns produced by their skin. Biol. Bull. 193: 207-208. Shashar, N., P. S. Rutledge, and T. W. Cronin. 1996. Polarization vision in cuttlefish a concealed communication channel? J. Exp. Biol. 199: 2077-2084. 62 R T. HANLON ET AL. Shashar, N., R. T. Hanlon, and A. M. Petz. 1998. Polarization vision alopods. Vol. I, N. Voss, M. Vecchione. R. B. Toll, and helps detect transparent prey. Nature 393: 222-223. M. J. Sweeney, eds. Smithsonian Institution Press. Washington. Stevenson, J. A. 1934. On the behavior of the long-finned squid (Loligo DC pealii. (Lesueur)). Can. Field-Nat. 48: 4-7. Verrill, A. E. 1880-1881. The cephalopods of the northeastern coast of Summers, W. C. 1983. Loligo pealei. Pp. 1 15-142 in Cephalopod Life America. Part II. The smaller cephalopods. including the 'squids' and Cycles. Vol. I, P. R. Boyle, ed. Academic Press. London. the octopi, with other allied forms. Trans. Conn. Acad. Sci. 5: 259- Vecchione, M., T. F. Brakoniecki, Y. Natsukari, and R. T. Hanlon. 446. 1998. 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. Although these signals influence the initiation of behaviors and pairing success, there appear to be many different pathways leading to pairing success, and no single behavior and perhaps no single signal is necessary for pairing success. Courtship behaviors and chemical sig- naling may operate in a more complex and flexible manner than previously demonstrated. Acknowledgments The author thanks Dr. Anson H. Mines for his assistance, support, and review of this manuscript. This work was funded through a Smithsonian Postdoctoral Fellowship to PB, and an NSF grant OCE-971 1843 to AHH. Literature Cited Ache, B. W. 1975. Anlcnnular mediated host location by symbiotic crustaceans. Mar. Behav. Phyxiul. 3: 125-130. Ameyaw-Akumfi, C., and B. A. Hazlett. 1975. Sex recognition in the crayfish Procambarus clarkii. Science 190: 1225-1226. Atema, J. 1985. Chemoreception in the sea: adaptations of chemorecep- tors and behavior to aquatic stimulus conditions. Soc. Exp. Biol. Semin. Ser. 39: 387-423. Atema, J., and D. G. Engstrom. 1971. Sex pheromone in the lobster. Hoinarus americanus. Nature 232: 261-263. Bamber, S. D., and E. Naylor. 1997. Sites of release of putative sex pheromone and sexual behaviour in female Carcinus maenas (Crusta- cea: Decapoda). Esttiarine. Coastal Shelf Set. 44: 195-202. Barren, L. C., and B. A. Hazlelt. 1989. Directed currents: a hydrody- namic display in hermit crabs. Mar. Behav. Physiol. 15: 83-87. Bastock, M. 1967. Cotirtxliip: an Elhological Study. Aldiline Publishing Company, Chicago. Berry, P. F. 1970. Mating behavior, oviposition and fertilization in the spiny lobster Paniilirux linnuinix (Linnaeus). South African Associa- tion for Marine Biological Research Investigational Report no. 24, 16 PP Borowsky, B. 1984. Effects of receptive females' secretions on some male reproductive behaviors in the amphipod crustacean Microdeuto- pus gryllotalpa. Mar. Biol. 33: 266-271. Borowsky, B. 1985. Responses of the amphipod crustacean Gammarus palustix to waterbome secretions of conspecifics and congenerics. J. Chem. Ecol. 11: 1545-1552. Breithaupt, T., and J. Atema. 1993. Evidence for the use of urine signals in agonistic interactions of the American lobster. Biol. Bull. 185: 318. Breithaupt, T., B. Schmit/., and J. Tautz. 1995. Hydrodynamic orien- tation of crayfish (Prucumharux clarkii) to swimming fish prey. J. Comp. Physiol. A 177: 481-491. Bushmann, P., and J. Atema. 1996. Nephropore rosette glands of the 70 P. J. BUSHMANN lobster Homarus americanus: possible sources of urine pheromones. J. Crustiic. Biol. 16(2): 221-231. Bushmann, P.. and J. Atema. 1997. Shelter sharing and chemical courtship signals in the lobster. Homarus americanus. Can. J. Fish. Ac/iuit. Sci. 54(3): 647-654. Carlson, A. D., and J. Copt-land. 1978. Behavioral plasticity in the flash communication systems of fireflies. Am. Sci. 66(3): 340-346. Charlton, R. E., and R. T. Card(943e. 1990. Behavioral interactions in the courtship of Lymantna dispur (Lepidoptera: Lymantnidae). Ann. Entinnol. SIM Am. 83(1): 89-96. Christy, J. H.. and M. Salmon. 1991. Comparative studies of reproduc- tive behavior in mantis shrimps and fiddler crabs. Am. Zool. 31: 329-337. Churchill, K. P., Jr. 1921. Life history of the blue crab. Bull. U.S. Bur. Fish. 36: 95-128. Cowan. I). F. 1991. The role of olfaction in courtship behavior of the American lobster Homarus americanus. Biol. Bull 181: 402-407. Dejean, A. 1987. Behavioral plasticity of hunting workers of Serras- truma serru/a (Hymenoptera: Formicidae. Myrmicinae) presented with different arthropods. Sociobiology 13(3): 191-208. Devine, D. V., and J. Atema. 1982. Function of chemoreceptor organs in spatial orientation of the lobster. Homarus americanus: differences and overlap. Biol. Bull. 163: 144-153. Drach, P. 1939. Mue et cycle d'intermue chez les crustaces decapodes. ,4iiii. Inst. Oceanogr. 19: 103-391. Dunham, P. J. 1978. Sex pheromones in Crustacea. Biol. Rev. Camb. Plains. Soc. 53: 555-583. Dunham, P. J. 1988. Pheromones and behaviour in Crustacea. Pp. 375-392 in Endocrinology of Selected Invertebrate Types, H Laufer and G. H. Downer, eds. Liss, New York. Eales, A. J. 1974. Sex pheromone in the shore crab Carcinus maenas. and the site of its release from females. Mar. Behav. Physiol. 2: 345-355. Elner, R. W., and P. G. Beninger. 1995. Multiple reproductive strate- gies in snow crab. Cliionoecetes opilio: Physiological pathways and behavioral plasticity. / Exp. Mar. Biol. Ecol. 193: 93-1 12. Fuzessery, Z. M., and J. J. Childress. 1975. Comparative chemosen- sitivity to amino acids and their role in the feeding activity of bathy- pelagic and littoral crustaceans. Biol. Bull. 149: 522-538. Gibhs III, D. S. 1996. Field and laboratory evidence of pheromone mediated mating behavior in the blue crab, Callinectes sapidus. M.S. thesis. The College of William and Mary. Gleeson, R. A. 1980. Pheromone communication in the reproductive behavior of the blue crab, Callinecles sapidiis. Mar. Behav. Pliysiol. 7: 119-134. Gleeson, R. A. 1982. Morphological and behavioral identification of the sensory structures mediating pheromone reception in the blue crab, Callinectes sapidus. Biol. Bull. 163: 162-171. Gleeson, R. A. 1991. Intrinsic factors mediating pheromone communi- cation in the crab Callinecles sapidus. Pp. 1 7-32 in Crustacean Sexual Biology, R. T. Baur and J. N. Martin, eds. Columbia University Press, New York. Gleeson, R. A., M. A. Adams, and A. B. Smith HI. 1984. Character- ization of a sex pheromone in the blue crab, Callinectes sapidus: Crustecdysone studies. J. Chem. Ecol. 10: 913-921. Hartnoll, R. G. 1969. Mating in the Brachyura. Crustaceana 16: 161- 181. Hay, VV. P. 1905. The life history of the blue crab (Callinecles sapidus). Rep. U.S. Bur. Fish. 1904: 395-413. Hazlett, B. A. 1982. Chemical induction of visual orientation in the hermit crab Clibanarius vittatus. Anim. Behav. 30(4): 1259-1260. Hazlett, B. A. 1995. Behavioral plasticity in Crustacea: why not more? / E.\p. Mar. Biol. Ecol. 193: 57-66. Hughes, M. 1996. The function of concurrent signals: visual and chem- ical communication in snapping shrimp. Anim. Behav. 52: 247-257. Jaccard, J. 1983. Statistics for the Behavioral Sciences. Wadsworth, Belmont, CA. Jachowski, R. L. 1974. Agonistic behavior of the blue crab. Callinectes sapidus Rathbun. Behaviour 50: 232-253. Jivoff, P. 1997a. The relative roles of predation and sperm competition on the duration of the post-copulatory association between the sexes in the blue crab. Callinectes sapidus. Behav. Ecol. Sociobiol. 40: 175- 185. Jivoff, P. 1997b. Sexual competition among male blue crab, Callinectes sapidus. Biol. Bull. 193: 368-380. Johnson. P. T. 1980. Histology of the Blue Crab. Callinectes sapidus. A Model for the Decapoda. Praeger Publishers, New York. Kaissling, K. E. 1979. Recognition of pheromones by moths, especially in saturnids and Bombyx mori. Pp. 43-56 in Chemical Ecolog\: Odour Communication in Animals. F. J. Ritter. ed. Elsevier. Amster- dam. Kamiguchi. Y. 1972. A histological study of the "sternal gland" in the female freshwater prawn, Palaemon paucidens, a possible site of origin of the sex pheromone. J. Fac. Sci. Hokkaido Univ. Ser. VI, Zool. 18(3): 356-365. Kaplan, L. J., C. Lowrance, J. Basil, and J. Atema. 1993. The role of chemical and visual cues in agonistic interactions of the American lobster. Biol. Bull. 185: 320-321. Linn. C. E., L. B. Bjostad, J. W. Du, and W. L. Roelofs. 1984. Redundancy in a chemical signal: behavioral responses of male Trichoplusia ni to a 6-component sex pheromone blend. J. Chem. Ecol. 10(11): 1635-1658. Lott, D. F. 1991. Intraspecific Variation in the Social Systems of Wild Vertebrates. Cambridge University Press, Cambridge. Mantel, L. H., and L. L. Farmer. 1983. Osmotic and ionic regulation. Pp. 53-161 in The Biology of Crustacea Vol. 5, L. H. Mantel, ed. Academic Press, New York. Ra'anan, Z., and D. Cohen. 1984. The effect of group interactions on the development of size distribution in Macrobrachium rosenbergii (de Man) juvenile populations. Biol. Bull. 166: 22-31. Rand, A. S., M. J. Ryan, and W. Wilczynski. 1992. Signal redundancy and receiver permissiveness in acoustic mate recognition by the Tun- gara frog, Physa/aemus pustii/osus. Am. Zool. 32: 81-90. Reynolds, J. D. 1993. Should attractive individuals court more? Theory and test. Am. Nat. 14: 914-927. Ryan, E. P. 1966. Pheromone: evidence in a decapod crustacean. Science 151: 340-341 Ryan, M. J. 1990. Sexual selection, sensory systems and sensory ex- ploitation. O.v/: Sun 1 . Evoi Biol. 7: 157-195. Salmon, M., and K. W. Horch. 1972. Acoustic signalling and detection by semi-terrestrial crabs of the family Ocypodidae. Pp 60-96 in Be- havior of Marine Animals. Current Perspectives in Research. Vol. I, Invertebrates. H. E. Winn and B. L. Olla. eds. Plenum Press, New York. Seifert, P. 1982. Studies on the sex pheromone of the shore crab. Carcinus maenas, with special regard to ecdysone excretion. Ophelia 12(2): 147-158. Siegel, S., and N. J. Castellan Jr. 1988. Nonparametric Statistics for the Behavioral Sciences. 2nd ed. McGraw-Hill, New York. Snyder, M. J., C. Ameyaw-Akumfi, and E. S. Chang. 1993. Sex recognition and the role of urinary cues in the lobster, Homarus americanus. Mar. Behav. Physio/. 24: 101-116. Stauffer, H. P., and R. D. Semlitch. 1993. Effects of visual, chemical and tactile cues of fish on the behavioural responses of tadpoles. Anim. Behav. 46: 355-364. Talbot, P., and D. Deniers. 1993. Tegumental glands of Crustacea. Pp. CONCURRENT SIGNALS IN BLUE CRABS 71 151-191 in The Crustacean Integument. M. N. Horst and J. A. Free- man, eds. CRC Press, Boca Raton. Teytaud, A. R. 1971. The laboratory studies of sex recognition in the blue crab, Callinectes sapidus Rathbun. Sea Grant Technical Bulletin No. 156. University of Miami Sea Grant Program. 62 pp. Tinbergen, N. 1953. Social Behaviour in Animals with Special Refer- ence to Vertebrates. Chapman and Hall. London. Van den Hurk, R., and J. G. D. Lambert. 1983. Ovarian steroid gluconurides function as sex pheromones for female zebrafish, Brachy- danio rerio. Can. J. Zool. 61: 2381-2387. Van Engel, W. A. 1958. The blue crab and its fishery in Chesapeake Bay. Part I Reproduction, early development, growth and migration. Commer. Fish. Rev. 20: 6-17. Vetter, R. S., and T. C. Baker. 1983. Behavioral responses of male Heliothis virescens in a sustained-flight tunnel to combinations of seven compounds identified from female sex pheromone glands. / Chem. Ecol. 9: 747-759. Waas, J. R., and P. W. Colgan. 1992. Chemical cues associated with visually elaborate aggressive displays of three-spine sticklebacks. J. Chem. 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. K., I. A. N. Lucas, and J. R. Turner. 1996. Carbon budgets in temperate anthozoan-dinoflagellate symhioses. Mar. Biol. 126: 773- 783. Davy, S. K., J. R. Turner, and I. A. N. Lucas. 1997. The nature of temperate anthozoan-dinoflagellate symbioses. Proc. 8 th Int. Coral Reef Symp. 2: 1307-1312. Dingman. H. C. 1998. Environmental influence on algal symbiont pop- ulations in the sea anemone Ant/iopleiiru elegantissima. Master's the- sis. Western Washington University. 92 pp Fitt, W. K., R. L. Pardy, and M. M. Littler. 1982. Photosynthesis, respiration, and contribution to community productivity of the symbi- otic sea anemone Anthopleura elegantissima (Brandt, 1835). / Exp. Mar. Biol. Eco/. 61: 213-232. Jensen, S., and G. Muller-Parker. 1994. Inorganic nutrient fluxes in anemone-dominated ddepools. Pac. Sci. 48: 3243. Lowry, O. H., N. J. Rosebrough, H. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chan. 193: 265-275. McCloskey, L. R., T. G. Cove, and E. A. Verde. 1996. Symbiont expulsion from the anemone Anthopleiint elegantissima (Brandt) (Cni- dana; Anthozoa). J. E\p. Mai. Biol. Ecol. 195: 173-186. Mews, L. K., and D. C. Smith. 1982. The green hydra symbiosis. VI. What is the role of maltose transfer from alga to animal? Proc. R. Soc. Loiul. B 216: 347-413. Muscatine, L. 1967. Glycerol excretion by symbiotic algae from corals and Tridacna and its control by the host. Science 156: 516-519. Muscatine, L. 1971. Experiments on green algae coexistent with zoo- xanthellae in sea anemones. Pac. Sci. 25: 13-21. Muscatine, L.. L. R. McCloskey, and R. E. Marian. 1981. Estimating the daily contribution of carbon from zooxanthellae to coral animal respiration. Liuinoi. Oceanogr. 26: 601-61 1. Muscatine, L., P. G. Falkowski, J. W. Porter, and Z. Duhinsky. 1984. Fate of photosynthetic fixed carbon in light- and shade-adapted colo- nies of the symbiotic coral Stylophora pistilUitu. Proc. R Soc. Ltmd. B 222: 181-202. O'Brien, T. L. 1980. The symbiotic association between intracellular CARBON TRANSLOCATION IN ANEMONES 81 zoochlorellae (Chlorophyceae) and the coelenterate Anthopleura \uii- thoxrammica. J. E\p. Zoo/. 211: 343-355. O'Brien, T. L., and C. R. Wyttenbach. 1980. Some effects of temper- ature on the symbiotic association between zoochlorellae (Chloro- phyceae) and the sea anemone Anthopleura xanthogrammica. Trans. Am. Microse. Sue. 99(2): 221-225. Parsons, T. R., Y. Malta, and C. M. Lalli. 1984. A Manual of Chemical and Biological Methods for Seawaler Analysis. Pergamon Press. Ox- ford, England. Pp. 115-119, 140-148. Saunders, B. K. 1995. The effects of temperature and light on popula- tions of symbiotic algae in the sea anemone Anthopleura elegontissima. Master's thesis. Western Washington University. 52 pp. Saunders, B. K., and G. Muller-Parker. 1997. The effects of temper- ature and light on populations of two algae in the temperate sea anemone Anthopleura elegantissima (Brandt, 1835). J. Exp. Mar. 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. Gen. 4(4): 199-484. Bowman, P. D., M. du Bois, R. R. Shivers, and K. Dorovini-Zis. 1992. Endothelial tight junctions. Pages 305-320 in Tix/ir Junctions. M. Cereijido. ed. CRC Press, Ann Arbor. MI. Bretag, A. H. 1987. Muscle chloride channels. Physiol. Rev. 67(2): 618-724. Brown, I)., and R. Montesano. 1980. Membrane specialization in the rat epididymis. I. Rod-shaped intramemhrane particles in the apical (mitochondria-rich) cell. J. Cell Sci. 45: 187-198. Biillesbach, E. E.. C. Schwabe, and E. R. Lacy. 1997. Identification of a glycosylated relaxin-like molecule from the male Atlantic stingray, Dasyatis sabina. Biochemistry 36: 10735-10741 Byers, S., and R. Marc-Pelletier. 1992. Sertoli-sertoli cell tight junc- tions and blood-testis barrier. Pages 279-307 in Tight Junctions. M. Cereijido, ed. CRC Press, Ann Arbor. MI. Carter, M. J., and D. S. Parsons. 1971. The isoenzymes of carbonic anhydrase: Tissue subcellular distribution and function significance, with particular reference to the intestinal tract. J. Physio/. 215: 71-94. Claude, P. 1978. Morphological factors influencing transepithelial per- meability: A model for the resistance of the zonula occludens. / Meiuhr. B,,,l. 39: 219-232. Claude, P., and 1). A. Goodenough. 1973. Fracture faces of zonulae occludente from "tight" and "leaky" epithelia. J. Cell Biol. 58: 390- 400. Curci, S., L. Debellis, and E. Frontier. 1987. Evidence for rheogenic sodium bicarbonate cotransport in the basolateral membrane of oxyntic cells of frog gastric fundus. Pflugerx Arch. 408: 497-504. Daniel, F. J. 1934. Urogenital system. Pages 300-303 in The Elasmo- hnuich Fishes. F, J. Daniel, ed. University of California, Berkeley. CA. Enns, T. 1967. Facilitation by carbonic anhydrase of carbon dioxide transport. Science 155: 44 \1 . Essig, M., C. Schoenfeld, R. D-Eletto, R. Amelar, L. Dubin, B. G. Steinetz, M. O'Bryne, and G. Weiss. 1982. Relaxm in human seminal plasma. Ann. NY Ac ad. Sci. 380: 224-230. Feldman, G. M., S. F. Berman, and R. L. Stephenson. 1988. Bicar- bonate secretion in the distal colon in vitro: A measurement technique. Am. J. Phyxiol. 254: C383-390. Fitz, J. G., M. Persico, and B. F. Scharschmidt. 1989. Electrophysio- logical evidence for sodium-coupled bicarbonate transport in cultured rat hepatocytes. Am. ./. Physiol. 256: G49I-500, Fritsche, C., J. G. Kleinman, J. L. W. Bain, R. R. Heinen, and D. A. Rilt'V. 1991a. Carbonic anhydrase in turtle bladder mitochondnal- rich lurmnal and suhluniinul cells. Am. J. Phvsio/. 260: F43 1-442. STINGRAY ALKALINE GLAND 93 Fritsche, C., J. G. Kleinman, J. L. W. Bain, R. R. Heinen, and D. A. Riley. 199lb. Carbonic anhdrase and proton secretion in turtle blad- der mitochondrial-nch cells. Am. J. Plivxiol. 260: F443-458. Gray, M. A., A. Harris, L. Coleman, J. R. Greenwell, and B. E. Argent. 1989. Two types of chloride channel on duct cells cultured from human fetal pancreas. Am. J. Physiol. 257: C240-2.M. Hansson, H. P. J. 1967. Histochemical demonstration of carbonic an- hydrase activity. Histocliemie 11: 112-128. Hansson, H. P. J. 1968. Histochemical demonstration of carbonic an- hydrase activity in some epitheliu noted for active transport. Ada Phy.siol. Scaml. 73: 427-434. Hannan, 1). 199(1. Lipofuscin and ceriod formation: The cellular recy- cling system. Adv. Exper. Med. Biol. 266: 3-18. Ito, S., and M. J. Karnovsky. 1968. Formaldehyde-glutaraldehyde fix- atives containing trinitro compounds. J. Cell Biol. 39: 168a. Kunzelmann, K., L. Gerlach, U. Frobe, and R. Greger. 1991. Bicar- bonate permeability of epithelial chloride channels. PflugerArch. 417: 616-621. Lacy, E. R. 1983a. Carbonic anhydrase localization in elastnobranch rectal gland. J. Exp. Zool. 226: 163-169. Lacy, E. R. 1983b. Histochemical and biochemical studies of carbonic anhydrase activity in the opercular epithelium of the euryhaline teleost. r'uiidu/ii.s heteroclitus. Am. J. Aiuit. 166: 19-39. Maren, T. H., J. A. Rawls. J. W. Burger, and A. C. Myers. 1963. The alkaline (Marshall's) gland of the skate. Comp. Biochem. Physiol. 10: 1-16. Maren, T. H. 1980a. Current status of membrane-bound carbonic anhy- drase. Ann. NY Acad. Sci. 341: 246-258. Maren, T. H. 1980b. Kinetics, equilibrium and inhibition in the Hansson histochemical procedure for carbonic anhydrase: A validation of the method. Hixlochem. J. 12: 183-190. Masur, S. K. 1984. Electron microscopy of the alkaline gland epithelium of the little skate. Raja erinacea. Bull. Mt. Desert Is. Hint. Luh. 24: 96-97. Maynard, J. R., and J. E. Coleman. 1971. Elasmobranch carbonic anhydrase. Purification and properties of the enzyme from two species of shark. J. Bin/. Client. 246: 4455-4464. Melvin, J. E., and R. J. Turner. 1992. Cl fluxes related to fluid secretion by the rat parotid: Involvement of Cr/HCO 3 ~ exchanger. Am. J. Physiol. 262: G393-398. Mitchinson, M. J., K. P. Sherman, and A. M. Stainer-Smith. 1975. Brown patches in the epididymis. J. Pathoi 115: 57-62. Nellens, H. N., R. A. Frizzell, and S. G. Schultz. 1975. Effect of acetazolamide on sodium and chloride transport by in vitro rabbit ileum. Am. J. Phyxiol. 228: 1808-1814. Nicander, L. 1958. Studies on the regional histology and cytochemistry of the ductus epididymis in stallions, rams, and bulls. ACTA Morplt. Neerl. Scuiul. 1: 337. Pappenheimer, A. M.. and J. Victor. 1954. "Ceroid" pigment in human tissues. Am. ,/. Putliol. 22: 395-413. Pirani. D., L. A. Evans, I). I. Cook, and J. A. Young. 1987. Intracel- lular pH in the rat mandihular salivary gland: The role of Na-H and Cl-HCO, antipons in secretion. Pftugers Arch. 408: 178-184. Reed, R., G. McMillan, S. Hartroft, and E. Porta. 1965. Progress of medical science: Pathology and bacteriology. Am. J. Med. Sci. 250: 1 16-137. Sevier, A. C., and B. L. Munger. 1965. A silver method for paraffin sections of neural tissue. J. Neuropathol. Exp. Neural. 24: 130-135. Smith, H. W. 1929. The composition of the body fluids of elasmo- branchs. J. Biol. Cliem. 81: 407-419. Smith, P. L. 1981. Electrolyte transport by the alkaline gland of the little skate, Raja erinacea. Mechanism of luminal alkalinization. Bull. Mt. Desert Is. Biol. Lah. 21: 80-83. Smith. P. L. 1985. Electrolyte transport by alkaline gland of little skate Raja erinacea. Am. ./. Phyxiol. 248: 346-352. Stewart, C. P., J. M. Winterhages, K. Heintze, and K. U. Petersen. 1989. Electrogenic bicarbonate secretion by guinea pig gallbladder epithelium: Apical membrane exit. Am. J. Physiol. 256: C736-749. Sugai, N., and S. Ito. 1980. Carbonic anhydrase, ultrastructural local- ization in the mouse gastric mucosa and improvements in the tech- nique. J. Histochcm, Cytoclwm. 28(61: 511-525. Tabcharani, J. A., T. J. Jensen, J. R. Riordan, and J. W. Hanrahan. 1989. Bicarbonate permeability of the outward rectifying anion chan- nel. ./. Mcmhr. Biol. 112: 104-122. Weiner, I. M., and G. H. Mudge. 1985. Pp. 887-907 in The Pharma- cological Basis of Therapeutics. 7 th Edition. MacMillan, New York. Weiderholt, M., T. J. Jentsch, and S. K. Kellar. 1985. Electrogenic sodium-bicarbonate symport in cultured corneal endothelial cells. PflugerArch. 405 (Suppl. 1): SI67-171. Weiss, G. 1989. Relaxin in the male. Biol. Repmd. 40: 197-200. Wistrand, P. J. 1984. Properties of membrane-bound carbonic anhy- drase. Ann. NY Acad Sci. 429: 1 95-206. Yoshitomi. K., B-Ch. Burckhardt, and E. Fromter. 1985. Rheogenic sodium-bicarbonate cotransport in the peritubular cell membrane of rat proximal tubule. Pflugers Arch. 405: 360-366. Reference: Biol. Bull. 197: 94-103. (August 1999) Waterborne and Surface-Associated Carbohydrates as Settlement Cues for Larvae of the Specialist Marine Herbivore Alderia modesta PATRICK J. 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 University of New Zealand on the processing of electrosensory information in the midbrain of the skate; and studies by Dr. James Zheng from the Robert Wood Johnson Medical School on the cellular mechanisms underlying the formation of nerve connections. Cell biologists included Dr. Mark Alliegro of Louisiana State University, who studied cells from sea urchins and other organisms to learn how cells differentiate, and Dr. John Eriksson of the Turku Center for Biotechnology in Finland, who studied the mitotic protein phosphatases in surf clam eggs. Fourteen other scientists whose research focused on topics ranging from global climate change to sensory physiology rounded out the group of 1998 fellows. Improvements Around Campus As always there is work to be done on the MBL's physical plant, all of it important, all of it requiring time. effort, and financial support. In 1998, renovations were made at the Loeb building and to the Neurobiology course laboratories. And the Lillie Auditorium got a new roof. I'm pleased to report that commitments are now in hand to install an air-conditioning system at the Library, and very soon the MBL will have a new emergency generator. One of the most the exciting changes to the MBL campus last summer was the creation of the new Robert W. Pierce Visitors Center, which shares 100 Water Street with the MBL and Satellite Clubs. This beautiful new facility, which is also home to the MBL Associates Gift Shop, was dedicated and opened in the summer of 1998. It has already introduced thousands of Woods Hole visitors to the Marine Biological Laboratory. MBL Trustees In 1998. the MBL Board of Trustees welcomed Dr. John E. Dowling as President of the Corporation. Dr. Dowling succeeded Dr. James D. Ebert, who retired after serving as President for seven years. Dr. Dowling is the Maria Moors Cabot Professor of Natural Science at Harvard University, as well as an MBL summer investigator and former MBL Trustee. His research focuses on the physiology of vision, especially the correlation between structure and function in the vertebrate retina. He also is interested in retinal development and uses the zebrafish as a model organism for these studies. Last year the MBL Board of Trustees elected Ronald P. O'Hanley, President of Dreyfus Institutional Investors in Boston, and Vincent J. Ryan, President, Chairman, and CEO of Schooner Capital Corporation, also of Boston, to membership in the Class of 2003. The Laboratory is most fortunate to welcome these dynamic and thoughtful individuals to help guide our progress over the next few years. Burton J. Lee, III, Laurie J. Landeau, Darcy Brisbane Kelley, and Jean Pierce were reappointed to the R6 Annual Report Board in November 1998 as members of the class of 2003. Directors Emeriti The Board of Trustees voted to name three former directors of the Marine Biological Laboratory "Directors Emeriti." James D. Ebert, Paul R. Gross, and Harlyn O. Halvorson were recognized for the contributions that each of these men made to the growth and strength of the Laboratory during their tenures as director. Each of these individuals has left a legacy of achievement that has earned the respect and gratitude of the MBL community. In closing, 1998 was an exciting time, and 1999 should be no less so. The Marine Biological Laboratory remains a wonderful gathering place for scientists and students from around the world. Anchored by a top-notch team of year-round investigators, enlivened by some of the best students anywhere, and stimulated by the summer influx of great researchers, the MBL continues to serve science in a unique and exciting way. John E. Burris Report of the Treasurer During 1998 the Marine Biological Laboratory continued a favorable trend in operations. This progress was due to healthy increases in five of the six areas of Operating Support. Government grants increased 9.6% and now represent 42.5% of the total support and revenues. Double digit increases in Private Contracts (38.3%), Fees for Conferences and Services ( 10.7%) and Miscellaneous Revenues (22.3% ) powered the year's success story. While there was an easing in the present value of Contributions this was predictable at the midpoint of our very successful Discovery Campaign. As already noted in the Report of the Director and CEO, the campaign is ahead of schedule. Focusing on the change in Unrestricted Net Assets, we enjoyed a three-year favorable trend. The change before nonoperating activity has improved from a deficit of $1 million in 1996, to a deficit of $753 thousand in 1997, to a deficit of only $256 thousand this year. This is particularly auspicious when one realizes these figures are after approximately $1.5 million in depreciation each year. While the Change in Net Assets before nonoperating activity was only half of 1997 results, it was still a robust $1.1 million. Total Investment Income and Earnings of only 820 thousand dollars were unsatisfactory when compared to the multi-million dollar returns in previous years. This was a result of the volatile markets and a revamping of our endowment management philosophy. As a result. Net Assets increased for the fourth year in a row, but the Return on Average Net Assets was only 1.1%. A review of the 1998 Balance Sheet demonstrates our continued strong liquidity and low and improving leverage. Property Plant and Equipment showed a slight decline (2.4%). but this is the smallest decline in the past four years as we are in the process of upgrading the physical plant. Plans are underway to expand our capital maintenance efforts and to build a new Environmental Sciences Building. Ultimately, this will make the Laboratory an even more attractive facility to conduct science. In summary, the Laboratory continues to demonstrate the ability to attract funds from the federal government, foundations and individuals. Our housing and conferences continue to generate surplus cash. Successful completion of the Discovery Campaign and a return to our history of very successful endowment performance will guarantee the financial strength of the Marine Biological Laboratory for the 21 st century. Mary B. Conrad R7 Financial Statements PrrcewaterhouseCoopers LIP One Post Offic e Square Boston MA 0_> 1 09 Telephone (hi 7) 478 5000 F.ii simile (111 7) 478 5900 REPORT OF INDEPENDENT ACCOUNTANTS To the Board of Trustees of Marine Biological Laboratory Woods Hole. Massachusetts In our opinion, the accompanying balance sheet of Marine Biological Laboratory (the "Laboratory") as of December 31, 1998 and the related statements of activities and cash flows for the year then ended present fairly, in all material respects, the financial position of the Laboratory as of December 31, 1998, and the changes in its net assets and its cash flows for the year then ended in conformity with generally accepted accounting principles. These financial statements are the responsibility of the Laboratory's management; our responsibility is to express an opinion on these financial statements based on our audit. We conducted our audit in accordance with generally accepted auditing standards. Those standards require that we plan and perform the audit to obtain reasonable assurance about whether the financial statements are free of material misstatement. An audit includes examining, on a test basis, evidence supporting the amounts and disclosures in the financial statements. An audit also includes assessing the accounting principles used and significant estimates made by management, as well as evaluating the overall financial statement presentation. We believe that our audit provides a reasonable basis for the opinion expressed above. Our audit was conducted for the purpose of forming an opinion on the basic financial statements taken as a whole. The supplemental schedule of functional expenses for the year ended December 31, 1998 is presented for the purpose of additional analysis and is not a required part of the basic financial statements. Such information has been subjected to the auditing procedures applied in the audit of the basic financial statements and, in our opinion, is fairly stated, in all material respects, in relation to the basic financial statements taken as a whole. April 9, 1999 R8 MARINE BIOLOGICAL LABORATORY BALANCE SHEETS December 31, 1998 (with comparative totals as of December 31. 1997) ASSETS Cash and cash equivalents Short-term investments, at market (Note C) Accounts receivable, net of allowance for doubtful accounts of $34,195 in 1998 and $36.782 in 1997 Current portion of pledges receivable (Note H) Receivables due for costs incurred on grants and contracts Other assets Total current assets Long-term investments, at market (Notes C and Di Pledges receivable, net of current portion (Note H) Plant assets, net (Notes B. E and F) Total long-term assets Total assets 1998 S 1,187,954 3.561,544 1,242,530 1 ,607.664 1,531,083 557.908 9.688.683 37.054.120 2.855.352 19.536.171 59,445.643 $69.134,326 1997 $ 560.801 4.408.046 1,221,781 2,219,056 1.157.165 560.269 10.127.118 35,614,151 2,238,826 20.026.580 57.879.557 $68.006.675 LIABILITIES AND NET ASSETS Current portion of long-term debt (Note E) Accounts payable and accrued expenses Deferred income and advances on contracts Total current liabilities Annuities and unitrusts payable Long-term debt, net of current portion (Note E) Advances on contracts Total long-term liabilities Total liabilities Commitments and contingencies (Notes F and H) 243,274 2,057,741 462.873 2.763.888 1.412,200 2.324.096 1.272.390 5.008.686 7.772,574 229,657 1.494,948 384.258 2.108.863 1.213.583 2,567.370 1.433.208 5.214.161 7,323,024 Net assets: Unrestricted Temporarily restricted Permanently restricted Total net assets (Note Bl Total liabilities and net assets 18,451.865 25.635.237 17.274.650 61.361.752 $69,134.326 18.729.311 25,596.656 16.357.684 60.683.651 $68.006,675 The accompanving notes are an integral part of the financial statements. R9 MARINE BIOLOGICAL LABORATORY STATEMENTS OF ACTIVITIES for the year ended December 31,1 998 (with comparative totals for the year ended December 31, 1997) Operating support and revenues: Government grants Private contracts Laboratory rental income Tuition Fees for conferences and services Contributions Investment income Miscellaneous revenue Present value adjustment to annuities Net assets released from restrictions Total operating support and revenues Expenses: Research Instruction Conferences and services Other programs (Note B) Total expenses Change in net assets before nonoperating activity Nonoperating revenue: Total investment income and earnings Less: investment earnings used tor operations Reinvested (utili/ed) investment earnings Total change in net assets Net assets, beginning of year Net assets, end of year Temporarily Permanently 1998 1997 Unrestricted Restricted Restricted Total Total $10,943,239 $ $ $10,943,239 $ 9.986.800 1.629,283 1.629,283 1.178,192 1.470.372 1.470.372 1.478.757 489,726 489.726 399.703 3.415,519 3,415,519 3.085,616 1.264,235 3,420,615 653.152 5,338,002 6.441.429 490,474 1,465,261 1.955.735 1 ,709,983 405,633 405.633 322,667 (68,849) (7.853) (76.702) (164.447) 4.100,624 (4,138,622) 37,998 24,209,105 678,405 683.297 25.570.807 24.438.700 12,666.746 12,666.746 11.031.914 4.433,789 4,433,789 4,144,508 1 ,999,433 1 ,999,433 1,487.705 5,365,530 5.365.530 5.440.808 24,465,498 24,465.498 22.104.935 (256,393) 678,405 683,297 1,105,309 2.333,765 27.353 558.683 233,669 819.705 4,869,035 (48.406) (1.198.507) (1.246.913) (1.056,211) (21,053) (639,824) 233.669 (427.208) 3,812,824 (277.446) 38,581 916,966 678.101 6,146,589 18.729,311 25,596,656 16,357,684 60.683.651 54,537.062 $18,451,865 $25.635 2^7 $17 274650 $61 361 75" 1 $60683 651 The accompanying notes are an integral part of the financial statements. RIO MARINE BIOLOGICAL LABORATORY STATEMENTS OF CASH FLOWS for the year ended December 31, 1998 (with comparative totals for the year ended December 31, 1997) Cash flows from operating activities: Change in net assets Adjustments to reconcile change in net assets to net cash provided by (used in) operating activities: Depreciation Unrealized (gain) loss on investments Realized (gain) loss on investments Present value adjustment to annuities and unitrusts payable Contributions restricted for long-term investment and annuities Provision for bad debt Provision for uncollectible pledges Change in certain balance sheet accounts: Accounts receivable Pledges receivable Grants and contracts receivable Other assets Accounts payable and accrued expenses Deferred income and advances on contracts Annuities and unitrusts payable Advances on contracts Net cash provided by operating activities Cash flows from investing activities: Purchase of property and equipment Proceeds from sale of investments Purchase of investments Net cash used in investing activities Cash flows from financing activities: Payments on annuities and unitrusts payable Receipt of permanently restricted gifts Annuity and unitrusts donations received Loan proceeds Payments on long-term debt Net cash provided by financing activities Net increase in cash and cash equivalents Cash and cash equivalents at beginning of year Cash and cash equivalents at end of year 1W8 $ 67S.10I 1.505.696 2,755.079 (2.805.560) 76.702 (682.817) 15,771 250.000 (36.520) (255.134) (373.918) 2.361 562,793 78.615 163.700 (160,818) 1.774.051 (1.015.287) 18.935,050 (19.478.036) (1.558.273) (41.785) 653,152 29,665 (229.657) 411.375 627.153 560.801 S 1.187.954 6.146,589 1.483.203 (1.740,501) (1.728,792) 164.447 (1.390,609) 21,781 89.620 (480.702) (314.658) (43,083) (77.277) (71.564) 62,260 120.052 222.258 2,463,024 (814,159) 23,450,218 (26.321.432) (3.685.373) (30.430) 1,321,302 69.307 250.000 (218.557) 1,391.622 169.273 391,528 560.801 The accompanying notes are an integral part of the financial statements. Rll R12 Annual Report Marine Biological Laboratory Notes to Financial Statements A. Background: The Marine Biological Laboratory (the "Laboratory") is a private, independent not-for-profit research and educational institution dedicated to establishing and maintaining a laboratory or station for scientific study and investigation, and a school for instruction in biology and natural history. The Laboratory was founded in 1888 and is located in Woods Hole. Massachusetts. B. Significant Accounting Policies: Basis of Presentation The accompanying financial statements have been prepared on the accrual basis of accounting and in accordance with the principles outlined in the American Institute of Certified Public Accountants' Audit Guide. "Not-For-Profit Organizations." The financial statements include certain prior-year summari/.ed comparative information in total but not by net asset class. Such information does not include sufficient detail to constitute a presentation in conformity with generally accepted accounting principles. Accordingly, such information should be read in conjunction with the Laboratory's financial statements for the year ended December 31, 1997, from which the summarized information was derived. The Laboratory classifies net assets, revenues, and realized and unrealized gains and losses based on the existence or absence of donor-imposed restrictions and legal restrictions imposed under Massachusetts State law. Accordingly, net assets and changes therein are classified as follows: Unrestricted Unrestricted net assets are not subject to donor-imposed restrictions of a more specific nature than the furtherance of the Laboratory's mission. Revenues from sources other than contributions are generally reported as increases in unrestricted net assets. Expenses are reported as decreases in unrestricted net assets. Gains and losses on investments and other assets or liabilities are reported as increases or decreases in unrestricted net assets unless their use is restricted by explicit donor stipulations or law. Expirations of temporary restrictions on net assets, that is, the donor-imposed stipulated purpose has been accomplished and or the stipulated time period has elapsed, are reported as reclassifications between the applicable classes of net assets. Temporarily Restricted Temporarily restricted net assets are subject to legal or donor-imposed stipulations that will be satisfied either by the actions of the Laboratory, the passage of time, or both. These assets include gifts plus monies for which the specific, donor-imposed restrictions have not been met, and pledges, annuities, and unitrusts for which the ultimate purpose of the proceeds is not permanently restricted. As the restrictions are met, the assets are released to unrestricted net assets. Also, realized/unrealized gains/losses associated with permanently restricted gifts which are not required to be added to principal by the donor are classified as temporarily restricted but maintain the donor requirements for expenditure. Permanently Restricted Permanently restricted net assets are subject to donor-imposed stipulations that they be invested to provide a permanent source of income to the Laboratory. These assets include gifts, pledges and trusts which require that the corpus be invested in perpetuity and only the income be made available for program operations in accordance with donor restrictions. Nonoperating revenues include realized and unrealized gains on investments during the year as well as investment income on the master pooled investments. Investment income from short-term investments and investments held in trust by others are included in operating support and revenues. To the extent that nonoperating investment income and gains are used for operations as determined by the Laboratory's total return utilization policy (see below), they are reclassified from nonoperating to operating on the statement of activities as "Investment earnings used for operations." All other activity is classified as operating revenue. The Laboratory recorded net realized gains of $2,805,560, net unrealized losses of $2.755,079 and dividend and interest income of $1,478,046 in 1998. Cash and Cash Equivalents Cash equivalents consist of resources invested in overnight repurchase agreements and other highly liquid investments with original maturities ot three months or less. Financial instruments which potentially subject the Laboratory to concentrations of risk consist primarily of cash and investments. The Laboratory maintains cash accounts with one banking institution. Investments Investments purchased by the Laboratory are carried at market value. Donated investments are recorded at fair market value at the date of the gift. For determination of gain or loss upon disposal of investments, cost is determined based on the first-in, first-out method. Investments with an original maturity of three months to one year are classified as short-term. All other investments are considered long-term. Investments are maintained primarily with five institutions. In 1924, the Laboratory became the beneficiary of certain investments, included in permanently restricted net assets, which are held in trust by others. The Laboratory has the continuing rights to the income produced by these funds in perpetuity, subject to the contractual restrictions on the use of such funds. Accordingly, the trust has established a process to conduct a review every ten years by an independent committee to ensure the Laboratory Financial Statements R13 continues to perform valuable services in biological research in accordance with the restrictions placed on the funds by the agreement. The committee met in 1994 and determined that the Laboratory has continued to meet the contractual requirements. The market values of such investments are $7.673.828 and $7,440.158 at December 31. 1998 and 1997, respectively. The dividend and interest income on these investments totaled $260.805 and $254.898 in 1998 and 1997, respectively. Investment Income and Distribution For the master pooled investments, the Laboratory employs a total return utilization policy that establishes the amount of the investment return made available for spending each year. The Finance Committee has approved a standing policy that the withdrawal will be based on a percentage of the latest three-year average ending market values of the funds. The market value includes the principal plus reinvested income, realized and unrealized gains and losses. Spending rates in excess of 5%, but not exceeding 1%, can be utilized if approved in advance by the Finance Committee of the Board of Trustees. For fiscal 1998 and 1997. the Laboratory obtained approval to expend 6% of the latest three-year average ending market values of the investments. The net appreciation on permanently and temporarily restricted net assets is reported together with temporarily restricted net assets until such time as all or a portion of the appreciation is distributed for spending in accordance with the total return utilisation policy and applicable state law. Investment income on the pooled investment account is allocated to the participating funds using the market value unit method (Note D). Plant Assets Buildings and equipment are recorded at cost. Donated facility assets are recorded at fair market value at the date of the gift. Depreciation is computed using the straight-line method over the asset's esiimated useful life. Estimated useful lives are generally three to ten years for equipment and 20 to 40 years for buildings and improvements. Depreciation expense for the years ended December 31. 1998 and 1997 amounted to $1.505.696 and $1.483,203. respectively, and has been recorded in the statement of activities in the appropriate functionalized categories. When assets are sold or retired, the cost and accumulated depreciation are removed from the accounts and any resulting gain or loss is included in unrestricted income for the period. Annuities tint! Unitnists Pavable Amounts due to donors in connection with gift annuities and unitrusts are determined based on remainder value calculations, with varied assumptions of rates of return and payout terms. Deferred Income and Advances on Contracts Deferred income includes prepayments received on Laboratory publications and advances on contracts to be utilized within the next year. Advances on contracts includes funding received for grants and contracts before it is earned. In certain circumstances, long-term advances are invested in the master pooled account until they are expended. Revenue Recognition Revenue is recognized at the time it is earned. The sources of revenue include grant payments from governmental agencies, contracts from private organizations, and income from the rental of laboratories and classrooms for research and educational programs. The tuition income is net of student financial aid of $523. 1 90 and 5536,097 in 1 998 and 1 997, respectively. Fees for conferences and other services include the following activities: housing, dining, library, scientific journals, aquatic resources and research services. Contributions Contribution revenue includes gifts and pledges. Gifts are recognized as revenue upon receipt. Pledges are recognized as temporarily or permanently restricted revenue in the year pledged and are recorded at the present value of expected future cash flows, net of allowance for unfulfilled pledges. Gifts and pledges, other than cash, are recorded at fair market value at the date of contribution. Expenses Expenses are recognized when incurred and charged to the functions to which they are directly related. Expenses that relate to more than one function are allocated among functions using various methodologies. Other programs expense consists primarily of fundraising, year-round labs and library room rentals, costs associated with aquatic resource sales and scientific journals. Total fundraising expense for 1998 and 1997 is $1.037,495 and $1.226,360, respectively. Use of Estimates The preparation of financial statements in conformity with generally accepted accounting principles requires management to make estimates and assumptions that affect the reported amounts of assets and liabilities and disclosure of contingent assets and liabilities at the date of financial statements and the reported amounts of revenues and expenses during the reporting period. Actual results could differ from those estimates. Tax-Exempt Status The Laboratory is exempt from federal income tax under Section 501(c)(3) of the Internal Revenue Code. R14 Annual Report C. Investments: The following is a summary of the cost and market value of investments at Dec ember 31. 1998 and 1997: Market Cost 199S 1997 1998 1997 Certificates of deposit $ 40.000 S 40.000 $ 40.000 $ 40,000 Money market securities 1.052.276 2,168.958 1.052,276 2.168,958 U.S. Government securities 1,397,686 1.292.600 1,136.219 1.098.526 Corporate fixed income 2.504.507 2,587,861 2.472.653 2.472,653 Common stocks 5,033.704 5,279,266 4,290,581 4,271,853 Mutual funds 29,548,89! 23.223,812 26.225.214 19,317,499 Limited partnerships 1,038.600 5.429,700 958.982 3,309.994 Total investments $40,615,664 $40,022,197 $36,175,925 $32,679,483 Investment portfolios tor the years ended December 31, 1998 and 1997 are as f< allows: Mark el Cost 1998 1997 1998 7997 Short-Term Investments Certificates of deposit $ 40,000 $ 40.000 $ 40,000 $ 40,000 Money market 1784 Fund 559,314 1 .759.589 559.314 1 .759.589 Common stocks 6.241 551.780 6,241 530.936 Mutual funds 2,955,989 2,056.677 2,940.929 2,056.679 Total 3.561,544 4,408,046 3.546.484 4.387,204 Mark et Cost 1998 1997 1998 1997 Long-Term Investments Pooled investments: Master pooled investments $27.057.909 $26,163,702 $23.723.343 $20,201,962 Separately invested: General Chase trust 6,038,153 5,846.916 5,433,574 4,986.443 Library Chase trust 1,635,675 1,593.242 1.477,462 1.358.149 Annuity and unitrust investments 2.322.383 2,010,291 1.995,062 1,745,725 Total 37.054,120 35,614,151 32.629.441 28.292,279 Total investments $40.615.664 $40.022.197 $36.175.925 $32.679.483 Financial Statements KI5 D. Accounting for Pooled Investments: Certain net assets are pooled for investment purposes. Investment income from the pooled investment account is allocated on the market value unit basis, and each fund subscribes to or disposes of units on the basis of the market value per unit at the beginning of the calendar quarter within which the transaction takes place. The unit participation of the funds at December 31. 1998 and 1997 is as follows: Unrestricted Temporarily restricted Permanently restricted Advances on contracts 199N 4.001 44,455 65.016 6.437 119.909 1997 4.192 42,693 65,411 6.506 118,802 Pooled investment activity on a per-unit basis was as follows: Unit value at beginning of year Unit value at end of year Total return on pooled investments 1998 $ 220.30 225.51 $ 5.21 1997 $ 186.35 220.30 $ 33.95 E. Long-Term Dchj: Long-term debt consisted of the following at December 31 : Variable rate (5.15% at December 31, 1998) Massachusetts Industrial Finance Authority Series 1992A Bonds payable in annual installments through 2012 6.63% Massachusetts Industrial Finance Authority Series 1992B Bonds, payable in annual installments through 2012 5.8% The University Financing Foundation. Inc.. payable in monthly installments through 2000 5.8% The University Financing Foundation. Inc.. payable in monthly installments through 2002 I99N $ 925,000 1,230.000 226.024 186.346 $2.567.370 1997 $ 960,000 1.280.000 325.210 231.817 S2.797.027 The aggregate amount of principal due on long-term debt for each of the next five fiscal years and thereafter is as follows: 1999 2000 2001 2002 2003 Thereafter Less current portion of long-term debt Long-term debt net of current return $ 243.274 267.404 173,664 148,028 125.000 1.610.000 2.567.370 (243.274) $2.324.096 In 1992. the Laboratory issued $1.100.000 Massachusetts Industrial Finance Authority (MIFA) Series 1992A Bonds with a variable interest rate and SI. 500.000 MIFA Series 1992B with an interest rate of 6.63%. Interest expense debt totaled $136.340 tor the year ended December 31. 1998. The Series 1992 A and B Bonds mature on December 1. 2012 and are collateralized by a first mortgage on certain Laboratory property. On March 17. 1998, the Laboratory entered into a ten-year interest rate swap contract in connection with the Series 1992A Bonds. This contract effectively fixes the interest rate at 6.30% through December 17. 200S. R16 Annual Report The agreements related to these bonds subject the Laboratory to certain covenants and restrictions. Under the most restrictive covenant of this debt, the Laboratory's operating surplus, exclusive of interest expense and depreciation expense, must be greater than or equal to 1.2 times all debt service payments, as defined by the agreement. The Laboratory was in compliance with these covenants and restrictions at December 31, 1998. In 1996. the Laboratory borrowed $500,000 with an interest rate of 5.8% per annum from the University Financing Foundation. Inc. The interest expense for the year ended December 31. 1998 was $16,253. The loan matures in 2000 and is collaterali/ed by 50.000 shares of a fixed income fund with a fair value of $595.000 at December 31. 1998. In 1997, the MBL borrowed $250.000 with an interest rate of 5.8% per annum from the University Financing Foundation. Inc. The interest expense for the year ended December 31, 1998 was $12,249. This loan matures in 2002 and is collateralized by 19.440 shares of a fixed income mutual fund with a fair value of $231,336 at December 31. 1998. The Laboratory has a line of credit agreement with BankBoston from which it may draw up to $1.000.000. No amounts were outstanding under this agreement as of December 31. 1998 and 1997. F. Plant Assets: Plant assets consist of the following at December 3 1 : 1W8 1997 Land Buildings Equipment Total Less: Accumulated depreciation Plant assets, net $ 702.908 33,334,107 4,401,184 $ 702,908 32,419,072 4,300,932 38,438.199 (18,902,028) 37,422.912 (17,396,332) $19.536.171 $20,026.580 G. Retirement Plan: The Laboratory participates in the defined contribution pension plan of TIAA-CREF (the "Plan"). The Plan is available to permanent employees who have completed two years of service. Under the Plan, the Laboratory contributes 10%< of total compensation for each participant. Contributions amounted to $737,156 and $715,858 for the years ended December 31, 1998 and 1997. respectively. H. Pledges: Unconditional promises to give are included in the financial statements as pledges receivable and the related revenue is recorded in the appropriate net asset category. Unconditional promises to give are expected to be realized in the following periods: In one year or less Between one year and five years After five years /W,S $1,607.664 3,110,354 146,586 7997 $2,219,056 2.485,851 80.000 Total 4,864.604 4.784.907 Less: discount of $301,588 in 1998 and $227,025 in 1997 and allowance of $100,000 in 199S and $100.000 in 1 997 (401.5X8) (327,025) $4,463.016 $4.457.882 Financial Statements R17 Pledges receivable at December 31 have the following restrictions: Research and education Permanently restricted net assets $3,933,988 529,028 1997 $3.787,882 670.000 $4.463.016 $4,457.882 I. Postretirement Benefits: The Laboratory accounts for its postretiremen! benefits under Statement No. 106, "Employers' Accounting tor Postretiremen! Benefits Other than Pensions." which requires employers to accrue, during the years that the employee renders the necessary service, the expected cost of benefits to be provided during retirement. As permitted, the Laboratory has elected to amortize the transition obligation over 20 years commencing on January 1 . 1994. The Laboratory's policy is that all current retirees and certain eligible employees who retired prior to June 1 . 1994 will continue to receive postretirement health benefits. The remaining current employees will receive benefits; however, those benefits will be limited as defined by the Plan. Employees hired on or after January 1. 1995 will not be eligible to participate in the postretirement medical benefit plan. The following tables set forth the Plan's funded status as of December 31: Benefit obligation at December 31 Fair value of plan assets at December 3 1 Funded status Accrued benefit cost Weighted-average assumptions as of December 3 1 : Discount rate Expected return on plan assets Compensation increase rate Benefit cost Employer contribution Benefits paid 1998 $ 2.171.119 820.645 $(1.350.474) $ (26.654) 6.75% 7.25% N/A 210,339 192.082 109.404 1997 $ 1.919.865 701,140 $(1,218.725) $ (8.397) 7.50% 8.00% N/A 192,082 192.082 111.255 For measurement purposes a 7.5% annual rate of increase in the per capita cost of covered health care benefits was assumed for 1999. The rate was assumed to decrease by half of 1 .00% per year to 4.25% in 2006 and remain at that level thereafter. Pension plan assets consist of investments in a money market fund. O OO ri -t r- ON r*~ t rn Os r) ^t od f', ff\ ri NO oo oo oo ri o r- ri NO o r*- ( r*~, -t r*- ( -t OO ON ri OC r i r i vC Os O OC ( ^ C ^ 06 ri ' * ' ' i > ^ i s a; "3 I -t r ' -t Os ! ? -f it", rr, r*~, r^ Os r O so -t -t r- r- sC a & so r^'t ^ 1 i/~" O so Cs r-' r-' Q C. -f Os 1 1 -t C *-J C?; _' Os ON so -f in -f r^i -, as r~ "- sC -tr*-i-t- r^-^-sOOO as UJ "^ g Os O OOC^r^r^iSO Ow~' NO NO ri oo so* so' oo as' Os 1 rf oc rK r*~i ^ ~ n \o as n f~> >^~i os 1 t^ n O\ Os *v uj ^ * Q- a. oc ^; '~ [-; -t t O X ON c -S E- W 5 ^ * ' ' & < -; Q 1 tl ^ < % II 1 1 r i u-, r-NO-TOOOmNOrr-, iri rf ro iy~j r- oo O <"") (^i r- -O ^C -T^r- m - roooOs-T Or} ri r- -t -j- r i r i hJ <-> 3 ^ -S _ ; : "*' ^' $ ^' o i 1 -s oo os u-, r- NO r- "n NO ^. ON \o n OO 3 2 s v. n-, r*-, <-| sC IT, r^, -(- O r- r*~, r-' O O oq 2 = 3^ 1 CQ ^ G -J > *^ >O Osr*-, O w-i I-^OO ' ON' r*"i O -t c: *T ~-f lO M w ^ ^2 z g - > a; 5 'S ^ Ori OsOO-t oc r~~ r~~ oon ^t r- ir> r^ co r~- r- ri oc w~, O r^l Wj OS -?t OO OO^ -t - O r- ^^ ' ^ C! I ^ I p , -^ . Xi "fOs'lm -? oo < \O JC -.' r-' oo f^i Os ri rj NO n-, -t c/) S torsion angles. Structure generation based on interproton distance restraints and torsion angle measurements yield convergent structures generated using distance geometry and simulated annealing methods. The goal of this project is to determine the structural role of y- carboxyglutamic acid in the Gla-containing conotoxins. Staff Barbara C. Furie, Scientist Bruce Furie, Scientist Johan Stenflo. Visiting Scientist Eva Czerwiec. Postdoctoral Fellow Gail Begley, Postdoctoral Fellow Alan Rigby. Postdoctoral Fellow Staff Hanlon. Roger, Senior Scientist Sussman, Raquel, Investigator Hatfield, Emma, Postdoctoral Fellow Maxwell, Michael, Postdoctoral Scientist Rurnmel, John, Visiting Scientist Shashar. Nadav. Postdoctoral Scientist Visiting Investigators Boal, Jean, Visiting Scientist Gabr, Howaida, Graduate Student. Suez Canal University. Egypt Cavanaugh, Joseph. Graduate Student, Boston University Marine Program Fern, Sophie, Graduate Student, Boston University Marine Program Wittenberg. Kim, Boston University Marine Program Laboratory of Shinya Inoue Scientists in this laboratory study the molecular mechanism and control of mitosis, cell division, cell motility, and cell morphogenesis, with emphasis on biophysical studies made directly on single living cells, especially developing eggs in marine invertebrates. Development of biophysical instrumentation and methodology, such as the centrifuge polarizing microscope, high-extinction polarization optical and video microscopy, digital image processing techniques, and exploration of their underlying theory are an integral part of the laboratory's efforts. Staff Inoue, Shinya. Distinguished Scientist Knudson. Robert. Instrument Development Engineer Maccaro, Jackie, Laboratory Assistant MacNeil, Jane. Executive Assistant Laboratory of Alan M. Kuzirian Research in the laboratory explores the functional morphology and infrastructure of various organ systems in molluscs. The program includes mariculture of the nudibranch. Hermissenda crassicnnus. with emphasis on developing reliable culture methods for rearing and maintaining the animal as a research resource. The process of metamorphic induction by natural and artificial inducers is being explored in an effort to understand the processes involved and as a R50 Annual Report video, and digital image processing for fast analysis of specimen birefringence over the entire viewing field. Examples of biological systems currently investigated with the Pol-Scope are: microtubule-based structures (asters, mitotic spindles, single microtubules); actin-based structures (acrosomal process, stress fibers, nerve growth cones); zona pellucida of vertebrate oocytes; and biopolymer liquid crystals. Staff Oldenbourg. Rudolf, Associate Scientist Katoh. Kaoru. Postdoctoral Research Associate Geer. Thomas. Research Assistant Knudson. Robert. Instrument Development Engineer Barahy, Diane. Laboratory Assistant means to increase the yield of cultured animals. Morphologic studies stress the ontogeny of neural and sensory structures associated with the photic and vestibular systems which have been the focus of learning and memory studies, as well as the spatial and temporal occurrence of regulatory and transmitter neurochemicals. Concurrent studies detailing the toxic effects of lead on Hermissenda learning and memory, feeding, and the physiology of cultured neurons are also being conducted. New studies include cytochemical investigations of the Ca :+ /GTP binding protein, calexcitin. and its modulation with learning and lead exposure. Collaborative research includes histochemical investigations on strontium's role in initiating calcification in molluscan embryos (shell and statoliths). immunocytochemical labelling of cell-surface antigens, neurosecretory products, second messenger proteins involved with learning and memory, as well as intracellular transport organelles using mono- and polyclonal antibodies on squid (Loligo pealei) giant axons and Hermissenda sensory and neurosecretory neurons. Additional collaborations involve studying neuronal development and defects, as well as nerve regeneration and repair in phylogenetically conserved nervous systems. Additional collaborative research includes DNA fingerprinting using RAPD-PCR techniques in preparation for isogenic strain development of laboratory-reared Hermissenda and hatchery-produced bay scallops (Argopectin irradiana) with distinct phenotypic markers tor the rapid field identification. Systematic and taxonomic studies of nudibranch molluscs, to include molecular phylogenetics, are also of interest. Staff Ku/.irian, Alan M.. Associate Scientist Visiting Scientists Chikarmane, Hemant. Assistant Scientist, MBL Clay. John R.. NINDS/NIH Gould, Robert. NYS Institute of Basic Research Laboratory of Rudolf Oldenbourg Laboratory for Reproductive Medicine, Brown University and Women and Infants Hospital, Providence Work in this laboratory centers on the investigation of the underlying mechanisms behind female infertility. Particular emphasis is placed on the physiology of the oocyte or early embryo, with the aim of assessing developmental potential and mitochondria dysfunction arising from mtDNA deletions. The studies taking place at the MBL branch of the Brown Laboratory use some of the unique instrumentation available through the resident programs directed by Rudolf Oldenbourg and Peter J. S. Smith. Most particularly, non-invasive methods for oocyte and embryo study are being sought. Of several specific aims, one is to use the Pol-Scope to analyze the birefringence of the preimplantation mammalian zona pellucida a structure most predictive of successful implantation. We also have used this instrument to examine meiotic spindles. An additional aim is to continue the studies on transmembrane ion transport using the non-invasive electro-physiological techniques available at the BioCurrents Research Center. Preliminary studies indicate that the calcium transport may form an accurate predictor of oocyte and embryo health. The newly developed oxygen probe also offers the possibility of looking directly at abnormalities in the mitochondria arising from accumulated mtDNA damage. Our laboratory has also focused on studying the mechanism underlying age-associated infertility in terms of oocyte quality, attempting to rescue the developmentally compromised oocytes or embryos through nuclear- cytoplasmic transfer technology. We have characterized oxidative stress- induced mitochondria! dysfunctions, developmental arrest and cell death in early embryos using animal models. Ultimately, in addition to investigating the mechanisms behind cellular aging underlying infertility, this laboratory aims to produce clinical methods for assessing preimplantation embryo viability, a development that will make a significant contribution to the health of women and children. Staff Keefe. David. Director Liu. Lin. Research Scientist Pepperell, John. Visiting Investigator Trimarchi. James. Postdoctoral Scientist The laboratory imotig.itcs the molecular architecture ot living cells and of biological model systems using optical methods for imaging and manipulating these structures. For imaging non-invasively and non- destructively cell architecture dynamically and at high resolution, we have developed a new polari/ed light microscope (Pol-Scope). The Pol- Scope combines microscope optics with new electro-optical components. Laboratory of Sensory Physiology Members of this laboratory have conducted research on various facets of vision since 1473. Current investigations use UV/V1S light microspectrophotometry on vertebrate retinal photoreceptors for the Yciir-Round Research R51 determination of visual pigment ahsorbance characteristics. One aim is to arrive at a better understanding of the method of spectral tuning that forms the chemical basis of color vision. Polarized light microscopic techniques are used to measure linear dichroism and linear birefringence aimed at revealing structure-function relationships and biophysical mechanisms. An area of interest is polarization discrimination, the mechanisms that could account for the ability of some fish species to detect the direction of polarization of light collected by their eyes. As a recent development, investigations are carried out on sickling in fish red blood cells due to hemoglobin polymerization, once again making extensive use of polarized light microscopic techniques. Staff Harosi, Ferenc I.. Senior Scientist, MBL. and Boston University School of Medicine Novales Flamarique. I., Postdoctoral Fellow Laboratory of Osainu Shinwmiira Biochemical mechanisms involved in the bioluminescence of various luminescent organisms are investigated. Based on the results obtained, various improved forms of bioluminescent and chemiluminescent probes are designed and produced for the measurements of intracellular free calcium and superoxide anion. Staff Shimomura, Osamu. Senior Scientist, MBL, and Boston University School of Medicine Shimomura, Akemi. Research Assistant Laboratory of Robert B. Silver The members of this laboratory study how living cells make decisions. The focus of the research, typically using marine models, is on two main areas: the role of calcium in the regulation of mitotic cell division (sea urchins, sand dollars, etc.) and structure and function relationships of hair cell stereociliary movements in vestibular physiology (oyster, toadfish). Other related areas of study, i.e. synaptic transmission (squid), are also, at times, pursued. Tools include video light microscopy, multispectral, subwavelength, and very high speed (sub-millisecond frame rate) photon counting video light microscopy, telemanipulation of living cells and tissues, and modeling of decision processes. A cornerstone of the laboratory's analytical efforts is high performance computational processing and analysis of video light microscopy images and modeling. With luminescent, fluorescent, and absorptive probes, both empirical observation and computational modeling of cellular, biochemical, and biophysical processes permit interpretation and mapping of space-time patterns of intracellular chemical reactions and calcium signaling in living cells. A variety of in vitro biochemical, biophysical, and immunological methods are used. In addition to fundamental biological studies, the staff designs and fabricates optical hardware, and designs software for large video image data processing, analysis, and modeling. Staff Silver. Robert. Associate Scientist Visiting Scientist Pearson, John, Los Alamos Nations Laboratory Interns King, Leslie A., REU Intern. Duke University Wise. Alyssa. REU Intern. Yale University Laboratory of Seymour Zigman This laboratory is investigating basic mechanisms of photooxidative stress to the ocular lens due to environmentally compatible UVA radiation. This type of oxidative stress contributes to human cataract formation. Other studies are the search for and use of chemical antioxidants to retard the damage that occurs. Cultured mammalian lens epithelial cells and whole lenses in vitro are exposed to environmentally compatible UVA radiation with or without previous antioxidant feeding. The following parameters of lens damage are examined: molecular excitation to singlet states via NADPH (the absorber); cell growth inhibition and cell death; calalase inactivation; cytoskeletal description (of actin. tubulin. integrins): and cell membrane damage (lipid oxidation, loss of gap junction integrity and intercellular chemical communications). Thus far. the most successful antioxidant to reduce these deficiencies is alpha-tocopherol (10 /j.g/ml) and tea polyphenols (especially from green tea). The preliminary phases of the research are usually carried out using marine animal eyes (i.e.: smooth dogfish) as models. Our goal is to provide information that will suggest means to retard human cataract formation. Staff Seymour Zigman. Laboratory Director, Professor of Ophthalmology. Boston University Medical School Keen Rafferty. Research Associate. Boston University Medical School Nancy S. Rafferty, Research Associate, Boston University Medical School Buiinie R. Zigman. Laboratory Manager, Boston University Medical School R52 Annual Report The Marine Resources Center The Marine Resources Center (MRC) is one of the world's most advanced facilities for maintaining and culturing aquatic organisms essential to advanced biological, biomedical, and ecological research. Service and education also play an important and complementary role in the modern. 32,000-square-foot facility. The MRC and its life support systems have already increased the ability of MBL scientists to conduct research and have inspired new concepts in scientific experiments. Vigorous research programs focusing on basic biological and biomedical aquatic models are currently being developed at the Center. The Program in Scientific Aquaculture was initiated in 1998. In addition to research, the MRC provides a variety of services to the MBl. community through its Aquatic Resources Division, the Water Quality and System Engineering Division, and the Administrative Division. Research and educational opportunities are available at the facility to established investigators, postdoctoral fellows, graduate, and undergraduate students. Investigators and students will tind that the MRC's unique life support and seawater engineering systems make this a favorable environment in which to conduct independent research and masters and doctoral theses using a variety of aquatic organisms and flexible tank space for customi/ed experimentation on live animals. Prospective investigators and students should contact the Director of the MRC for further information. Staff Hanlon, Roger. Director and Senior Scientist Sussman. Raquel. Investigator Ku/.irian, Alan, Associate Scientist Maxwell. Michael, Postdoctoral Scientist Shashar, Nadav. Postdoctoral Scientist Visiting Invextiguturx Adamo, Shelly, Dalhousie University. Canada Baker. Robert. New York University Boal. Jean. Visiting Scientist Cavanaugh, Joseph, Boston University Marine Program Gabr, Howaida. Graduate Student, Sue?- Canal University, Egypt Gilland, Edwin, Staff Scientist Kier, William. University of North Carolina Spotte, Stephen. University of Connecticut Wittenberg, Kim, Boston University Marine Program Honors Friday Evening Lectures June \<-> June 26 July 3 July 10 July 17 July 23, 24 July 31 August 7 August 14 Stephen L. Hajduk, School of Medicine and Dentistry, University of Alabama at Birmingham "Carriers of Death: Civil War and Tsetse Flies" Mary Lidstrom. College of Engineering, University of Washington, Seattle "Borrowing Genes to Create New Metabolism" (Glassman Lecture) David Garbers. Howard Hughes Medical Center, Dallas "From Sea Urchins To High Blood Pressure: Smell and Vision" Donald Brown. Department of Embryology, Carnegie Institution of Washington "How Tadpoles Turn Into Frogs" Irene Pepperberg, Department of Ecology and Evolutionary Biology, University of Arizona, Tucson "In Search of King Solomon's Ring: Studies on Cognitive and Communicative Abilities of Grey Parrots" (Lang Lecture) Nicolas Spitzer, Department of Biology, University of California, San Diego 1. "The Development of Electrical Excitability in Nerve and Muscle" 2. "Breaking the Code: Regulation of Differentiation by Patterns of Calcium Transients" (Forbes Lectures) Eric Kandel. Center for Neurobiology and Behavior, Howard Hughes Medical Institute, Columbia University "Genes, Synapses and Long-Term Memory" Peter Raven. Missouri Botanical Gardens, St. Louis "Biodiversity, the Global Environment, and the New Millennium" Matthew Meselson. Harvard University "Evolution Without Sexual Reproduction and Genetic Recombination" Fellowships and Scholarships Robert Day Allen Fellowship Fund Drs. Joseph and Jean Sanger MBL Associates Endowed Scholarship Fund MBL Associates Mr. and Mrs. Douglas P. Amon Dr. and Mrs. Leonard Laster Frank A. Brown, Jr. Memorial Readership Dr. and Mrs. Francis D. Carlson C. Lalor Burdick Scholarship Fund The Lalor Foundation Gary Nathan Calkins Scholarship Fund Ms. Sarah A. Calkins Charles R. Crane Fellowship Fund Friendship Fund Mr. Thomas S. Crane John O. Crane Fellowship Fund Friendship Fund Mr. Thomas S. Crane Jean and katsuma Dan Fellowship Fund Drs. Joseph and Jean Sanger Mrs. Eleanor Steinbach Bernard Davis Fellowship Fund Mrs. Elizabeth M. Davis E. E. Just Research Fellowship Fund Ayco Charitable Foundation Dr. Jewel Plummer Cobb Mr. and Mrs. Jonathan Conrad Fiduciary Trust Company International Daniel Grosch Scholarship Fund Ms. Alice C. Leech Ms. Lena T. Lord Ms. Enid K. Sichel Dr. Margaret W. Taft Aline D. Gross Scholarship Fund Dr. and Mrs. Benjamin Kaminer Technic, Inc. R53 R54 Annual Report Keffer Hartline Fellowship Fund Dr. Lloyd M. Beidler Dr. Lawrence Eisenberg Dr. Paul Rosen Mr. Robert L. Schoenfeld Dr. and Mrs. Jonathan D. Victor Dr. Earl Weidner Dr. Torsten Wiesel and Ms. Jean Stein Fred Karush Endowed Library Readership Dr. and Mrs. Laszlo Lorand Dr. and Mrs. Arthur M. Silverstein Stephen W. Kuffler Fellowship Fund Dr. and Mrs. Edward A. Kravitz Frank R. Lillie Fellowship and Scholarship Fund Dr. and Mrs. George H. Acheson Mr. and Mrs. John J. Valois Josiah Macy, Jr. Research Fellowship Fund Josiah Macy, Jr. Foundation James A. and Faith Miller Fellowship Fund Drs. David and Virginia Miller Frank Morrell Scholarship Fund Dr. and Mrs. Maynard M. Cohen Dr. Leyla de Toledo Morrell Mr. Paul Morrell Mountain Memorial Fund Dr. and Mrs. Dean C. Allard, Jr. Ms. Brenda J. Bodian Dr. and Mrs. Benjamin Kaminer Ms. Anne C. Kimball, Ph.D. Mr. and Mrs. Amos L. Roberts Mr. and Mrs. William B. Sanford Mr. and Mrs. Hans L. Schlesmger Dr. and Mrs. R. Walter Schlesinger Neural Systems & Behavior Scholarship Fund Dr. Ronald Calabrese and Dr. Christine Cozzen Dr. and Mrs. Alan Gelperin Dr. Warren M. Gnll Dr. Ronald Hoy and Dr. Margaret Nelson Drs. Darcy B. Kelley and Richard M. Bockman Dr. William Kristan and Dr. Kathleen French Dr. Richard and Mrs. Jane Levine Dr. Janis C. Weeks and Dr. William M. Roberts Nikon Fellowship Fund Nikon. Inc. The Ann Osterhout Edison/Theodore Miller Edison and Olga Osterhout Sears/Harold Bright Sears Endowed Scholarship Fund Mrs. Jean S. Holden Dr. Susan M. Plourde William Townsend Porter Scholarship Fund William Townsend Porter Foundation Phillip H. Presley Scholarship Fund Carl Zeiss, Inc. Ruth Sager Endowed Scholarship Dr. Arthur B. Pordee Science Writing Fellowships Program Association for Research in Vision and Ophthalmology American Society for Biochemistry and Molecular Biology American Society for Cell Biology American Society for Photobiology Charles A. Dana Foundation Federation of American Society for Experimental Biology Foundation for Microbiology Friendship Fund New York Times Foundation Nicholas B. Ottaway Foundation Society for Integrative and Comparative Biology The Times Mirror Foundation The Washington Post Company Milton L. Shifman Endowed Scholarship Milton L. Shifman Scholarship Trust The Evelyn and Melvin Spiegel Fellowship Fund Drs. Joseph and Jean Sanger Drs. Melvin and Evelyn Spiegel The Sprague Foundation H. B. Steinbach Fellowship Fund Mrs. Eleanor Steinbach Marjorie R. Stetten Scholarship Fund Ms. Pauline F. Blanchard Mr. and Mrs. John C. Campbell Cognos Corporation Cognos Inc. Ms. Ann P. B. Fit/.gerald Mr. and Mrs. Douglas W. Lucy Mr. and Mrs. William Morton Ms. Linda A. O'Donnel Mrs. Jane Lazarow Stetten Mrs. Janet L. Vanderweil Ms. Ann M. White Horace W. Stunkard Scholarship Fund Dr. Albert Stunkard and Dr. Margaret Maurin Walter L. Wilson Endowed Scholarship Dr. Paul N. Chervin Mr. and Mrs. Rexford A. English Dr. Jean R. Wilson Honors R55 Fellowships Awarded MBL Summer Research Fellows Mark C. Alliegro, Ph.D.. Louisiana State University. Dr. Alliegro uses sea urchins, and a variety of mammalian cells in culture to study the mechanisms of cell differentiation. He was supported by the Frederik B. Bang Fellowship Fund, the James A. and Faith Miller Memorial Fund, and an MBL Associates Fellowship. Brian D. Bovard, Ph.D., Duke University. Dr. Bovard worked at a field site located in Abisko, Sweden, this summer. He studies relations between plants and water as part of a climate change project being conducted by scientists at the MBL's Ecosystem Center. He was supported by the William Townsend Porter Fellowship for Minority Investigators. Wei-Jun Cai, Ph.D.. University of Georgia. Dr. Cai is developing microelectrodes to aid in the study of benthic carbon recycling. He was supported by the Lucy B. Lemann Fellowship. William Cohen, Ph.D.. Hunter College. Dr. Cohen uses blood clams in his studies of the formation and function of the cellular framework known as the cytoskeleton. He was supported by the Erik B. Fries Endowed Fellowship. John Costello, Ph.D., Providence College. Dr. Costello studies the feeding behavior in the comb jelly, Mnemiopsis leidyi. He was supported by the Erik B. Fries Endowed Fellowship, the Lucy B. Lemann Fellowship, and an MBL Associates Fellowship. ' John E. Eriksson. Ph.D.. Turku Center for Biotechnology, Finland. Dr. Eriksson is studying mitotic protein phosphatases in the eggs of the surf clam, Spisula. He was a Herbert W. Rand Fellow. Andrew F. Giusti. University of California. Santa Barbara. Mr. Giusti investigates the role of the SRC tyrosine kinase during egg activation at fertilization. He was supported by the Frederik B. Bang Fellowship Fund. Matthew Halstead. Ph.D.. University of Auckland, New Zealand. Dr. Halstead studies sensory processing of electrosensory information in the midbrain of the little skate. Raja. He was supported by the M.G.F. Fuortes Fellowship, the Frank R. Lillie Fellowship, and an MBL Associates Fellowship. ' Jonathan J. Henry. Ph.D.. University of Illinois. Dr. Henry examines the cellular and molecular mechanisms involved in embryonic cell fate and axis determination using barnacles as his research model. He was supported by the Evelyn and Me/vin Spiegel Fellowship Fund and the NASA Life Science Program Fellowship. Elizabeth Jonas, Ph.D.. Yale University School of Medicine. Dr. Jonas studies the intracellular channels that regulate synaptic function. She was supported by the Ann E. Kammer Memorial Fellowship Fund, the H. B. Steinbach Fellowship, an MBL Associates Fellowship, the Charles R. Crane Fellowship, and the John O. Crane Fellowship Fund. Nicholas Lartillot, Universite Paris Sud. Mr. Lartillot conducted a molecular study of mesoderm specification in marine spiralians. He was an MBL Associates Fellow. Guy Major, a Research Fellow from Lucent Technologies. Mr. Major took voltage-sensitive dye recordings from multiple parts of single brain cells. He was a Herbert W. Rand Fellow. Mark Martindale, Ph.D., University of Chicago. Dr. Martindale studies the evolution of development, in particular axial specification and the role of the cleavage program in body plan evolution. He was a NASA Life Sciences Program Fellow. Paul McNeil, Ph.D., Medical College of Georgia. Dr. McNeil hopes to discover how cells reseal tears in their outer covering, the plasma membrane, and to demonstrate that such membrane tears are physiological events. He uses sea urchins, starfish eggs, and squid in his studies. He was supported by a Robert Day Allen Fellowship and a NASA Life Science Program Fellowship, Allen Mensinger. Ph.D.. Washington University School of Medicine. Dr. Mensinger is developing an acoustical transmitter tag for neural telemetry. He was a NASA Life Science Program Fellow and an MBL Associates Fellow. ' Inigo Novales Flamarique, Ph.D.. University of Victoria, Canada. Dr. Novales Flamarique studies the functional organization of visual pathways from the retina to the brain in fishes. He was a Herbert W. Rand Fellow and an MBL Associates Fellow. Elaine C. Seaver, Ph.D., University of Texas, Austin. Dr. Seaver studies the mechanism of segmentation in polychaetes. She was supported by the Evelyn and Melvin Spiegel Fellowship Fund. Matt Wachowiak, Ph.D.. University of California. Berkeley. Dr. Wachowiak studies the transmission of olfactory information from sensory cells to the central nervous system. He was supported by a Stephen W. Kuffler Fellowship and an MBL Associates Fellowship. James Q. Zheng. Ph.D., Robert Wood Johnson Medical School. Dr. Zheng studies the cellular mechanisms underlying the formation of nerve connections. He was a Nikon Fellow. Grass Fellows Pamela England, Ph.D.. California Institute of Technology. Project: Probing the role of the protein tyrosine kinase SRC in long-term potentiation. Alexander Gimelbrant. Ph.D., University of Kentucky Medical Center. Project: Characterization of cDNAs specific to individual lobster olfactory receptor neurons. Kathryn Jessen-Eller, Ph.D., Tufts University School of Veterinary Medicine. Project: Serotonergic growth and p53 expression in developing embryos. Jane Roche King, Ph.D.. University of Arizona. Project: Vestibular contribution to escape turning and orientation to prey in the leopard frog. Rana pipiens. Maria Fabiana Kubke. Ph.D., University of Maryland. Project: Analysis of early position as a function of best frequency in the hindbrain auditory nuclei of the chicken. David P. Len/.i. Ph.D.. University of Oregon. Project: The role of the synaptic ribbon at sensory cell output synapses. Andrey Loboda, University of Pennsylvania. Project: Elucidation of the role of the S4-S5 linker in gating of the shaker potassium channel by site-directed crosslinking and gating current measurements. Matthias Lorez. University of Zurich. Project: The role of HRS- 2 in synaptic transmission in the giant synapse of the squid Loligo pealei. Kimberly McAllister. Ph.D.. The Salk Institute for Biological Studies. Project: Properties of synapse formation between cultured cortical neurons. Kristina S. Mead, Ph.D., University of California, Berkeley. Project: The biomechanics and neurobiology of chemoreception in stomatopods. Hong-Sheng Wang, Ph.D., SUNY at Stony Brook. Project: Angiotensin modulation of transient outward current of cardiac myocytes. MBL Science Writing Fellowships Program Fellows Monica Allen. Bangor Daily News Kevin P. Carmody. Chicago Daily South/own R56 Annual Report Thomas Carney. Des Moines Register Randall J. Edwards. Columbus Dispatch Don Finley, Sun Antonio Express-News Joel Greenberg. The Los Angeles Times Ralph K. M. Haurwitz, Austin American-Statesman Diedtra Henderson. Seattle Times Edie Lau. Sacramento Bee Larry Proulx. The Washington Post Frank D. Roylanee, Baltimore Sun Angela Swafford, Mas Vida/CBS Diane Toomey. WUNC Radio Ulysses Torassa. Cleveland Plain Dealer Karin Vergoth. National Public Radio/Science Friday Joby S. Warrick. The Washington Post Philip Yam, Scientific American Program Directors Robert D. Goldman, Northwestern University Boyce Rensberger. Knight Science Journalism Program Htinds-On Laboratory Course Directors Rex Chisholm, Northwestern University (Biomedical) John Hobbie. Marine Biological Laboratory (Environment) Jerry Melillo. Marine Biological Laboratory (Environment) Robert Palazzo, University of Kansas (Biomedical) SPINES Summer Program in Neuroscience Ethics and Survival SPINES is a month-long program directed by Joe L. Martinez, Jr., and James Townsel. The program is supported by grants from NIMH administered by the American Psychological Association and the Association of Neuroscience Departments and Programs. SPINES offers an introduction to the opportunities available at the MBL and in the field of neuroscience in general. Fellows are taught responsible conduct in research and other survival skills such as scientific writing, poster construction, presentations, grant mechanisms, and how to seek a postdoctoral or job position. Fellows Carlos Bolanos-Guzman Morry Brown Winfred Monica Bryan Damani Nabet-Yero Bryant Jameel Dennis Cynthia Gentry Karen Gilliums Caterina Maria Hernandez William Meilandt Silke Monn Nivia Perez Acevedo Osceola Whitney Scholarships Awarded Aline D. Gross Scholarship Fund Tao, Haiyang, Ohio University American Society for Cell Biology Minorities Affairs Committee Anderson, Tonya, University of California. Los Angeles Foster, Andrea, Stanford University Freeman, Antoinette, Boston University School of Medicine Hinojos, Cruz, University of Texas, Houston Tafari. Tsahai, University of California. San Diego Arthur Klorfein Scholarship and Fellowship Fund Garcia. Ana Anton, Universidad Miguel Hernandez. Spain Jacobson, Eyal. Technion, Israel Rossi. Francesco, Scuola Normale Superiore, Pisa Tarlera, Silvana, Universidad de la Republica of Uruguay Hubby. Bolyn, University of Georgia Lambert, Laurence, Universitat Miinchen Lovett, Jennie. Washington University Lyons, Emily, Indiana University Matuschewski, Kai, New York University Nde, Pius, Humboldt University, Germany Oli, Monica, Auburn University Paul, Kimberly, Princeton University Burroughs Wellcome Fund Frontiers In Reproduction Course Arechavaleta-Velasco. Fabian, National Institute of Nutrition, Mexico Beg, Mohd, National Institute of Immunology, India Chen, Chie-Pein, MacKay Memorial Hospital Moreno, Ricardo, Oregon Regional Primate Research Center Kumar, Ramasamy Sampath, University of Western Ontario Sanchez-Partida. Luis. University of Adelaide Santos. Joao, Oregon Regional Primate Research Center Biology Club of the College of the City of New York Belluscio, Leonardo, Columbia University Burroughs Wellcome Fund Biology of Parasitism Course Alves. Fabio. Fundaeao Oswaldo Cruz, Brazil Arevalo, Myriam, Universidad del Valle Cali Artis, David, University of Manchester Henze, Katrin. The Rockefeller University Burroughs Wellcome Fund Molecular Mycology Course Cisalpino. Patricia, Universidade Federal de Minas Gerais Edens, Heather, Montana State University Haycocks, Neil, University of Texas Medical Branch Lee, Samuel, Yale University School of Medicine Nagabhushan, Moolky, Loyola University, Chicago Santangelo, Rosaria. Public Health Research Institute, Italy Schaffrath, Raffael, University of Halle. Germany Sheppard. Don, McGill University Honors R57 C. Lalor Burdick Scholarship Shirasaki, Ryuichi, Osaka University Caswell Grave Scholarship Fund Champion, Mia, University of California, Davis Pollack, Anne, University of Arizona Tao, Haiyang, Ohio University Wagner, Eric, Duke University Charles Baker Metz and William Metz Scholarship Fund in Reproductive Biology Arechavaleta-Velasco, Fabian, National Institute of Nurtrition, Mexico Carabatsos, Mary Jo, Tufts University Euling, Susan, US Environmental Protection Agency Grazul-Bilska, Anna. North Dakota State University Halvorson, Lisa, Brigham and Women's Hospital Rulli, Susan, Hospital General de Ninos Ricardo Gutierrez Sanchez-Partida, Luis, University of Adelaide Daniel S. Grosch Scholarship Fund Castro, Hector. University of Florida Edwin Grant Conklin Memorial Fund Wagner. Eric, Duke University Frank R. Lillie Fellowship and Scholarship Fund Lee, Agnes, Yale University Strieker, Jesse, Duke University Vos. Johannes, University of Massachusetts Gary N. Calkins Memorial Scholarship Fund Bjornsson, Christopher, University of Manitoba Herbert W. Rand Fellowship and Scholarship Fund Azouz, Rony. University of California, Davis Battaglia, Francesco, SISSA, Italy Bi, Guoqiang, University of California. San Diego Brenner, Naama, NEC Research Institute Buschbeck, Elke. Cornell University Cai, Rick, University of California, Los Angeles d'Avella. Andrea, Massachusetts Institute of Technology Fairhall, Adrienne, Weizmann Institute. Israel Fellows, Matthew, Brown University Hartings. Jed, University of Pittsburgh Kepecs, Adam, Brandeis University Klug, Achim, University of Texas Lee, Ann, Brown University Machens, Christian, Humboldt University, Germany Majewska. Anna, Columbia University Pollack, Anne, University of Arizona Ruggiano, Stephanie, Boston University Shaub, Amy, University of North Carolina. Chapel Hill Scares, Daphne, University of Maryland Van Rossum. Mark, University of Pennsylvania Weber, Stacy, Ohio University Wright, Brian, University of California, San Francisco Zhang, Ying, Harvard Medical School Howard A. Schneiderman Endowed Scholarship Buschbeck, Elke, Cornell University Shirasaki, Ryuichi. Osaka University Tahmci, Emilios. Boston University Wonsettler, Angela, Marshall University School of Medicine Howard Hughes Medical Institute Educational Scholarship Funding Berggren, Kirsten, University of Vermont Bjornsson, Christopher, University of Manitoba Castro, Hector, University of Florida Chang, Sunghoe, University of Illinois Gladfelter, Amy, Duke University Jacobson. Eyal, Technion, Israel Locke, Emily, Johns Hopkins University Mansharamani, Malini, Texas Tech University Health Science Center Marchant. Jonathan. University of California. Irvine Ober, Elke. Max-Planck-Institut Tubingen Pappu. Kartik. Wesleyan University Roch. Fernando. Wellcome/CRC Institute. United Kingdom Rozowski. Marion, Wellcome/CRC Institute. United Kingdom Runt't. Linda, University of Connecticut Shaub, Amy, George Washington University Medical Center Strieker, Jesse, Duke University Wagner, Eric, Duke University Zhou. Ming. State University of New York. Buffalo Indo-U.S. Contraceptive and Reproductive Health Initiative Program Award Beg. Mohd. National Institute of Immunology. India International Brain Research Organization Scholarships Burzio, Veronica, University of Chile Concha, Miguel, University of Chile Gallo. Gianluca, University of Minnesota Miiller, Ferenc, IGBMC. Strasbourg St. Amant, Louis. McGill University Wang, Feng, Yale University Jacques Loeb Founders' Scholarship Fund Gladfelter, Amy. Duke University Marjorie W. Stetten Scholarship Fund Avila. Antonia. CINVESTAV-IPN, Mexico Pepi, Milva, University of Siena Speirs, Kendra, University of Pennsylvania Massachusetts Space Grant Consortium Awards Monteiro, Antonia, Harvard University Rocha-Olivares, Axayacatl. Scripps Institution of Oceanography Rosenthal, Benjamin. Harvard University R58 Annual Report MBL Pioneers Scholarship Fund Geraci, Fabiana, Dip di Biologia Cellulare e dello Sviluppo, Italy Lupo. Guiseppe. University of Pisa Ober, Hike, Max-Planck-Institut Tubingen Vonica, Alin, Cornell University Medical Center Merck & Company, Inc. Scholarships Locke, Emily, Johns Hopkins University Paul, Kimberly, Princeton University Runt't, Linda. University of Connecticut Saxowsky, Tina, Johns Hopkins School of Medicine Speirs, Kendra, University of Pennsylvania Waller, Ross, University of Melbourne Zaph, Colby, University of Victoria Billing. Susan. US Environmental Protection Agency Grazul-Bilska. Anna, North Dakota State University Halvorson, Lisa, Brigham and Women's Hospital McBnde. Helen. University of Utah McCauley, David. Pennsylvania State University Smith, Katherine, University of Virginia Stimson, Laura, University of Arizona Surdna Foundation Scholarship Ghazi, Arjuman, National Centre for Biological Sciences, India Lupo, Giuseppe, University of Pisa Pappu. Kartik. Wesleyan University Roch, Fernando, Wellcome/CRC Institute, United Kingdom Tahinci, Emilios, Boston University Mountain Memorial Fund Scholarship Chenevert, Janet, CNRS, France Deavours. Bettina, Virginia Tech Lam, Phoebe, Princeton University Lanntina. Samuel, Emory University Omara, Felix, Universite de Quebec Zaarour, Rania. Yale University Pfizer Inc. Endowed Scholarship Fund Locke, Emily. Johns Hopkins University Runft, Linda, University of Connecticut Phillip H. Presley Scholarship Award, Funded by Carl Zeiss, Inc. Kappler, Andreas, University of Konstanz Paemeleire. Koen, University of Ghent Paliulis. Leocadia. Duke University Pepi. Milva. University of Siena Rossi. Francesco, Scuola Normale Superiore, Pisa Takasu, Mari, Harvard University Planetary Biology Internship Awards Klappenbach. Joel, Michigan State University Spear. John. Colorado School of Mines Ruth Sager Memorial Scholarship Weber, Stacy. Ohio University S. O. Mast Memorial Fund d'Avella, Andrea, Massachusetts Institute of Technology Komarova, Svetlana. NASA Ames Research Center Society for Developmental Biology Scholarships Carabatsos. Mary Jo. Tufts University Chen. James, Harvard University Walter L. Wilson Endowed Scholarship Fund Mansharamani. Malini. Texas Tech University Health Science Center William F. and Irene C. Diller Memorial Scholarship Fund Champion. Mia. University of California. Davis William Morton Wheeler Family Founders' Scholarship Bjomsson. Christopher. University of Manitoba Soares. Daphne. University of Maryland Zhou, Ming, State University of New York. Buffalo William Randolph Hearst Foundation Scholarships Wang, Jing. Bell Laboratories Lee, Agnes, Yale University William Townsend Porter Fellowship and Scholarship Fund Anderson, Tonya. University of California, Los Angeles Foster. Andrea, Stanford University Freeman, Antoinette. Boston University School of Medicine Hinojos, Cruz, University of Texas, Houston McFarlane, Matthew, Stanford University McGiffert. Christine, University of California, San Diego Tafan, Tsahai, University of California, San Diego World Health Organization Scholarships Arechavaleta-Velasco, Fabian. National Institute of Nutrition, Mexico Cohen. Debora, IBYME, Argentina Rulli, Susan. Hospital General de Ninos. Argentina Honors R59 Post Course Research Awards Brinda Dass, Texas Tech University Health Sciences Center, Physiology Jonathan Marchant, University of California, Irvine. Physiology Bettina Deavours, Virginia Tech, Physiology David McCauley. Penn State University, Embryology James Hitt, SUNY Health Science Center. Syracuse, Neural Systems Linda Runft, University of Connecticut, Physiology and Behavior Tshai Tafari, University of California. San Diego, Physiology Adam Kepecs. Brandeis University. Neural Systems and Behavior Sinju Tauhata. Dep. de bioquimica FMRP/USP. Brazil. Shann Kim. University of Illinois. Chicago. Physiology Physiology Malini Mansharamani. Texas Tech University Health Science Center. Johannes Vos. University of Massachusetts. Physiology Physiology Board of Trustees and Committees Corporation Officers & Trustees Chairman of the Board of Trustees, Sheldon J. Segal. The Population Council Co-Vice Chair of the Board of Trustees, Frederick Bay, Josephine Bay Paul and C. Michael Paul Foundation Co-Vice Chair of the Board of Trustees, Mary J. Greer, New York. NY President of the Corporation, John E. Dowling. Harvard University Director and Chief Executive Officer, John E. Burris, Marine Biological Laboratory* Treasurer of the Corporation, Mary B. Conrad. Fiduciary Trust International* Clerk of the Corporation, Neil Jacobs, Hale and Dorr Chair of the Science Council, Kerry S. Bloom. University of North Carolina* Class of 2002 Class of 1999 Mary-Ellen Cunningham, Grosse Pointe Farms. MI Darcy Brisbane Kelley, Columbia University Laurie J. Landeau. Marinetics. Inc. Burton J. Lee, III, Vero Beach, FL Robert E. Mainer, The Boston Company Jean Pierce, Boca Grande. FL Class of 2000 Alexander W. Clowes, University of Washington School of Medicine Story C. Landis, Case Western Reserve University Irwin B. Levitan, Brandeis University G. William Miller. G. William Miller and Co.. Inc. Frank Press, The Washington Advisory Group Christopher M. Weld. Sullivan and Worcester Class of 2001 Porter Anderson. North Miami Beach. FL Frederick Bay, Josephine Bay Paul and C. Michael Paul Foundation, Inc. Martha W. Cox. Hobe Sound, FL Mary J. Greer. New York. NY William C. Steere, Jr.. Pnzer Inc. Gerald Weissmann. New York L'niversity School of Medicine *Ex officio Sydney M. Cone, III. Cleary. Gottlieb. Steen & Hamilton John R. Lakian. The Fort Hill Group. Inc. Joan V. Ruderman. Harvard Medical School Sheldon J. Segal, The Population Council William T. Speck, New York Presbyterian Hospital Alfred Zeien. The Gillette Company Honorary Trustees James D. Ebert. Baltimore. MD William T. Golden. New York. NY Ellen R. Grass. The Grass Foundation Trustees Emeriti Edward A. Adelberg, Yale University John B. Buck. Sykesville. MD Seymour S. Cohen, Woods Hole, MA Arthur L. Colwin, Key Biscayne, FL Laura Hunter Colwin, Key Biscayne, FL Donald Eugene Copeland, Woods Hole, MA Sears Crowell, Jr., Indiana Lmiversity Alexander T. Daignault. Falmouth. MA (deceased) Teru Hayashi, Woods Hole, MA Ruth Hubhard, Cambridge. MA Lewis Kleinholz, Reed College Maurice Krahl. Tucson, AZ C. Ladd Prosser. University of Illinois W.D. Russell-Hunter. Syracuse University John W. Saunders, Waquoit. MA D. Thomas Trigg. Wellesley, MA Walter S. Vincent, Woods Hole. MA Directors Emeriti James D. Ebert, Baltimore, MD Paul R. Gross, Falmouth, MA Harlyn O. Halvorson, Woods Hole, MA K60 Trustees and Committees R61 Executive Committee of the Board of Trustees Sheldon J. Segal. Chair Frederick Bay. Co-Vice Chair Mary J. Greer, Co- Vice Chair John E. Burris* Ronald L. Calabrese (1998) Kerry S. Bloom Mary B. Conrad Mary Ellen Cunningham Robert Mainer Joan V. Rudemian Gerald Weissmann Science Council Ronald L. Calabrese. Chair (8/98) Donald Abt (1999) Clay M. Armstrong (8/98-8/2000) Peter Armstrong (8/98-8/2000) Vincent E. Dionne (1999) John Dowling (8/98) Barbara Ehrlich (1999) Laurinda Jaffe (8/98-8/99) Charles Hopkinson (8/98-8/2000) Bruce J. Peterson (8/98) Mitchell Sogin (8/98-8/2000) Standing Committees of the Board of Trustees Development Mary Ellen Cunningham. Chair Porter W. Anderson Robert Barlow Fred Bay Mary B. Conrad Martha Cox James Ebert Philip Grant Neil Jacobs John Lakian Burton Lee G. William Miller Jean Pierce William Speck William Steere Christopher Weld Facilities & Capital Equipment Joan Ruderman, Chair Porter W. Anderson Frederick Bay Lawrence Cohen Neal Cornell Story Landis Irwin Levitan Jean Pierce Frank Press Christopher Weld Investment Robert Mainer. Chair Svdnev M. Cone Mary B. Conrad John R. Lakian G. William Miller Sheldon Segal Alfred Zeien Finance Robert Mainer. Chair Alexander Clowes Sydney M. Cone Mary B. Conrad Donald DeHart Neil Jacobs Darcy Kelley John R. Lakian Laurie Landeau Werner Loewenstem Robert Manz G. William Miller Ronald O'Hanley Alfred Zeien Nominating Gerald Weissmann Ronald L. Calabrese Alexander Clowes Martha Cox Mary Ellen Cunningham Mary Greer Story Landis Sheldon Segal William Steere Standing Committees of the Corporation and Science Council Buildings and Grounds Lawrence B. Cohen. Chair Barbara C. Boyer Alfred B. Chaet Richard Cutler* William R. Eckberg *Ex officio R62 Annual Report Barry Fleet* Ferenc Harosi Joe Hayes* Bruce J. Peterson Kenyon S. Tweedell Ivan Valiela Education Committee John Dowling, Chair Kerry S. Bloom Elaine Bearer Vincent Dione Paul Dunlap Rachel Fink Roger Hunlon Holger Jannasch George M. Langford Michael Mendelsohn Steve Zottoli Ron Calahrese* E.A. Dawidowicz* Dorianne Chrysler Mebane* LouAnn King* Robert P. Malchow Darrell R. Stokes Ann E. Stuart Janis C. Weeks MBL/WHOI Library Joint Advisory Committee David Shepro, Chair. MBL Judy Ashmore, MBL* David Dow, NMFS Daniel Fornan. WHOI G. Richard Harbison, WHOI John Hobbie, MBL Sylvia Kane, NMFS Mark Kurz, WHOI Colleen Hurter, WHOI* Cathy Norton, MBL* James Robb. USGS Birgit Rose, MBL Peter J.S. Smith. MBL Bruce Warren. WHOI Fellowships Thoru Pederson. Chair Linda Deegan Barbara Ehrlich George M. Langford Jose Lemos Cindy Lee VanDover E.A. Dawidowicz* Sandra Kautmann* Research Services and Space Housing, Food Service and Child Care Carole Browne, Chair Kerry S. Bloom Hans Laufer, Chair Peter B. Armstrong Neal Cornell Richard Cutler* E.A. Dawidowicz* Kenneth Foreman Louis M. Kerr* David Landowne Andrew Mattox* Merle Mizell Peter J.S. Smith Paul Steudler Ivan Valiela Discovery: The Campaign for Science at the Marine Biological Laboratory Steering Committee Frederick Bay, Campaign Chair William T. Golden. Honorary Chair Ellen R. Grass, Honorary Chair Alexander W. Clowes. Vice-Chair Martha W. Cox, Vice-Chair G. William Miller, Vice-Chair Gerald Weissmann, Vice-Chair Porter W. Anderson Robert B. Barlow. Jr. Norman Bernstein Jewell Plummer Cobb Mary B. Conrad Mai> I'llen Cunningham *Ex < John E. Dowling James D. Ebert Gerald D. Fischbach Robert D. Goldman Mary J. Greer M- Howard Jacobson Laurie J. Landeau George M. Langford Burton J. Lee, III Jean Pierce Robert A. Prcndergast David Shepro William T. Speck William C. Steere, Jr. Christopher M. Weld Alfred M. Zeien Trustees and Committees R63 Council of Visitors Norman B. Asher, Esq., Hale and Dorr, Boston. MA Mr. Donald J. Bainton. Chairman & CEO, Conlinental Can Co., Boca Raton, FL Mr. David Bakalar, Chestnut Hill. MA Mr. Charles A. Baker, The Liposome Company, Inc., Princeton, NJ Dr. George P. Baker, Massachusetts General Hospital, Boston, MA Dr. Sumner A. Barenberg. Bernard Technologies, Chicago, 1L Mr. Robert P. Beech. President/CEO. Component Software International. Inc., Mason, Ohio Mr. George Berkowitz. Chairman and Founder, Legal Sea Foods, Allston, MA Dr. Elkan R. Blout, Harvard Medical School, Boston. MA Mr. and Mrs. Philip Bogdanovitch. Lake Clear. New York Mr. Malcolm K. Brachman. Northwest Oil Company. Dallas, TX Dr. Goodwin M. Breinin. New York University Medical Center, New York, NY Mr. John Callahan, President, Carpenter. Sheperd & Warden, New London, NH Mrs. Elizabeth Campanella, West Falmouth, MA Thomas S. Crane, Esq., Mintz Levin Cohen Ferris Glovsky & Popeo, PC, Boston, MA Dr. Stephen D. Crocker. Chief Technology Officer, Cyber Cash Inc., Reston. VA Ms. Lynn W. Piasecki Cunningham, Film and Videomaker, Piasecki Productions, Brookline, MA Dr. Anthony J. Cutaia, Sr. Director, Office of Health Issues. Anheuser-Busch. Inc.. St. Louis, Missouri Dr. Georges de Menil. DM Foundation. New York, NY Mrs. Sara Greer Dent, Chevy Chase, MD Mr. D. H. Douglas-Hamilton. Vice President, Research and Development. Hamilton Thorne Research, Beverly. MA Mr. Benjamin F. Du Pont. Du Pont Company, Deepwater, New Jersey Dr. Sylvia A. Earle, Founder, Deep Ocean Engineering. Oakland. CA Mr. Anthony B. Evnin, General Partner, Venrock Associates, New York, NY Stuart Feiner, Esq., Vice President and Secretary, General Counsel. Inco Limited. Toronto, Ontario, Canada Mrs. Hadley Mack French, Consultant, Edsel & Eleanor Ford House, Grosse Pointe Farms. MI Mr. William J. Gilbane. Jr.. Gilbane Building Company, Providence. Rl Dr. Michael J. Goldblatt, Intelligent Biocides, Tewksbury. MA Mr. Maynard Goldman, President, Maynard Goldman & Associates, Boston, MA Ms. Charlotte I. Hall, Edgartown. MA Mr. Thomas J. Hynes, Jr.. President, Meredith & Grew, Inc., Boston, MA Mr. M. Howard Jacobson, Bankers Trust Westborough. MA Mrs. Elizabeth Ford Kontulis, New Canaan. CT Mr. and Mrs. Robert Lambrecht, Boca Grande. FL Dr. Catherine C. Lastavica. Tufts University School of Medicine. Boston. MA Mr. Joel A. Leavitt, Boston, MA Mr. Stephen W. Leibhol/. President. TechLabs. Inc.. Huntingdon. PA Mrs. Margaret Lilly, West Falmouth, MA Mr. George W. Logan, Chairman, Valley Financial Corp., Roanoke, VA Mr. Michael T. Martin, SportsMark, Inc.. New York, NY Mrs. Christy Swift Maxwell. Grosse Pointe Farms. MI Mr. Ambrose Monell. G. Unger Vetlesen Foundation, Palm Beach, FL Dr. Mark Novitch, Washington, DC Ms. Julie Packard, Executive Director, Monterey Bay Aquarium, Monterey, CA Mr. David R. Palmer, Founder & Managing Director, David Ross Palmer & Associates, Waquoit, MA Dr. Roderic B. Park, Richmond. CA Mr. Santo P. Pasqualucci, President/CEO Falmouth Co-Operative Bank. Falmouth. MA Mr. Robert Pierce, Jr., Pierce Aluminum Co., Canton, MA Mr. Richard Reston, Editor and Publisher, Vineyard Gazette, Edgartown, MA Mr. Marius Robinson, Managing Partner, Fundamental Investors Ltd., Key Biscayne, FL John W. Rowe, M.D., President, Mt. Sinai School of Medicine and Mt. Sinai Medical Center, New York, NY Mr. Edward Rowland, Tucker, Anthony, Inc.. Boston. MA Mr. Gregory A. Sandomirsky, Mintz Levin Cohen Ferris Glovsky & Popeo, PC, Boston, MA Mrs. Mary Schmidek. Marion. MA Dr. Cecily C. Selby. New York. NY Mr. Robert S. Shifman. St. Simon's Island, GA Mr. and Mrs. Gregory Skau, Grosse Pointe Farms. MI Mr. Malcolm B. Smith. Vice Chairman. General American Investors Co., New York, NY Mr. John C. Stegeman, Owner, Campus Rentals, Ann Arbor, MI Mr. Joseph T. Stewart. Jr.. Skillrnan. NJ Mr. John W. Stroh. Ill, Chief Executive Officer. The Stroh Brewery Company, Detroit. MI Mr. Gerard L. Swope. Washington. DC Mr. John F. Swope, Concord, NH Mr. and Mrs. Stephen E. Taylor, Boston, MA Mrs. Donna Vanden Bosch-FIynn, Spring Lake. NJ Mrs. Carolyn W. Verbeck, Vineyard Haven, MA Mr. Benjamin S. Warren, III, Grosse Pointe Farms, MI Nancy B. Weinstein, R.N., The Hospice, Inc., Glen Ridge, NJ Stephen S. Weinstein, Esq., Morristown, NJ Mr. Frederick J. Weyerhaeuser, Beverly, MA Mr. Tony L. White, The Perkin Elmer Corporation. Norwalk. CT Dr. Torsten N. Wiesel. President Emeritus, The Rockefeller University, New York, NY Administrative Support Staff 1 Biological Bulletin Greenberg, Michael J., Editor-in-Chief Hinkle, Pamela Clapp. Managing Editor Burns, Patricia Gibson. Victoria R. Schachinger. Carol H. Financial Sen'ices Office Lane, Jr., Homer W., Chief Financial Officer Roddy, Timothy, Chief Financial Officer Bowman, Richard, Controller Arbnso, Janis Barry, Maureen Dwyer, Patricia E. Eidelman, Dana Hopkins, Ann E. Lancaster, Cindy Poravas. Maria Ranzinger. Laura Sprague, Patricia A. Stark, Judy M. Stellrecht, Lynette Slock Room Schorer, Timothy M., Supervisor Capano, Holly 2 O'Connor-Lough. Susan Purchasing Hall Jr., Lionel E., Supervisor Shamon, Lynne R. Stone, Janice G. 2 Director's Office Burris, John E.. Director and Chief Executive Officer Donovan, Marcia H. MacNeil, Jane L. External Affairs Carotenuto, Frank C., Director Butcher. Valerie 1 Including persons who joined or left the staff during 1998. : Summer or temporary. Callahan Jr., John L. : Faxon. Wendy P. Martin, Theresa H. Maxwell, Thanh L. 2 Patch-Wing. Dolores Quigley. Barbara A. Scibek, John C. Shaw. Kathleen L. Associates Program Bohr. Kendall B. Brown. Shannon K. 2 Gault, Miciah Bay 2 Communications Office Hinkle, Pamela Clapp, Director Burton. Anne E. Flynn, Bridget Hinkle. Kristen" Joslin, Susan Liles. Beth R Housing and Conferences King. LouAnn D.. Director Barry, Maureen J. Grasso, Deborah Hanlon, Arlene K. 2 Johnson-Herman. Frances N. Masse, Todd C. Perito, Diana Switchboard Baker, Ida M. : Ridley, Alberta W. 2 Human Resources Goux. Susan P., Director Cox, Sarah 2 Orange. Stacey B. Houser, Carmen Renaud, Nina L. Marine Resources Center Hanlon, Roger T.. Director Moni/., Priscilla R64 Administrative Support Staff R65 Aquatic Resources Department Enos, Jr., Edward G., Superintendent Bourque, Ryan M. 2 Chappell, P. Dreux 2 DeGiorgis, Joseph A. 2 Grossman, William M. Gudas. Christopher N. 2 Kilpamck. Brian 2 Klimm III, Henry W. Luther. Herbert Mansfield, Darren P. 2 Sexton, Andrew W. Smith, Gary 2 Sullivan, Daniel A. Tassinari, Eugene MRC Life Support System Mebane. William N., Systems Operator Hanley, Janice S. Kuzirian, Alan Solbo, Jr.. Steven 2 Stukey, Jetley M. Till, Geoffrey A. MBLAVHOI Library Norton, Catherine N., Director Ashmore, Judith A. Costa, Marguerite E. Crocker, Daniel 2 Cullen. Cynthia M. 2 Deveer, Joseph M. Duda, Laurel E. Farrar, Stephen R. L. Medeiros, Melissa Monahan. A. Jean Moniz, Kimberly L. Nelson. Heidi Riley, Jacqueline Swasey, Anne E. 2 Copy Center Mountford, Rebecca J., Supervisor Abisla. Richard L. 2 Clark, Tamara L. Delaney. Elizabeth S. (Suwijn) 2 Kefeauver, Lee LaPlante, Robert F. Mancini. Mary E. Sorocco. Debra 2 Wallace, Jennifer 2 Warner, Kathleen 2 Information Systems Division Smith, Adrian P., Assistant Director Berrios, Kelly 2 Ennis, Douglas E. 2 Gage, Timothy J. 2 Katz, Corey 2 Malchow, Robert 2 Mountford, Rebecca J. Moynihan, James V. Remsen, David P. Renna, Denis J. Space. David B. Safety Sen'ices Mattox, Andrew H.. Environmental. Health, and Safety Manager Bradley. Margaret 2 O'Neill, Maureen D. 2 Sen'ice, Projects and Facilities Cutler. Richard D.. Director Enos, Joyce B. Apparatus Baptiste, Michael G. Barnes. Franklin D. Haskins, William A. Building Senices & Grounds Hayes, Joseph H., Superintendent Anderson. Lewis B. Atwood, Paul R. Baker, Harrison S. Barnes. Susan M. Berrios, Jessica L. 2 Boucher, Richard L. Brenerman, Brian 2 Brereton, Richard S. 2 Callahan. John J. Cameron, Lawrence M. 2 Collins. Paul J. Cowan, Matthew B. 2 Cutler, Matthew D. 2 Dirnond, Jay 2 Dorris. John J. Eldridge, Myles 2 Fernandez, Peter R. 2 Gibbons, Roberto G. Gonsalves, Nelson Gray, Joshua 2 Hannigan. Catherine Harrington. James D. Illgen. Robert F. Lawrence. Adam 2 Ledwell. L. Patrick 2 Luther, Herbert Lynch, Henry L. Maccaro. Jackie Mayock, Michael J. 2 McNumara, Noreen M. McQuillan, Jeffrey 2 Plant, Stephen W. Rattacasa, Frank" Ryan. Timothy A. : Sholkovitz.. David 2 Silva. Cynthia C. Stites, Clint 2 Tardif. Joseph G. R. 2 Ware, Lynn M. Plant Operations anil Maintenance Fleet. Barry M.. Superintendent Cadose. James W., Maintenance Supervisor R66 Annual Report Barnes, John S. Blunt, Hugh F. Bourgoin. Lee E. Carini, Robert J. Carroll, James R. Deree. Dana J. Fish Jr., David L. Fuglister, Charles K. Goehl, George Gonsalves, Jr.. Walter W. Hathaway, Peter J. Henderson. Jon R Justason, C. Scott Langill. Richard Lochhead, William M. McAdarns III, Herbert M. McHugh. Michael O. Mills, Stephen A. Olive, Jr.. Charles W. Schoepf, Claude Settlemire, Donald Shepherd. Denise M. Sylvia, Frank E. 2 Toner, Michael Wetzel, Ernest D. 2 Photolab Nelson, Linda M. Research Administration & Educational Programs Dawidowicz, Eliezar A.. Director Hamel. Carol C. Kaufmann. Sandra J. Kefauver, Lee Iwaszko, Nicole 2 Lynn, Rebecca Mebane. Dorianne C. Malmude-Davis, Anna 2 Palmer, Pamela 2 Patten. Brooke A. 2 Stukey, Jetley Central Microscopy Facility and General Use Rooms Kerr, Louis M.. Supervisor DeProto, Jamin E. 2 Luther, Herbert Peterson. Martha B. Josephine Bay Paul Center for Comparative Molecular Biology and Evolution Administrative Staff Harris, Marian Lim, Pauline Journal of Membrane Biology Loewenstein. Werner R., Editor Fay, Catherine H. Howard Isenberg. Linda L. Lynch, Kathleen F. Satellite/Periwinkle Children 's Programs Robinson. Paulina H. 2 Browne, Jennifer L. 2 Collins, Anne E. 2 Curran, Kelly 2 Douglas, Alicia D. 2 Fitzelle. Annie 2 Gallant, Carolyn A. 2 Gallant, Cynthia 2 Guiffrida, Beth 2 Griffin, Courtney A. 2 Jenkins, Michelle 2 Laundy, Jennifer 2 McCusker. Stephanie 2 Robinson, Jayma L. 2 NASA Center for Advanced Studies in the Space Life Sciences Dawidowicz. Eliezar A., Administrator Amit, Udem P. Ecosvstems Center Administrative Staff Berthel. Dorothy J. Donovan, Su/.anne J. Nunez, Guillermo Seifert, Mary Ann Members of the Corporation Life Members Acheson, George H., 25 Quissett Avenue. Woods Hole, MA 02543 Adelberg, Edward A., 204 Prospect Street. New Haven. CT 065 1 1 - 2107 Afzelius. Bjorn, University of Stockholm, Wenner-Gven Institute. Department of Ultrastructure Research. Stockholm, SWEDEN Amatniek, Ernest, address unknown Arnold, John M., 329 Sippewissett Road, Falmouth. MA 02540 Bang, Betsy G., 76 F. R. Lillie Road. Woods Hole, MA 02543 Bartlett. James H., University of Alabama. Department of Physics. Box 870324. Tuscaloosa. AL 35487-0324 Berne, Robert M., University of Virginia School of Medicine, Department of Physiology. Box 1116, MR4. Charlottesville, VA 22903 Bernheimer, Alan W., New York University Medical Center, Department of Microbiology, 550 First Avenue. New York. NY 10016 Bertholf, Lloyd M., Westminster Village. #2114, 2025 East Lincoln Street, Bloomington. IL 61701-5995 Bosch, Herman F., P.O. Box 353, Woods Hole, MA 02543 Buck, John B., Fairhaven C-020, 7200 Third Avenue. Sykesville, MD 21784 Burbanck, Madeline P., P.O. Box 15134. Atlanta. GA 30333 Burbanck. \\illiam D.. P.O. Box 15134. Atlanta. GA 30333 Carlson, Francis D., Johns Hopkins University. Biophysics Department Jenkins Hall, North Charles Street, Baltimore. MD 21218 (deceased) Clark, Arnold M., 53 Wilson Road, Woods Hole, MA 02543 Clark, James M., 258 Wells Road. Palm Beach. FL 33480-3625 Cohen, Seymour S., 10 Carrot Hill Road, Woods Hole, MA 02543- 1206 Colwin, Arthur L., 320 Woodcrest Road, Key Biscayne, FL 33149- 1322 Colwin, Laura Hunter, 320 Woodcrest Road. Key Biscayne, FL 33149-1322 Cooperstein. Sherwin J., University of Connecticut. School of Medicine, Department of Anatomy, Farmington. CT 06030-3405 Copeland. D. Eugene, Marine Biological Laboratory. Woods Hole. MA 02543 Corliss, John O., P.O. Box 2729, Bala Cynwyd, PA 19004-21 16 Costello, Helen M., Carolina Meadows, Villa 137, Chapel Hill, NC 27514-8512 Crouse, Helen, Rte. 3, Box 213. Hayesville. NC 28904 DeHaan, Robert I,., Emory University School of Medicine, Department of Anatomy & Cell Biology. 1648 Pierce Drive, Room 108. Atlanta. GA 30322 Dudley, Patricia L., 3200 Alki Avenue SW. #401. Seattle. WA 98116 Edwards, Charles, 3429 Winding Oaks Drive, Longboat Key, FL 34228 Elliott, Gerald F., The Open University Research Unit. Foxcombe Hall. Berkeley Road. Boars Hill. Oxford OX1 5HR, ENGLAND Failla, Patricia M., 2 1 49 Loblolly Lane, Johns Island, SC 29455 Ferguson, James K. W., 56 Clarkehaven Street. Thornhill, Ontario L4J 2B4, CANADA Glusman, Murray, New York State Psychiatric Institute. 722 W. 168th St.. Unit #70, New York, NY 10032 Goldman, David E., 140 Ter Heun Drive, Room 212. Falmouth. MA 02540 (deceased) Graham, Herbert, 36 Wilson Road, Woods Hole, MA 02543 Hamburger, Viktor, Washington University. Department of Biology. 740 Trinity Avenue. St. Louis. MO 63 1 30 Hamilton. Howard L., University of Virginia, Department of Biology, 238 Gilmer Hall, Charlottesville. VA 22901 Harding, Jr., Clifford V., 54 Two Ponds Road, Falmouth. MA 02540 Haschemeyer, Audrey E. V'., 21 Glendon Road, Woods Hole, MA 02543-1406 Hauschka, Theodore S., 333 Fogler Road, Bremen, ME 0455 1 Hayashi, Teru, 1 5 Gardiner Road, Woods Hole, MA 02543- 1113 Hisaw, Frederick L.. 1 765 SW Tamarack Street, Apt 11, McMinnville, OR 97128-7416 Hoskin, Francis C. G., c/o Dr. John E. Walker, U.S. Army Natick RD&E Center. SAT NC-YSM, Kansas Street, Natick, MA 01760- 5020 Humes, Arthur G., Marine Biological Laboratory. Boston University Marine Program, Woods Hole. MA 02543 Hunter, W. Bruce, 305 Old Sharon Road, Peterborough, NH 03458- 1736 R67 R6S Annual Report Hurwitz, Charles, Stratum VA Medical Center. Research Service. Albany. NY 1220S Kalz, George, Merck. Sharp and Dohme. Fundamental & Experimental Research Laboratory. PO Bo\ 2000, Rahway, NJ 07065 Kingsbury, John M., Cornell University, Department of Plant Biology. Plant Science Building. Ithaca. NY 14853 Kleinholz. Lewis, Reed College. Department of Biology, 3203 SE Woodstock Boulevard. Portland, OR 97202 Kusano, Kiyoshi, National Institutes of Health, Building 36, Room 4D- 20, Bethesda, MD 20892 Laderman, Ezra, Yale University, New Haven. CT 06520 LaMarche, Paul H., Eastern Maine Medical Center, 489 State Street, Bangor, ME 04401 Lauffer, Max A., Penn State University Medical Center, Department of Biophysics & Physiology. Hershey. PA 1 7033 LeFevre, Paul G., 1 5 Agassiz Road, Woods Hole. MA 02543 (deceased) Lochhead, John H., 49 Woodlawn Road, London SW6 6PS. UK Loevvus, Frank A., Washington State University, Institute of Biological Chemistry, Pullman, WA 99164 Loftfield, Robert B., University of New Mexico, School of Medicine. Albuquerque, NM 87131 Malkiel, Saul, 174 Queen Street, #9A, Falmouth. MA 02540 Marsh, Julian B., 9 Eliot Street. Chestnut Hill. MA 02467-1407 Martin, Lowell V., 10 Buzzards Bay Avenue, Woods Hole, MA 02543 Mathews, Rita W., East Hill Road, P.O. Box 237. Southfield. MA 01259-0237 Moore, John A., University of California. Department of Biology. Riverside, CA 92521 Moscona, Aron A., University of Chicago, Department Molecular Genetics & Cell Biology, Chicago. IL 60637 Musacchia, X. J., P.O. Box 5054, Bella Vista, AR 72714-0054 Nasatir, Maimon, P.O. Box 379, Ojai, CA 93024 Passano, Leonard M., University of Wisconsin, Department of Zoology. Birge Hall. Madison. WI 53706 Prosser, C. Ladd, University of Illinois, Department of Physiology, 524 Burrill Hall, Urbana. IL 61801 Prytz, Margaret McDonald, address unknown Ratner, Sarah, Public Health Research Institute, Department ol Biochemistry. 455 First Avenue, New York. NY 10016 Renn, Charles E., address unknown Reynolds, George T., Princeton University, Department of Physics, Jadwin Hall. Princeton, NJ 08544 Rice, Robert V., 30 Burnham Drive, Falmouth, MA 02540 Rockstein, Morris, 600 Biltmore Way. Apt. 805, Coral Gables. FL 33134 Ronkin, Raphael R., 3212 McKinley Street. NW. Washington. DC 20015-1635 Sanders, Howard L., Woods Hole Occanographic Institution, Woods Hole, MA 02543 Sato, Hidemi, Nagova University, 3-24-101. Oakinishi Machi, Toha Mie 517-0023, JAPAN Saz, Arthur K., Cieorgi )<: n University Medical School, Department of Immunology, Washington. DC 20007 Schlesinger, R. Walter, 7 Langley Road, Falmoulh, MA 02540-1809 Scott, Allan C., Colby College, Waterville. ME 04901 Silverstein, Arthur M.. Johns Hopkins University. Institute of the History of Medicine. 1900 E. Monument Street, Baltimore, MD 21205 Sjodin, Raymond A., University of Maryland. Department of Biophysics. Baltimore. MD 21201 Smith. Paul F., P.O. Box 264. Woods Hole. MA 02543-0264 Speer, John VV., 293 West Main Road, Portsmouth, RI 02871 Sperelakis, Nicholas, University of Cincinnati, Department of Physiology/Biophysics, 231 Bethesda Avenue, Cincinnati, OH 45267- 0576 Spiegel, Evelyn, Dartmouth College, Department of Biological Sciences, 204 Oilman, Hanover. NH 03755 Spiegel, Melvin, Dartmouth College, Department of Biological Sciences. 204 Gilman, Hanover, NH 03755 Steinhardt, Jacinto, 1508 Spruce Street, Berkeley, CA 94709 (deceased) Stephens, Grover C., University of California, School of Biological Sciences, Department of Ecolocy and Evolution/Biology, Irvine, CA 92717 Strehler, Bernard L., 2310 N. Laguna Circle Drive, Agoura, CA 9130I-2SS4 Sussman, Maurice, 72 Carey Lane, Falmouth, MA 02540 Sussman, Raquel B., Marine Biological Laboratory. Woods Hole, MA 02543 Szent-Gyorgyi, Gwen P., 45 Nobska Road, Woods Hole, MA 02543 Taylor, Robert E., 339 Gifford Street. Apt. 303, Falmouth, MA 02540 (deceased) Thorndike, W. Nicholas, Wellington Management Company, 200 State Street, Boston, MA 02104 Trager, William, The Rockefeller University, 1230 York Avenue. New York. NY 10021-6399 Trinkaus, J. Philip, 870 Moose Hill Road, Guilford, CT 06437 Villee, Jr., Claude A., Harvard Medical School, Carrel L, Countway Library. 10 Shattuck Street. Boston, MA 021 15 Vincent, Walter S., 16 F.R. Lillie Road, Woods Hole, MA 02543 Wald, Ruth, Harvard University. Biological Laboratories. Cambridge, MA 02138 Waterman. Talbot H., Yale University, Box 208103, 912 KBT Biology Department, New Haven, CT 06520-8103 Wigley, Roland L., 35 Wilson Road, Woods Hole, MA 02543 Wilber, Charles G., Colorado State University, Department of Biology, Forensic Science Laboratory, Fort Collins. CO 80523 (deceased) Members Aht, Donald A., Marine Biological Laboratory, Laboratory of Aquatic Animal Medicine and Pathology. Woods Hole, MA 02543 Adams, James A., 348 1 Paces Ferry Road. Tallahassee. FL 32308 Adelman, William J., 160 Locust Street. Falmoulh. MA 02540 Alkon, Daniel L., N1H Laboratory of Adaptive Systems. 36 Convent Drive. MSC 4124, 36/4A2I, Bethesda, MD 20892-4124 Allen, Garland E., Washington University. Department of Biology, Box 1 137, One Brookings Drive, Street Louis, MO 63130-4899 Allen, Nina S., North Carolina State University, Department of Botany, Box 7612, Raleigh. NC 27695 Alliegro, Mark C., Louisiana State University Medical Center. Department of Cell Biology and Anatomy. 1901 Perdido Street, New Orleans. LA 701 12 Anderson, Everett, Harvard Medical School, Department of Cell Biology, 240 Longwood Avenue, Boston, MA 021 15-6092 Anderson, John M., 110 Roat Street, Ithaca, NY 14850 Members of the Corporation R6M Anderson, Porter W., 100 Bayview Drive #2224. North Miami Beach, FL 33160 Armett-Kihel, Christine, University of Massachusetts. Dean of Science Faculty. Boston. MA 02125 Armstrong, Clay M., University of Pennsylvania School of Medicine. B701 Richards Building. Department of Physiology. 3700 Hamilton Walk. Philadelphia. PA 19104-6085 Armstrong, Ellen Prosser, 57 Milltield Street. Woods Hole, MA 02543 Arnold, William A., Oak Ridge National Laboratory. Biology Division. 102 Balsalm Road. Oak Ridge. TN 37830 Ashton, Robert W., Bay Foundation, 1 7 West 94th Street. New York, NY 10025 Atema, Jelle, Boston University Marine Program. Marine Biological Laboratory. Woods Hole. MA 02543 Baccetti, Baccio, University of Sienna. Institute of Zoology, 53100 Siena. ITALY Baker. Robert G., New York University Medical Center, Department Physiology and Biophysics, 550 First Avenue, New York, NY 10016 Baldwin, Thomas O., Texas A and M University. Department of Biochemistry and Biophysics. College Station, TX 77843-2128 Baltimore, David, California Institute of Technology, 204-31. Pasadena, CA 91125 Barlow, Robert B., SUNY Health Science Center at Syracuse, 750 East Adams Street, Center for Vision Research, 3258 Weiskotten Hall. Syracuse, NY 13210 Barry, Daniel T., 2415 Fairwmd Drive, Houston, TX 77062-4756 Barry. Susan R., Mount Holyoke College. Department of Biological Sciences. South Hadley, MA 01075 Bass. Andrew H., Cornell University, Department of Neurobiology and Behavior. Seely Mudd Hall, Ithaca, NY 14853 Battelle, Barbara-Anne, University of Florida, Whitney Laboratory, 9505 Ocean Shore Boulevard, Street Augustine. FL 32086 Bay, Frederick, Bay Foundation. 17 W. 94th Street. First Floor. New York. NY 10025-7116 Baylor, Martha B., P.O. Box 93. Woods Hole. MA 02543 Bearer, Elaine L., Brown University. Division of Biology and Medicine. Department of Pathology. Box G. Providence. RI 02912 Beatty, John M., University of Minnesota. Department of Ecology and Behavioral Biology, 1987 Gortner, Street Paul, MN 55108 Beauge, Luis Alberto, Instituto de Investigacion Medica. Department of Biophysics. Casilla de Correo 389. 5000 Cordoba, ARGENTINA Begenisich, Ted, University of Rochester, Medical Center, Box 642, 601 Elmwood Avenue. Rochester, NY 14642 Begg, David A.. University of Alberta, Faculty of Medicine, Department of Cell Biology and Anatomy. Edmonton. Alberta T6G 2H7, CANADA Bell, Eugene, Tissue Engineering, Inc.. 451 D Street, Boston, MA 02210 Benjamin, Thomas L., Harvard Medical School, Pathology. D2-230, 200 Longwood Avenue, Boston. MA 021 15 Bennett. Michael V. L., Albert Einstein College of Medicine. Department of Neuroscience, 1300 Morris Park Avenue, Bronx. NY 10461 Bennett, Miriam F., Colby College, Department of Biology. Waterville, ME 04901 Berg, Carl J., P.O. Box 681. Kilauea. Kauai. HI 96754-0681 Berlin, Suzanne T., 5 Highland Street, Gloucester. MA 01930 Bernstein, Norman, Columbia Realty Venture. 5301 Wisconsin Avenue, NW. #600. Washington. DC 20015-2015 Bezanilla, Francisco, Health Science Center, Department of Physiology, 405 Hilgard Avenue, Los Angeles, CA 90024 Biggers, John D., Harvard Medical School. Department of Physiology. Boston. MA 02115 Bishop. Stephen H., Iowa State University, Department of Zoology, Ames, IA 50010 Blaustein, Mordecai P., University of Maryland, School of Medicine, Department of Physiology, Baltimore. MD 21201 Blennemann, Dieter, 1117 East Putnam Avenue, Apt. #174. Riverside, CT 06878-1333 Bloom, George S., The University of Texas Southwestern Medical Center. Department of Cell Biology and Neuroscience. 5323 Harry Hmes Boulevard. Dallas. TX 75235-9039 Bloom, Kerry S., University of North Carolina. Department of Biology. 623 Fordham Hall CB#3280. Chapel Hill, NC 27599-3280 Bodznick, David A., Wesleyan University, Department of Biology, Lawn Avenue. Middletown. CT 06497-0170 Boettiger, Edward G.. 17 Eastwood Road, Storrs, CT 06268-2401 Boolootian, Richard A., Science Software Systems. Inc.. 3576 Woodcliff Road. Sherman Oaks. CA 91403 Borgese, Thomas A., Lehman College, CUNY. Department of Biology. Bedford Park Boulevard. West. Bronx. NY 10468 Borst, David W., Illinois State University. Department of Biological Sciences, Normal, 1L 61790-4120 Bowles, Francis P., Marine Biological Laboratory, Ecosystems Center, Woods Hole. MA 02543 Boyer, Barbara C., Union College, Biology Department, Schenectady. NY 12308 Brandhorst, Bruce P., Simon Fraser University, Institute of Molecular Biology/Biochemistry, Barnaby. B.C. V5A 1S6. CANADA Brinley, F. J., NINCDS/NIH. Neurological Disorders Program. Room 812 Federal Building, Bethesda, MD 20892 Bronner-Fraser, Marianne, California Institute of Technology, Beckman Institute Division of Biology. 139-74. Pasadena. CA 91125 Brown, Stephen C., SUNY. Department of Biological Sciences. Albany. NY 12222 Brown, William L., 80 Black Oak Road. Weston, MA 02193 Browne, Carole L., Wake Forest University. Department of Biology. Box 7325 Reynolds Station, Winston-Salem. NC 27109 Browne, Robert A., Wake Forest University. Department of Biology, Box 7325. Winston-Salem. NC 27109 Bucklin, Anne C., University of New Hampshire, Ocean Process Analysis Laboratory. 142 Morse Hall, Durham. NH 03824 Bullis, Robert A., Manne Biological Laboratory. 7 MBL Street, Woods Hole. MA 02543 Burger, Max M., Friedrich Miescher Institute. P.O. Box 2543. CH 4002 Basel, SWITZERLAND Burgess, David R., Boston College. Academic Vice President and Dean of Facilities, Bourneuf House, 84 College Road, Chestnut Hill. MA 02467-3838 Burgos, Mario H., IHEM Medical School, UNC Conicet, Casilla de Correo 56, 5500 Mendoza, ARGENTINA Burky, Albert, University of Dayton. Department of Biology. Dayton, OH 45469 Burris, John E., Marine Biological Laboratory, 7 MBL Street, Woods Hole, MA 02543 Burstyn, Harold Lewis, United States Air Force. Air Force Materiel Command, Rome Research Site RL/JA, 26 Electronic Parkway, Rome, NY 13441-4514 Bursztajn. Sherry, LSU Medical Center, 1501 Kings Highway, Building BR1F 6-13. Shreveport, LA 71130 Calabrese, Ronald L., Emory University, Department of Biology. 1510 Clifton Road. Atlanta. GA 30322 Callaway, Joseph C., New York Medical College. Department of Physiology. Basic Sciences Building. Valhalla. NY 10595 Cameron, R. Andrew, California Institute of Technology. Division of Biology 156-29, Pasadena, CA 91 125 R70 Annual Report Campbell, Richard H., Bang-Campbell Associates, Eel Pond Place, Box 402. Woods Hole, MA 02543 Candelas, Graciela C., University of Puerto Rico, Department of Biology. P.O. Box 23360, UPR Station. San Juan. PR 00931-3360 Cariello, Lucio, Stazione Zoologica "A. Dohm", Villa Comunale. 80121 Naples, ITALY Case, James F., University of California. Marine Science Institute. Santa Barbara. CA 93106 Cassidy, Joseph D., Providence College, Priory of Street Thomas Aquinas. Providence, Rl 02918-11001 Cavanaugh, Colleen M., Harvard University, Biological Laboratories, 16 Divinity Avenue, Cambridge, MA 02138 Chaet, Alfred B., University of West Florida, Department of Cell and Molecular Biology, 1 1000 University Parkway, Pensacola, FL 32514 Chambers, Edward L., University of Miami School of Medicine. Department of Physiology and Biophys., P.O. Box 016430, Miami. FL 33101 Chang, Donald C., Hong Kong University, Science and Technology, Department of Biology, Clear Water Bay, Kowloon, HONG KONG Chappell, Richard L., Hunter College, CUNY. Department of Biological Sciences. Box 210. 695 Park Avenue. New York, NY 10021 Child III, Frank M., 28 Lawrence Farm Road, Woods Hole. MA 02543-1416 Chisholm, Rex Leslie, Northwestern University, Medical School, Department of Cell Biology, Chicago, IL 6061 1 Citkowitz, Elena, Hospital of Street Raphael, Lipid Disorders Clinic, 1450 Chapel Street, New Haven, CT 0651 1 Clark, Eloise E., Bowling Green State University, Biological Sciences Department. Bowling Green, OH 43403 Clark, Hays, 150 Gomez Road, Hobe Sound, FL 33455 Clark, Wallis H., 12705 NW I 12th Avenue, Alachua, FL 32615 Claude, Philippa, University of Wisconsin, Department of Zoology, Zoology Research Building 125. 1 1 17 W Johnson Street, Madison, Wl 53706 Clay, John R., NIH-NINDS. Building 36. Room 2-CO2. Bethesda. MD 20892 Clowes, Alexander W., University of Washington, School of Medicine. Department of Surgery, Box 356410. Seattle. WA 98195-6410 Cobb, Jewel Plummer, California State University, 5151 University Drive, Health Center 205, Los Angeles, CA 90032-8500 Cohen, Carolyn, Brandeis University. Rosenstiel Basic Medical, Sciences Research Center, Waltham, MA 02254 Cohen, Lawrence B., Yale University School of Medicine, Department of Physiology. 333 Cedar Street, New Haven. CT 06520 Cohen, Maynard M., Rush Medical College, Department of Neurological Sciences, 600 South Paulina, Chicago, IL 60612 Cohen, William D., Hunter College. Department Biological Sciences, New York, NY 10021 Coleman, Annette W., Brown University, Division of Biology and Medicine, Providence. Rl 02912 Colinvaux, Paul, Smithsonian Tropical Research Institute. Unit 0948. Apo AA 34002-0948, USA Collier. Jack R., 3431 Highway. #107. P.O. Box 139. Effie. LA 71331 Collier, Marjorie McCann, 3431 Highway 107. P.O. Box 139. Effie, LA 71331 Cook, Joseph A., Edna McConnell Clark Foundation, 250 Park Avenue, New York. NY 10177-0026 Cornell, Neal W., Marine Biological Laboratory, Woods Hole. MA 02543 Cornwall, Melvin C., Boston University School of Medicine, Department of Physiology L714, Boston. MA 021 18 Corson, D. Wesley, Storm Eye Institute, Room 537, 171 Ashley Avenue. Charleston, SC 29425 Corwin, Jeffrey T., University of Virginia, School of Medicine, Department Otolaryngology and Neuroscience, Box 396. Charlottesville, VA 22908 Couch, Ernest F., Texas Christian University. Department of Biology, TCU Box 298930, Fort Worth. TX 76129 Cox, Rachel Llanelly, Woods Hole Oceanographic Institute, Biology Department. Woods Hole. MA 02543 Crane, Sylvia E., 438 Wendover Drive, Princeton, NJ 08540 Cremer-Bartels, Gertrud, Universitats Augenklinik, 44 Munster, GERMANY Crow, Terry J., University of Texas Medical School, Department of Neurobiology and Anatomy. Houston, TX 77225 Crowell, Sears, Indiana University, Department of Biology. Bloomington. IN 47405 Crowther, Robert J., Shriners Hospitals for Children. 51 Blossom Street. Boston, MA 021 14 Cunningham, Mary-Ellen, 62 Cloverly Road, Grosse Pointe Farms, MI 48236-3313 Cutler. Richard D., Marine Biological Laboratory. Woods Hole. MA 02543 Daignault, Alexander T., Edgewood #6308. 575 Osgood Street, North Andover, MA 01845 (deceased) Davidson, Eric H., California Institute of Technology. Division of Biology. 156-29. 391 South Holliston. Pasadena. CA 91 125 Davison, Daniel B., Bristol-Myers Squibb PR1. Biomformatics Department, 5 Research Parkway, Wallingford. CT 06492 Daw, Nigel W., 5 Old Pawson Road, Branford, CT 06405 Dawidowicz, Eliezar A., Marine Biological Laboratory. Office of Research Administration and Education, Woods Hole, MA 02543 De Weer, Paul J., University of Pennsylvania, B400 Richards Building. Department of Physiology, 3700 Hamilton Walk, Philadelphia. PA 19104-6085 Deegan, Linda A., Marine Biological Laboratory, The Ecosystems Center, Woods Hole, MA 02543 DeGroof, Robert C., 145 Water Crest Drive. Doylestown, PA 18901- 3267 Denckla, Martha Bridge, Johns Hopkins University. School of Medicine. Kennedy-Krieger Institute. 707 North Broadway, Baltimore. MD 21205 DePhillips, Henry A., Trinity College, Department of Chemistry, 300 Summit Street. Hartford, CT 06106 DeSimone, Douglas \V., University of Virginia, Department of Cell Biology. Box 439, Health Sciences Center. Charlottesville, VA 22908 Dettbarn, Wolf-Dietrich, Vanderbilt University. School of Medicine, Department of Pharmacology, Nashville. TN 37232 Dionne, Vincent E., Boston University Marine Program, Marine Biological Laboratory. Woods Hole. MA 02543 Dowling, John E., Harvard University, Biological Laboratories, 16 Divinity Street, Cambridge, MA 02138 Drapeau, Pierre, Montreal General Hospital, Department of Neurology, 1650 Cedar Avenue. Montreal. Que. H3G 1A4. CANADA DuBois, Arthur Brooks, John B. Pierce Foundation Laboratory. 290 Congress Avenue, New Haven. CT 06519 Duncan, Thomas K., Nichols College, Environmental Sciences Department. Dudley, MA 01571 Dunham, Philip B., Syracuse University, Department of Biology. 1 30 College Place. Syracuse. NY 13244-1220 Dunlap, Paul V., University of Maryland Biotechnology Institute. Center of Marine Biotechnology. Columbus Center. Suite 236, 701 East Pratt Street, Baltimore, MD 21202 Ebert, James I)., The Johns Hopkins University. Department of Members of the Corporation R71 Biology. Homewood, 3400 North Charles Street. Baltimore. MD 21218-2685 Eckberg, William R., Howard University. Department of Biology, P.O. Box 887. Administration Building. Washington, DC 20059 Edds, Kenneth T., R & D Systems, Inc., Hematology Division. 614 McKinley Place, NE, Minneapolis, MN 55413 Eder, Howard A., Albert Einstein College of Medicine, 1300 Morris Park Avenue. Bronx, NY 10461 Edstrom, Joan, 53 Two Ponds Road, Falmouth, MA 02540 Egyud, Laszlo G., Cell Research Corporation, P.O. Box 67209, Chestnut Hill. MA 02167-0209 Ehrlich, Barbara E., Yale University Medical School, B207 SHM. New Haven, CT 06473 Eisen, Arthur Z., Washington University, Division of Dermatology, Street Louis, MO 63110 Eisen, Herman N., Massachusetts Institute of Technology. Center for Cancer Research, El 7- 128, 77 Massachusetts Avenue, Cambridge, MA 02139-4307 Elder, Hugh Young, University of Glasgow, Institute of Physiology, Glasgow G12 8QQ, SCOTLAND Englund, Paul T., Johns Hopkins Medical School, Department of Biological Chemistry. 725 North Wolfe Street. Baltimore, MD 21205 Epel, David, Stanford University, Hopkins Marine Station, Ocean View Boulevard, Pacific Grove, CA 93950 Epstein, Herman T., 18 Lawrence Farm Road, Woods Hole, MA 02543 Epstein, Ray L., 1602 W. Olympia Street, Hernando. FL 34442 Farb, David H., Boston University School of Medicine, Department of Pharmacology L603, 80 East Concord Street, Boston. MA 02 1 1 8 Farmanfarmaian, A. Verdi, Rutgers University. Department of Biological Sciences. Nelson Biology Laboratory FOB 1059, Piscataway. NJ 08855 Feldman, Susan C., University of Medicine and Dentistry, New Jersey Medical School, 100 Bergen Street. Newark, NJ 07103 Festoff, Barry William, VA Medical Center, Neurology Service (151), 4801 Linwood Boulevard. Kansas City. MO 64128 Fink, Rachel D., Mount Holvoke College, Department of Biological Sciences, Clapp Laboratories, South Hadley, MA 01075 Finkelstein, Alan, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461 Fischbach, Gerald D., National Institute of Health, Neurological Disorders and Strokes, 31 Center Drive. MSC 2540, Bldg 31, Rm 8A03. Bethesda, MD 20892-2540 Fishman, Harvey M., University of Texas Medical Branch, Department of Physiology and Biophysics, 301 University Boulevard, Galveston, TX 77555-0641 Flanagan, Dennis, 12 Gay Street, New York, NY 10014 Fluck, Richard Allen, Franklin and Marshall College, Department of Biology, Box 3003, Lancaster, PA 17604-3003 Foreman, Kenneth H., Marine Biological Laboratory, Woods Hole, MA 02543 Fox, Thomas Oren, Harvard Medical School, Division of Medical Sciences. MEC 435. 260 Longwood Avenue, Boston, MA 021 15 Franzini-Armstrong, Clara, University of Pennsylvania, School of Medicine. 330 South 46th Street, Philadelphia, PA 19143 Fraser, Scott, California Institute of Technology, Beckman Institute 139-74, 1201 East California Boulevard, Pasadena, CA 91 125 Frazier, Donald T., University of Kentucky Medical Center, Department of Physiology and Biophysics, MS501 Chandler Medical Center. Lexington, KY 40536 French, Robert J., University of Calgary, Health Sciences Centre, Alberta, T2N 4NI, CANADA Fulton, Chandler M., Brandeis University, Department of Biology, MS 008, Waltham, MA 02454-91 10 Furie, Barbara C., Beth Israel Deaconess Medical Center. BIDMC Cancer Center, Kirstein 1, 330 Brookline Avenue, Boston, MA 02215 Furie, Bruce, Beth Israel Deaconess Medical Center, BIDMC Cancer Center, Kirstein 1, 330 Brookline Avenue, Boston, MA 02215 Furshpan, Edwin J., Harvard Medical School. Department of Neurobiology. 220 Longwood Avenue. Boston, MV\ 021 15 Futrelle, Robert P., Northeastern University, College of Computer Science, 360 Huntmgton Avenue, Boston, MA 021 15 Gabr, Howaida, Sue/. Canal University, Department of Marine Science. Faculty of Science, Ismailia, EGYPT Gabriel, Mordecai L., Brooklyn College. Department of Biology, 2900 Bedford Avenue, Brooklyn, NY 11210 Gadsby, David C., The Rockefeller University, Laboratory of Cardiac Physiology, 1230 York Avenue, New York, NY 10021-6399 Gainer, Harold, NIH. NINDS.BNP.DIR, Neurochemistry. Building 36, Room 4D20, Bethesda. MD 20892-4130 Galatzer-Levy, Robert M., 180 North Michigan Avenue. Suite 2401, Chicago, IL 60601 Gall, Joseph G., Carnegie Institution, 1 15 West University Parkway, Baltimore, MD 21210 Garber. Sarah S., Allegheny University of the Health Sciences, Department of Physiology, 2900 Queen Lane, Philadelphia, PA 19124 Gascoyne, Peter, University of Texas. M. D. Anderson Cancer Center, Experimental Pathology. Box 89, Houston, TX 77030 Gelperin, Alan, Bell Labs Lucent, Department Biology Comp., Rm 1C464, 600 Mountain Avenue, Murray Hill, NJ 07974 German, James L., The New York Blood Center, Laboratory of Human Genetics, 310 East 67th Street, New York, NY 10021 Gibbs, Martin, Brandeis University, Institute for Photobiology of Cells and Organelles, Waltham. MA 02254 Giblin, Anne E., Marine Biological Laboratory. The Ecosystems Center. Woods Hole. MA 02543 Gibson, A. Jane, Cornell University, Department of Biochemistry. Biotech. Building. Ithaca, NY 14850 Gifford, Prosser, 540 North Street, SW. Apt. #S-903. Washington, DC 20024-4557 Gilbert, Daniel L., National Institutes of Health, Biophysics Sec., BNP, Building 36, Room 5A-27, Bethesda, MD 20892 Giudice, Giovanni, Universita di Palermo, Dipartimento di Biologia, Cellulare e Dello Sviluppo, 1-90123 Palermo. ITALY Giuditta, Antonio, University of Naples, Department of General Physiology, Via Mezzocannone 8, Naples, 80134, ITALY Glynn, Paul, P.O. Box 6083, Brunswick, ME 04011-6083 Golden, William T., Chairman Emeritus, American Museum of Natural History, Rm. 4201. 40 Wall Street, New York, NY 10005 Goldman, Robert D., Northwestern University Medical School, Department of Cell and Molecular Biology. 303 E. Chicago Avenue, Chicago, IL 60611-3008 Goldsmith, Paul K., National Institutes of Health. Building 10, Room 9C-101. Bethesda. MD 20892 Goldsmith. Timothy H., Yale University, Department of Biology, New Haven, CT 06510 Goldstein, Moise H., The Johns Hopkins University, ECE Department, Barton Hall, Baltimore. MD 21218 Goodman, Lesley Jean (deceased) Gould, Robert Michael, NYS Institute of Basic Research, 1050 Forest Hill Road, Staten Island, NY 10314-6399 Govind, C. K., Scarborough College, Life Sciences Division, 1265 Military Trail, West Hill, Ontario MIC 1A4, CANADA Grace, Dick, Doreen Grace Fund, The Brain Center, Promontory Point, New Seabury, MA 02649 Graf, Werner M., College of France. 1 1 Place Marcelin Berthelot. 75231 Paris Cedex 05, FRANCE R72 Annual Report Grant, Philip, National Institutes of Health. NINDS.BN.DIR.Neurochemistry. Building 36. Room 4D20. Bethesda. MD 20892-4130 Grass, Ellen R., The Grass Foundation, 77 Reservoir Road. Quincy. MA 02 170-3610 Grassle, Judith P., Rutgers University, Institute of Marine and Coastal Studies, Box 23 1 , New Brunswick, NJ 08903 Graubard, Katherine G., University of Washington, Department of Zoology, NJ-15. Box 351800, Seattle, WA 98195-1800 Greenberg, Everett Peter, University of Iowa. College of Medicine. Department of Microbiology. Iowa City, IA 52242 Greenberg, Michael J., University of Florida, The Whitney Laboratory, 9505 Ocean Shore Boulevard. St. Augustine, FL 32086-8623 Greer, Mary J., 176 West 87th Street, #12A, New York. NY 10024- 2402 Griffin, Donald R., Harvard University. Concord Field Station, Old Causeway Road, Bedford, MA 01730 Gross, Paul R., 1 1 1 Perkins Street. Apt. 45. Jamaica Plain, MA 02130- 4320 Grossman, Albert, New York University Medical Center, 550 First Avenue. New York, NY 10016 Grossman, Lawrence, The Johns Hopkins University. Department of Biochemistry. 615 North Wolfe Street. Baltimore, MD 21205 Gruner, John A., Cephalon, Inc.. 145 Brandyw'ine Parkway, West Chester, PA 19380-4245 Gunning. A. Robert, P.O. Box 165. Falmouth. MA 02541 Gwilliam, G. F., Reed College, Department of Biology, Portland, OR 97202 Haimo, Leah T., University of California. Department of Biology. Riverside. CA 92521 Hajduk, Stephen L., University of Alabama. School of Medicine/Dentistry, Department of Biochemistry/Molecular Genetics. University Station. Birmingham. AL 35294 Hall, Linda M., SUNY. Department of Biochemstry Pharmacology, 329 Huchstetter Hall, Buffalo, NY 14260-1200 Hall, /;K h YV., University of California, Department Physiology, San Francisco. CA 94114 Halvorson, Harlyn O., University of Massachusetts, Policy Center for Marine Biosciences and Technology. 100 Morrissey Boulevard. Boston. MA 02125-3393 Haneji, Tatsuji. Kyushu Dental College. Department of Anatomy, 2-6- 1, Mana/.uru. Kokurakita-Ku, Kitakyushu 803. JAPAN Hanlon, Roger T., Marine Biological Laboratory. Woods Hole, MA 02543 Harosi, Ferenc, New College of the USF, Division of Natural Sciences, 5700 North Tamiami Trail. Sarasota, FL 34243-2197 Harrigan, June F., 7415 Makaa Place, Honolulu. HI 96825 Harrington, Glenn W., Weber State University, Department of Microbiology, Ogden. UT 84408 Harrison, Stephen C., Harvard University, Department of Molecular and Cell Biology, 7 Divinity Avenue. Cambridge. MA 02138 Haselkorn, Robert, University of Chicago. Department of Molecular Genetics and Cell Biology, Chicago, IL 60637 Hastings, J. Woodland, Harvard University. The Biological Laboratories. 16 Divinity Avenue, Cambridge. MA 02138-2020 Huydnn-Baillie, Wensley G., Porton Institute, 2 Lowndes Place, I ondon SW1X 8Dd, ENGLAND Hayes, Raymond L., Howard University, College of Medicine. 520 W Street. NW, Washington. DC 20059 Heck. Diane E.. F.OHSI. Department of Pharmacology/Toxicology, 681 Frelinghuysen Road, Piscataway, NJ 08855 Henry, Jonathan Joseph, University of Illinois, Department of Cell and Struct. Biology. 601 South Goodwin Avenue #BI07, Urbana, II. 61801-3709 Hepler. Peter K., University of Massachusetts. Department of Biology. Morrill III. Amherst, MA 01003 Herndon, Walter R., University of Tennessee. Department of Botany, Knoxville, TN 37996-1 100 Herskovits, Theodore T.. Fordham University. Department of Chemistry. John Mulcahy Hall. Room 638. Bronx. NY 1045S Hiatt. Howard H., Bngham and Women's Hospital. Department of Medicine. 75 Francis Street, Boston, MA 021 15 Highstein, Stephen M., Washington University. Department of Otolaryngology. Box 8115. 4566 Scott Avenue, Street Louis, MO 63110 Hildehrand, John G., University of Arizona, ARL Division of Neurobiology. P.O. Box 210077. Tucson, AZ 85721-0077 Hill, Richard W., Michigan State University, Department of Zoology, East Lansing. MI 48824 Hill, Susan D., Michigan State University. Department of Zoology, East Lansing. MI 48824 Hillis, Llewellya W., Smithsonian Tropical Research Institute. Unit 0948. APO. AA 34002-0948 Hinkle, Gregory J., Bioinformatics Group, Cereon Genomics, One Kendall Square, Building 200. Cambridge. MA 02139 Hinsch, Gertrude W., University of South Florida. Department of Biology. Tampa, FL 33620 Hinsch, Jan, Leica. Inc.. 1 10 Commerce Drive, Allendale. NJ 07401 Hobhie, John E., Marine Biological Laboratory. The Ecosystems Center. Woods Hole. MA 02543 Hodge, Alan J., 3843 Mount Blackburn Avenue. San Diego, CA 921 1 1 Hoffman, Joseph F., Yale University School of Medicine, Cellular and Molecular Physiology. 333 Cedar Street. New Haven. CT 06520-8026 Hollyfield, Joe G. address unknown Holz IV, George G., New York University Medical Center, Medical Sciences Building Room 442, 550 First Avenue. New York. NY 10016 Hopkinson, Charles S., Marine Biological Laboratory, Woods Hole, MA 02543 Houk, James C., Northwestern University Medical School, 303 East Chicago Avenue, Ward 5-315. Chicago. IL 6061 1-3008 Hoy, Ronald R., Cornell University. Section of Neurobiology and Behavior, 215 Mudd Hall. Ithaca, NY 14853 Huang, Alice S., California Institute of Technology. Mail Code 1-9. Pasadena, CA 91125 Hufnagel-Zackroff. Linda A., University of Rhode Island, Department of Microbiology, Kingston. RI 02881 Hummon, William D., Ohio University. Department of Biological Sciences. Athens. OH 45701 Humphreys, Susie H., Food and Drug Administration, HFS-308, 200 C Street. SW, Washington. DC 20204-0001 Humphreys, Tom, University of Hawaii. Kewalo Marine Laboratory. 41 Ahui Street. Honolulu, HI 96813 Hunt, Richard T., ICRF, Clare Hall Laboratories. South Minims Potter's Bar, Herb EN6-3LD. ENGLAND Hunter, Robert D., Oakland University, Department of Biological Sciences. Rochester. MI 48309-4401 Huxley, Hugh E., Brandeis University, Rosenstiel Center, Biology Department. Waltham. MA 02154 1 1. HI. Joseph, Case Western Reserve University, School of Medicine. Department of Anatomy, Cleveland. OH 44106 Ingoglia, Nicholas A., New Jersey Medical School, Department of Pharmacology/Physiology, 185 South Orange Avenue. Newark. NJ 07103 Inoue, Saduyki, McGill University. Department of Anatomy, 3640 University Street, Montreal.PQ H3A 2B2, CANADA Inoue, Shinya, Marine Biological Laboratory. Woods Hole, MA 02543 Members of the Corporation R73 Isselbacher, Kurt J., Massachusetts Genera] Hospital Cancer Center. Charlestown. MA 02129 Issidorides, Marietta Radovic, University of Athens, Department of Psychiatry, Monis Petraki 8. Athens, 140, GREECE Izzard, Colin S., SUNY-Albany. Department of Biological Sciences, 1400 Washington Avenue. Albany, NY 12222 Jacobs, Neil, Hale and Dorr, 60 State Street, Boston, MA 02109 Jaffe, Laurinda A., University of Connecticut Health Center, Department of Physiology. Farmington Avenue. Farmington, CT 06032 Jaffe, Lionel, Marine Biological Laboratory, Woods Hole, MA 02543 Jannasch. Holger W., Woods Hole Oceanographic Institute. Department of Biology. Woods Hole, MA 02543 (deceased) Jeffery, William R., University of Maryland. Department of Biology. College Park. MD 20742 Johnston, Daniel, Baylor College of Medicine. Division of Neuroscience, Baylor Plaza. Houston. TX 77030 Josephson, Robert K., University of California, School of Biological Science. Department of Psychobiology, Irvine. CA 92697 Kaczmarek, Leonard K., Yale University School of Medicine, Department of Pharmacology, 333 Cedar Street. New Haven, CT 06520 Kaley, Gabor, New York Medical College. Department of Physiology. Basic Sciences Building. Valhalla. NY 10595 Kaltenbach, Jane, Mount Holyoke College. Department Biological Sciences, South Hadley, MA 01075 Kaminer, Benjamin, Boston University Medical School, Physiology Department, 80 East Concord Street, Boston, MA 021 18 Kaneshiro, Edna S., University of Cincinnati, Biological Sciences Department, JL 006. Cincinnati. OH 45221-0006 Kaplan, Ehud, 450 E 63 rd Street. New York. NY 10021-7928 Karakashian, Stephen J., Apartment 16-F. 165 West 91st Street. New York. NY 1 0024 Karlin, Arthur, Columbia University, Center for Molecular Recognition, 630 West 168th Street, Room 1 1-401. New York, NY 10032 Keller, Hartmut Ernst, Carl Zeiss. Inc.. One Zeiss Drive. Thomwood. NY 10594 Kelley, Darcy B., Columbia University. Department of Biological Sciences. 911 Fairchild. Mailcode 2432. New York. NY 10027 Kelly, Robert E., 5 Little Harbor Road, Woods Hole, MA 02543 Kemp, Norman E., University of Michigan, Department of Biology, Ann Arbor. Ml 48109 Kendall, John P., Faneuil Hall Associates, 176 Federal Street, 2nd Floor. Boston. MA 02110 Kerr, Louis M.. Marine Biological Laboratory. Woods Hole, MA 02543 Keynan, Alexander, Israel Academy of Science and Humanity. P.O. Box 4040, Jerusalem. ISRAEL Khan, Shahid M.M., Albert Einstein College of Medicine. Department of Physiology and Biophysics. 1300 Morris Park Avenue. Room U273. Bronx, NY 10461 Khodakhah, Kamran, University of Colorado School of Medicine. Department of Physiology and Biophysics, 4200 East 9th Avenue. C-240, Denver, CO 80262 Kiehart, Daniel P., Duke University Medical Center. Department of Cell Biology. Box 3709, 308 Nanalme Duke Building. Durham. NC 27710 Kleinfeld, David, University of California. Department of Physics. 0319 9500 Oilman Drive. La Jolla. CA 92093 Klessen, Rainer, Address unknown. Klotz, Irving M., Northwestern University. Department of Chemistry. Evanston. II. WI20I Knudson, Robert A., Marine Biological Laboratory. Woods Hole. MA 02543 Koide, Samuel S., The Rockefeller University. The Population Council. 1230 York Avenue. New York. NY 10021 Kornberg, Hans, Boston University. The University Professors, 745 Commonweath Avenue, Boston. MA 02215 Kosower, Edward M., Tel-Aviv University, Department of Chemistry. Ramat-Aviv. Tel Aviv, 69978, ISRAEL Krahl. Maurice E., 27X3 West Casas Circle, Tucson, AZ 85741 Krane. Stephen M., Massachusetts General Hospital. Arthritis Unit, Fruit Street, Boston. MA 021 14 Krauss, Robert, P.O. Box 291. Demon. MD 21629 Kravitz, Edward A., Harvard Medical School. Department of Neurobiology, 220 Longwood Avenue, Boston. MA 02 1 1 5 Kriebel, Mahlon E., SUNY Health Science Center, Department of Physiology. Syracuse. NY 13210 Kristan Jr., William B., University of California. Department of Biology 0357, 9500 Oilman Drive, La Jolla, CA 92093-0357 Kropinski, Andrew M., Queen's University, Department of Microbiology/Immunology, Kingston. Ontario K7L 3N6. CANADA Kuffler. I). inn, 11 P., Institute of Neurobiology. 201 Boulevard del Valle. San Juan 00901. PR Kuhns. William J., Hospital for Sick Children, Biochemistry Research, 555 University Avenue. Toronto, Ontario M5G 1X8, CANADA Kunkel, Joseph G., University of Massachusetts, Department of Biology, Amherst, MA 01003 Kuzirian, Alan M., Marine Biological Laboratory, Woods Hole, MA 02543-1015 Laderman, Aimlee D., Yale University. School of Forestry and Environmental Studies. 370 Prospect Street, New Haven. CT 065 1 1 Landeau. Laurie J., Listowel. Inc.. 2 Park Avenue. Suite 1525. New York. NY 10016 I .nulls. Dennis M.D., University Hospital of Cleveland, Department Neurology. I 1 100 Euclid Avenue, Cleveland. OH 44106 Landis, Story C., National Institutes of Health, Building 36. Room 5A05, 36 Convent Drive. Bethesda. MD 20892-4150 Landowne, David, University of Miami Medical School, Department of Physiology, P.O. Box 016430. Miami. FL 33101 Langford, George M., Dartmouth College. Department of Biological Sciences. 6044 Oilman Laboratory. Hanover, NH 03755 Laskin, Jeffrey, University of Medical and Dentistry of New Jersey, Robert Wood Johnson Medical School, 675 Hoes Lane. Piscataway, NJ 08854 Lasser-Ross. Nechama, New York Medical College. Department of Physiology. Valhalla. NY 10595 Laster, Leonard. University of Massachusetts Medical School. 55 Lake Avenue. North, Worcester. MA 01655 Laties, Alan, Scheie Eye Institute, Myrin Circle, 51 North 39th Street, Philadelphia, PA 19104 Laufer, Hans, University of Connecticut. Department of Molecular and Cell Biology. U-125. 75 North Eagleville Road Storrs. CT 06269- 3125 Lazarow, Paul B., Mount Sinai School of Medicine. Department of Cell Biology and Anatomy, 1190 Fifth Avenue. Box 1007, New York, NY 10029-6574 Lazarus, Maurice, Federated Department Stores, Sears Crescent, City Hall Pla/a. Boston. MA 02108 Leadhetter, Edward R., University of Connecticut, Department of Molecular and Cell Biology, U-131. Beach Hall. Room 249. 354 Mansfield Road. Storrs. CT 06269-2 1 3 1 Lederberg, Joshua. The Rockefeller University. 1230 York Avenue. New York, NY 10021 R74 Annual Report Lee, John J.. City College of CUNY. Department of Biology, Convent Avenue and 138th Street. New York, NY 10031 Lehy, Donald B., 35 Willow Field Drive, North Falmouth. MA 02556 Leibovitz, Louis, 3 Kettle Hole Road, Falmouth. MA 02540 (deceased) Leighton, Joseph, Aeron Biotechnology. Inc., 1933 Davis Street #310. San Leandro. CA 44577 (deceased) Leighton. Stephen B.. National Institutes of Health, Building 13, 3WI3. Bethesda, MD 20842 Lemos, Jose Ramon, University of Massachusetts Medical Center. Worcester Foundation Campus. 222 Maple Avenue. Shrewsbury. MA 01545-2737 Lerner, Aaron B.. Yale University School of Medicine, Department of Dermatology. P.O. Box 3333, New Haven, CT 06510 Levin, Jack, Veterans Administration. Medical Center. 1 1 1 H2. 4150 Clement Street, San Francisco. CA 94121 Levine, Michael, University of California. Department MCB. 401 Barker Hall. Berkeley. CA 94720 Levine, Richard B., University of Arizona. Division of Neurobiology. Room 611. Gould Simpson Building. P.O. Box 210077. Tucson. AZ 85721-0077 Levinthal, Francoise, Columbia University, Department of Biological Sciences, Broadway and 116th Street. New York, NY 10026 Levitan, Herbert, National Science Foundation. 4201 Wilson Boulevard, Room 835, Arlington. VA 22230 Levitan. Irwin B., Brandeis University. Volen Center for Complex Systems. 415 South Street, Waltham. MA 02254 Linck. Richard VV., University of Minnesota School of Medicine. Cell Biology and Neuroanatomy Department, 4-135 Jackson Hall. 321 Church Street. Minneapolis. MN 55455 Lipicky. Raymond J., FDA/CDER/ODEI/HFD- 1 10. 5600 Fishers Lane. Rockville. MD 20857 Lisman, John E., 199 Coolidge Avenue, #902, Watertown. MA 02172- 1572 Liuzzi. Anthony, 320 Beacon Street, Boston. MA 021 16 Llinas, Rodolf'o R., New York University Medical Center. Department of Phsyiology/Biophysics. 550 First Avenue. Room 442. New York. NY 10016 Lobel, Phillip S., Boston University Marine Program. Marine Biological Laboratory, Woods Hole. MA 02543 Loew, Franklin M., Becker College. 61 Sever Street. Worcester. MA 01615-0071 Loewenstein, Birgit Rose, Marine Biological Laboratory. Woods Hole. MA 02543 Loewenstein, Werner R., Marine Biological Laboratory, Woods Hole. MA 02543 London, Irving M., Harvard-MIT. Division. E-25-551. Cambridge. MA 02 1 39 Longo, Frank J., University of Iowa. Department of Anatomy. Iowa City. IA 52442 Lorand, Laszlo, Northwestern University Medical School. CMS Biology. Searle 4-555. 303 East Chicago Avenue, Chicago. 1L 60611- 3008 Luckenbill, Louise M., Ohio University. Department of Biological Sciences, Irvine Hall, Athens, OH 45701 Macagno, Fduardo R., Columbia University. 109 Low Memorial Library, Mail Code 4306. New York, NY 10027 MacNichol Jr., Kdward F., Boston University School of Medicine. Department of Physiology, 80 East Concord Street, Boston, MA 021 IS Maglott-Dul'lield, Donna R., American Type Culture Collection, 12301 Parklawn Drive. Roik \ille. MD 20852-1776 Maienschein, Jane Ann, Ari/ona State University, Department of Philosophy. P.O. Box 872004. Tempe. AZ 85287-2004 Mainer. Robert E., The Boston Company. Inc.. One Boston Place, OBP-15-D, Boston. MA 02108 Malhon, Craig C., SUNY, University Medical Center. Pharmacology- HSC. Stony Brook, NY 11794-8651 Malchow, Robert P., University of Illinois, Department of Ophthalmology, 1855 West Taylor Street N/C 648, Chicago. IL 60612 Man. ilis, Richard S., Indiana-Purdue University, Department of Biological Science, 2101 Coliseum Boulevard East, Fort Wayne. IN 46805 Mangum, Charlotte P., College of William and Mary. Department of Biology. Williamsburg. VA 23187-8795 (deceased) Manz, Robert D., 304 Adams Street, Milton. MA 02186 Margulis, Lynn, University of Massachusetts. Department of Geosciences. Morrill Science Center, Box 35820, Amherst. MA 01003-5820 Marinucci, Andrew C., 102 Nancy Drive, Mercerville, NJ 08619 Martinez, Joe L., The University of Texas, Division of Life Sciences, 6900 North Loop 1604 West, San Antonio. TX 78249-0662 Martinez-Palomo, Adolfo, CINVESTAV-IPN, Sec. de Patologia Experimental. 07000 Mexico. D.F.A. P. 140740, MEXICO Mastroianni, Luigi, Hospital of University of Pennsylvania. 106 Dulles. 3400 Spruce Street, Philadeplna, PA 19104-4283 Mauzerall, David, Rockefeller University, 1230 York Avenue, New York, NY 10021 McC'ann, Frances V., Dartmouth Medical School, Department of Physiology. Lebanon. NH 03756 McLaughlin, Jane A., Marine Biological Laboratory. Woods Hole, MA 022543 McMahon, Robert F., University of Texas, Arlington. Department of Biology. Box 19498. Arlington, TX 76019 Meedel, Thomas, Rhode Island College. Biology Department. 600 Mount Pleasant Avenue, Providence, RI 02908 Meinertzhagen, Ian A., Dalhousie University, Department of Psychology, Halifax. NS B3H 4J 1 . CANADA Meiss, Dennis E., Immunodiagnostic Laboratories. 488 McCormick Street, San Leandro, CA 94577 Melillo, Jerry M., Marine Biological Laboratory. Ecosystems Center, Woods Hole. MA 02543 Mellon Jr., DeForest, University of Virginia. Department of Biology. Gilmer Hall, Charlottesville, VA 22903 Mellon. Richard P.. P.O. Box 187. Laughlintown, PA 15655-0187 Mendelsohn, Michael E., New England Medical Center, Molecular Cardiology Laboratory. NEMC Box 80, 750 Washington Street. Boston. MA 021 I I Merriman, Melanie Pratt, 751 1 Beach View Drive, North Bay Village. FL 33141 Meselson, Matthew, Harvard University. Fairchild Biochemistry Building, 7 Divinity Avenue. Cambridge. MA 02138 Metuzals, Janis, University of Ottawa. Department of Pathology and Laboratory Medical. 451 Smyth Road, Ottawa. Ontario K1H 8M5. CANADA Miledi. Ricardo, University of California. Irvine. Department of Psychobiology. 2205 Biology Sci. II. Irvine. CA 92697-4550 Milkman. Roger D., University of Iowa. Department of Biological Sciences, Biology Buiilding, Room 318, Iowa City, IA 52242-1324 Miller, Andrew L., Hong Kong University of Science and Technology. Department of Biology, Clearwater Bay. Kowloon, HONG KONG Mills, Robert, 10315 44th Avenue. W 12 H Street. Brandenton. FL 34210 Misevic, Gradimir, University Hospital of Basel. Department of Research. Mebelstr. 20. CH-403 1 Basel. SWITZERLAND Mitchell. Ralph. Harvard University, Division of Applied Sciences, 29 Oxford Street. Cambridge. MA 02 1 38 Members of the Corporation R75 Miyakawa, Hiroyoshi, Tokyo College of Pharmacy. Laboratory of Cellular Neurobiology. 1432-1 Horinouchi, Hachiouji, Tokyo 192-03, JAPAN Miyamoto. David M., Drew University. Department of Biology. Madison, NJ 07940 Mi/. II. Merle, Tulane University. Depanment of Cell and Molecular, Biology. New Orleans. LA 70118 Moore, John W., Duke University Medical Center. Department of Neurobiology, Box 3209. Durham, NC 27710 Moreira, Jorge E., NIH/NICHD. Department of Cell and Molecular Biol., Bethesda, MD 20852 Morin, James G., address unknown Morrell. Leyla de Toledo, Rush-Presbyterian-Street Lukes, Medical Center, 1653 West Congress Parkway, Chicago, IL 60612 Morse, M. Patricia, National Science Foundation, Room 885, Esie. Arlington, VA 22230 Morse, Stephen S., DARPA/DSO, 3701 North Fairfax Drive. Arlington, VA 22203-1714 Mote, Michael I., Temple University. Department of Biology, Philadelphia, PA 19122 Muller, Kenneth J., University of Miami School of Medicine, Department of Physiology and Biophysics. 1600 NW 10th Avenue. R-430. Miami, FL 33136 Murray, Andrew W., University of California. Department of Physiology. Box 0444, 513 Parnassus Avenue. San Francisco. CA 94143-0444 Nabrit, S. M., 686 Beckwith Street, SW, Atlanta, GA 30314 Nadelhoffer, Knute J., Marine Biological Laboratory, 7 MBL Street. Woods Hole, MA 02543 Naka, Ken-ichi, 2-9-2 Tatumi Higashi, Okazaki, 444, JAPAN Nakajima, Yasuko, University of Illinois, College of Medicine. Anatomy and Cell Biology Department. M/C 512. Chicago, IL 60612 Narahashi, Toshio, Northwestern University Medical School, Department of Pharmacology. 303 East Chicago Avenue. Chicago. IL 60611 Nasi, Enrico, Boston University School of Medical. Department of Physiology, R-406. 80 East Concord Street, Boston, MA 02118 Neill, Christopher, Marine Biological Laboratory. 7 MBL Street, Woods Hole, MA 02543 Nelson, Leonard, Medical College of Ohio. Department of Physiology. CS 10008. Toledo. OH 43699 Nelson, Margaret C., Cornell University. Section of Neurobiology and Behavior. Ithaca, NY 14850 Nicholls, John G., University of Basel, Department of Pharmacology Biocenter. Klingelbergstrasse 70. Basel, CH-4056, SWITZERLAND Nickerson, Peter A., SUNY, Buffalo, Department of Pathology, Buffalo, NY 14214 Nicosia, Santo V., University of South Florida, College of Medicine. Box 1 1. Department of Pathology, Tampa, FL 33612 Noe, Bryan D., Emory University School of Medicine. Department of Anatomy and Cell Biology, Atlanta. GA 30322 Norton, Catherine N., Marine Biological Laboratory, 7 MBL Street. Woods Hole, MA 02543 Nusbaum, Michael P., University of Pennsylvania School of Medicine, Department of Neuroscience, 215 Stemmler Hall, Philadelphia. PA 191(14-6074 O'Herron, Jonathan, Lazard Freres and Company. 30 Rockefeller Plaza. 59th Floor. New York. NY 10020-1900 Obaid, Ana Lia, University of Pennsylvania School of Medicine, Neuroscience Department, 234 Stemmler Hall, Philadelphia, PA 19104-6074 Ohki, Shinpei, SUNY at Buffalo, Department of Biophysical Sciences, 224 Cary Hall. Buffalo. NY 14214 Oldenbourg, Rudolf, Marine Biological Laboratory. 7 MBL Street. Woods Hole. MA 02543 Olds, James L., George Mason University. Krasnow Institute for Advanced Studies, Mail Stop 2A1, Fairfax, VA 22030-4444 Olins, Ada L., 45 Eastern Promenade, #7-D, Portland. ME 04101 Olins, Donald E., 45 Eastern Promenade. #7-D, Portland, ME 04101 Oschman, James L., Nature's Own Research Association, P.O. Box 5101. Dover, NH 03X20 Palazzo, Robert E., University of Kansas, Department of Physiology and Cell Biology, Lawrence, KS 66045 Palmer, John D., University of Massachusetts, Department of Zoology, 221 Morrill Science Center, Amherst, MA 01003 Pant, Harish C., NINCDS/NIH. Laboratory of Neurochemistry, Building 36, Room 4D20. Bethesda. MD 20892 Pappas, George D., University of Illinois. College of Medicine, Department of Anatomy. Chicago. IL 60612 Pardee, Arthur B., Dana-Farber Cancer Institute. D810, 44 Binney Street, Boston, MA 02 1 1 5 Pardy, Rosevelt L., University of Nebraska, School of Life Sciences, Lincoln, NE 68588 Parmentier, James L., 175 S. Great Road, Lincoln, MA 01773-41 12 Pederson, Thoru, University of Massachusetts Medical Center, Worcester Foundation Campus. 222 Maple Avenue. Shrewsbury, MA 01545 Perkins, Courtland I)., 400 Hilltop Terrace, Alexandria, VA 22301 Person, Philip, 137-87 75th Road, Flushing, NY 11367 Peterson, Bruce J., Marine Biological Laboratory, 7 MBL Street, Woods Hole. MA 02543 Pethig. Ronald, University College of North Wales. School of Electronic Engineering. Bangor. Gwynedd. LL 57 IUT, UNITED KINGDOM Pfohl, Ronald J.. Miami University, Department of Zoology, Oxford, OH 45056 Pierce, Sidney K., University of Maryland. Department of Zoology, College Park. MD 20742 Pleasure. David E., Children's Hospital. Neurology Research, 5th Floor. Ambramson Building. Philadelphia, PA 19104 Poindexter, Jeanne S., Barnard College. Columbia University, 3009 Broadway. New York, NY 10027-6598 Pollard, Harvey B., NIH/NIDDKD, Building 8, Room 401, Bethesda. MD 20892 Pollard, Thomas D., Salk Institute for Biological Studies. 10010 N. Torrey Pines Road. La Jolla. CA 92037 Porter, Beverly H., 5542 Windysun Court, Columbia, MD 21045 Porter, Mary E., University of Minnesota, Department of Cell Biology and Neuroanatomy. 4-135 Jackson Hall, 321 Church Street SE, Minneapolis, MN 55455 Potter, David D., Harvard Medical School. Department of Neurobiology, 25 Shattuck Street. Boston, MA 02115 Potts, William T., LIniversity of Lancaster. Department of Biology, Lancaster, ENGLAND Powers, Maureen K.. Vanderbilt University. Department of Psychology. 301 Arts and Science Psychology Building, Nashville. TN 37240 Prendergast, Robert A., Wilmer Institute, Johns Hopkins Hospital. 600 North Wolfe Street, Baltimore. MD 21287-9142 Price, Carl A., Rutgers University, Waksman Institute of Microbiology, P.O. Box 759. Piscataway, NJ 08855-0759 Prior, David J., Northern Arizona University. Arts and Sciences Dean's Office, Box 5621, Flagstaff, AZ 8601 1 Prusch. Robert D., Gonzaga University, Department of Life Sciences, Spokane, WA 99258 R76 Annual Report Purves, Dale, Duke University Medical Center. Department of Neurobiology. Box 3209. 101-1 Bryan Research Building, Durham. NC 27710 Quigley, James P., SUNY Health Sciences Center, Department of Pathology. BHS Tower 4. Room 140. Stony Brook. NY 1 1794-8691 Rahb, Irving VV., 1010 Memorial Drive. Cambridge. MA 02138 Rabin, Harvey. P.O. Box 4022. Wilmington. DE 19807 Rabinowitz, Michael B., Marine Biological Laboratory, 7 MBL Street. Woods Hole. MA 02543 Rafferty, Nancy S., Marine Biological Laboratory. 7 MBL Street. Woods Hole, MA 02543 Rakowski. Robert F., UHS/The Chicago Medical School, Department of Physiology and Biophysics, 3333 Greenbay Road. N. Chicago, IL 60064 Ramon, Fidel, Universidad Nacional Autonoma de Mexico. Division EStreet Posgrado E Invest.. Facultad de Medicina, 04510, D.F., MEXICO Ranzi, Silvio, Sez. Zoologia Scienze Naturali, Dip. di Biologia. Via Celoria, 26, 20133 Milano. ITALY (deceased) Rastetter, Edward B., Marine Biological Laboratory, The Ecosystems Center. Woods Hole, MA 02543 Ri'bhun. Lionel I., University of Virginia. Department of Biology, Gilmer Hall 45, Charlottesville. VA 22901 Reddan. John R., Oakland University. Department of Biological Sciences. Rochester. MI 48309-4401 Reese, Thomas S., NIH. N1NDS. Building 36. Room 2A29. Bethesda, MD 20892 Reinisch, Carol L., Marine Biological Laboratory, 7 MBL Street. Woods Hole. MA 02543 Rickles, Frederick R., 2633 Danforth Lane, Decatur, GA 30033 Rieder, {.'only L., Wadsworth Center, Division of Molecular Medicine. P.O. Box 509. Albany. NY 12201-0509 Riley, Monica, Marine Biological Laboratory, 7 MBL Street, Woods Hole. MA 02543 Ripps, Harris, University of Illinois at Chicago. Department of Ophthalmology/Visual Sciences. 1855 West Taylor Street, Chicago, IL 60612 Ritchie, J. Murdoch, Yale LIniversity School of Medicine. Department of Pharmacology, 333 Cedar Street. New Haven. CT 06510 Rome, Lawrence C., University of Pennsylvania. Department of Biology. Philadelphia. PA 19104 Rosenhluth, Jack, New York University School of Medical, Department of Physiology and Biophysics. RR 714. 400 East 34th Street, New York, NY 10016 Rosenhluth, Raja, Simon Fraser University. Institute of Molecular Biology and Biochemistry. Burnaby, BC. BC V5A IS6. CANADA Rosenh'eld, Allan, Columbia University School of Public Health. 600 West IfiSih Street. New York. NY 10032-3702 Ro.senkranz. Herbert S., 130 Desoto Street. Pittsburgh. PA 15213-2535 Roslansky, John D., 57 Buzzards Bay Avenue. Woods Hole. MA 02543 Roslansky, Priscilla F., Associates of Cape Cod. Inc., P.O. Box 224, Woods Hole, MA 02543 Ross, William N., New York Medical College. Department of Physiology. Valhalla. NY 10595 Roth, Jay S., P.O. Box 692. Woods Hole. MA 02543-0692 Rottenfusser, Rudi, Marine Biological Laboratory, 7 MBL Street. Woods Hole. MA 02543 Rowland. Lewis P., Neurological Institute. 710 West 168th Street, New York. NY 10032 Riiderman, Joan V., Harvard Medical School. Department of Cell Biology. 240 Longwood Avenue, Boston, MA 021 15 Rummel. John I)., Marine Biological Laboratory, 7 MBL Street, Woods Hole, MA 02543 Rushforth, Norman B.. Case Western Reserve University, Department of Biology. Cleveland, OH 44106 Russell-Hunter, W. D., 71 1 Howard Street, Easton. MD 21601-3934 Saffo, Mary Beth, Arizona State University West. Life Science Department, MC 2352. P.O. Box 37100. Phoenix. AZ 85069-7100 Salama, Guy, University of Pittsburgh, Department of Physiology. Pittsburgh. PA 15261 Salmon, Edward D., University of North Carolina. Department of Biology. CB 3280. Chapel Hill, NC 27514 Salvers, Abigail, University of Illinois. Department of Microbiology. 407 South Goodwin Avenue, Urbana. IL 61801 Salzberg, Brian M., University of Pennsylvania School of Medicine, Department of Neuroscience. 215 Stemmler Hall. Philadelphia. PA 19104-6074 Sanger, Jean M., University of Pennsylvania School of Medicine. Department of Anatomy. 36th and Hamilton Walk. Philadelphia, PA 19104 Sanger. Joseph W., University of Pennsylvania Medical Center, Department of Cell and Developemental Biology, 36th and Hamilton Walk. Philadelphia. PA 19104-6058 Saunders Jr., John W., Marquette University, P.O. Box 3381. Wauuoit. MA 02536 Schachman, Howard K., University of California. Molecular and Cell Biology Department. 229 Stanley Hall. #3206. Berkeley. CA 94720- 3206 Schatten, Gerald P., Oregon Health Sciences University. Oregon Regional Primate Research Center. 505 N.W. 185th Avenue. Beaverton, OR 97006 Schatten, Heide, University of Wisconsin. Department of Zoology, Madison, WI 53706 Schmeer, Arlene C., Mercenene Cancer Research Institute, 790 Prospect Street, New Haven, CT 065 1 1 Schuel. Herbert. SUNY at Buffalo, Department of Anatomy/Cell Biology, Buffalo, NY 14214 Schwartz, James H., New York State Psychiatric Institute, Research Annex, 722 West 168th Street, 7th floor, New York, NY 10032 Schwartz, Lawrence, University of Massachusetts. Department of Biology, Morrill Science Center, Amherst, MA 01003 Schweitzer. A. Nicola, Brigham and Women's Hospital. Immunology Division, Department of Pathology, 221 Longwood Avenue, LMRC 521. Boston. MA 02115 Segal, Sheldon J.. The Population Council, One Dag Hammarskjold Pla/a. New York, NY 10036 Senl't, Stephen Lamont, Neuroengineering/Neuroscience Center, P.O. Box 208205. New Haven. CT 06520-8205 Shanklin. Douglas R., University of Tennessee. Department of Pathology, Room 576, 800 Madison Avenue, Memphis, TN 381 17 Shashoua, Victor E., Harvard Medical School, Ralph Lowell Labs. McLean Hospital. I 15 Mill Street. Belmont. MA 02178 Shaver, Gaius R.. Marine Biological Laboratory. The Ecosystems Center, Woods Hole. MA 02543 Shaver, John R., Michigan State University. Department of Zoology, East Lansing, MI 48824 Sheetz, Michael P.. Duke University Medical Center. Department of Cell Biology, Bx 3709, 388 Nanalmc Duke Building. Durham. NC 27710 Slii'pro, David, Boston University. CAS Biology. 5 Cummington Street, Boston, MA 02215 Shimomura. Osamii, Marine Biological Laboratory. 7 MBL Street. Woods Hole. MA 02543 Shipley, Alan M., P.O. Box 2036. Sandwich. MA 02563 Members of the Corporation R77 Silver, Robert B., Marine Biological Laboratory. 7 MBL Street, Woods Hole, MA 0254.1 Siwicki, Kathleen K., Swarthmore College, Biology Department. 500 College Avenue. Swarthmore. PA 19081-1397 Skinner, Dorothy M., Oak Ridge National Laboratory, Biology Division. P.O. Box 2009. Oak Ridge. TN 37831 Sloboda, Roger D., Dartmouth College. Department of Biological Science. 6044 Oilman. Hanover. NH 03755-1893 Sluder, Greenfield, University of Massachusetts Medical School, Room 324. 377 Plantation Street. Worcester. MA 01605 Smith, Peter J.S., Marine Biological Laboratory. 7 MBL Street. Woods Hole. MA 02543 Smith, Stephen J., Stanford University School of Medicine, Department of Molecular and Cellular Physiology. Beckman Center. Stanford, CA 94305 Smolowitz, Roxanna S., Marine Biological Laboratory, 7 MBL Street. Woods Hole. MA 02543 Sogin, Mitchell L., Marine Biological Laboratory. 7 MBL Street. Woods Hole. MA 02543 Sorenson, Martha M., Cidade Universitana-UFRJ. Department Bioquimica Medica-ICB. 21941-590 Rio de Janerio. BRAZIL Speck. William T., Columbia-Presbyterian Medical Center. 161 Fort Washington Avenue. 14th Floor. Room 1470, New York. NY 10032- 3784 Spector, Abraham, Columbia University. Department of Ophthalmology. 630 West 168th Street. New York. NY 10032 Speksnijder. Johanna E., University of Groningen, Department of Genetics, Kerklaan 30. 9751 NN Haren, THE NETHERLANDS Spray, David C., Albert Einstein College of Medicine, Department of Neuroscience, 1300 Morris Park Avenue. Bronx, NY 10461 Spring, Kenneth R., National Institutes of Health, 10 Center Drive. MSC 1598. Building 10. Room 6N260. Bethesda. MD 20892-1603 Steele, John H., Woods Hole Oceanographic Institution, Woods Hole, MA 02543 Steinacker, Antoinette, University of Puerto Rico, Instituet of Neurobiology, 201 Boulevard Del Valle. San Ian, PR 00901 Steinberg. Malcolm, Princeton University, Department of Molecular Biology. M-18 Moffett Laboratory. Princeton, NI 08544-1014 Stemmer, Andreas C., Institut fur Robotik, ETH-Sentrum. 8092 Zurich. SWITZERLAND Stenflo, Johan, University of Lund. Department of Clinical Chemistry. Malmo General Hospital, S-205 02 Malmo. SWEDEN Stetten, Jane Lazarow, 4701 Willard Avenue. #1413. Chevy Chase. MD 20815-4627 Steadier, Paul A., Marine Biological Laboratory. The Ecosystems Center. Woods Hole, MA 02543 Stokes, Darrell R., Emory University, Department of Biology. 1510 Clifton Road NE, Atlanta. GA 30322-1100 Stommel, Elijah W., Darmouth Hitchcock Medical Center. Neurology Department. Lebanon. NH 03756 Stracher, Alfred, SUNY Health Science Center. Department of Biochemistry, 450 Clarkson Avenue. Brooklyn. NY 1 1 203 Strumwasser, Felix, P.O. Box 2278. East Falmouth. MA 02536-2278 Stuart, Ann E., University of North Carolina. Department of Physiology, Medical Research Building 206H, Chapel Hill. NC 27599-7545 Sugimori. Mutsuyuki, New York University Medical Center. Department of Physiology and Neuroscience, Room 442, 550 First Avenue, New York. NY 10016 Summers, William C., Western Washington University, Huxley College of Environmental Studies, Bellingham. WA 98225-9181 Suprenant, Kathy A., University of Kansas. Department of Physiology and Cell Biology. 4010 Haworth Hall. Lawrence. KS 66045 Swenson. Katherine I., Duke University Medical Center. Department of Molecular Cancer Biology, Box 3686, Durham. NC 27710 Sydlik, Mary Anne, Hope College, Peale Science Center. 35 East 1 2th St./P.O. Box 9000, Holland. MI 49422 Szent-Gyorgyi, Andrew G., 9 Westgate Road. Wellesley. MA 02181 Tabares, Lucia, University of Seville School of Medicine. Department of Physiology. Avda. Sanchez Pizjuan 4, Seville 41009, Spain Tamm, Sidney L., Boston University. 725 Commonwealth Avenue. Boston. MA 02215 Tanzer, Marvin L., University of Conn School of Dental Medicine. Department of Biostructure and Function. Farmmgton, CT 06030- 3705 Tasaki, Ichiji, NIMH/NIH, Laboratory of Neurobiology, Building 36. Room 2B-16. Bethesda. MD 20892 Taylor, D. Lansing, Carnegie Mellon University, Center for Flurorescence Research. 4400 Fifth Avenue. Pittsburgh, PA 15213 Taylor, Edwin W., University of Chicago, Department of Molecular Genetics, 920 E. 58th Street, Chicago, IL 60637 Teal, John M., Woods Hole Oceanographic Institute. Department of Biology. Woods Hole. MA 02543 Telfer, William H., University of Pennsylvania. Department of Biology. Philadelphia. PA 19104 Telzer, Bruce. Pomona College, Department of Biology, Thille Building, 175 West 6th Street, Claremont, CA 91711 Townsel, James G., Meharry Medical College. Department of Physiology. 1005 DB Todd Boulevard. Nashville, TN 37208 Travis, David M., 19 High Street, Woods Hole, MA 02543-1221 Treistman, Steven N., University of Massachusetts Medical Center, Department of Pharmacology. 55 Lake Avenue North, Worcester. MA 01655 Trigg, D. Thomas, One Federal Street. 9th Floor. Boston. MA 0221 1 Troll, Walter, NYU Medical Center. 550 First Avenue. New York, NY 10016 Troxler, Robert F., Boston University School of Medicine. Department of Biochemistry, 80 East Concord Street, Boston, MA 021 18 Tucker, Edward B., Baruch College. CUNY, Department of Natural Sciences, 17 Lexington Avenue. New York, NY 10010 Turner, Ruth D., Harvard University, Museum of Comparative Zoology, Mollusk Department, Cambridge, MA 02138 Tweedell, Kenyon S., University of Notre Dame, Department of Biological Sciences. Notre Dame, IN 46556-0369 Tykocinski, Mark L., Case Western Reserve University, Institute of Pathology, 2085 Adelbert Road, Cleveland. OH 44106 Tytell, Michael, Wake Forest University, Bowman Gray School of Medicine, Department of Anatomy and Neurobiology. Winston- Salem, NC 27157 Ueno, Hiroshi, Kyoto University. AGR Chemistry. Faculty of Agriculture, Sakyo. Kyoto 606-8502. JAPAN Valiela, Ivan, Boston University Marine Program. Marine Biological Laboratory. Woods Hole. MA 02543 Vallee. Richard, University of Massachusetts Medical Center. Worcester Foundation Campus, 222 Maple Avenue. Shrewsbury, MA 01545 Valois, John J., 420 Woods Hole Road, Woods Hole, MA 02543 Van Holde, Kensal E., Oregon State University, Biochemistry and Biophysics Department. Corvallis. OR 97331-7503 Van Dover, Cindy Lee, University of Alaska, P.O. Box 757220. Fairbanks. AK 99775 Vogl. Thomas P., Environmental Research Institute of Michigan. 1101 Wilson Boulevard. Arlington, VA 22209 R78 Annual Report Wainvvright, Norman R., Marine Biological Laboratory, 7 MBL Street, Woods Hole. MA 02543 Waksman, Byron H., New York University Medical Center. Department of Pathology. 550 First Avenue, New York, NY 10016 Wall, Betty, 9 George Street, Woods Hole, MA 02543 Wang, Hsien-Yu, State University of New York, University Medical Center, Physiology and Biophysics-HSC, Stony Brook. NY 1 17^4- 8633 Wangh, Lawrence J., Brandeis University, Department of Biology. 415 South Street, Waltham, MA 02254 Warner, Robert C., University of California, Irvine. Molecular Biology and Biochemistry, Irvine. CA 92717 Warren, Leonard, Wistar Institute, 36th and Spruce Streets. Philadelphia, PA 191(14 Waterbury, John B., Woods Hole Oceanographic Institution. Department of Biology, Woods Hole. MA 02543 Waxman. Stephen G., Yale Medical School. Neurology Department, 333 Cedar Street, P.O. Box 208018, New Haven, CT 06510 Webb, H. Marguerite, 184 Chestnut Street, Foxboro, MA 02035-1548 Weber, Annemarie, University of Pennsylvania School of Medicine, Department of Biochemstry and Biophysics. Philadelphia, PA 19066 Weeks, Janis C., University of Oregon, Institute of Neuroscience, Eugene, OR 97403- 1 254 Weidner, Earl, Louisiana State University, Department of Biological Sciences. 508 Life Sciences Building, Baton Rouge, LA 70803-1715 Weiss, Alice Sara, 105 University Boulevard West, Silver Spring, MD 20901 Weiss, Dieter, University of Rostock. Institute of Zoology. D- 18051 Rostock. GERMANY Weiss, Leon P., University of Pennsylvania School of Vet Medicine, Department of Animal Biology. Philadelphia, PA 19104 Weiss, Marisa C., Paoli Memorial Hospital, Department of Radiation Oncology, 255 W. Lancaster Avenue. Paoli, PA 19301 Weissmann, Gerald, New York University Medical Center, Department of Medicine/Division Rheumatology. 550 First Avenue. New York, NY 10016 Westerh'eld, Monte, University of Oregon, Institute of Neuroscience, Eugene, OR 97403 Whittaker, J. Richard, University of New Brunswick, Department of Biology, BS 4511, Frederiction, NB E3B 6E1, CANADA Wilkens, Lon A., University of Missouri, Department of Biology. 8001 Natural Bridge Road, Street Louis, MO 63121-4499 MBL Associates Wilson, Darcy B., San Diego Regional Cancer Center. 3099 Science Park Road, San Diego, CA 92 1 2 1 Wilson, T. Hastings, Harvard Medical School. Department of Physiology, 25 Shattuck Street, Boston, MA 02 1 1 5 Witkovsky, Paul, NYU Medical Center, Department of Ophthalmology, 550 First Avenue. New York, NY 10016 Wittenberg, Beatrice, Albert Einstein College of Medicine, Department of Physiology and Biophysics, Bronx, NY 10461 Wittenberg, Jonathan B., Albert Einstein College of Medicine, Department of Physiology and Biophysics, Bronx, NY 10461 Wolken, Jerome J., Carnegie Mellon University, Department of Biological Sciences, 440 Fifth Avenue. Pittsburgh, PA 15213 (deceased) Wonderlin, William F., West Virginia University, Pharmacology and Toxicology Department. Morgantown, WV 26506 Worden, Mary Kate, University of Virginia, Department of Neuroscience, McKim Hall Box 230, Charlottesville. VA 22908 Worgul, Basil V., Columbia University, Department of Ophthalmology. 630 West 16X Street. New York, NY 10032 Wu, Chau Hsiung, Northwestern University Medical School. Department of Pharmacology (S215), 303 East Chicago Avenue, Chicago, II. 6061 1-3008 Wyttenbach, Charles R., University of Kansas, Biological Sciences Department, 2045 Haworth Hall. Lawrence. KS 66045-2106 Yen, Jay Z., Northwestern University Medical School, Department of Pharmacology, Chicago. IL 6061 1 Zacks, Sumner I., 65 Saconesset Road, Falmouth. MA 02540-1851 Zigman, Seymour, University of Rochester Medical School, Ophthalmology Research, Box 314, 601 Elmwood Avenue. Rochester. NY 14640 Zigmond, Michael J., Lmiversity of Pittsburgh. S-526 Biomedical Science Tower, 3500 Terrace Street. Pittsburgh. PA 15213 Zimmerberg, Joshua J., National Institutes of Health, LCMB, NICHD, Building 10. Room 10D14, 10 Center Drive. Bethesda, MD 20892 Zottoli, Steven J., Williams College, Department of Biology, Williamstown, MA 01267 Zucker, Robert S., University of California. Neurobiology Division, Molecular and Cellular Biology Department. Berkeley. CA 94720 Zukin, R. Suzanne, Albert Einstein College of Medicine, Department of Neuroscience, 1410 Pelham Parkway South, Bronx. NY 10461 Executive Board Ruth Ann Laster, President Jack Pearce, Vice President Hanna Hastings, Treasurer Molly Cornell. Secretary Elizabeth Farnham, Membership Chair Tammy Smith Amon Duncan Aspmwall Barbara Atwood Kitty Brown Julie Child Seymour Cohen Michael Fenlon Alice Knowles Rebecca Lash Barbara Little Cornelia McMurtne Jack Moakley Joan Pearlman Virginia R. Reynolds Volker Ulbnch John Valois Kensal E. Van Holde Patrons Mr. and Mrs. David Bakalar Josephine B. Crane Foundation Dr. and Mrs. James J. Ferguson, Jr. Sustaining Associate Mr. Robert A. Jaye George Frederick Jewett Foundation Dr. and Mrs. Edward F. MacNichol. Jr. Plymouth Savings Bank Mr. and Mrs. William A. Putnam, III Supporting Associate Mrs. George H.A. Clowes Dr. and Mrs. James D. Ebert Mr. and Mrs. David Fausch Dr. and Mrs. Prosser Gifford Mr. and Mrs. Lon Hocker Mrs. Mary D. Janney Drs. Luigi and Elaine Mastroianni Dr. and Mrs. William M. McDennott Drs. Matthew & Jeanne Meselson Dr. and Mrs. Courtland D. Perkins Ms. Linda Sallop and Mr. Michael Fenlon Mrs. Anne W. Sawyer Dr. Maxine F. Singer Members of the Corporation R79 Dr. John Tochko and Mrs. Christina Myles-Tochko Mr. and Mrs. John J. Valois Drs. Walter S. Vincent and Dore J. Butler Fumilv Membership Dr. Frederick W. Ackroyd Dr. and Mrs. Edward A. Adelberg Mr. and Mrs. Douglas F. Allison Drs. Peggy and Fred Alsup Drs. James and Helene Anderson Dr. and Mrs. Samuel C. Armstrong Mr. and Mrs. Duncan P. Aspinwall Mr. and Mrs. Donald R. Aukamp Mr. and Mrs. John M. Baitsell Mr. and Mrs. William L. Banks Dr. and Mrs. Robert B. Barlow. Jr. Mr. and Mrs. John E. Barnes Dr. and Mrs. Robert M. Berne Drs. Harriet and Alan Bernheimer Mr. and Mrs. Robert O. Bigelow Dr. and Mrs. Edward G. Boettiger Mr. and Mrs. Kendall B. Bohr Dr. and Mrs. Alfred F. Borg Dr. and Mrs. Thomas A. Borgese Mr. and Mrs. Richard M. Bowen Dr. and Mrs. Francis P. Bowles Dr. and Mrs. John B. Buck Dr. and Mrs. John E. Burns Mr. and Mrs. William O. Burwell Mr. and Mrs. G. Nathan Calkins. Jr. Mr. and Mrs. D. Bret Carlson Prof, and Mrs. James F. Case Dr. and Mrs. Alfred B. Chaet Dr. and Mrs. Richard L. Chappell Dr. and Mrs. Frank M. Child, III Dr. and Mrs. Arnold M. Clark Mrs. LeRoy Clark Mr. and Mrs. James Cleary Dr. and Mrs. Laurence P. Cloud Mr. and Mrs. Lawrence H. Coburn Dr. and Mrs. Neal W. Cornell Mr. and Mrs. Norman C. Cross Mr. and Mrs. Bruce G. Daniels Mr. and Mrs. Joel P. Davis Mr. and Mrs. Richard C. Dierker Dr. and Mrs. Arthur Brooks DuBois Mr. and Mrs. John Eustis. II Mr. and Mrs. Harold Frank Mr. and Mrs. Howard G. Freeman Dr. and Mrs. Robert A. Frosch Dr. and Mrs. John J. Funkhouser Dr. and Mrs. Mordecai L. Gabriel Dr. and Mrs. David Garber Dr. and Mrs. Sydney Gellis Dr. and Mrs. James L. German, III Mr. and Mrs. Robert S. Gillette Dr. and Mrs. Murray Glusman Drs. Alfred and Joan Goldberg Mr. and Mrs. Charles Goodwin Dr. and Mrs. Philip Grant Dr. and Mrs. Thomas C. Gregg Prof, and Mrs. Lawrence Grossman Dr. and Mrs. Antoine P.O. Hadamard Mr. and Mrs. Peter A. Hall Dr. and Mrs. Harlyn O. Halvorson Capt. and Mrs. Frederick J. Hancox Drs. Alexander and Carol Hannenberg Mrs. Janet Harvey and Dr. Richard Harvey Dr. and Mrs. J. Woodland Hastings Mr. and Mrs. Gary G. Hayward Dr. and Mrs. Howard H. Hiatt Mr. and Mrs. David Hibbitt Dr. and Mrs. John E. Hobbie Drs. Francis C. G. Hoskin and Elizabeth M. Farnham Dr. and Mrs. Robert J. Huettncr Dr. and Mrs. Shinya Inoue Dr. and Mrs. Kurt J. Isselbacher Dr. and Mrs. Gary Jacobson Dr. and Mrs. Benjamin Kaminer Mr. and Mrs. Paul W. Knaplund Mr. and Mrs. A. Sidney Knowles, Jr. Dr. and Mrs. S. Andrew Kulin Dr. and Mrs. George M. Langford Dr. and Mrs. Leonard Laster Dr. and Mrs. Hans Laufer Mr. William Lawrence and Mrs. Barbara Buchanan Mr. and Mrs. Stephen R. Levy Mr. and Mrs. Robert Livingstone, Jr. Mr. and Mrs. James E. Lloyd Mr. and Mrs. Bernard Manuel Dr. and Mrs. Julian B. Marsh Mr. and Mrs. Joseph C. Martyna Mr. and Mrs. Frank J. Mather, III Mr. and Mrs. John E. Matthews Dr. and Mrs. Robert T. McCluskey Mr. Paul McGonigle Dr. and Mrs. Jerry M. Melillo Dr. Martin Mendelson Mr. and Mrs. Richard Meyers Dr. and Mrs. Daniel G. Miller Dr. and Mrs. Merle Mizell Dr. and Mrs. Charles H. Montgomery Mr. and Mrs. Charles F. Murphy Dr. and Mrs. John E. Naugle Dr. Pamela Nelson and Mr. Christopher Olmsted Mr. and Mrs. Frank L. Nickerson Dr. and Mrs. Clifford T. O'Connell Mr. and Mrs. David R. Palmer Dr. and Mrs. George D. Pappas Mr. and Mrs. Robert Parkinson Mr. and Mrs. Richard M. Paulson, Jr. Mr. and Mrs. William J. Pechilis Mr. and Mrs. John B. Peri Dr. and Mrs. Philip Person Mr. and Mrs. Frederick S. Peters Mrs. and Mr. Grace M. Peters Mr. and Mrs. George H. Plough Dr. and Mrs. Aubrey Pothier, Jr. Dr. and Mrs. Carl A. Price Mr. Allan Ray Putnam Dr. and Mrs. Lionel I. Rebhun Dr. and Mrs. George T. Reynolds Mr. and Mrs. John Ripple Dr. and Mrs. Harris Ripps Ms. Jean Roberts Drs. Priscilla and John Roslansky Dr. and Mrs. John D. Rummel Dr. and Mrs. John W. Saunders, Jr. Dr. and Mrs. R. Walter Schlesinger Mr. and Mrs. Harold H. Sears Mr. John Seder and Ms. Frances Plough Dr. and Mrs. Sheldon J. Segal Dr. and Mrs. Douglas R. Shanklin Dr. and Mrs. David Shepro Mr. and Mrs. Bertram R. Silver Mr. and Mrs. Jonathan O. Simonds Drs. Frederick and Marguerite Smith Dr. and Mrs. Hein/. Specht (Dr. Specht deceased) Drs. William and Phoebe Speck Dr. and Mrs. William K. Stephenson Mr. and Mrs. E. Kent Swift. Jr. Mr. and Mrs. Gerard L. Swope, III Mr. and Mrs. Emil D. Tietje, Jr. Mr. Norman N. Tolkan Dr. and Mrs. Walter Troll Mr. and Mrs. Volker Ulbrich Drs. Claude and Dorothy Villee Mr. and Mrs. Samuel Vincent Dr. and Mrs. Samuel Ward Mr. J. Ware and Ms. Sharon McCarthy Dr. and Mrs. Henry B. Warren Dr. and Mrs. Gerald Weissmann Dr. and Mrs. Paul S. Wheeler Dr. Martin Keister White Mr. and Mrs. Geoffrey G. Whitney, Jr. Mr. and Mrs. Leonard M. Wilson Mr. and Mrs. Leslie J. Wilson Mr. and Mrs. Dick S Yeo Dr. and Mrs. Sumner I. Zacks Individual Membership Mr. David C. Ahearn Mr. Henry Albers Mrs. Constance M. Allard Dr. Nina S. Allen Mrs. Tammy Amon Mr. Dean N. Arden Mrs. Ellen Prosser Armstrong Mrs. Kimball C. Atwood, III Dr. Serena Baccetti Mr. Everett E. Bagley Mr. C. John Berg Ms. Avis Blomberg Mrs. Elinor W. Bodian Mr. Thomas C. Bolton Mrs. Jennie P. Brown Mrs. M. Kathryn S. Brown Ms. Hennete Bull Dr. Alan H. Burghauser Mrs. Barbara Gates Burwell Mr. Bruce E. Buxton Mr. Patrick J. Calie Dr. Graciela C. Candelas Mrs. Winslow G. Carlton (deceased) Mr. Frank C. Carotenuto R80 Annual Report Dr. Robert H, Currier Mrs. Patricia A. Case Dr. Sallie Chisholm Mrs. Octavia C. Clement Mr. Allen W. Clowes Dr. Jewel Plummer Cohh Mrs. Margaret H. Cohurn Dr. Seymour S. Cohen Ms. Anne S. Concannon Mr. Robert J. Cook Prof. D. Eugene Copeland Dr. Helen M. Costello Dr. Vincent Cowling Mrs. J. Sterling Crandall Ms. Dorothy Crossley Ms. Helen M. Crossley Mrs. Villa B. Crowell Mr. Norman Dana Dr. Morton Davidson Ms. Carol Reimann De Young Dr. Mane A. DiBerardino Mrs. Shirley Dierolt Mr. David L. Donovan Mr. Stephen Doyle Ms. Suzanne Drohan Mr. Roy A. Duffus Mrs. Charles Eastman Dr. Frank Egloff Mr. Raymond Eliott Mr. William M. Ferry Mr. Robert Fitzpatrick Ms. Sylvia M. Flanagan Mr. Robert P. Flynn. Jr. Mr. John W. Folino, Jr. Mr. John H. Ford Dr. Krystyna Frenkel Mr. Paul J. Freyheit Mrs. Paul M. Fye Mr. Joseph C. Gallagher Miss Eleanor Garrield Dr. Patricia E. Garrett Mr. Charles Gilford Mrs. James R. Glazebrook Mrs. Mary L. Goldman Mr. Michael P. Goldring Mrs. Phyllis Goldstein Ms. Muriel Gould Mrs. Deborah Ann Green Dr. B. Herold Griffith Mrs. Edith T. Grosch (deceased) Mrs. Barbara Grossman Mrs. Valerie A. Hall Dr. Peter J. Hamre Ms. Mary Elizabeth Hamstrom Ms. Elizabeth E. Hathaway Dr Robert R. Haubrich Mrs Jane M. Heald Mr. Michael W. Herlihy Mrs. Nalhan Hirschfeld Mrs. Eleanor D. Hodge Mr. Roger W Huhbell Ms. Susan A. Huettner Miss Eli/ubelh B. Jackson Dr. Joseph Jacohson Mr. Raymond L. Jewett Mrs. Barbara W. Jones Mrs. Margaret H. Jones Mrs. Barbara Kanellopoulos Mrs. Joan T. Kanwisher Mrs. Sally Karush Mrs. Marcella Katz Ms. Patricia E. Keoughan Dr. Peter N. Kivy Lady Haber Kornberg Dr. Bruno P. Kremer Ms. Norma Kumin Mr. Bernard H. Labitt Mrs. Janet W. Larcom Ms. Rebecca Lash Dr. Marian E. LeFevre Dr. Mortimer Levitz Mr. Edwin M. Libbm Mr. Lennart Lindberg Mrs. Barbara C. Little Mrs. Sarah J. Loessel Mrs. Ermine W. Lovell Mr. Richard C. Lovering Mrs. Margaret M. Macleish Ms. Anne Camille Maher Mrs. Annemarie E. Mahler Mr. Patrick J. Mahoney Dr. Phillip B. Maples Mr. Daniel R. Manin Dr. G. C. Matthiessen Dr. Miriam Jacob Mauzerall Mrs. Mary Hartwell Mavor Mrs. Jane C. McCormack Ms. Suzanne McDermott Mrs. Nella W. McElroy (deceased) Dr. Susan Gerbi Mcllwain Ms. Mary W. McKoan Ms. Geraldine G. McLean Ms. Cornelia Hanna McMurtrie Mrs. Ellen L. Meigs Mr. Ted Mehllo Mrs. Grace S. Metz Mrs. Mary G. Miles Mrs. Florence E. Mixer Mr. John T. Moaklcy Mrs. Mary E. Montgomery Ms. Cynthia Moor Mr. Stephen A. Moore Mr. Alan F. Morrison Dr. M. Patricia Morse Mrs. Eleanor M. Nace Mr. William G. Neall Mrs. Anne Nelson Mrs. Catherine N. Norton Mr. Thomas J. Novitsky Mr. John J. O'Connor Dr. Renee Bennett O'Sullivan Miss Carolyn L. Parmenter Mrs. Dolores Patch-Wing Dr. John B. Pearce Ms. Joan Pearlman Dr. Judith Pederson Ms. Joyce S. Pendery Dr. Murray E. Pendleton Mr. Raymond W. Peterson Ms. Victoria A. Powell Mrs. Julia S. Rankin Mr. Fred J. Ravens, Jr. Mrs. Adell R. Rawson Dr. Robert M. Reece Ms. Anecia Kathy Regis Dr. Renato A. Ricca Dr. Mary Esther Rice Mr. John Riina Dr. Monica Riley Mrs. Alison A. Robb Mrs. Lola E. Robertson Mrs. Ruth J. Robinson Mrs. Arlene Rogers Mrs. Wendy E. Rose Ms. Hilde Rosenthal Mrs. Atholie K. Rosett Dr. Virginia F. Ross Mr. Raymond A. Sanborn Mrs. Joyce Waksman Schambacher Ms. Elaine Schott Mrs. Elsie M. Scott Sea Education Association, Inc. Dr. Cecily C. Selby Mrs. Deborah G. Senft Mrs. Charlotte Shemin Mrs. Phyllis J. Silver Mrs. Cynthia C. Smith Mrs. Perle Sonnenblick Dr. Evelyn Spiegel Dr. Guy L. Steele, Sr. Dr. Robert E. Steele Mrs. Eleanor Steinbach Mrs. Judith G. Stetson Mrs. Jane Lazarow Stetten Dr. Dorothy A. Stracher Mr. Robert Stump Dr. Maurice Sussman Mr. Albert H. Swain Mr. James K. Taylor Mrs. Alice Todd Mr. Arthur D. Trauh Ms. Natalie Trousof Ms. Ciona Ulbrich Mrs. Barbara Van Holde Dr. Kensal E. Van Holde Ms. Sylvia Vatuk Ms. Susan Veeder Mr. Lee D. Vincent Mr. Arthur D. Voorhis Mrs. Eve Warren Mr. John T. Weeks Ms. Lillian Wendorff Dr. William M. Wheeler Ms. Mabel E. Whelpley Mrs. Barbara Whitehead Mrs. A. A. Wickersham Mrs. Clare M. Wilber Mrs. Ann S. Wilke Mr. Albert Wilson Dr. T. Hastings Wilson Ms. Nancy Woitkoski Mrs. Eli/abeth S. Yntema Members (if the Corporation R81 Mrs. Donald J. Zinn Pat Hancox Arlene Rogers Hanna Hastings Lil Saunders MBL Gift Shop Volunteers Sally Karush Alice Knowles Louise Specht Cynthia Smith Marion Adelberg Donna Kornberg Peggy Smilh Barbara Atwood Evelyn Laufer Jane Stetten Caroline Banks Barbara Little Elaine Troll Harriet Bernheiiner Sally Loessel Natalie Trousof Avis Blomberg Winnie Mackey Barbara Van Holde Gloria Borgese Miriam Mauzerall Susan Veeder Kitty Brown Mary Mavor Carol Ann Wagner Elisabeth Buck Jane McCormack Mabel Whelpley Vera Clark Louise McManus Clare Wilbcr Peggy Clowes Phyllis Meyers Jewel Cobb Polly Miles Janet Daniels Florence Mixer MBL Summer Tour Guides Carol DeYoung Lorraine Mizell Fran Eastman Elizabeth Moseley Sears Crowell Alma Ebert Stacia Palmer Barbara Little Jane Foster Bertha Person Steve Oliver Becky Glazebrook Margareta Pothier Julie Rankin Muriel Gould Julie Rankm Pnscilla Roslansky Barbara Grossman Millie Rebhun Mary Ulbrich Jean Halvorson Jean Ripps John Valois Certificate of Organization Articles of Amendment Bylaws Certificate of Organization Articles of Amendment (On File in the Office of the Secretary of the Commonwealth) No. 3170 We, Alpheus Hyatt, President. William Stanford Stevens. Treasurer, and William T. Sedgwick, Edward G. Gardiner, Susan Mims and Charles Sedgwick Minot being a majority of the Trustees of the Marine Biological Laboratory in compliance with the requirements of the fourth section of chapter one hundred and fifteen of the Public Statutes do hereby certify that the following is a true copy of the agreement of association to constitute said Corporation, with the names of the subscribers thereto: We, whose names are hereto subscribed, do, by this agreement, associate ourselves with the intention to constitute a Corporation according to the provisions of the one hundred and fifteenth chapter of the Public Statutes of the Commonwealth of Mas- sachusetts, and the Acts in amendment thereof and in addition thereto. The name by which the Corporation shall be known is THE MARINE BIOLOGICAL LABORATORY. The purpose for which the Corporation is constituted is to establish and maintain a laboratory or station for scientific study and investigations, and a school for instruc- tion in biology and natural history. The place within which the Corporation is established or located is the city of Boston within said Commonwealth. The amount of its capital stock is none. In Witness Whereof, we have hereunto set our hands, this twenty seventh day of February in the year eighteen hundred and eighty-eight. Alpheus Hyatt, Samuel Mills. William T. Sedgwick, Edward G. Gardiner, Charles Sedgwick Minot, William G. Farlow. William Stanford Stevens, Anna D. Phillips, Susan Mims, B. H. Van Vteck. That the tirst meeimy nt the subscribers to said agreement was held on the thirteenth day of March in the year eighteen hundred and eighty-eight. In Witness Whereof, we have hereunto signed our names, this thirteenth day of March in the year eighteen hundred and eighty-eight, Alpheus Hyatt, President, William Stanford Stevens, Treasurer, Edward G. Gardiner. William T Sedgwick, Susan Minis, Charles Sedgwick Minot. (Approved on March 20, 1888 as follows: I hereby certify that it appears upon an examination of the within wntten certificate and the records of the corporation duly submitted to my inspection, that the require- ments of sections one. two and three of chapter one hundred and fifteen, and sections eighteen, twenty and twenty-one of chapter one hundred and six, of the Public Statutes, have been complied with and I hereby approve said certificate this twentieth day of March A,D. eighteen hundred and eighty-eight. Charles Endicoit Commissioner of Corporations) (On File in the Office of the Secretary of the Commonwealth) We, James D. Ebert. President, and David Shepro, Clerk of the Marine Biological Laboratory, located at Woods Hole, Massachusetts 02543, do hereby certify that the following amendment to the Articles of Organization of the Corporation was duly adopted at a meeting held on August 15, 1975, as adjourned to August 29. 1975, by vote of 444 members, being at leasi two-thirds of its members legally qualified to vote in the meeting of the corporation: Voted: That the Certificate of Organization of this corporation be and it hereby is amended by the addition of the following provisions: "No Officer, Trustee or Corporate Member of the corporation shall be personally liable for the payment or satisfaction of any obligation or liabilities incurred as a result of, or otherwise in connection with, any commitments, agreements, activities or affairs of the corporation. "Except as otherwise specifically provided by the Bylaws of the corporation, meet- ings of the Corporate Members of the corporation may be held anywhere in the United States. "The Trustees of the corporation may make, amend or repeal the Bylaws of the corporation in whole or in part, except with respect to any provisions thereof which shall by law, this Certificate or the bylaws of the corporation, require action by the Corporate Members." The foregoing amendment will become effective when these articles of amendment are filed in accordance with Chapter 180, Section 7 of the General Laws unless these articles specify, in accordance with the vote adopting the amendment, a later effective date not more than thirty days after such riling, in which event the amendment will become effective on such later date. In Witness whereof and Under the Penalties of Perjury, we have hereto signed our names this 2nd day of September, in the year 1975, James D. Ehert, President; David Shepro, Clerk. (Approved on October 24, 1975, as follows: I hereby approve the within articles of amendment and, the riling fee m the amount of $10 having been paid, said articles are deemed to have been filed with me this 24th day of October. 1975, Paul Guzzi Secretary' of the Commonwealth) Bylaws (Revised August 7, 1992 and December 10. 1992) ARTICLE 1 THE CORPORATION A. Name iintl Piirpiat: The name of the Corporation shall he The Marine Biolog- ical Laboratory. The Corporation's purpose shall he to establish and maintain a K82 Bylaws of the Corporation R83 laboratory or station tor scientific study and investigation and a school lor instruction in biology and natural history. B. Nondiscrimination. The Corporation shall not discriminate on the basis of age, religion, color, race, national or ethnic origin, sex or sexual preference in its policies on employment and administration or in its educational and other programs. ARTICLE II MEMBERSHIP A. Members. The Members of the Corporation ("Members") shall consist of persons elected by the Board of Trustees (the "Board"), upon such terms and conditions and in accordance with such procedures, not inconsistent with law or these Bylaws, as may be determined by the Board. At any regular or special meeting of the Board, the Board may elect new Members. Members shall have no voting or other rights with respect to the Corporation or its activities except as specified in these Bylaws, and any Member may vote at any meeting of the Members in person only and not by proxy. Members shall serve until their death or resignation unless earlier removed with or without cause by the affirmative vote of two-thirds of the Trustees then in office. Any Member who has retired from his or her home institution may, upon written request to the Corporation, be designated a Life Member. Life Members shall not have the right to vote and shall not be assessed for dues. B. Meetings. The annual meeting of the Members shall be held on the Friday following the first Tuesday in August of each year, at the Laboratory of the Corpo- ration in Woods Hole, Massachusetts, at 9:30 a.m. The Chairperson of the Board shall preside at meetings of the Corporation. If no annual meeting is held in accordance with the foregoing provision, a special meeting may be held in lieu thereof with the same effect as the annual meeting, and in such case all references in these Bylaws, except in this Article II. B.. to the annual meeting of the Members shall be deemed to refer to such special meeting. Members shall transact business as ma\ properly come before the meeting. Special meetings of the Members may be called by the Chair- person or the Trustees, and shall be called by the Clerk, or in the case of the death, absence, incapacity or refusal by the Clerk, by any other officer, upon written application of Members representing at least ten percent of the smallest quorum of Members required for a vote upon any matter at the annual meeting of the Members, to be held at such time and place as may be designated. C. Quorum. One hundred (100) Members shall constitute a quorum at any meeting. Except as otherwise required by law or these Bylaws, the affirmative vote of a majonty of the Members voting in person at a meeting attended by a quorum shall constitute action on behalf of the Members. D. Notice of Meetings. Notice of any annual meeting or special meeting of Members, if necessary, shall be given by the Clerk by mailing notice of the time and place and purpose of such meeting at least 15 days before such meeting to each Member at his or her address as shown on the records of the Corporation. E. Wavier of Notice. Whenever notice of a meeting is required to be given a Member, under any provision of the Articles or Organization or Bylaws of the Corporation, a written waiver thereof, executed before or after the Meeting by such Member, or his or her duly authorized attorney, shall be deemed equivalent to such notice. F. Adjournments. Any meeting of the Members may be adjourned to any other time and place by the vote of a majority of those Members present at the meeting, whether or not such Members constitute a quorum, or by any officer entitled to preside at or to act as Clerk of such meeting, if no Member is present or represented. It shall not be necessary to notify any Members of any adjournment unless no Member is present or represented at the meeting which is adjourned, in which case, notice of the adjournment shall be given in accordance with Article II. D. Any business which could have been transacted at any meeting of the Members as originally called may be transacted at an adjournment thereof. ARTICLE III ASSOCIATES OF THE CORPORATION Associates of the Corporation. The Associates of the Marine Biological Laboratory shall be an unincorporated group of persons (including associations and corporations) interested in the Laboratory and shall be organized and operated under the general supervision and authority of the Trustees. The Associates of the Marine Biological Laboratory shall have no voting rights. ARTICLE IV BOARD OF TRUSTEES A. Powers. The Board of Trustees shall have the control and management of the affairs of the Corporation. The Trustees shall elect a Chairperson of the Board who shall serve until his or her successor is elected and qualified. They shall annually elect a President of the Corporation. They shall annually elect a Vice Chairperson of the Board who shall be Vice Chairperson of the meetings of the Corporation. They shall annually elect a Treasurer. They shall annually elect a Clerk, who shall be a resident of Massachusetts. They shall elect Trustees-at-Large as specified in this Article IV. They shall appoint a Director of the Laboratory for a term not to exceed five years, provided the term sh.ill not exceed one year if the candidate has attained the age of 65 years prior to the date of the appointment. They shall choose such other officers and agents as they shall think best. They may fix the compensation of all officers and agcnls D| the Corporation and may remove them at any time. They may fill vacancies occurring in any of the offices. The Board shall have the power to choose an Executive Committee from their own number as provided in Article V, and to delegate to such Committee such of their own powers as they may deem expedient in addition to those powers conferred by Article V. They shall, from time to time, elect Members to the Corporation upon such terms and conditions as they shall have determined, not inconsistent with law or these Bylaws. B. Composition anJ Elt'clion. ( 1 1 The Board shall include 24 Trustees elected by the Board as provided below: (a) At least six Trustees I "Corporate Trustees") shall be Members who are scientists, and the other Trustees I "Trustees-at-Large" ) shall he individuals who need not be Members or otherwise affiliated with the Corporation. (b) The 24 elected Trustees shall be divided into four classes of six Trustees each, with one class to be elected each year to serve for a term of four years, and with each such class to include at least one Corporate Trustee. Such classes of Trustees shall be designated by the year of expiration of their respective terms. (2) The Board shall also include the Chief Executive Officer, Treasurer and the Chairperson of the Science Council, who shall be ex officia voting members of the Board. (3) Although Members or Trustees may recommend individuals for nomination as Trustees, nominations for Trustee elections shall be made by the Nominating Committee in its sole discretion The Board may also elect Trustees who have not been nominated by the Nominating Committee. C. Eligibility. A Corporate Trustee or a Trustee-at-Large who has been elected to an initial four-year term or remaining portion thereof, of which he/she has served at least two years, shall be eligible for re-election to a second four-year term, but shall be ineligible for re-election to any subsequent term until one year has elapsed after he/she has last served as a Trustee. D. Removal. Any Trustee may be removed from office at any time with or without cause, by vote of a majonty of the Members entitled to vote in the election of Trustees: or for cause, by vote of two-thirds of the Trustees then in office. A Trustee may be removed for cause only if notice of such action shall have been given to all of the Trustees or Members entitled to vote, as the case may be. prior to the meeting at which such action is to be taken and if the Trustee to be so removed shall have been given reasonable notice and opportunity to be heard before the body proposing to remove him or her. E. Vacancies. Any vacancy in the Board may be filled by vote of a majority of the remaining Trustees present at a meeting of Trustees at which a quorum is present. Any vacancy in the Board resulting from the resignation or removal of a Corporate Trustee shall be tilled by a Member who is a scientist. F. Meetings. Meetings of the Board shall be held from time to time, not less frequently than twice annually, as determined by the Board. Special meetings of Trustees may be called by the Chairperson, or by any seven Trustees, to be held at such lime and place as may be designated. The Chairperson of the Board, when present, shall preside over all meetings of the Trustees. Written notice shall be sent to a Trustee's usual or last known place of residence at least two weeks before the meeting. Notice of a meeting need not be given to any Trustee if a written waiver of notice executed by such Trustee before or after the meeting is filed with the records of the meeting, or if such Trustee shall attend the meeting without protesting prior thereto or at its commencement the lack of notice given to him or her. G. Quorum and Action by Trustees. A majority of all Trustees then in office shall constitute a quorum. Any meeting of Trustees may be adjourned by vote of a majonty of Trustees present, whether or not a quorum is present, and the meeting ma\ be held as adjourned without further notice. When a quorum is present at any meeting of the Trustees, a majority of the Trustees presenl and voting (excluding abstentions) shall decide any question, including the election of officers, unless otherwise required by law. the Articles of Organization or these Bylaws. H. Transfers of Interests in Land. There shall be no transfer of title nor long-term lease of real properly held by the Corporation without prior approval of not less than two-thirds of the Trustees. Such real property transactions shall he finally acted upon at a meeting of the Board only if presented and discussed at a prior meeting of the Board. Either meeting may be a special meeting and no less than four weeks shall elapse between the two meetings. Any property acquired by the Corporation after December 1. 1989 may be sold, any mortgage or pledge of real property (regardless of when acquired) lo secure bonowings by the Corporation may he granted, and any transfer of title or interest in real property pursuant to the foreclosure or endorsement R84 Annual Report of any such mortgage or pledge of real property may be effected by any holder of a mortgage or pledge of real property of the Corporation, with the prior approval of not less ihan two-thirds of the Trustees {other than any Trustee or Trustees with a direct or indirect financial interest in the transaction being considered tor approval) who are present at a regular or special meeting of the Board at which there is a quorum. ARTICLE V COMMITTEES A. Executive Committee. There shall be an Executive Committee of the Board of Trustees which shall consist of not more than eleven (II) Trustees, including ex officio Trustees, elected by the Board. The Chairperson uf the Board shall act as Chairperson of the Executive Committee and the Vice Chairperson as Vice Chairperson. The Executive Committee shall meet at such times and places and upon such notice and appoint such subcommittees as the Committee shall determine. The Executive Committee shall have and may exercise all the powers of the Board during the intervals between meetings of the Board except those powers specifically withheld, from lime to time, by vote of the Board or by law. The Executive Committee may also appoint such committees, including persons who are not Trust- ees, as it may, from time to time, approve to make recommendations wilh respect to matters to be acted upon by the Executive Committee or the Board. The Executive Committee shall keep appropriate minutes of its meetings, which shall be reported to the Board. Any actions taken by the Executive Committee shall also be reported to the Board. B. Nominating Committee. There shall be a Nominating Committee which shall consist of not fewer than four nor more than six Trustees appointed by the Board in a manner which shall reflect the balance between Corporate Trustees and Trustees- at-Large on the Board. The Nominating Committee shall nominate persons for election as Corporate Trustees and Trustees-at-Large. Chairperson of the Board. Vice Chairperson of (he Board, President, Treasurer, Clerk, Director of the Laboratory and such other officers, if any, as needed, in accordance with (he requirements of these Bylaws. The Nominating Committee shall also be responsible for overseeing the training of new Trustees. The Chairperson of the Board of Trustees shall appoint the Chairperson of the Nominating Committee, The Chairperson of the Science Council shall be an ex officio voting member of the Nominating Committee. C. Science Council. There shall be a Science Council (the "Council") which shall consist of Members of the Corporation elected to the Council by vote of the Members of the Corporation, and which shall advise the Board with respect to matters con- cerning the Corporation's mission, its scientific and instructional endeavors, and the appointment and promotions of persons or committees with responsibility for matters requiring scientific expertise. Unless otherwise approved by a majority of the mem- bers of the Council, the Chairperson of the Council shall be elected annually by the Council. The chief executive officer of the Corporation shall be an c.\ officio voting member of the Council D. Board of Overseers. There shall be a Board of Overseers which shall consist of not fewer than five nor more than eight scientists who have expertise concerning matters with which the Corporation is involved. Members of the Board of Overseers may or may not be Members of the Corporation and may be appointed by the Board of Trustees on the basis of recommendations submitted from scientists and scientific organizations or societies. The Board of Overseers shall be available to review and offer recommendations lo the officers. Trustees and Science Council regarding scientific activities conducted or proposed by the Corporation and shall meel from time to time, not less frequently than annually, as determined by the Board of Trustees. E. Board Committees Generallv. The Trustees may elect or appoint one or more other committees (including, but not limited to, an Investment Commiltee, a Devel- opment Committee, an Audit Committee, a Facilities and Capital Equipment Com- mittee and a Long-Range Planning Committee) and may delegate to am sin.li committee or committees any or all of their powers, except those which by law, the Arliclcs of Organization or these Bylaws the Trustees are prohibited from delegating; provided thai any committee to which the powers of the Trustees are delegated shall consist solely of Trustees. The members of any such committee shall have such tenure and duties as the Trustees shall determine. The Investment Committee, which shall oversee (he management of the Corporation's endowment funds and marketable securities sh.ill include as e.\ officio members, the Chairperson of the Board, the Treasurer and the Chairperson of the Audit Committee, together with such Trustees as may be requiixo fur not less than two-thirds of the Investment Committee to consist of Trustees. Except a> otherwise provided by these Bylaws or determined by the Trustees, any such committee may make rules lor the conduct of its business, but, unless otherwise provided by the Trustees or in such rules, its business shall he conducted as nearly as possible in the same manner as is provided by these Bylaws for the Trustees. F. Actitms Without n Meeting. Any action required or permitted to be taken at any meeting of the Executive Committee or any other committee elected by the Trustees may be taken without a meeting if all members of such committees consent to the action in writing and such written consents are filed with the records of meetings. Members of the Executive Committee or any other committee elected by the Trustees may also participate in any meeting by means of a telephone conference call, or otherwise lake action in such a manner as may, from time to time, be permitted by law. G. Manual of Procedures. The Board of Trustees, on the recommendation of the Executive Committee, shall establish guidelines and modifications thereof to be recorded in a Manual of Procedures. Guidelines shall establish procedures for: (1) Nomination and election of members of the Corporation, Board of Trustees and Executive Commiltee; (2) Election of Officers; (3) Formation and Function of Standing Committees. ARTICLE VI OFFICERS A. Enumeration. The officers of the Corporation shall consist of a President, a Treasurer and a Clerk, and such other officers having the powers of President, Treasurer and Clerk as the Board may determine, and a Director of the Laboratory. The Corporation may have such other officers and assistanl officers as the Board may determine, including (without hmiialion) a Chairperson of the Board, Vice Chairper- son and one or more Vice Presidents. Assistant Treasurers or Assistanl Clerks. Any two or more offices may be held by the same person. The Chairperson and Vice Chairperson of the Board shall be elected by and from the Trustees, but other officers of the Corporation need not be Trustees or Members. If required by the Trustees, any officer shall give the Corporation a bond for the faithful performance of his or her duties in such amount and with such surely or sureties as shall be satisfactory to the Truslees. B. Tenure. Except as otherwise provided by law, by the Articles of Organization or by these Bylaws, the President. Treasurer, and all other officers shall hold office until the first meeting of the Board following the annual meeting of Members and thereafter, until his or her successor is chosen and qualified. C. Resignation. Any officer may resign by delivering his or her written resignation to the Corporation at its principal office or to the President or Clerk and such resignation shall be effective upon receipt unless it is specified to be effective at some other time or upon Ihe happening of some other event. D. Removal. The Board may remove any officer with or withoul cause by a vote of a majority of the entire number of Trustees then in office, at a meeting of the Board called for thai purpose and for which notice of the purpose thereof has been given, provided that an officer may be removed for cause only after having an opportunity to be heard by the Board at a meeting of the Board at which a quorum is personally present and voting. E. Vacancy. A vacancy in any office may be filled for the unexpired balance of the term by vote of a majority of the Trustees present at any meeting of Trustees at which a quorum is present or by written consent of all of Ihe Truslees, if less than a quorum of Trustees shall remain in office. F. Chairperson. The Chairperson shall have such powers and duties as may be determined by the Board and, unless otherwise determined by the Board, shall serve in thai capacity for a term coterminous with his or her term as Trustee. G. Vice Chairperson. The Vice Chairperson shall perform Ihe duties and exercise the powers of the Chairperson in Ihe absence or disability of the Chairperson, and shall perform such other duties and possess such other powers as may be determined by the Board. Unless otherwise determined by the Board, the Vice Chairperson shall serve for a one-year term. H. Director. The Director shall be the chief operating officer and, unless otherwise voted by the Trustees, the chief executive officer of the Corporation. The Director shall, subject to the direction of the Trustees, have genera! supervision of the Laboratory and control of (he business of the Corporation. Al Ihe annual meeting, the Director shall submit a report of the operations of the Corporation for such year and a statement of its affairs, and shall, from time to time, report to the Board all matters \\ ithin his or her knowledge which the inlerests of the Corporation may require to he brought to its notice I. Depin\ Director The Deputy Director, if any. or if there shall be more than one, the Deputy Directors in the order determined by the Truslees, shall, in the absence or disability of the Director, perform the duties and exercise the powers of the Director and shall perform such other duties and shall have such other powers as the Truslees may. from lime lo lime, prescribe J President- The President shall have Ihe powers and duties as may he vested in him or her by the Board. K. Treasurer and Assistant Treasurer. The Treasurer shall, subject to the direction of the Trustees, have general charge of the financial affairs of the Corporation, Bylaws of the Corporation R85 including us long-range financial planning, and shall cause to he kept accurate books of account. The Treasurer shaJl prepare a yearly report on the financial status of the Corporation to be delivered at the annual meeting. The Treasurer shall also prepare or oversee all filings required by the Commonwealth of Massachusetts, the Internal Revenue Service, or other Federal and State Agencies. The account of the Treasurer shall be audited annually by a certified public accountant. The Assistant Treasurer, if any, or if there shall be more than one, the Assistant Treasurers in the order determined by the Trustees, shall, in the absence or disability of the Treasurer, perform the duties and exercise the powers of the Treasurer, shall perform such other duties and shall have such other powers as the Trustees may, from time to time, prescribe. L. Clerk and Assistant Clerk. The Clerk shall be a resident of the Commonwealth of Massachusetts, unless the Corporation has designated a resident agent in the manner provided by law. The minutes or records of all meetings of the Trustees and Members shall be kept by the Clerk who shall record, upon the record books of the Corporation, minutes of the proceedings at such meetings. He or she shall have custody of the record books of the Corporation and shall have such other powers and shall perform such other duties as the Trustees may, from time to time, prescribe. The Assistant Clerk, if any, or if there shall be more than one, the Assistant Clerks in the order determined by the Trustees, shall, in the absence or disability of the Clerk, perform the duties and exercise the powers of the Clerk and shall perform such other duties and shall have such other powers as the Trustees may, from time to time, prescribe. In the absence of the Clerk and an Assistant Clerk from any meeting, a temporary Clerk shall be appointed at the meeting. M. Other Powers unj Dunes. Each officer shall have in addition to the duties and powers specifically set forth in these Bylaws, such duties and powers as are custom- arily incident to his or her office, and such duties and powers as the Trustees may. from time to time, designate. ARTICLE VII AMENDMENTS These Bylaws may be amended by the affirmative vote of the Members at any meeting, provided that notice of the substance of the proposed amendment is stated in the notice of such meeting. As authori/.ed by the Articles of Organization, the Trustees, by a majority of their number then in office, may also make, amend or repeal these Bylaws, in whole or in part, except with respect to (a) the provisions of these Bylaws governing d) the removal of Trustees and (ii) the amendment of these Bylaws and (b) any provisions of these Bylaws which by law, the Articles of Organization or these Bylaws, requires action by the Members. No later than the time of giving notice of meeting of Members next following the making, amending or repealing by the Trustees of any Bylaw, notice thereof stating the substance of such change shall be given to all Members entitled to vote on amending the Bylaws. Any Bylaw adopted by the Trustees may be amended or repealed by the Members entitled to vote on amending the Bylaws. ARTICLE VIII INDEMNITY Except as otherwise provided below, the Corporation shall, to the extent legally permissible, indemnify each person who is, or shall have been, a Trustee, director or officer of the Corporation or who is serving, or shall have served at the request of the Corporation as a Trustee, director or officer of another organization in which the Corporation directly or indirectly has any interest as a shareholder, creditor or otherwise, against all liabilities and expenses (including judgments, fines, penalties, and reasonable attorneys' fees and all amounts paid, other than to the Corporation or such other organization, in compromise or settlement) imposed upon or incurred by any such person in connection with, or arising out of, the defense or disposition of any action, suit or other proceeding, whether civil or criminal, in which he or she may be a defendant or with which he or she may be threatened or otherwise involved, directly or indirectly, by reason of his or her being or having been such a Trustee, director or officer. The Corporation shall provide no indemnification with respect to any matter as to which any such Trustee, director or officer shall be finally adjudicated in such action, suit or proceeding not to have acted in good faith in the reasonable belief that his or her action was in the best interests of the Corporation. The Corporation shall provide no indemnification with respect to any matter settled or comprised unless such matter shall have been approved as in the best interests of the Corporation, after notice that indemnification is involved, by (i) a disinterested majority of the Board of the Executive Committee, or (ii) a majority of the Members. Indemnification may include payment by the Corporation of expenses in defending a civil or criminal action or proceeding in advance of the final disposition of such action or proceeding upon receipt of an undertaking by the person indemnified to repay such payment if it is ultimately determined that such person is not entitled to indemnification under the provisions of this Article VIII, or under any applicable law. As used in the Article VIII, the terms "Trustee," "director," and "officer" include their respective heirs, executors, administrators and legal representatives, and an "interested" Trustee, director or officer is one against whom in such capacity the proceeding in question or another proceeding on the same or similar grounds is (hen pending. To assure indemnification under this Article VIII of all persons who are determined by the Corporation or otherwise to be or to have been "fiduciaries" of any employee benefits plan ol the Corporation which may exist, from time to lime, this Article VIII shall be interpreted as follows: (i) "another organization" shall be deemed to include such an employee benefit plan, including without limitation, any plan of the Corpo- ration which is governed by the Act of Congress entitled "Employee Retirement Income Security Act of 1974," as amended, from time to time, ("ERISA"); (ii) "Trustee" shall be deemed to include any person requested by the Corporation to serve as such for an employee benefit plan where the performance by such person of his or her duties to the Corporation also imposes duties on, or otherwise involves services by, such person to the plan or participants or beneficiaries of the plan; (iii) "fines" shall be deemed to include any excise tax plan pursuant to ERISA; and (iv) actions taken or omitted by a person with respect to an employee benefit plan in the performance of such person's duties for a purpose reasonably believed by such person to be in the interest of the participants and beneficiaries of the plan shall be deemed to be for a purpose which is in the best interests of the Corporation. The right of indemnification provided in this Article VIII shall not be exclusive of or affect any other rights to which any Trustee, director or officer may be entitled under any agreement, statute, vote of Members or otherwise. The Corporation's obligation to provide indemnification under this Article VIII shall be offset to the extent of any other source of indemnification of any otherwise applicable insurance coverage under a policy maintained by the Corporation or any other person. Nothing contained in the Article shall affect any rights to which employees and corporate personnel other than Trustees, directors or officers may be entitled by contract, by vote of the Board or of the Executive Committee or otherwise. ARTICLE IX DISSOLUTION The consent of every Trustee shall be necessary to effect a dissolution of the Manne Biological Laboratory. In case of dissolution, the property shall be disposed of in such a manner and upon such terms as shall be determined by the affirmative vote of two-thirds of the Trustees then in office in accordance with the laws of the Com- monwealth of Massachusetts, ARTICLE X MISCELLANEOUS PROVISIONS A. Fiscal Year, Except as otherwise determined by the Trustees, the fiscal year of the Corporation shall end on December 31st of each year. B. Seal. Unless otherwise determined by the Trustees, the Corporation may have a seal in such form as the Trustees may determine, from time to time. C. Execution of Instruments. All checks, deeds, leases, transfers, contracts, bonds, notes and other obligations authorized to be executed by an officer of the Corporation in its behalf shall be signed by the Director or the Treasurer except as the Trustees may generally or in particular cases otherwise determine. A certificate by the Clerk or an Assistant Clerk, or a temporary Clerk, as to any action taken by the Members, Board of Trustees or any officer or representative of the Corporation shall as to all persons who rely thereon in good faith be conclusive evidence of such action. D. Corporate Records. The original, or attested copies, of the Articles of Organi- zation, Bylaws and records of all meetings of the Members shall be kept in Massa- chusetts at the principal office of the Corporation, or at an office of the Corporation's Clerk or resident agent. Said copies and records need not all he kept in the same office. They shall be available at all reasonable times for inspection by any Member for any proper purpose, but not to secure a list of Members for a purpose other than in the interest of the applicant, as a Member, relative to the affairs of the Corporation. E. Articles of Organization. All references in these Bylaws to the Articles of Organization shall be deemed to refer to the Articles of Organization of the Corpo- ration, as amended and in effect, from time to time F. Transactions with Interested Parties. In the absence of fraud, no contract or other transaction between this Corporation and any other corporation or any firm, association, p;irtncislup or pcrsnn shall be affected or invalidated by the fact that any Trustee or officer of this Corporation is pecuniarily or otherwise interested in or is a director, member or officer of such other corporation or of such firm, association or partnership or in a party to or is pecuniarily or otherwise interested in such contract or other transaction or is in any way connected with any person or person, firm, association, partnership, or corporation pecuniarily or otherwise interested therein; provided that the fact that he or she individ- ually or as a director, member or officer of such corporation, firm, association or RS6 Annual Report partnership tn such a party or is so interested shall be disclosed to or shall have been authorizing any such contract or transaction with like force and effect as if he/she were not known by the Board ot Trustees or a majority of such Members thereof as shall be present so interested, or were not a director, member or officer of such other corporation, firm, at a meeting of the Board of Trustees at which action upon any such contract or association or partnership, provided that any vote with respect to such contract or transaction shall be taken; any Trustee may be counted in determining the existence of a transaction must be adopted by a majority of the Trustees then in office who have no quorum and may vote at any meeting of the Board of Trustees for the purpose of interest in such contract or transaction. VOLUME 197 THE NUMBER 2 BIOLOGICAL BULLETIN BIOI OGICAL BUUJH'IN CENTENNIAL ISSUE OCTOBER 1899-1999 llll BIOLOGICAL BU LI J.TIN Published by the Marine Biological Laboratory It's Wort. i_ook Brilliant: and easy. The Axioskop 2 is the microscope that brings new levels of comfort and productiv- ity to biomedical research. Renowned ICS infinity optics provide top quality images, and its operating convenience adds extra efficiency to your work. Fluorescence 4-fold. Carry out demanding fluorescence applica- tions with flexibility and optimal illumina- tion. Reflector turret for 4 fluorescence modules, 6-place excitation filter slide, plus the power of 100 watts. Motorized automation. In the Axioskop 2 MOT option, motorized functions include stage focusing and uni- versal condenser setting (including Koehler). These and other functions can be automated for high productivity and preci- sion when examining large numbers of specimens. Contact Zeiss today for the full stor , le Axioskop 2. It's smart microscopy m ea r . Carl Zeiss, Inc. Microscopy & Imaging Systems One Zeiss Drive Thornwood, NY 10594 800.233.2343 Fax 91 4. 681. 7446 micro@zeiss.com www.zeiss.com ZEISS THE BIOLOGICAL BULLETIN OCTOBER 1999 Editor Associate Editors Section Editor Online Editors Editorial Board Editorial Office MICHAEL J. GREENBERG Louis E. BURNETT R. ANDREW CAMERON CHARLES D. DERBY MICHAEL LABARBERA SHINYA INOUE, Imaging ami Microscopy JAMES A. BLAKE, Keys to Marine Invertebrates of the Woods Hole Region WILLIAM D. COHEN, Marine Models Electronic Record and Compendia PETER B. ARMSTRONG ERNEST S. CHANG THOMAS H. DIETZ RICHARD B. EMLET DAVID EPEL GREGORY HINKLE MAKOTO KOBAYASHI DONAL T. MANAHAN MARGARET MCFALL-NGAI MARK W. MILLER TATSUO MOTOKAWA YOSHITAKA NAGAHAMA SHERRY D. PAINTER J. HERBERT WAITE RICHARD K. ZIMMER PAMELA CLAPP HINKLE VICTORIA R. GIBSON CAROL SCHACHINGER PATRICIA BURNS The Whitney Laboratory, University of Florida Grice Marine Biological Laboratory. College of Charleston California Institute of Technology Georgia State University University of Chicago Marine Biological Laboratory ENSR Marine & Coastal Center. Woods Hole Hunter College, City University of New York University of California, Davis Bodega Marine Lab., University of California, Davis Louisiana State University Oregon Institute of Marine Biology, Univ. of Oregon Hopkins Marine Station, Stanford University Cereon Genomics, Cambridge, Massachusetts Hiroshima University of Economics, Japan University of Southern California Kewalo Marine Laboratory, University of Hawaii Institute of Neurobiology, University of Puerto Rico Tokyo Institute of Technology, Japan National Institute for Basic Biology, Japan Marine Biomed. Inst., Univ. of Texas Medical Branch University of California, Santa Barbara University of California, Los Angeles Managing Editor Staff Editor Editorial Associate Subscription & Advertising Secretary Published by MARINE BIOLOGICAL LABORATORY WOODS HOLE, MASSACHUSETTS Cover The three-dimensional stereo anaglyph on the cover is a ventral view of a brachiolaria larva of Patiriella regularis, a starfish; the brachiolaria de- picted is about 1500 jtim in length. Serotonergic neurons in the larva were stained with a rabbit antiserum and appear, in confocal fluorescent mi- croscopy, as bright dots lining the ciliated bands and brachiolar arms. The image (which should be viewed through the stereo glasses provided with this issue) is composed of 145 optical sections and was reconstructed as described in the article by Francis Chee and Maria Byrne (p. 123). Immunoreactive serotonergic cells are already visible in the gastrulae of echinoderms; but they increase in number and form an increasingly com- plex neural system as development proceeds. Be- cause the immunoreactivity is associated with the ciliary bands of free-swimming, planktotrophic lar- val forms as well as with their sensory structures and buccal cavity the serotonergic system has been thought to coordinate the locomotory and feeding behaviors of these larvae. In Iheir paper. Chee and Byrne focus on the larval stages of Patiriella regularis, which are all plank- totrophic; thus the development of the serotonergic system can be monitored throughout development, from the gastrula. through the brachiolaria (the last larval stage), and on to metamorphosis. The authors have used confocal fluorescence microscopy to re- construct the development of the serotonergic ner- vous sytsem in three dimensions and have related the increase in complexity to morphogenetic changes in the larvae. They have demonstrated a complex network of cells with varicose processes that connect the preoral and postoral ciliated bands, supporting the hypothesis that this network is reg- ulating larval feeding and swimming. In a related article in this issue (see p. 115), Michael Dailey and his colleagues use the mamma- lian brain as a model to show how multidimensional confocal fluorescence microscopy can enhance studies of biological structure and function. The images in this article are fine examples of the tech- niques described, and readers should use the stereo glasses to examine them. This is the third in a series of papers on Concepts in Imaging and Microscopy; the series is supported by the Optical Imaging Association, which has also provided the stereo glasses. Finally, this issue marks the end of The Biolog- ical Bulletin's first century of publication and the beginning of its second. The four small images on the cover, below the anaglyph, show how the face of the journal changed as the decades passed, biol- ogy expanded, the world shrank, and scientific pub- lishing entered its greatest revolution since the in- vention of movable type. A metamorphosis is certainly at hand, but the nature of the imago re- mains unresolved. CONTENTS VOLUME 197, No. 2: OCTOBER 1999 EDITORIAL IMMUNOLOGY Greenberg, Michael J. A century of science: The Biological Bulletin looks back and forward . 113 IMAGING AND MICROSCOPY Dailey, Michael, Glen Marrs, Jakob Satz, and Marc Waite Concepts in Imaging and Microscopy: Exploring biolog- ical structure and function with confocal micros- copy 115 NEUROBIOLOGY AND BEHAVIOR Chee, Francis, and Maria Byrne Development of the larval serotonergic nervous sys- tem in the sea star Patiriella regu/aris as revealed by confocal imaging 123 Hartline, O.K., E.J. Buskey, and P.H. Lenz Rapid jumps and bioluminescence elicited by con- trolled hydrodynamic stimuli in a mesopelagic cope- pod, Pleuromamma xiphica 132 Harrison, Paul J.H., and David C. Sandeman Morphology of the nervous system of the barnacle cypris larva (Balanus amplutnte Darwin) revealed by light and electron microscopy 144 PHYSIOLOGY Gainey, Louis F., Jr., Kelly J. Vining, Karen E. Doble, Jennifer M. Waldo, Aurora Candelario-Martinez, and Michael J. Greenberg An endogenous SCP-related peptide modulates cili- ary beating in the gills of a venerid clam, Mercenaria mercenaria 159 DEVELOPMENT AND REPRODUCTION Saigusa, Masayuki, and Hiroshi Iwasaki Ovigerous-hair stripping substance (OHSS) in an es- tuarine crab: purification, preliminary characteriza- tion, and appearance of the activity in the developing embrvos 174 Shirae, Maki, Euichi Hirose, and Yasunori Saito Behavior of hemocytes in the allorejection reaction in two compound ascidians, Bottyllus scalaris and Sywz- plegma replant 188 ECOLOGY AND EVOLUTION Skorokhod, Alexander, Vera Gamulin, Dietmar Gun- dacker, Vadini Kavsan, Isabel M. Muller, and Werner E.G. Muller Origin of insulin receptor-like tyrosine kinases in marine sponges 198 Grain, Jennifer A. Functional morphology of prey ingestion by Placetron wosnessenskii Schalfeew Zoeae (Crustacea: Anomura: Lithodidae): 207 SHORT REPORTS FROM THE 1999 GENERAL SCIENTIFIC MEETINGS OF THE MARINE BIOLOGICAL LABORATORY FEATURED ARTICLE Rome, Lawrence C. Introduction. Bringing the script to life: the role of muscle in behavior 225 Rome, Lawrence C., Andrei A. Klimov, and Iain S. Young A new approach for measuring real-time calcium pumping and SR function in muscle fibers 227 PHYSIOLOGY Malchow, Robert Paul, and David J. Ramsey Responses of retinal Muller cells to neurotransmitter candidates: a comparative study 229 Clay, John R., and Alan M. Kuzirian Fluorescence localization of K + channels in the membrane of squid giant axons 231 Ruta, Vanessa J., Frederick A. Dodge, and Robert B. Barlow Evaluation of circadian rhvthms in the Limnlus eve. . . 233 CONTENTS: VOLUME Novales Flamarique, Iriigo, and Ferenc I. Harosi Photoreceptor pigments of the blueback herring (Aloia aestevalis, Clupeidae) and the Atlantic silver- side (Mfnitiiii mi'iii/lin, Atherinidae) 235 Hanley, Janice S., Nadav Shashar, Roxanna Smolowitz, William Mebane, and Roger T. Hanlon Soft-sided tanks improve long-term health of cul- tured cuttlefish 237 King, Alison J., Shelley A. Adamo, and Roger T. Hanlon Contact with squid eggs increases agonistic behavior in male squid (Loligo f>ealei) 256 CELL MOTILITY PISCINE NEVROBIOLOGY A.\L> BEHAVIOR Zottoli, S.J., F.R. Akanki, N.A. Hiza, D.A. Ho-Sang, Jr., M. Motta, X. Tan, K.M. Watts, and E.-A. Seyfarth Physiological characterization of supramedullary/ dor- sal neurons of the cunner, Tuutogolfilmis adspersus. . . . Fay, R.R., and P.L. Edds-Walton Sharpening of directional auditory input in the descend- ing octaval nucleus of the toadfish, Opasnus tau ....... Kaatz, Ingrid M., and Phillip S. Lobel Acoustic behavior and reproduction in five species of Corycoras catfishes (Callichthvidae) .............. Lobel, Phillip S., and Lisa M. Ken- Courtship sounds of the Pacific damselfish, Abudefduf sordidus (Pomacentridae) ..................... Oliver, Steven J., and Elise Watson Threat-sensitive nest defense in domino damselfish 239 240 241 Price, Nichole N., and Allen F. Mensinger Predator-prey interactions of juvenile toadfish, Opsa- iiu\ Ian .................................... Tang, Kathleen Q., Nichole N. Price, Maureen D. O'Neill, Allen F. Mensinger, and Roger T. Hanlon Temperature effects on first-year growth of cultured oyster toadfish, Opsanus tau .................... 24(i Bearer, E.L., M.L. Schlief, X.O. Breakefield, D.E. Schu- back, T.S. Reese, and J.H. LaVail Squid axoplasm supports the retrograde axonal transport of herpes simplex virus 257 Gould, Robert, Concetta Freund, Frank Palmer, Pam- ela E. Knapp, Jeff Huang, Hilary Morrison, and Doug- las L. Feinstein Messenger RNAs for kinesins and a dvnein are lo- cated in neural processes 259 Fukui, Yoshio, Taro Q.P. Uyeda, Chikako Kitayama. and Shinya Inoue Migration forces in Dictyostelium measured by centri- fuge DIG microscopy 260 Tran, P.T., P. Maddox, F. Chang, and S. Inoue Dynamic confocal imaging of interphase and mitotic microtubnles in the fission yeast, S. pombe 262 Maddox, Paul, Arshad Desai, E.D. Salmon, T.J. Mitchi- son, Karen Oogema, Tarun Kapoor, Brian Matsumoto, and Shinya Inoue Dynamic confocal imaging of mitochondria in swim- ming Tftrahymena and of microtubule poleward flux in Xenopus extract spindles 263 Wollert, Torsten, Ana S. DePina, and George M. Lang- ford Effects of vanadate on actin-dependent vesicle motil- ity in extracts of clam oocytes 265 CHEMORECEPTION AND BEHAVIOR Mjos, Katrin, Frank Grasso, and JeUe Atema Antennule use by the American lobster, Homann americanus, during chemo-orientation in three turbu- lent odor plumes 249 Hanna, John P., Frank W. Grasso, and Jelle Atema Temporal correlation between sensor pairs in differ- ent plume positions: A study of concentration infor- mation available to the American lobster, Humartis inni'rn(tnti\, during chemotaxis 250 Zetder, Erik, and Jelle Atema Chemoreceptor cells as concentration slope detec- tors: preliminary evidence from the lobster nose . . . 252 Berkey, Ci istin, and Jelle Atema Individual recognition and memory in HII//KIIIH amem/i//ii\ > >l<--female interactions 253 McLaughlin, L she C., Jennifer Walters, Jelle Atema, and Norman V aimvrighl Urinary protein coiuenlration in connection with agonistic interactions m Haimini*, nmmcanus 254 CELL AND DEVELOPMENTAL BIOLOGY Billack, Blase, Jeffrey D. Laskin, Michael A. Gallo, and Diane E. Heck Effects of a-bungarotoxin on development of the sea urchin Arbacia puncttdatu 267 Silver, Robert B., and Nicole M. Deming Leukotriene B 4 as calcium agonist for nuclear enve- lope breakdown: an enzymological sur\'ey of endo- membranes of mitotic cells 268 Weidner, Earl, and Ann Findley Extracellular survival <>1 an intracellular parasite (Spraffiii'ii l/>/ih/i, Microsporea) 270 Kaltenbach, Jane C., William J. Kuhns, Tracy L. Simp- son, and Max M. Burger Intense concanavalin A staining and apoptosis of peripheral flagellated cells in larvae of the marine sponge Microfionti prulifrnt: significance in relation to morphogenesis 271 CONTENTS: VOLUME COMPARATIVE BIOCHEMISTRY Harrington, John M., and Peter B. Armstrong A cuticular secretion of the horseshoe crab, Limulus polyphemus: a potential anti-fouling agent Asokan, Rengasamy, and Peter B. Armstrong Cellular mechanisms of hemolysis by the protein limu- lin, a sialic-acid-specific lectin from the plasma of the American horseshoe crab. Limiting polyphermis Biswas, Chhanda, and Peter B. Armstrong Identification of a hemolvtic activity in the plasma of the gastropod Sustain canaliculatum Kiihns, William J., Max M. Burger, and Eva Turley Hyaluronic acid: a component of the aggregation factor secreted by the marine sponge, Microciona pro- lifera Popescu, Octavian, Key Interior, Gradimir Misevic, Max M. Burger, and William J. Kuhns Biosynthesis of tyrosine O-sulfate by cell proteoglycan from the marine sponge, Microciona frrolifrra Vasse, Aimee, Alice Child, and Norman Wainwright Prophenoloxidase is not activated by microbial sig- nals in Limulus poliiplirnnis Ogunseitan, O.A., S.L. Yang, and E. Scheinbach The 8-aminolevulinate dehydratase of marine Vibrio alginolyticus is resistant to lead (Pb) Hoskin, Francis C.G., Diane M. Steeves, and John E. Walker Substituted cyclodextrin as a model for a squid en- zyme that hydrolyzes the nerve gas soman Zigman, Seymour, Nancy S. Rafferty, Keen A. Rafferty, and Nathaniel Lewis Effects of green tea polyphenols on lens photooxida- tive stress ECOLOGY AND EVOLUTION Mondrup, Thomas Salinity effects on nutrient dynamics in estuarine sediment investigated by a plug-flux method 275 276 283 285 Pease, Katherine M., L. Claessens, C. Hopkinson, E. Rastetter, J. Vallino, and N. Kilham Ipswich River nutrient dynamics: preliminary assess- ment of a simple nitrogen-processing model Wolfe, Felisa L., Kevin D. Kroeger, and Ivan Valiela Increased labiliiv of estuarine dissolved organic ni- trogen from urbanized watersheds Evgenidou, A., A. Konkle, A. D'Ambrosio, A. Corcoran, J. Bowen, E. Brown, D. Corcoran, C. Dearholt, S. Fern, A. Lamb, J. Michalowsky, I. Ruegg, and J. Cebrian Effects of increased nitrogen loading on the abun- dance of diatoms and dinoflagellates in estuarine phytoplanktonic communities Cubbage, Andrea, David Lawrence, Gabrielle Tomasky, and Ivan Valiela Relationship of reproductive output in Acartia tonsa, chlorophyll concentration, and land-derived nitrogen loads in estuaries in Waquoit Bay, Massachusetts Canfield, Susannah, Luc Claessens, Charles Hopkinson Jr., Edward Rastetter, and Joseph Vallino Long-term effect of municipal water use on the water budget of the Ipswich River Basin Young, Talia, Sharon Komarow, Linda Deegan, and Robert Garritt Population size and summer home range of the green crab, Carriniu nu'tiii.\, in salt marsh tidal creeks Komarow, Sharon, Talia Young, Linda Deegan, and Robert Garritt Influence of marsh flooding on the abundance and growth of Fundulus hettrvclitus in salt marsh creeks . . . Widener, Justin W., and Robert B. Barlow Decline of a horseshoe crab population on Cape Cod Kerr, Lisa M., Phillip S. Lobel, and J. Mark Ingoglia Evaluation of a reporter gene system biomarker for detecting contamination in tropical marine sedi- ments. . 289 290 294 ORAL PRESENTATIONS 287 Pl'BLISHED BY TlTLE ONLY 297 299 300 303 307 THE BIOLOGICAL BULLETIN THE BIOLOGICAL BULLETIN is published six times a year by the Marine Biological Laboratory. 7 MBL Street. Woods Hole, Massachusetts 02543. Subscriptions and similar matter should be addressed to Subscription Manager. THE BIOLOGICAL BULLETIN. Marine Biological Laboratory, 7 MBL Street, Woods Hole, Massachusetts 02543. Subscription per year (six issues, two volumes): $205 for libraries; $95 for individuals. Subscription per volume (three issues): $102.50 for libraries; $47.50 for individuals. Back and single issues (subject to availability): $40 for libraries; $20 for individuals. Communications relative to manuscripts should be sent to Michael J. Greenberg, Editor-in-Chief, or Pamela Clapp Hinkle, Managing Editor, at the Marine Biological Laboratory, 7 MBL Street, Woods Hole. Massachusetts 02543. Telephone: (508) 289-7428. FAX: 508-289-7922. E-mail: pclapp@mbl.edu. http://www.mbl.edu/BiologicalBulletin/ The home page for the electronic companion to THE BIOLOGICAL BULLETIN the Marine Models Electronic Record and other BIOLOGICAL BULLETIN publications is available on the World Wide Web at the address shown above. THE BIOLOGICAL BULLETIN is indexed in bibliographic services including lnde.\ Medicits and MEDLINE. Chemical Abstracts. Current Contents. Elsevier BIOBASE/Ciirrent Awareness in Biological Sciences, and Geo Abstracts. Printed on acid free paper, effective with Volume 180, Issue 1. 1991. POSTMASTER: Send address changes to THE BIOLOGICAL BULLETIN. Marine Biological Laboratory. 7 MBL Street, Woods Hole, MA 02543. Copyright 1999, by the Marine Biological Laboratory Periodicals postage paid at Woods Hole, MA, and additional mailing offices. ISSN 0006-3185 INSTRUCTIONS TO AUTHORS The Biological Bulletin accepts outstanding original research reports of general interest to biologists throughout the world. Papers are usually of intermediate length (10-40 manuscript pages). A limited number of solicited review papers may be accepted after formal review. A paper will usually appear within four months after its acceptance. Very short, especially topical papers (less than 9 manuscript pages including tables, figures, and bibliography) will be published in a separate section entitled "Research Notes." A Research Note in The Biological Bulletin follows the format of similar notes in Nature. It should open with a summary paragraph of 150 to 200 words comprising the introduction and the conclusions. The rest of the text should continue on without subheadings, and there should be no more than 30 references. References should be referred to in the text by number, and listed in the Literature Cited section in the order that they appear in the text. Unlike references in Nature, references in the Research Notes section should conform in punctuation and arrangement to the style of recent issues of The Biological bulletin. Materials and Methods should be incorpo- rated into appropriate figure legends. See the article by Loh- mann ct at. (October 1990, Vol. 179: 214-218) for sample style. A Researcl: Note will usually appear within two months after its acceptance. The Editorial Board requests that regular manuscripts con- form to the requirements set below; those manuscripts that do not conform will be returned to authors for correction before review. 1 . Manuscripts. Manuscripts, including figures, should be submitted in quadruplicate, with the originals clearly marked. (Xerox copies of photographs are not acceptable for review pur- poses.) The submission letter accompanying the manuscript should include a telephone number, a FAX number, and (if possible) an E-mail address for the corresponding author. The original manu- script must be typed in no smaller than 12 pitch or 10 point, using double spacing (including figure legends, footnotes, bibliography, etc.) on one side of 16- or 20-lb. bond paper, 8 by 1 1 inches. Please, no right justification. Manuscripts should be proofread carefully and errors corrected legibly in black ink. Pages should be numbered consecutively. Margins on all sides should be at least 1 inch (2.5 cm). Manuscripts should conform to the Council of Biology Editors Snle Manual. 5th Edition (Council of Biology Editors, 1983) and to American spelling. Unusual abbreviations should be kept to a minimum and should be spelled out on first reference as well as defined in a footnote on the title page. Manuscripts should be divided into the following components: Title page. Abstract (of no more than 200 words). Introduction, Materials and Methods, Results, Discussion, Acknowledgments, Literature Cited, Tables, and Figure Legends. In addition, authors should supply a list of words and phrases under which the article should be indexed. 2. Title page. The title page consists of a condensed title or running head of no more than 35 letters and spaces, the manuscript title, authors' names and appropriate addresses, and footnotes listing present addresses, acknowledgments or contribution num- bers, and explanation of unusual abbreviations. 3. Figures. The dimensions of the printed page. 7 by 9 inches, should be kept in mind in preparing figures for publication. We recommend that figures be about 1 times the linear dimensions of the final printing desired, and that the ratio of the largest to the smallest letter or number and of the thickest to the thinnest line not exceed 1:1.5. Explanatory matter generally should be included in legends, although axes should always be identified on the illustra- tion itself. Figures should be prepared for reproduction as either line cuts or halftones. Figures to be reproduced as line cuts should be unmounted glossy photographic reproductions or drawn in black ink on white paper, good-quality tracing cloth or plastic, or blue-lined coordinate paper. Those to be reproduced as halftones should be mounted on board, with both designating numbers or letters and scale bars affixed directly to the figures. All figures should be numbered in consecutive order, with no distinction between text and plate figures and cited, in order, in the text. The author's name and an arrow indicating orientation should appear on the reverse side of all figures. Color: The Biological Bulletin will publish color figures and plates, but must bill authors for the actual additional cost of printing in color. The process is expensive, so authors with more than one color image should consistent with editorial concerns, especially citation of figures in order combine them into a single plate to reduce the expense. On request, when supplied with a copy of a color illustration, the editorial staff will provide a pre-publi- cation estimate of the printing cost. 4. Tables, footnotes, figure legends, etc. Authors should follow the style in a recent issue of The Biological Bulletin in preparing table headings, figure legends, and the like. Because of the high cost of setting tabular material in type, authors are asked to limit such material as much as possible. Tables, with their headings and footnotes, should be typed on separate sheets, num- bered with consecutive Roman numerals, and placed after the Literature Cited. Figure legends should contain enough informa- tion to make the figure intelligible separate from the text. Legends should be typed double spaced, with consecutive Arabic numbers, on a separate sheet at the end of the paper. Footnotes should be limited to authors' current addresses, acknowledgments or contri- bution numbers, and explanation of unusual abbreviations. All such footnotes should appear on the title page. Footnotes are not normally permitted in the body of the text. 5. Literature cited. In the text, literature should be cited by the Harvard system, with papers by more than two authors cited as Jones et al.. 1980. Personal communications and material in prep- aration or in press should be cited in the text only, with author's initials and institutions, unless the material has been formally accepted and a volume number can be supplied. The list of references following the text should be headed Literature Cited, and must be typed double spaced on separate pages, conforming in punctuation and arrangement to the style of recent issues of Tin- Biological Bulletin. Citations should include complete titles and inclusive pagination. Journal abbreviations should normally follow those of the U. S. A. Standards Institute (USASI). as adopted by BIOLOGICAL ABSTRACTS and CHEMICAL ABSTRACTS, with the minor differences set out below. The most generally useful list of bio- logical journal titles is that published each year by BIOLOGICAL ABSTRACTS (BIOSIS List of Serials; the most recent issue). Foreign authors, and others who are accustomed to using THE WORLD LIST OF SCIENTIFIC PERIODICALS, may find a booklet published by the Biological Council of the U.K. (obtainable from the Institute of Biology. 41 Queen's Gate, London, S.W.7, England, U.K.) useful, since it sets out the WORLD LIST abbreviations for most biological journals with notes of the USASI abbreviations where these differ. CHEMICAL ABSTRACTS publishes quarterly supplements of addi- tional abbreviations. The following points of reference style for THE BIOLOGICAL BULLETIN differ from USASI (or modified WORLD LIST) usage: A. Journal abbreviations, and book titles, all underlined (for italics) B. All components of abbreviations with initial capitals (not as European usage in WORLD LIST e.g.. J. Cell. Comp. Physiol. NOT J. cell. comp. Physiol. ) C. All abbreviated components must be followed by a period, whole word components must not (i.e.. J. Cancer Res.) D. Space between all components (e.g.. J. Cell. Comp. Physiol., not J.Cell.Comp.Physioi) E. LInusual 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: 113-114. (October 1999) A Century of Science: The Biological Bulletin Looks Back and Forward The first number of The Biological Bulletin was pub- lished in October 1899; so with the current issue we cele- brate the 100th anniversary of this journal. The founder was Prof. C. O. Whitman, director of the Marine Biological Laboratory at Woods Hole, Massachusetts (MBL), who aimed to publish short articles with simple illustrations as rapidly as possible. Progress was halting at first: a prede- cessor, the Zoological Bulletin (with C. O. Whitman and W. M. Wheeler as editors, but unassociated with the MBL). failed in 1898 after two volumes: and The Biological Bul- letin itself ceased publication for a time, also after two volumes. 1 But guided by general policies set out in a pro- spectus written in June 1902, 2 the Bulletin began to function smoothly under the editorship of Frank R. Lillie. Those policies, paraphrased below, have informed the operation of the journal ever since: The Bulletin will be published under the auspices of the Marine Biological Laboratory. Its scope will include "Zoology, General Biology, and Physiology": it is a general interest journal. It will contain original articles, occasional reviews, re- ports of work and lectures at the MBL: and preliminary statements of important results will be special feature. It will meet the need for rapid publication of results. It will be open to contributions from any source. A general journal. The Biological Bulletin was meant to be, in some measure, an institutional journal; and indeed, the Annual Report of the MBL Corporation has been pub- lished by the Bulletin since 1908. Moreover, the scientific agenda of the MBL has always been very broad, so the close association of the Bulletin with the Laboratory has ensured that the scope of the journal would also be so. Probably every editor has considered this characteristic of the journal. Indeed, Alfred C. Redrield ( 1941 ) called it "one of the most perplexing problems of policy with which the ... editor 1 The history of The Biological Bulletin has been described thoroughly twice before: by Alfred C. Redfield ( 1941 ). who was Managing Editor of this journal from 1930 to 1942; and (on the occasion of the 100th anni- versary of the founding of the MBL) by Pamela L. Clapp ( 1988), who was Editorial Assistant to Charles B. Metz. the Editor from 1980-1989. 2 The prospectus is published in the Tenth Report for the Years 1903- 1906 including Financial Report from 1900-1906, Section XI Publica- tions, pp. 42-44. (1907) Marine Biological Laboratory. Woods Holl. Massachusetts. must deal." and he concluded that "in this day of special- ization [in 1941!} one journal at least should present a rather broad cross-section of biology as a whole." This is not a necessary conclusion, but it has been accepted by the seven editors that succeeded Lillie. Today we expect beyond technical competence that a publishable submission will contain data resulting from the experimental testing of some hypothesis, and that it will advance its own area signifi- cantly. Moreover, we suppose that if such an investigation is not too narrowly focused, it is likely to interest a generous fraction of our diverse readership. Of course, the actual scope of The Biological Bulletin has always been determined by those who contribute manu- scripts: and the composition of that pool of potential au- thors which is unconstrained by policy has changed markedly through the years. In the decade 1930-1940, roughly 700 papers were published in the Bulletin; of these, 29% originated from the MBL, and 40% were written by members of the Corporation. These values began to decline during the late '60s, as investigators at the MBL (as else- where) began to send their work to specialty journals. The pace of specialization increased markedly, so that in the most recent decade (1989-1999), only about 10% of arti- cles were authored by members of the Corporation. More- over, the rate of acceptance by foreign authors, which began to increase in the '60s. reached 30% of papers published in the last decade. So today more than ever before, the con- tributors to the Bulletin are widely distributed throughout a shrinking world. We are able, in a way, to ask these contributors whether The Biological Bulletin is actually a general journal, be- cause authors select the headings under which their papers are published. The profile varies from issue to issue, but over 50% of recent articles have appeared under the rubric of either Physiology or Development & Reproduction; 20% are listed under Ecology & Evolution; and a quarter are distributed between Cell Biology and Neurobiology & Be- havior. These data suggest that, although the pool of authors is different than it was a century ago. the scope of published material is similar to that in 1903 or 1941. On the other hand, of the animals reported on in the past two years, all but 6 were invertebrates from 13 different phyla, mostly marine, and mostly molluscs, crustaceans, cnidarians, and echinoderms. Insects are virtually absent, only one paper is about nematodes, and of 37 molluscan articles, only one is 114 about Aplysia: current marine biomedical models appear largely in specialty journals. The subject matter in Bulletin articles is diverse and thus difficult to characterize in brief. But it is predominantly experimental and functional, and the functions tend to be complex, sometimes appearing in un- usual, primitive, or extremeophilic animals, and often at the intersection of fields, e. g.. the development of symbiosis; or neural or pheromonal regulation of development, activity, or metamorphosis. In summary. The Biological Bulletin, like all general journals, has its special focus. We might predict that so long as its publication is under the auspices of the MBL, the scope of the Bulletin will remain general; but its focus might well shift with time or new leadership. Enhancing the contents. Although The Biological Bul- letin contains primarily research reports, successive editors have leavened the diet with reviews, some based on lec- tures. The series of lectures on Concepts in Imaging and Microscopy, one of which appears in this issue, is exem- plary. During the past decade, the Bulletin has also pub- lished symposia and workshops, about one per year, on a variety of topics. These proceedings have also served to broaden the scope of the journal. As part of its association with the MBL, The Biological Bulletin has, since 1936, published the abstracts of the General Scientific Meetings held at the Laboratory each summer. Investigators, postdoctoral fellows, and students present their work at these meetings, so the abstracts pro- vide a snapshot of the research taking place at the MBL. The abstracts have been enhanced since 1991: they are longer and contain a figure or table; moreover, they are reviewed and are thus more credible and valuable than the old ab- stracts were. In the past nine years, about 420 of these short reports have been published, which has also served to widen the focus of the Bulletin. Response to electronic publishing. Printing, design, graphic techniques, and the quality of paper have improved slowly over the past 100 years, brightening and enhancing the data published in The Biological Bulletin, as in other journals. This improvement in appearance is symbolized by the images on the cover of this issue. But these technical advances are trivial compared with the sea change that electronic commu- nication has brought in the last decade. This revolution is not close to peaking, but it has already fundamentally altered the way that scientists do business. To date. The Biological Bulletin has responded to the potential for electronic publishing with three online prod- ucts: the Compendia, the Marine Models Electronic Record (MMER), and the Keys to the Invertebrates of the Woods Hole Region (The Keys). These products, or their contents, are composed of relatively independent units of data or methods. I he) are therefore well adapted to navigation and viewing on-screen and are amenable to the advantages of online editing a,"d publication, especially continual updat- ing. The Ct>iupeii:lia consist of tabulated data: e.g., compo- sition of physiological solutions, breeding seasons and ga- mete characteristics, and invertebrate anesthetics and relaxants (in review). The MMER is a completely electronic journal devoted to the collection, culture, and preparation of marine animals for experimentation. The Keys, first pub- lished in 1954, were particularly useful to researchers who are not systematists; unfortunately, this guide is now out- of-date, but it is undergoing revision online. These three products are accessible on the home page of Biological Bulletin Publications at: www.mbl.edu/BiologicalBulletin/. Biological Bulletin Publications also manages the classical print journal. Tables of contents and the abstracts of articles published in each issue of the Bulletin are published elec- tronically as soon as they have all been accepted and proof- read. Moreover, videos and data supplemental to published articles are also maintained online. The full text of the papers in The Biological Bulletin are still not available electronically; but this state of affairs cannot continue forever. Our readers do not want to store paper journals. They want to store a collection of articles, selected from a variety of journals, and tailored to their specific needs. Overwhelmingly, now, these papers are ob- tained, not as reprints, but by photocopying or, where avail- able, by downloading from the internet. More important, our authors expect that, when the paper is accepted and the editorial process is complete, their paper will be distributed as rapidly and widely as possible. If this expectation is to be met, then The Biological Bulletin should be published elec- tronically, and since fees limit distribution, access should be free of cost to readers. The present Biological Bulletin Publications could pro- duce an online journal, but two major, well-ventilated ques- tions remain: First, who should pay for this significant service to authors? Probably authors at least in part. At present, however. The Biological Bulletin has no page charges, and revenue comes almost entirely from libraries. Second, although paper documents last for hundreds of years, electronic storage technology turns over in about five; so if we only publish electronically, how do we solve the problem of archiving? Clearly paper archival copies must be produced. But who will pay for them? Probably the librar- ies at least in part. In any event, scientific publication will be revolutionized in the next decade, and The Biological Bulletin if it survives its inevitable transmogrification will bear about as much physical resemblance to its earlier life as a butterfly does to a caterpillar. MICHAEL J. GREENBERG, Editor-in-Chief References Clapp, P. L. 1988. The history of The Biological Bulletin. Bwl. Bull. 174: 1-3. Kcdtield, A. C. 1941. Annual report of the Marine Biological Laboratory for the year 1940. Report of the Managing Editor. Biol. Bull. 81: 12-17. Reference: Bio/. Bull- 197: 115-122. (October 1999) Concepts in Imaging and Microscopy Exploring Biological Structure and Function with Confocal Microscopy MICHAEL DAILEY. GLEN MARRS 1 , JAKOB SATZ 1 , AND MARC WAITE Department of Biological Sciences and l Program in Neitroscience. The University of lomi. Iowa Citv, Iowa 52242 Abstract. Confocal microscopy is providing new and ex- citing opportunities for imaging cell structure and physiol- ogy in thick biological specimens, in three dimensions, and in time. The utility of confocal microscopy relies on its fundamental capacity to reject out-of-focus light, thus pro- viding sharp, high-contrast images of cells and subcellular structures within thick samples. Computer controlled focus- ing and image-capturing features allow for the collection of through-focus series of optical sections that may be used to reconstruct a volume of tissue, yielding information on the 3-D structure and relationships of cells. Tissues and cells may also be imaged in two or three spatial dimensions over time. The resultant digital data, which encode the image, are highly amenable to processing, manipulation and quantita- tive analyses. In conjunction with a growing variety of vital fluorescent probes, confocal microscopy is yielding new information about the spatiotemporal dynamics of cell mor- phology and physiology in living tissues and organisms. Here we use mammalian brain tissue to illustrate some of the ways in which multidimensional confocal fluorescence imaging can enhance studies of biological structure and function. Received 26 March 1999: accepted 21 July 1999. To whom correspondence should be addressed: Dr. Michael Dailey. Dept. of Biological Sciences. 335 Biology Building, University of Iowa. Iowa City, IA 52242. E-mail: michael-e-dailey@uiowa.edu This is the third in a series of articles entitled "Concepts in Imaging and Microscopy." This series is supported by the Optical Imaging Association (OPIA) and was introduced with an editorial in the April 1998 issue of this journal (Bio/. Bull. 194: 99). Other articles in the series are listed on Tin- Biological Bulletin'f, website at . ll namics of glial cells responding to neural tissue injury (U. ms et a!.. 1996; Dailey and Waite. 1999). In each case, i ! -.mimic features of cell structure and movement could be \ < i-d in a near-native tissue environment. One of i and i -utilized features of confocal microscopy is the ability to image dynamic cell and tissue structures in four dimensions (4-D); that is. in three spatial dimensions over time (e.g., Kriete and Wagner. 1993; Konijn et ai. 1996; Errington et ul.. 1997; Zimmermann and Siegert. 1998). This can be accomplished by collecting stacks of confocal images at set time intervals The resultant time series of confocal image stacks can be used to reconstruct 3-D views of dynamic cell and tissue development. Mark Cooper's group has elegantly applied this approach to early CONFOCAL MICROSCOPY 119 Figure 3. Time-lapse sequence shows the dynamics of axon growth and contact with a dendrite in a developing rat hippocampal slice. Neurons were labeled with a fluorescent membrane dye, Dil. To image growth of neuronal processes in three dimensions, stacks of 16 optical sections spanning 30 ^xm in the axial dimension (2-^im ;-steps) were collected at time intervals of 6 min. Images in the top sequence represent a simple axial projection of the 16 images in the through-focus stack. The bottom series of images are red-green stereo images of the same data to provide depth information (viewing requires red-green or red-blue stereo glasses). A thin axon (arrow) extends parallel to a dendrite (arrowhead). Note the long thin filopodia at the leading edge of the growth cone (0 min), which advances (18 min) and bifurcates (arrows, 36 min). The left branch of the growth cone contacts the adjacent dendrite, and the axon growth is subsequently reoriented in that direction (54 min). A time-lapse movie of the axon growth is available for viewing on The Biological Bulletin Website at . zebrafish development (Cooper, 1999), demonstrating the power of time-resolved, 4-D confocal imaging in a fully intact, experimental vertebrate preparation. In Figure 3 we illustrate the use of 4-D confocal imaging to capture the dynamic behavior of an axonal growth cone extending and contacting a dendrite within a rat hippocampal brain slice. Imaging Cell and Tissue Physiology With increasing frequency, it is becoming necessary and feasible to gather information about both the structure and physiology of the biological specimen. This is espe- cially essential for studies on neural tissue, where spatial and temporal patterns of electrical and chemical signals play critical roles in brain function. Optical imaging of the phys- iology of individual cells within the context of a 3-D tissue can provide a powerful means of exploring tissue organiza- tion and function. Within a single field of view, the activity of many tens or hundreds of cells may be observed simul- taneously. This can help elucidate physiological features of populations of cells, reveal distinct functional properties and relationships of different cell types, and define func- tional domains within a tissue. In conjunction with the various fluorescent probes used in cell physiology, confocal imaging can provide information on absolute values of, as well as transient changes in, membrane potential, pH, intracellular calcium, and several other ions and physiological factors. For example, fluores- cent calcium indicator dyes (such as fluo-3) have been used often to investigate the dynamics of intracellular calcium fluctuation in a variety of cell and tissue preparations. Such studies have helped define the spatiotemporal aspects of intra- and inter-cellular calcium signals (Cornell-Bell et al., 1990; Cleemann et al.. 1998; Wier et al.. 1997). Confocal physiological imaging also has been feasible for studies in thick brain tissue slices (Dani e t al., 1993; van den Pol et al., 1992; Dailey and Smith, 1994; Komuro and Rakic, 1996; Guerineau et al.. 1998) and in other complex neural prep- arations, such as the intact zebrafish (Cox and Fetcho, 1996) and the neuromuscular junctions of frog (Reist and Smith. 1992) and fly (Karunanithi et al., 1997). Figure 4 illustrates the use of confocal imaging to examine, in cultured brain tissue, the spatiotemporal patterns of intra- and inter-cellu- lar activity in neuroglial cells in response to a physiological perturbation. Many calcium imaging experiments that use laser confo- cal microscopy have employed nonratiometric calcium in- dicator dyes (e.g., fluo-3, calcium green), primarily because the most popular ratiometric dyes (fura-2 and indo-1) re- M. DAILEY ET AL .ICTOsec 200 sec 200um Figure 4. Physiological time-lapse imaging reveals changing spatiotemporal patterns of intracellular cal- cium (Ca 2+ ) activity in brain tissue in response to potassium (K. f ) depolarization. The slice was loaded with fluo-4 AM (Molecular Probes, Eugene, OR), a membrane-permeant fluorescent indicator of intracellular calcium, and mounted in an open chamber for imaging. Single confocal scans were collected at 7-s intervals to detect changes in fluorescence intensity, which reflect changes in intracellular calcium levels. Each panel (left, center, right) is a composite of three images acquired at three slightly different time-points (7 s apart) and encoded red, green, or blue. Thus, the colors represent points in time when cells are active. Inactive cells appear black, and cells with sustained high calcium levels appear white. The left panel, corresponding to a time-point prior to K f depolarization, shows a low level of spontaneous calcium activity (few colored cells). The center panel, taken just after addition of medium containing high (9 mM) K ' . shows a much higher level of calcium activity in cells. Note that the small colored patches (corresponding to individual, active cells; arrows) are dispersed across the field of view. In the right panel, taken about 100 s later, the isolated cell activity has diminished, and a new pattern of activity emerges corresponding to groups of 5-15 synchronously active cells within patches that are 100-200 fiin in diameter. The active cells are probably astrocytes, and the emergence of synchronously active groups of neighboring cells probably represents electrical (gap junction) coupling among astrocytes (Charles, 1998; Harris-White et ni, 1998). A lime-lapse movie is available for viewing on The Biological Bulletin Website at . quire excitation wavelengths in the ultraviolet (UV) range. Such short-wavelength lasers are expensive and thus less widely available; moreover, chromatic aberration problems associated with UV excitation make confocal microscope design very challenging (Blinton and Lechleiter. 1995). However, several studies have shown that ratiometric phys- iological data can be obtained by visible wavelength con- focal imaging. These studies utilize two calcium-sensitive dyes (fluo-3 and fura red) simultaneously (Lipp and Niggli. 1993, 1994; Schild et a!., 1994), or a calcium sensitive (fluo-3) and a calcium-insensitive (rhodamine) dye in com- bination (Strieker. 1996). Since many physiological events occur on a very fast time-scale, an imaging system must sample at a sufficiently high rate to resolve such events. Many of the early confocal systems were severely limited by the rate at which they "ere able to collect and store images. This limitation is inc; , singly being overcome in two ways. First, with stan- dard r scanning confocal microscopes, the sampling rate can hi jd by reducing the size of the field over which image d;n . t'lected. In the extreme case, the "field size" is reduced to a vigle line that can be repeatedly scanned at high rates ( Mi Hz), a so-called line-scanning mode. This yields limited spatial information, but provides the ex- tremely high time-resolution necessary for resolving fast physiological events such as neural synaptic activity (Schild etal.. 1994; Korkotian and Segal, 1998; Yuste etui.. 1999). Second, several video rate or "real-time" confocal sys- tems have been developed, some of which are capable of collecting over one hundred .v-v (2-D) images per second. These systems have been utilized to study preparations as diverse as individual mesenchymal cells (Vesely and Boyde. 1996), perfused whole rat heart (Hama ft 100) and larvae were mounted on welled slides in a drop of Fluoro- guard antifade reagent (Bio-Rad). and the coverslips were sealed with nail polish. Slides were viewed immediately or stored at 4C in the dark. Slides refrigerated for several months showed no sign of fading when examined. All times quoted are postfertilization. The specimens were examined with a confocal laser scanner coupled to an epifluorescence microscope (a Bio Rad MRC600 scanner and a Zeiss Axiophot microscope or an MRC1024 and an Olympus BX 60). The 488-nm line of the krypton/argon laser was used with a 520DF32-nm filter block. Various numbers of optical sections were collected at different depth intervals. The depth of collection was deter- mined by the thickness of the specimen and the degree of immunolabeling. The number of immunoreactive cell bod- ies was determined by optically sectioning the various lar- vae. All images are displayed in ventral or lateral view, anterior at the top of the page. Image projections (extended focal length) were created using Confocal Assistant (Soft- ware version 4.02), and three-dimensional (3D) stereo ana- glyphs were produced using Laser Sharp (Bio-Rad Labora- tories) and Confocal Assistant. Computer animations were produced using a Silicon Graphics XS24 4000 with Voxel View Ultra software. Patiriella regularis larvae were prepared for scanning electron microscopy according to Byrne and Barker ( 1991 ) and examined with a Philips XL30 at 10 Kv. Results Giistmlii The first cells exhibiting specific 5-HT-like immunoreac- tivity occurred in mid gastrulae (Fig. 1A), about 24 h postfertilization. As the gastrulae began to elongate, these cells formed a partial dome-like array across the animal region and included monopolar. bipolar, and tripolar cells (Fig. IB). Varicosities were occasionally observed on these processes (Fig. 1C). Both the soma and the neurites of these cells were immunopositive (Fig. 1C). In cross section the cells spanned the epithelium (Fig. ID). With 3D computer reconstructions or extended focus projections, these cells were shown to be pyramidal. Control gastrulae (H = 15), were nonfluorescent. 5-HT NEUROGENESIS IN A LARVAL SEA STAR 125 Figure 1. Confocal images showing bodies and processes of cells with 5-HT-like immunoreactivity in early and advanced gastrula. (A) A projection from 16 optical sections taken at 4.5-/j,m intervals of a mid gastrula shows cells with 5-HT-like immunoreactivity (arrowheads) scattered in the epithelium. C, cilia. Bar. 63 jam. (B) Advanced gastrula. projection created from 14 images at 4.5-ju.m depth intervals showing the concentration of immunoreactive cells (arrowheads) in the animal half. Bar. 95 /am. (C) 5-HT-like immunoreactivity in a tripolar nerve cell in a 34-h gastrula. ax, axonal-like processes; v, varicosities; neb. nerve cell body. Bar. 16 (j,m. (D) Advanced gastrula/early bipinnaria epithelium (e) showing nerve cell bodies (neb) and axonal-like processes (ax) traveling along the basal lamina in a single confocal section. Bar. 20 ju.m. Bipinnaria Prior to the opening of the mouth, early bipinnariae had a distinct stomodaeal invagination while the blastopore was still located at the vegetal pole (Fig. 2A). The larvae could now be orientated according to their dorsoventral axis (Fig. 2 A). A 144-/xm-thick projection reconstructed from 32 op- tical sections showed that cells with 5-HT-like immunore- activity were abundant on both sides of the larva (Fig. 2A). On the ventral surface, the cells around the stomodaeum, about 10 in number, were monopolar and marked the posi- tion at which the adoral ciliated band will form (Fig. 2 A). A collection of bipolar ovoid immunoreactive cells on the dorsal surface was positioned roughly opposite the stomo- daeal invagination (Fig. 2A). At about 48 h postfertilization, the mouth opened. The larvae were further elongated and the anus opened ven- trally. With completion of the gut, the larvae were able to feed. As seen above, 5-HT-like immunoreactivity was conspicuous in the cells surrounding the mouth, which marked the position of the developing adoral ciliated band (Fig. 2B). A few immunoreactive cell bodies were also observed on the upper right region of the buccal cavity (Fig. 2B). Immunoreactive cells and processes on the mid-dorsal surface formed an incomplete ring that wrapped partially around the larva but did not extend to the ventral surface (Fig. 2C). Axonal-like processes from these cell bodies extended towards the posterior end of the larva (Fig. 2C). Although the fate of the immunore- active cells in this ring could not be followed, their mid-body position indicates that they were subsequently 126 F. CHEE AND M. BYRNE Figure 2. Confocal optical projections of early hipinnariae. (A) Early bipinnaria reconstructed from 32 optical sections. Cells with 5-HT-like immunoreactivity can be clearly seen around the stomodaeal invagination (si) and the dorsal surface (arrowheads) of the larva. (B) Projection of 5 confocal sections from the ventral side of a 48-h bipinnaria. 5-HT-like immunoreactivity is present in cell bodies (arrowheads) in the adoral ciliated band (adcb), and a few immunoreactive cells are also present on the upper left-hand side of the mouth (m). (C) Projection of 5 optical sections from the dorsal side of the larva in panel B. Immunoreactive cell bodies (neb) and axonal-like processes (ax) form a band partially wrapping around the larva. Bars. 95 /j.m. incorporated into the serotonergic tracts associated with the preoral and postoral ciliated bands. As the preoral, postoral and adoral ciliated bands devel- oped, the oral hood was also beginning to form (Fig. 3 A, B). Serotonin-like immunoreactivity was observed along the ciliated bands in the form of monopolar cell bodies with axonal-like tracts following the path of these bands (Fig. 3 A). By this stage, a ganglion was evident at the anterior end of the larva. This anterior ganglion ( AG) consisted of the immunopositive cells and processes innervating the preoral and postoral ciliated bands and processes intercon- necting these bands (Fig. 3A). Advanced bipinnaria (about 18 days old) underwent a distinct shape change with the formation of an extension at the anterior end of the larva (Fig. 4A, B). The three ciliated bands were well developed in these larvae (Fig. 4A, B). Internally, the larva had a well-developed gut. and the right and left enterocoels had formed. The distribution of immu- noreactive cells in these bands, discussed below, was con- sistent in all larvae examined (n = 100). Adoral cilititeil huiul The adoral ciliated band was located along the posterior margin of the mouth and was characteristically paraboloidal (Fig. 5A). Along this band were densely packed cells with 5-HT NEUROGENESIS IN A LARVAL SEA STAR 127 Figure 3. Three-dimensional red/green anaglyph and a scanning electron micrograph (false colored) of early bipinnariae. (A) A 3-D lateral view of an early bipinnaria showing immunoreactive cell bodies and axonal-like tracts following the ciliated bands. The anterior ganglion (ag) has formed and connects the preoral (procb) and postoral (pocb) ciliated bands, adocb. adoral ciliated band; o. esophagus; s, stomach; i, intestine. Bar, 95 /j.m. (B) Ventral view of an early bipinnaria at the same stage as in panel A. The ciliated bands are developing, but the anterior extension has not yet formed. Black arrowheads, preoral ciliated band; white arrowheads, postoral ciliated band; white arrow, adoral ciliated band; in. mouth; a. anus. Bar, 100 /u,m. 5-HT-like immunoreactivity; the apical ends of these cells extended to the edge of the ciliated epithelium. These cells were pyramidal and connected basally \-ui a thick immu- nopositive tract (Fig. 6A). Compared with the other ciliated bands, the adoral ciliated band had the highest concentration Figure 4. Scanning electron micrographs of a hipinnaria showing fully developed ciliated bands (arrowheads and arrows): (A) Ventral view of a bipinnaria with a flexed oral hood and mouth (m) open showing the position of the adoral ciliated band (adcb). (B) Lateral view of a bipinnaria showing the anterior extension of the oral hood, top right-hand side. Double-ended arrow indicates the anterior region where the anterior gan- glion links the preoral and postoral ciliated bands. Arrowheads, preoral ciliated band; arrows, postoral ciliated band: Bar, 200 /urn. of immunoreactive cells and processes, forming the adoral nerve plexus (ADNP). The ADNP innervated the epithe- lium of the adoral ciliated band. Confocal optical sectioning revealed that the apical region of these cells protruded to the exterior of the ciliated band epithelium. Computer anima- tions (data not illustrated) and a 3D anaglyph (Fig. 5A| showed that this plexus was also connected by serotonergic processes with the nerve plexus in the preoral transverse band via two thin (approximately 2.5 pirn) lateral immuno- reactive tracts. Preoral ciliated band The preoral ciliated band was located on the ventral surface of the larva and outlined the oral hood (Fig. 4A). Where this band traversed the larva above the mouth (pre- oral transverse region), a large number of flask-shaped cell bodies with 5-HT-like immunoreactivity (x = 21, SE = 0. 1 n = 10 larvae) were found in the epithelium (Fig. 4B). From these cell bodies, confocal sectioning into the larva from the ventral surface revealed axonal-like processes from the basal portion of the cells extending inwards toward the buccal cavity (Fig. 4B). In its lateral region, the preoral ciliated band contained a few immunopositive cells scat- tered along its path. Occasionally, a collection of cell bodies forming a pair of lateral ganglia were seen in the lateral region of the postoral ciliated band. These structures were not seen in all larvae and appear to be ephemeral. In the late bipinnaria, a ganglion developed at the anterior end of the larva. This ganglion consisted of immunoreactive cells 128 F. CHEE AND M. BYRNE Figure 5. Bipinnaria: 3-D anaglyph and a high-magnification confocal image projection of the anterior ganglion. Images were constructed from a series of optical sections covering a distance of 132 /urn. (A) 3-D anaglyph of a bipinnaria detailing the serotonergic nervous system following the pathway of the ciliated bands. White arrowheads, serotonergic connection between preoral and adoral ciliated bands; s, stomach; m, mouth; o, esophagus; arrows, immunoreactive coelomic cells. Bar, 200 JJITI. ( B ) A projection of the anterior ganglion in a late bipinnaria. Parallel axonal-like tracts on the opposing sides of the preoral ciliated band (prcb) and the postoral ciliated bands (pocb) interconnecting in a fine network of processes with 5-HT-like immunoreactivity. axt, axonal-like processes; neb, nerve cell bodies. Bar, 50 /im. associated with the preoral and postoral ciliated bands and a network of varicose processes spanning the two bands. This structure innervated the two bands and is a prominent neu- roanatomical feature in the bipinnaria of Patiriella rei>iultiiix with a similar shape. Three-dimensional recon- structions of the tripolar cells revealed that they were pyramidoid. a structure not previously reported. This ob- servation would, however, be dependent on the imaging 5-HT NELIROGENESIS IN A LARVAL SEA STAR 129 Figure 6. Confocal images detailing immunnreactive cells in ciliated bands. (Al Image from 15 optical sections (total thickness 139 /im) showing nerve cell bodies (neb) with 5-HT-like immunoreactivity and an axonal-like tract (axt and arrowsl in the adoral ciliated band. The entire band is immunoreactive. Note that the apical region of the neuron-like cells extend to the edge of the epithelium of the ciliated band. Arrowheads, cilia projecting into the buccal cavity. Bar. 20 /nm. (B) The preoral and postoral ciliated bands of a fully developed bipinnaria. Note the greater number of cells with 5-HT-like immunoreactivity (neb) present in the preoral ciliated band (procb) compared with the postoral ciliated band (pocb). The preoral ciliated band has immunoreactive processes (ip and lateral arrowheads) extending towards the buccal cavity, c and arrowheads, cilia. Bar, 63 fm. (C) Nerve cell bodies (ncbl and arrowheads) in the right lateral postoral ciliated band. Bar, 50 |um. technique employed. Monopolar cells were the most com- mon type of immunoreactive cell in the preoral and postoral ciliated hands, whereas multipolar pyramidal cells were the most common cell type in the adoral nerve plexus (ADNP) of the adoral ciliated band. The presence of the apical projection arising from the cell bodies in the ADNP suggests that this plexus may have a sensory role. This interpretation is similar to that of Ko- matsu et til. (1991), who defined sensory neurons in the bipinnaria of Luidia senegalensis as neurons whose apical surface contacts the external environment. Strathmann ( 1975) demonstrated that the cilia of the adoral ciliated band in bipinnariae are involved in carrying food particles into the esophagus. It is possible that the adoral ciliated band of P. regularis plays a gustatorial function under the influence of serotonergic activity in the ADNP. In the bipinnaria of P. regularis. serotonin-like immuno- reactivity was conspicuous in the adoral ciliated band, in the preoral and postoral ciliated bands, and in the anterior ganglion. The adoral ciliated band was strongly fluorescent and connected to the preoral ciliated band by an immuno- reactive tract. Detection of this connection was possible through generation of 3D anaglyphs from confocal optical sections, which allowed visualization, and tracing of the complex immunostained network with respect to larval anatomy. On-screen animations (Chee and Byrne, 1997) were also employed to view immunolabeled larvae to de- termine the structure and direction of the immunolabeled 130 F. CHEE AND M. BYRNE Figure 7. Confocal image showing immunoreactive cells (arrow- heads) in the intestinal wall (il and surrounding the anus (a): Bar. 50 jum. processes. Although serotonergic immunoreactivity of the adoral ciliated band has been described in several studies (Nakajima, 1988; Moss et ai, 1994). the connections be- tween the adoral and preoral ciliated bands have not been seen before. The conventional epi fluorescence microscopy used in these earlier studies would not, however, have allowed resolution of this fine structure. Our observations demonstrate the presence of an extensive serotonergic com- munication network that connects all the ciliated bands and may govern reactions to stimuli and generate the behavioral patterns associated with feeding and swimming. Optical sections through the oral region revealed that the immunoreactive cells in the preoral ciliated band gave rise to basal immunoreactive processes that project dorsally along the roof of the buccal cavity. The high density of immunoreactive cells in the region of the preoral ciliated band along the buccal opening suggests that these cells may play a sensory role in feeding. Selection and rejection of particles during feeding is thought to be associated with sensory cells in the buccal cavity (Strathmann, 1975). The cell processes in the roof of the buccal cavity in the larvae of P. regularis may connect with receptor sites that lie within the buccal cavity and are involved in particle selec- tion in feeding. The 5-HT immunopositive tract connecting the adoral and preoral ciliated bands indicates a serotonergic link between the adoral ciliated band and the preoral ciliated band; this link could be important in feeding. The anterior ganglion (AG) is first seen in early bipinna- ria prior to formation of the anterior extension. As this extension develops, the ganglion becomes more intricate, forming a highly complex network. In advanced bipinnaria the AG consisted of prominent, strongly fluorescent tracts traversing the anterior region of the preoral and postoral ciliated bands. The anterior ganglion was the only seroto- nergic connection between these ciliated bands. In contrast to that suggested for sea stars (Lacalli, 1994), the AG of P. regularis does not split to form a pair of lateral ganglia. On the contrary, the pair of lateral ganglia occa- sionally seen in P. regularis (Chee and Byrne, 1997) are distinct from the AG. This bilateral collection of nerve cells may be homologous to those seen in other sea star larvae (Nakajima, 1988; Moss et al, 1994), and it also corresponds to the position of peptidergic GFNSALMFamide (SI ) gan- glia seen in P. regularis (Byrne et ai, 1999). At the brachiolaria stage, the AG is incorporated into the attachment complex, which contains many serotonergic neuronal-like cells suggested to be involved in the settle- ment process (Chee and Byrne, 1999). What appears to be a serotonergic AG has been observed in other echinoderms. Immunocytochemical labeling with anti-serotonin in the auricularia larvae of a holothuroid produced a structure described as an apical ganglion (Burke et al., 1986). Unlike the AG of P. regularis, this structure was not composed of many immunoreactive tracts. Serotonergic AG that differ structurally from that of sea stars but are still anterior in position have been extensively described for sea urchin plutei (Bisgrove and Burke, 1987; Bisgrove and Raff, 1989; Nakajima et al., 1993). The AG in the bipinnaria of P. regularis is similar to the anterior concentration of serotonergic neurons, variously called apical organs or apical ganglia, characteristic of many invertebrate larvae (Lacalli, 1994). These appear to be highly conserved structures in marine invertebrate larvae and are thought to have a sensory function (Lacalli, 1994; Marois and Carew, 1997). The function of the AG and the significance of the connection between the preoral and postoral ciliated bands of P. regularis are not known. The position of the AG, considered together with the bipinna- ria' s anterior direction of swimming, suggests that it may have a sensory role in directional swimming; a similar function has been suggested for the apical ganglion of other invertebrate larvae (Marois and Carew, 1997). Moreover, in an ultrastructural study of the bipinnaria of Luiilia seuega- lensis, sensory cells were found in the preoral and postoral ciliated bands ( Komatsu et ai, 1991 ) in the region where the AG is located in P. regularis. Immuno-electron microscopic examination of thin sections from the anterior region of P. regularis would be needed to determine whether similar cells are present in this species. The anterior ganglion in P. regularis is also similar to non-serotonergic neuronal structures in other asteroids. Similar catecholaminergic anterior structures in the bipin- naria of Archaster typicits were described as a "fluorescent anastomosis" (Chen ct ai, 1995). Nakajima (1987) de- scribed a similar catecholaminergic structure as a "fibrous network" in the bipimiariae of Asterias ainurensis. We 5-HT NEUROGENESIS IN A LARVAL SEA STAR 131 believe that confocal imaging would reveal that these struc- tures are similar to the AG in P. regultiris. This study presents the most detailed immunocytochem- ical description of the development of the serotonergic system in a larval sea star. The organization of the seroto- nergic nervous system in the bipinnaria of Patiriella regu- luris reflects the bilateral symmetry of the larva. A striking bilateral symmetry is also seen in the SI -like peptidergic system in P. ref>ularis (Byrne et ai, 1999). For a complete picture of the expression of serotonin in nerve-like cells during development, we will continue this study in the brachiolaria of P. regultiris through metamorphosis. Inter- estingly, serotonin has never been localized immunocyto- chemically in the nervous system of adult sea stars. It appears that complex serotonergic innervation is a feature common to the swimming and feeding larval form across a range of marine invertebrate phyla. Changes in expression of serotonin in the lecithotrophic larvae of the other Patiri- ella species are being examined to document the evolution of neurogenesis in these asteroids. Acknowledgments We thank Paulina Selvakumaraswamy, Anna Cerra and Paula Cisternas, and Gillian Anderson for their comments and help with the manuscript. Ray Ritchie kindly supplied the algal cultures. Tony Romeo at the Electron Microscope Unit at the University of Sydney also provided assistance. This work was supported by an Australian Research Coun- cil grant. Literature Cited Bisgrove, B. YV., and R. D. Burke. 1986. Development of serotonergic neurons in embryos of the sea urchin. Strongylocentrotus purpiiraitis. Dev. Growth Differ. 28: 569-574. Bisgrove, B. W., and R. D. Burke. 1987. Development of the nervous system of the pluteus larvae of Strongylocentrotus droebachiensis. Cell Tissue Res. 248: 335-343. Bisgrove, B. W., and R. A. Raff. 1989. Evolutionary conservation of the larval serotonergic nervous system in a direct developing sea urchin. Dev. Growth Differ. 31: 363-370. Burke, R. D. 1983a. Development of the larval nervous system of the sand dollar. Dendmsler e.\centricus. Cell Tissue Res. 229: 145-154. Burke, R. D. 1983b. The structure of the larval nervous system of Pisaster ochraceus Echinodermata: Asteroidea. J. Morphol. 178: 23- 35. Burke, R. D., D. G. Brand, and B. VV. Bisgrove. 1986. Structure of the nervous system of the auricularia larva of Parasticopus ctilifoniicus. Biol Bull. 170: 450-460. Byrne, M., and M. F. Barker. 1991. Embryogenesis and larval devel- opment of the asteroid Puliriella regularis viewed by light and scan- ning electron microscopy. Biol. Bull. 180: 332-345. Byrne, M., and A. C'erra. 1996. Evolution of intrugonadul development in the diminutive asterinid sea stars Patiriella vivipara and P. par- vivipara with an overview of development in the Asterinidae. Biol. Bull. 191: 17-26. Byrne, M., F. Chee, P. Cisternas, and M. Thorndyke 1999. Localisa- tion of the neuropeptide SI in the larval and adult nervous system of the sea star Patiriella regularis. Pp. 187-191 in Echinoderm Research 1998, M.D. Candia Carneval and F. Bonasoro. eds. Balkema, Rotter- dam. Chee, F., and M. Byrne. 1997. Visualization of the developing seroto- nergic nervous system in the larvae of the sea star. Patiriella regularis. using confocal microscopy and computer generated 3D reconstructions. Im-ertehr. Reprod. Dev. 31: 151-158. Chee, F., and M. Byrne. 1999. Serotonin-like immunoreactivity in the brachiolaria larvae of Patiriella regularis. Invertebr. Reprod. Dev. 36: 1 I 1-115. Chen, C. P., C. H. Tseng, and B. Y. Chen. 1995. The development of the catecholaminergic nervous system in starfish and sea cucumber larvae. Zool. Snul. 34: 248-256. Collier, H. (). J. 1958. The occurrence of 5-hydroxytryptamme in na- ture. Pp. 5-19 in The Third Symposia on Drug Action. G. P. Lewis, ed. Pergamon Press. London. Hart, M. W., M. Byrne, and M. J. Smith. 1997. Molecular phyloge- netic analysis of life-history evolution in asterinid starfish. Evolution 51: 1848-1X61. Komatsu, M., F. Chia. and R. Koss. 1991. Sensory neurons in the bipinnaria larvae of the sea star, Luiilia senegalensis. Invertebr. Re- prod. Dev. 19: 203-21 1. Lacalli. T. 1994. Apical organs, epithelial domains, and the origin of the chordate central nervous system. Am. Zool. 34: 533-541. Lacalli, T. C., T. H. J. (iilmour. and J. E. West. 1990. Ciliary band innervation on the bipinnariu larva of Pisaster ochraceus. Phil/is. Trans. R. Sue. Loml. B. Biol. Sci. 330: 371-390. Marois, R., and T. J. Carew. 1997. Fine structure of the apical ganglion and its serotonergic cells in the larva of Aplysia californica. Biol. Bull. 192: 388-398. Moss, C., R. D. Burke, and M. C. Thorndyke. 1994. Immunocyto- chemical localization of the neuropeplide SI and serotonin in larvae of the starfish Pisaster ochruceus and Asterias nibeits. J. Mar. Biol. Assoc. UK. 74: 61-71. Nakajima, Y. 1987. Localisation of catecholaminergic nerves in larval echmoderms. Zool. Sci. 4: 293-299. Nakajima, Y. 1988. Serotonergic nerve cells of starfish larvae. Pp. 235-239 in Echinoderm Biology, R. D. Burke, P. V. Mlaldenov, P. Lambert, and R. L. Parsley, eds. Balkema. Rotterdam. Nakajima, Y., R. D. Burke, and Y. Noda. 1993. The structure and development of the apical ganglion in the sea urchin pluteus larvae of Strong\locentrotus droebachiensis and Mesipilia globtihis. Dev. Growth Differ. 35: 531-538. Strathmann, R. R. 1975. Larval feeding in echinoderms. Am. Zool. 15: 717-730. Strathmann, R. R. 1978. The evolution and loss of feeding larval stages of marine invertebrates. Evolution 32: 894-906. Welsh, J. H., and M. Moorehead. 1960. The quantitative distribution of 5-hydroxytryptamine in the invertebrates, especially in their nervous systems. J. Ncurochem. 6: 146-169. Reference: Biol. Bull. 197: 132-143. (October 1999) Rapid Jumps and Bioluminescence Elicited by Controlled Hydrodynamic Stimuli in a Mesopelagic Copepod, Pleuromamma xiphias D. K. HARTLINE 1 *, E. J. BUSKEY 2 , AND P. H. LENZ 1 1 Bekesy Laboratory of Neumbiology, Pacific Biomedical Research Center, Universitv of Hawaii at Manoa, 1993 East-West Rd., Honolulu, Hawaii 96822; and 2 Marine Science Institute, University of Texas at Austin. 750 Channelview Drive, Port Aransas, Texas 78373 Abstract. Actively vertically migrating mesopelagic copepods are preyed upon by a wide variety of fishes and invertebrates. Their responses to predatory attacks in- clude vigorous escape jumps and discharge of biolumi- nescent material. Escape jumps and bioluminescent dis- charges in the calanoid copepod Pleuromamma xiphias were elicited by quantified hydrodynamic disturbances. Brief weak stimuli (peak water velocity 64 21 /im s~') elicited weak (peak force 6.5 dynes) propulsive responses ("jumps") and no bioluminescence. Moderate stimuli (1580 780 ju,m s~') produced strong propulsive re- sponses consisting of long trains of coordinated power strokes by the four pairs of swimming legs ("kicks"). Peak forces averaged 42 dynes. Strong stimuli (5520 3420 ju,m s~') were required to elicit both a jump and a bioluminescent discharge. In several cases, multiple stimuli were needed to evoke bioluminescence, given the limits on stimulus magnitude imposed by the apparatus. Repeated bioluminescent discharges could be evoked, but this responsiveness waned rapidly. Latencies for the jump response (14 4 ms) were shorter than for the accompanying bioluminescent discharge (49 26 ms). The higher threshold for eliciting bioluminescent dis- charge compared to escape jumps suggests that the cope- pods save this defense mechanism for what is perceived to be a stronger threat. Received 13 May 1999; accepted 6 August 1999. * Author to whom correspondence should be addressed. E-mail: danh(<"phrc lunv.ui.edu Introduction Planktonic copepods are preyed upon by a wide variety of fishes and invertebrates (Hopkins and Baird, 1985; Hopkins ct a/.. 1996). Thus, predator evasion strategies are key to the survival of these animals in pelagic communities. Plank- tonic copepods respond to perceived attacks with rapid and powerful escape "jumps" (Singarajah, 1969, 1975; Strick- ler, 1975). The Augaptiloidea (Calanoida), which typically inhabit the mesopelagic region, possess the ability to dis- charge bioluminescent material (Clarke et /., 1962; Her- ring, 1988). These discharges are thought to either startle a potential predator away or misdirect a possible attack (David and Conover, 1961; Morin, 1983; Young, 1983). Although we have a qualitative understanding that biolumi- nescent discharges in these calanoids are used as a defense mechanism, we know less about how these discharges are triggered in the natural environment. In the laboratory, electrical stimulation and mechanical agitation are routinely used to elicit bioluminescent discharges (e.g., Latz et al., 1987. 1990; Widder, 1992). However, we know little about the magnitude of stimuli required to elicit this behavior. Neither do we understand the relationship between the escape jump and the bioluminescence. We addressed some of these questions in a laboratory study, working with tethered Plettromamma xiphias. This calanoid is a metridi- nid (Augaptiloidea) and belongs to a widespread and abun- dant genus in this group. Here we report on the minimum hydrodynamic stimuli necessary to elicit a jump response, and how this compares to the minimum stimulus that trig- gers bioluminescence. By concurrently monitoring jump behavior with a force transducer and bioluminescence with 132 COPEPOD JUMPS AND BIOLUMINESCENCE 133 a photomultiplier tube, we are able to describe the temporal sequences for the two behaviors following a quantitative stimulus. Materials and Methods Collection Animals were collected at night (2000 to 2200 h). about 1 mile offshore from Keauhou Bay, Kona, Island of Hawaii, at a depth of 70 to 100 m. A plankton net (0.5-m diam, 333-/j.m mesh) was towed from a small boat at idle speeds (<2 knots) for 15 to 20 min. Within 2 h of collection the animals were sorted into jars with clean seawater, cooled to 6C, and flown, in coolers, to Oahu. Once the animals were brought into the laboratory (within 16 h of collection), they were kept in the dark at 6 to 8C. Every 2 to 3 days the copepods were fed under dim red light with a mixture of Artemia nauplii and Isochrysis galbana cells. Tethering Copepods were affixed to aluminum wire tethers with cyanoacrylate glue (Borden or Loctite) under red light in an otherwise darkened room. They were corralled in a droplet of seawater, which was then drawn down until a portion of the dorsal prosome was briefly exposed to air. The wire, with some glue on its tip, was applied and held in place while the animal was reimmersed. During this procedure the animals typically bioluminesced in response to the tactile stimulation. Once a copepod was glued and transferred to the experimental setup, 3 h were allowed to elapse before it was tested for mechanical sensitivity. Good experimental animals had high mechanical sensitivity, maintained their swimming appendages in the promoted position (tucked under the body, anteriorly directed), and were biolumines- cently competent. In the experiments presented, the animals maintained their mechanical sensitivity for at least 2 days, although the force produced during the jump typically de- clined. Toward the end of the experiments we observed either a loss in sensitivity or a failure to maintain the swimming appendages in the promoted position. All ani- mals were still bioluminescently competent at the end of the experiments and responded to direct tactile stimulation with a discharge. While on the tether, copepods were fed Iso- chrysis galbana. Health To test the bioluminescent competence of P. xiphias, five specimens were tested for total mechanically stimulable luminescence (TMSL) using methods described in Buskey and Swift ( 1990). A single P. xiphias was placed in each of five liquid scintillation vials containing 10 ml of filtered seawater. After allowing the copepods to recover for about 2 h from the disturbance of being transferred, we placed a vial inside an integrating sphere (Labsphere, Polane coated) and stimulated bioluminescence by stirring the vial with a battery-powered test-tube stirrer until no additional biolu- minescence was detected. Bioluminescence was quantified using a photomultiplier tube (PMT; Hamamatsu R464) and a photon-counting photometer (Hamamatsu C1230). Values for TMSL of P. xiphias ranged from 5.3 X 10 10 to 5.5 X 10" photons, with a mean of 2.4 X 10" photons. These results are similar to previously measured values of TMSL for P. .xiphias (Buskey and Swift, 1990; Latz el al, 1990) and indicate that our experimental animals were capable of full bioluminescence and were in good physiological con- dition. Experimental protocol The experimental setup is diagrammed in Figure 1 and is described in detail in Lenz and Hartline (1999). After the tethered copepod was positioned in the apparatus, red back- ground lights were turned off, and illumination was switched to infrared light from four Optek OP-293A LEDs emitting 875 20 nm and positioned about 1 cm behind the animal, outside of the field of view of the video camera. Hydrodynamic stimuli were generated using a piezoelectric pusher to control movement of a plastic sphere of either 3- or 5-mm diameter positioned about 3 mm in front of the animal. At maximum amplitude, the experimental sphere was displaced vertically by 40 jum. A behavioral response was elicited at threshold by vertical movements of the larger sphere of less than 0.5 /AID. Water displacement at the rostrum, approximately parallel to the long axes of the first antennae, was calculated based on the dipole attenuation expected of near-field laminar water flow (Kalmijn, 1988; Gassie et al., 1993). Although there are some errors and approximations inherent in this indirect approach to deter- mining stimulus magnitude (see Gassie et al., 1993, and Lenz and Hartline, 1999, for detailed discussion), it is widely used in behavioral and physiological studies on hydrodynamic reception in aquatic organisms (e.g., Coombs etal., 1989: Bleckmann, 1994; Coombs, 1994) and provides a reasonable measure given uncertainties in such factors as the location of receptors. Computer-controlled stimuli in- cluded short and long sinusoidal movements ranging in frequency from 50 to more than 1000 Hz. Force measurement During a rapid swim the copepod exerted a force on the tether. The displacement this produced along a horizontal axis, roughly parallel to the copepod' s body axis, was measured with a fiberoptic displacement sensor (Philtec 88N) positioned opposite to a small reflective disk mounted on the tether (Fig. 1). The force was calibrated by pushing 134 A From Computer D. K. HARTLINE ET AL To Computer B Photometer To Computer To VCR Microscope Figure 1. Diagram of the experimental setup. (A) Side view showing the positions of the dipole (sphere) and the glued copepod. The sphere used in the stimulus was either 3 or 5 mm in diameter. The distance between the center of the sphere and the rostrum of the animal ranged from 3 to 5 mm. (B) View from the top. Location of the photometer, dissecting microscope, and camera are shown. The experimental dish was made out of microscope slides and designed to allow positioning of the equipment at right angles to the glass. against the tether with a wire, the deflection of which had been calibrated using weights. Force-transducer responses were monitored with an oscilloscope, digitized at 42 kHz per channel, and stored on computer. Resonance frequency of the transducer ( 1.5-2 kHz) was kept as high as possible while maintaining sufficient sensitivity for measurements. The transducer was underdamped, with an overshoot of around 20% to abruptly applied (0.5 ms rise) forces; it had a dampn time-constant of 4 ms. Force signals were filtered at 2 kHz wilt an 8-pole Bessel filter. Further details of the recording system are given in Gassie et til. ( 1993) and Lenz and Hartline (1999). Mounted perpendicular to the view presented in Figure 1 A were a photometer, a dissecting microscope, and a video camera (Fig. IB). Each of these instruments faced one of the five sides of the experimental chamber. Light from the I-R LEDs was blocked from the photometer with an interfer- ence filter (center wavelength 480 nm), and background recordings were very low. The spatial and temporal patterns of bioluminescent emission of P. xiphitis were recorded on videotape using a Cohu monochrome CCD (charge-cou- pled-device) camera (30 fps) fitted with a 55-mm Micro- N1KKOR macro lens, coupled to a Videoscope Interna- tional KS-1381 microchannel plate image intensifier. The video output signal was recorded on a Mitsubishi HU-770 videocassette recorder. The stimulus-trigger from the com- puter also triggered a 30-ms-long flash in an I-R LED, producing a single video frame with an elevated light level. This was used to correlate video with force and PMT records, which thus had an uncertainty of 30 ms. Charac- teristics of the bioluminescence monitored by the PMT could frequently be used to estimate the relative timing with higher temporal resolution. Lii;ht measurement The bioluminescent emissions of P. xipliicis were mea- sured in two ways: with a photomultiplier photometer and with an image intensifier. In early experiments, photometer measurements were made using a Hamamatsu C1230 pho- ton counter and a Hamamatsu R464 PMT. This system was convenient for measuring the total integrated biolumines- cence emitted by P. xiphias. but it did not provide the temporal resolution necessary to accurately measure flash kinetics since it integrates counts over 0.1 -s intervals. It was replaced with a Pacific Instruments model 126 wide-range photometer using an EMI QL-30 PMT. Amplified voltage from the PMT was sent directly to the computer and digi- tized along with other components of the data stream. Be- fore and after being shipped to Hawaii, both photometer systems were calibrated using cultures of bioluminescent bacteria (Photobacterium sp.) and a calibrated Quantalum 2000 luminescence photometer with a highly stable silicon photodiode sensor. A secondary standard ( U C emission standard made from Sylvania Type 132 blue phosphor, peak wavelength 455 nm) was also calibrated. The secondary standard was measured frequently to allow for calibration of readings of bioluminescence. Results Sudden hydrodynamic disturbances were capable of elic- iting behavioral responses in Plciironuiinmn xiphias; we interpret these responses as "rapid swims." or "jumps." In COPEPOD JUMPS AND B1OLUMINESCENCE 135 tethered animals, a complex temporal pattern of force de- velopment followed closely on the presentation of such a stimulus. Figure 2A shows a typical response to a brief (2-ms) water movement of peak-to-peak amplitude com- puted at 3.83 jum at the copepod's rostrum. Following a short latency ("L"), there was an abrupt rise ("R") in for- ward propulsive force. Then a relatively rapid return past zero force to a smaller reverse force ("Rv") led to the development of a second forward component. As in a pre- vious study on the epipelagic copepod Umlinula vulgaris (Lenz and Hartline, 1999), we interpret these propulsive units to be kicks generated by the combined power strokes of the four pairs of swimming legs (pereiopods). The fea- tures of strong locomotor responses in P. xiphias were 20 30 Time (ms) 50 100 150 Time (ms) 200 Figure 2. Force record of a fast swim response of a Pleiiminnniiiui xiphias adult female to a suprathreshold hydrodynamic stimulus. (A) Expanded temporal scale showing the first four kicks of the response. (B) Record showing the complete response to the stimulus. Stimulus: 700 H/,, 1.5 cycles, maximum water velocity of 8400 /mi s~' at the rostrum. Piezoelectric transducer: PZL-060; vertical movement of sphere: 40 ;um; sphere diameter: 5. 1 mm; distance from center of ball to rostrum: 4.4 mm (BPL97-8.D04, second trace). similar in most respects to those of U. vnlgaris (Lenz and Hartline, 1999). They were characterized by short latencies, measured from the onset of the stimulus to the onset of the forward propulsion, typically around 10 ms (minimum: 6 ms). A weak brief backward propulsion, or "preparatory movement." was observed in some animals immediately preceding the forward propulsion (e.g.. Fig. 3A, "Pr"). Following the peak of forward propulsion, there was often an irregular pattern of peaks and valleys for the remainder of the short stroke duration (mean: 8.7 ms. Table I). As in U. vulgaris (Lenz and Hartline, 1999) and Calanux helgo- landicus (Svetlichnyy, 1987), the major peaks can be as- signed to the individual strokes of pereiopod pairs. Minor peaks caused by resonance in the underdamped force-trans- ducer system were also often apparent (Fig. 2A "res"). The distinct reverse propulsion following the termination of the forward phase was a feature found consistently in P. xiphius but not in previous studies on U. vulgaris. A pattern of multiple kicks in quick succession characterized a strong response to a stimulus. This is illustrated in Figure 2B, which shows the same response as Figure 2A on a com- pressed time scale. In P. xiphius, a train of kicks was typical, producing a cohesive propulsive response we term a "jump." Within the train, kicks occurred at repetition rates of 80 Hz (Table I; range 59 to 98 Hz). Response depended on stimulus magnitude With the experimental setup described, we were able to monitor jumps and bioluminescence simultaneously. As with other copepods we have tested, P. xiphias is very sensitive to water movement. Figure 3 shows records from the PMT and the force transducer at three stimulus intensi- ties. We observed several degrees of response, graded with the intensity of the stimulus (Table II). Figure 3A shows a "weak" response given to the lowest intensity of a 1.5-cycle stimulus that elicited a measurable response in this animal. Peak water velocity produced by this stimulus at the rostrum was calculated to be 50 jum s ' (BPL97-10: Table II). Neither the PMT nor the image intensifier recorded any sign of bioluminescence. The force trace shows first one small 12-dyne kick followed by a 100-ms delay and then three additional kicks. The cumulative force impulse generated by these kicks (the integral of force over time; related to total distance moved in a linear viscous medium) reaches only 0.2 dyne-second. In general, a weak response consisted of a brief force transient, which often barely registered on the force transducer (e.g., mean of 6.5 dynes. Table 1). These weak responses consisted of a small number of pro- pulsive events (e.g., 1-3) with moderate latencies (15-20 ms). We term them "weak kicks," but determining what is involved in their production awaits high-resolution cinema- tography. As in Figure 3A, a weak kick was sometimes 136 D. K HARTLINE ET AL. I- CO CO 2. io 9 o cf 40 1g 20 I 8-20 o LL 50 100 150 ^* CO I 109 -^ I- 1 40 'c? 20 I 8-20 o LL 200 Time (ms) 50 100 1 50 200 w 50 100 200 Time (ms) 50 100 150 200 Figure 3. Behavioral responses of a Pleuromamma xiphias adult female to three stimulus intensities. Hydrodynamic stimulus was produced hy a piezoelectric transducer (PZL-060) with a 5.1 -mm sphere, the center of which was located 4.3 mm from the animal's rostrum. (A) Photomultiplier tube (PMT) and force records showing response to a small stimulus: 700 H/., 1.5 cycles, maximum water velocity of 50 iim s ' at the rostrum (vertical peak-to-peak movement of sphere: (1.22 /xm). (B) PMT and force records showing response to a moderate stimulus: 700 Hz, 1.5 cycles, maximum water velocity of IdOO ,um s ' at the rostrum (vertical peak-to-peak movement of sphere: 7.1 /j,m). (C) PMT and force records showing response to a large stimulus: 700 Hz, 8 cycles, maximum water velocity of XWO iim s" 1 at rostrum (vertical peak-to-peak movement ol sphere: 40 jtxm). Response is to fourth stimulus in a series of five presented at l-s intervals (the animal also luminesced to the fifth presentation). Estimated times of video frames shown in Figure 5 indicated with marks along the time axis. (Dl Integral of force over time for the force records shown in A, B. and C. Arrows indicate stimulus presentation Bar in C indicates the length of time the stimulus was on ( 1 1.5 ms). Stimulus length in A and B: 2 ms (BPL97-IO.D02, D04, D0f. followed ( :' SO-200 ms period of quiescence and then a cluster oi elayed, sometimes stronger, kicks. As stimulus intensity was progressively increased above the threshold level, a point was passed at which the intensity of the response increased abruptly (Tables I, II). Figure 3B shows force and PMT records for a stimulus intensity that is COPEPOD JUMPS AND BIOLUMINESCENCE Table I Characteristics of escape response elicited bv a hydrodynamic stimulus 137 Experiment Sex Weak kick force (dynes) Max kick force (dynes) Latency (ms) Kick duration (ms) Kick frequency (Hz) BPL97-3 M 7.5 1.7 60.0 6.4 12.9 9.1 8.1 1.2 89 3 (5) (5) (6) (4) (5) BPL97-6 M 4.7 24.8 6.8 11.5 1.0 6.9 0.6 98 5 (1) (5) (6) (5) (6) BPL97-8 F 4.3 55.9 3.5 7.4 0.6 8.5 0.7 89 5 (1) (6) (6) (6) (6) BPL97-10 F 10.4 3.2 36.8 6.8 1 1 . 1 3.3 11.3 0.8 75 4 (8) (19) (16) (8) (8) BPL97-II F 5.5 1.5 34.2 6.8 15.6 1.7 8.8 2.4 56 5 (3) (4) (4) (6) (7) Weak kick forces were measured from escape responses to near-threshold stimuli. Maximum kick force, latency, kick duration, and kick frequency were all measured from responses to suprathreshold stimuli. Maximum kick force refers to the largest force produced in a train of kicks. Latency and kick duration were measured as shown in Figure 2 (L. D). Kick frequency was calculated by averaging the number of kicks over time either for the complete jump or over the data record (200 ms) in the cases where the jumps extended beyond the sampling window. Means and standard deviations are given; sample size (in parentheses) indicates the number of measurements used for the mean and SD. 30 times higher than that shown in Figure 3A. The PMT record shows no sign of bioluminescence. However, many characteristics of the force record are substantially aug- mented (Fig. 3B). The response typically involved multiple strong kicks with maximum forces produced by individual kicks registering nearly 40 dynes (Fig. 3B). The force impulse produced in the example shown in Figure 3B over a 75-ms interval approached 1 dyne-second (Fig. 3D). In general, such "strong" responses were elicited at stimulus strengths 15 to 30 times above threshold for the weak kicks (Table II). Peak amplitudes (mean = 42 dynes) were greater by a factor of 5 or more than for the weak kicks (Table I). Duration of individual kicks averaged 8.7 ms (Table I). The overall envelope of peak forces during a jump was "spindle" shaped (Fig. 2B). The first few kicks increased progres- sively in amplitude, then continued with several (sometimes 35 or more) kicks, and finally tapered off somewhat before ending. Figure 4 shows an example of the initial phase of one of these very long spindle-shaped jumps. In a multiple- stimulus protocol, the first or second stimulus of a train of five at 1.5-s intervals usually evoked the longest spindle- shaped jump. Further increase in stimulus intensity would in some cases result in a bioluminescent discharge. Figure 3C shows Table II Calculated water velocities that elicited behavioral responses: weak kicks, strong kicks, and strong kicks and bioluminescent discharges Weak kick response Strong kick response Jump + biolum response Expt Sex Stim Velocity (ju.m s ') Stim Velocity ( yum s ' ) Stim Velocity (u.m s~') BPL97-3 M S700 70 S700 2220 S700 2220 BPL97-6 M S700 66 S700 1170 S700 6590 BPL97-7 F ND ND ND ND S700 8420 BPL97-8 F S700 84 S700 840 ND ND BPL97-9 F S700 28 S700 890 ND ND BPL97-10 F S700 50 S700 1570 F700 8860 BPL97-11 F S700 89 S700 2770 F700 8860 BPL97-1 F F700 58 ND ND F700 1830 BPL96-1 M ND ND ND ND F700 1830 Mean 64 1580 5520 SD 21 780 3420 Water velocities at the copepod were calculated using dipole equations. Sinusoidal vertical movements of sphere at 700 Hz were either short (S700. 1.5 cycles) or long (F700. 8 cycles). ND = not determined: threshold could not be established. 138 D. K. HARTLINE ET AL. 50 100 150 Time (ms) 200 B Q. E 50 100 Time (ms) 150 200 Figure 4. (A) Force record of a long series of multiple kicks in response to a large stimulus in Pteuromamma .\iphias. adult female. (B) Integral of force over time for the force record shown in A. Stimulus: 700 Hz. 1.5 cycles, maximum water velocity of 8900 jim s~ ' at rostrum. Piezoelectric transducer: PZL-060; sphere diameter: 5.1 mm: distance from center of ball to rostrum: 4.4 mm: vertical peak-to-peak movement of sphere: 40 /uni. the response of the same animal as in Figures 3A and 3B to a stimulus with an amplitude 180 times greater (and of longer duration) than threshold for eliciting the weak jump. Both a jump and a bioluminescent discharge were produced. The jump response was initiated well before (18 ms) the bioluminescence (Fig. 3C, top panel). The bioluminescent discharge started at the end of the second kick in a train of five and lasted for about 200 ms. The integrated force for the train of kicks was about 0.8 dyne-second (Fig. 3D). Biolu- minescence was usually accompanied by strong spindle- shaped jumps. Near threshold for bioluminescence. dis- charges were likely to be given in response to one of the later stimuli in a sequence of five, and were thus not clearly associated with the strongest (= longest) jump. We were not able to elicit bioluminescent discharges to hydrodynamic stimuli in all cases. This was not due to a lack of bioluminescent competence, as electrical stimuli or more vigorous mechanical disturbance would invariably elicit bioluminescence even if our strongest hydrodynamic stimulus would not. In five experiments, we obtained thresholds for both jump and bioluminescence. and the mean and standard deviations for the stimulus intensities are shown in Table II. The mean threshold of computed peak water velocity for a jump response was 64 /urn s~ ', whereas that for eliciting bioluminescence was 5520 jam s~'. The variability of the threshold for bioluminescence was greater than that for the jump. On average the stimulus magnitude had to be 90 times greater to elicit bioluminescent discharge than to produce a weak jump, but this ratio ranged from 30 to 180 in the five experiments. Once we established a threshold for bioluminescence for an experimental animal, we usually were able to elicit bioluminescence multiple times at that stimulus level, sometimes within half an hour from the previous discharge. Water velocity was not the only stimulus characteristic that affected the likelihood of a bioluminescent discharge, as shown in Table II. Stimulus length was important: the multi-cycle sinusoidal stimulus (F700) was more effective than the 1.5-cycle one (S700; Table II). Furthermore, re- peated presentation of stimuli in quick succession was even more effective. In these cases, the animals would respond with only a jump to the first and second stimulus presenta- tions, but would bioluminesce as well as jump to the sub- sequent one or two stimuli. Characteristics of evoked bioluminescence In our tethered animals, bioluminescence typically (though not always) outlasted the jump. The PMT record in Figure 3C shows that bioluminescence was initiated at about 30 ms post-stimulus, corresponding to the second kick. It lasted throughout the recording period, although by 200 ms post-stimulus it was well along an exponential decay. Excerpts from the corresponding video record are shown in Figure 5. Taken at 30 frames per second (fps), with the frame following stimulus delivery tagged by a light flash, the first frame shows no bioluminescence and the onset of the major kick transients occur in this interval. Bioluminescence begins to appear from the region of the abdomen in the next frame, and reaches a peak in the third. Its near-absence from the last two frames is partly a result of decay and partly that much of the material has left the field of view. Thirty-five minutes later a second trial for the same animal as in Figures 3 and 5 elicited an escape as well as a bioluminescent discharge from both head and abdomen (Fig. 6). The animal bioluminesced in response to the sec- COPEPOD JUMPS AND BIOLUMINESCENCE 139 Figure 5. Video frames showing the bioluminescent discharge associated with the records in Figure 3C. The pre-stimulus frame is a composite of the 10 frames preceding stimulus presentation. The next five video stills are from frames 3-7, counting the first post-stimulus frame as 1 (30 fpsl. Ventral-posterior aspect of animal faces camera. Discharge is primarily from abdominal glands. Broken outlines up to frame 3 indicate position of body prior to stimulation. ond stimulus of a train of five. It was somewhat more delayed (50-ms latency) and shorter (100-ms duration) than the earlier response (peak amplitude could not be measured owing to saturation of the PMT), but the jump was twice the length (10 kicks versus 5). Records of jumps and bioluminescent discharges from a male Pleuromamma xiphias are shown in Figure 7. In this case the animal completed its jump before the biolumines- cence. This example was chosen to illustrate a double re- action. The animal responded with two sets of kicks and matching bioluminescent discharges. The discharges were small and short in duration. The animals routinely push the bolus of bioluminescence away from them by flicking their urosomes. This can be seen in Figure 7 as the streaks of bioluminescence move across the screen. The force gener- ated by this behavior is very small compared to the pereio- pod power strokes and does not register on the force record. Comparing this record with the data from the female of Figures 3. 5. and 6 shows the differences that occur when the pereiopods beat during emission of bioluminescent ma- terial. The combined kicking and bioluminescence produce the explosion of bioluminescence seen in the video frames. This is in contrast to the male (Fig. 7). in which the lumi- nescent material clung to the urosome, presenting a streaky appearance. Temporal relations bet\\'een jump and bioluminescence The rapid swim was always initiated before the biolumi- nescence, as illustrated in Figure 8. a scatter plot of jump latencies versus bioluminescence latencies. All points are above the line with a slope of one. Rapid swims were initiated within 7 to 20 ms (mean SD = 14 4 ms), whereas bioluminescence latencies ranged from 20 to 50 ms (with one very delayed response that started at 110 ms; mean SD = 49 26 ms). In general, the longer the rapid swim latency the greater the delay for the bioluminescence, although the correlation coefficient was not significant (/ = 0.508, n = 8). Bioluminescent discharges in response to the hydrodynamic stimulus were typically short, lasting from 50 to 350 ms. Luminescence often (e.g.. Figs. 3C; 7), but not always (Fig. 6), extended well after the termination of the jump. Discussion Escape jumps Like all pelagic calanoids, mesopelagic Pleuromamma xiphias has an impressive escape jump at its disposal. When sensitivity to water perturbations and jump kinematics mea- sured in tethered animals are compared to similar data for neritic Undinula vulgaris (Lenz and Hartline, 1999), a pat- 140 D K HARTLINE ET AL 50 100 150 Time (ms) 200 Figure 6. (A) Force record of a second response from the animal in Figures 3 and 5. Comparison shows variability in propulsion and bioluminescence. Note the greater duration of the jump and the shorter duration of the bioluminescent discharge. Stimulus: second in a train of live identical to that for Figures 3 and 5, delivered 35 min following. (B) Video frames 3-7 and 10 following stimulus, showing bioluminescent discharge associated with records of A (30 fps). Broken outline indicates position of body prior to stimulation. Note luminescent discharge from cephalic gland. tern of characteristics emerges that is similar in broad scope but distinctive in detail. P. xiphias sensitivities (60 /xm s~') are similar to. though perhaps somewhat lower than, those in U. vulgaris (40 /xm s~'). Minimum latencies for P. xiphias (6 ms) were distinctly longer than for U. vulgaris ( -2 ms). This difference in reaction times is in part explained by the lack of myelination of nerve fibers in the Augaptiloidea (Davis et al., 1999). Peak forces of kicks from U. vulgaris showed a small gradation in magnitude as a function of the strength of the triggering stimulus and over the course of an escape jump. In contrast, those of P. xiphias exhibited a much wider range, with a 5- to 10-fold differ- ence between the weak kicks produced to near-threshold stimuli and the strongest kicks in the middle of a spindle- shaped jump. The strongest kicks registered in our apparatus by U. vulgaris (100 dynes) were almost twice the peak forces measured from P. xiphias (Table I). In U. vulgaris, the initial one or two kicks were the strongest, whereas in P. xiphias, the strength of kick built up over several cycles, and then waned, giving rise to the spindle-shaped enve- lope. Although both species produced multiple kicks in response to threshold and well supra-threshold stimuli. U. vulgaris consistently produced fewer (2-3 typical; up to 9) than did P. xiphias (5-10 typical; up to 35). For comparably sized animals, this should result in longer jump distances in the latter species. This expectation is in agreement with casual observations made while attempt- ing to catch P. xiphitis in an open vessel: jumps of tens of centimeters are not atypical, while those of U. vulgaris are shorter (3 to 5 cm). Bioluminescent discharges can he evoked hv hydmdynaniii stimuli Pleuromamma xiphias will produce a bioluminescent dis- charge to a brief water disturbance; tactile stimulation is not COPEPOD JUMPS AND BIOLUMINESCENCE 141 B o o .c Q. O 3< 0.5 31 41 61 71 81 1 00 200 Time (ms) 300 Figure 7. Response of Pleuromamma xiphuis adult male to a large hydrodynamic stimulus. (A) PMT and force records. (B) Video frames of bioluminescent discharge. The pre-stimulus frame is a composite ot 10 frames preceding stimulus presentation. The next five video stills are post-stimulus frames 3-8. Approximate times of frames are indicated with marks along time axis of A. Note the occurrence of two jumps and two separate bioluminescent discharges, spaced 150 ms apart. Stimulus: 700 Hz, 1.5 cycles, maximum water velocity of 6600 p,m s~ ' at rostrum. Piezoelectric transducer: PZL-060; sphere diameter: 5. 1 mm; distance from center of ball to rostrum: 4.8 mm, vertical peak-to-peak movement of sphere: 40 ^m. Broken outlines indicate position of body prior to stimulation and a portion of the stimulating sphere in lower left corner; posterior view of animal with dorsal toward lower right corner of frames. Glowing material appears associated with abdominal glands (BPL97-6.D03). required. The magnitude of the stimulus required varied among experimental animals, but in general was signifi- cantly greater than that sufficient to trigger strong escape jumps (velocities of 2000 to 9000 /urn s ~ ' ). When presented with a threat, P. xiphias preferentially responds with an escape jump. However, if the threat is prolonged or persists as in the case of repetitive strong stimulation, then the jump is more likely to be accompanied by a bioluminescent discharge. Widder ( 1992) found a similar pattern for Gaus- sia princeps. During a train of electrical stimulation (3 s~') G. princeps would respond with an escape alone until the fifth stimulus, when it finally produced a bioluminescent discharge as well. Bioluminescence is delayed compared to the jump Bioluminescence was always initiated after the onset of a jump sequence. Although the numbers of animals tested were insufficient for complete reliability, in two animals of our study (both males), jumps were completed before the bioluminescence began. In four others (all females), the bioluminescent discharge commenced during the train of kicks. This resulted in a qualitative difference in the visual effect of the bioluminescence. the luminescent bolus being swept along by water propelled posteriorly by the power strokes. An animal that bioluminesces after it has stopped swimming would seem more likely to become a victim of a predatory attack if the luminescent bolus attracts a predator. 142 D. K. HARTLINE ET AL. C/) O c Q) CO 120 80 E 40 _ o CO Fern X Male I I I 1 10 20 Jump Latency (ms) Figure 8. Scatter plot of fast swim latencies (x axis) versus biolumi- nescence latencies (v axis). Squares: adult females (H = 7); crosses: adult male (n = 1 1. All points lie above the line, which has a slope of one. The possibility that there might be a sexual difference in the response patterns is intriguing. Characteristics of biolitminescence and its relation to other cases reported in the literature The kinetics and spatial patterns of bioluminescence re- leased by copepods have been studied for copepods stimu- lated with electrical pulses (Latz et al. 1987; Bowlby and Case. 1991) and for copepods stimulated by mechanical disturbance of undefined frequency and intensity (Latz et ai, 1990). For the large mesopelagic copepod Gaussiu princeps, Bowlby and Case ( 1991 ) identified three types of flash in response to single electrical stimuli: a fast flash of about 2-s duration, a long flash of 7-s duration, and a slow flash of 17-s duration. Latz et al. (1987) found two compo- nents to flashes in P. xiphias stimulated with a single electrical pulse: a fast component that reached maximum intensity in < 100 ms and a slow component that reached peak intensity in > 600 ms. Double flashes with fast and slow characteristics were also observed. Using an intensi- fied video system, he observed that the fast component originated from thoracic and abdominal glands, without obvious discharge of bioluminescent material away from the body; the slow component of flashes was caused by the discharge of luminescent fluid from the abdominal organ. Flashes with similar kinetics were observed for P. xiphiax exposed to mechanical stimulation from a stirring paddle with three tines rotated at 2000 rpm for < 1 s. Since the spatial relationship between the copepod and the rotating tines is unknown during the stimulation period, neither the frequency nor the intensity of mechanical stimulation is known. In addition to strong hydrodynamic stimulation caused by the velocity of the water and the shear created by the spinning tines, mechanical stimulation is possible through direct contact of the copepods with the tines or by contact with the walls of the scintillation vial following an escape jump. In our observations of bioluminescence evoked by hydrodynamic stimuli of known intensity, only fast flashes were observed. In contrast to the observations of Latz et al. (1990). we observed bioluminescence having fast flash kinetics originating from abdominal glands, and with obvious discharge of bioluminescent material away from the body. We have noted that a copepod' s ability to produce a second bioluminescent discharge shortly after a previous one is not necessarily precluded. Thus recovery times mea- sured in TMSL protocols (8-24 h) are probably overly long for most natural situations. Ecological significance Vertically migrating copepods such as Pleuromamma xiphias are important components of mesopelagic food webs, and Pleuromamma spp. are often preferred prey of mesopelagic fish (Hopkins and Baird. 1985; Hopkins et ai. 1996). To help them avoid predation, these copepods have evolved several defensive behaviors, including vertical mi- gration (Bennett and Hopkins, 1989), strong escape jumps (Buskey et al., 1987; present study), and bioluminescence (Clarke et al.. 1962). In contrast to the diversity of strategies possessed by P. xiphias. neritic Undinula vtilgaris appears to have relied on enhancing the speed and strength of the escape response itself as a survival mechanism (Davis et al., 1999; Lenz and Hartline, 1999). The production of light in an otherwise dark environment may at first seem counter- intuitive as a defense mechanism against visual predators; discharge of bioluminescence while the predator is still remote might help the predator locate its prey. However, the higher stimulus threshold for eliciting bioluminescence compared to escape jumps suggests that copepods save this defense for what are perceived to be the strongest threats by predators in close proximity. Mesopelagic predators have sensitive eyes adapted to low light levels, and the discharge of bioluminescence when the predator is nearby may serve to temporarily blind and confuse the predator (Buck, 1978; Morin, 1983). Since copepods initiate escape jumps prior to release of bioluminescence, and leave behind distinct drop- lets or clouds of bioluminescent material (Widder, 1992), the bioluminescent discharge may also serve as a decoy to confuse visual predators (Morin, 1983). Acknowledgments We thank P. Couvillon, B. Kodama. and S. Lum for major alterations to the experiment room: C. Kosaki tor administrative assistance; H. Akaka and A. Davis for tech- COPEPOD JUMPS AND BIOLUMINESCENCE 143 nical assistance; C. Unabia for providing the algal cultures; and P. Cunningham for making the copepod collections possible. The Natural Energy Laboratory of Hawaii pro- vided access to the laboratory facilities at Keahole Point, Hawaii. This is University of Texas Marine Science Insti- tute Contribution number 1118. The research was supported by NSF grant OCE 95-21375. Literature Cited Bennett, J. L., and T. L. Hopkins. 1989. Aspects of the ecology of the calanoid copepod genus Pleuromamma in the eastern Gulf of Mexico. Conlrih. Mar. Sci. 31: 119-136. Bleckmann, H. 1994. Reception of Hydrodynamic Stimuli in Aquatic and Semiaquatic Animals. G. Fischer, New York, 1 15 pp. (Progress in Zoology vol. 41). Bowlby, M. R., and J. F. Case. 1991. Flash kinetics and spatial patterns of bioluminescence in the copepod Gaussia princeps. Mar. Biol. 110: 329-336. Buck, J. B. 1978. Functions and evolutions of bioluminescence. Pp. 419-460 in Bioluminescence in Action. P. J. Herring, ed. Academic Press, New York. Buskey, E. J., and E. Swift. 1990. An encounter model to predict natural bioluminescence. Limnol. Oceanogr. 35: 1469-1485. Buskey, E. J., C. G. Mann, and E. Swift. 1987. Photophobic responses of calanoid copepods: possible adaptive value. J. Plankton Res. 9: 857-870. Clarke, G. L., R. J. Conover, C. N. David, and J. A. C. Nicol. 1962. Comparative studies of luminescence in copepods and other pelagic marine animals. J. Mar. Biol. Assoc. UK 42: 541-564. Coombs, S. 1994. Neartield detection of dipole sources by the goldfish (Carassius auratus) and the mottled sculpin (Coitus bairdi). J. Exp. Biol. 190: 109-129. Coombs, S., R. R. Fay, and J. Janssen. 1989. Hot-film anemometry for measuring lateral line stimuli. J. Acoust. Soc. Am. 85: 2185-2193. David, C. N., and R. J. Conover. 1961. Preliminary investigation on the physiology and ecology of luminescence in the copepod Metridia lucens. Biol. Bull. 121: 92-107. Davis, A. D., T. M. Weatherby, D. K. Hartline, and P. H. Lenz. 1999. Myelin-like sheaths in copepod axons. Nature 398: 571. Gassie, D. V.. P. H. Lenz, J. Yen, and D. K. Hartline. 1993. Mech- anoreception in zooplankton first antennae: electrophysiological tech- niques. Bull. Mar. Sci. 53: 96-105. Herring, P. J. 1988. Copepod luminescence. Hydrobiologia 167/168: 183-195. Hopkins, T. L., and R. C. Baird. 1985. Aspects of the trophic ecology of the mesopelagic lish Lampan\ctus a/alits (Family Myctophidae) in the eastern Gulf of Mexico. Biol. Oceanogr. 3: 285-313. Hopkins, T. L., T. T. Sutton, and T. M. Lancroft. 1996. The trophic structure and predation impact of a low latitude midwater fish assem- blage. Prog. Oceanogr. 38: 205-239. Kalmijn, A. J. 1988. Hydrodynamic and acoustic field detection. Pp. 83-130 in Sensory Bio/ogv of Aquatic Animals, J. Atema, R. R. Fay, A. N. Popper, and W. N. Tavolga, eds. Springer Verlag, New York. Latz, M. R., T. M. Frank, M. R. Bowlby, E. A. Widder, and J. F. Case. 1987. Variability in Hash characteristics of a bioluminescent cope- pod. Biol. Bull- 173: 489-503. Latz, M. R., M. R. Bowlby, and J. F. Case. 1990. Recovery and stimulation of copepod bioluminescence. J. Exp. Mar. Biol. Ecol. 136: 1-22. Lenz, P. H., and D. K. Hartline. 1999. Reaction times and force production during escape behavior of a calanoid copepod. Undinula vulgaris. Mar. Biol. 133: 249-258. Morin, J. G. 1983. Coastal bioluminescence: patterns and functions. Bull. Mar. Sci. 33: 787-817. Singarajah, K. V. 1969. Escape reactions of zooplankton: avoidance of a pursuing siphon tube. J. Exp. Mar. Biol. Ecol. 3: 171-178. Singarajah, K. V. 1975. Escape reactions of zooplankton: effects of light and turbulence. J. Mar. Biol Assoc. UK 55: 627-639. Strickler, J. R. 1975. Swimming of planktonic Cyclops species (Cope- poda, Crustacea): pattern, movements and their control. Pp. 599-613 in Swimming and Flying in Nature, Vol. 2, T. Y.-T. Wu, C. J. Brokaw, and C. Brennan. eds. Plenum Press, New York. Svetlichnyy, L. S. 1987. Speed, force and energy expenditure in the movement of copepods. Oceanology 27: 497-502. Widder, E. A. 1992. Mixed light imaging system for recording biolu- minescence behaviours. J. Mar. Biol. Assoc. UK 72: 131-138. Young, R. E. 1983. Oceanic bioluminescence: an overview of general functions. Bull. Mar. Sci. 33: 829-845. Reference: Biol. Bull. 197: 144-158. (October 1999) Morphology of the Nervous System of the Barnacle Cypris Larva (Balanus amphitrite Darwin) Revealed by Light and Electron Microscopy PAUL J. H. HARRISON* AND DAVID C. SANDEMAN School of Biological Science, University of New South Wales, Sydney, Australia 2052 Abstract. The central nervous system of the cypris larva of Balanus amphitrite consists of a brain and posterior ganglion. The neuropil of the brain includes protocerebral and deutocerebral divisions, with nerve roots from the pro- tocerebrum extending to the eyes and frontal filaments, and nerve roots from the deutocerebrum extending to the first antennae (antennules) and cement glands. The neuropil of the posterior ganglion includes subesophageal and thoracic divisions, with nerve roots from subesophageal divisions extending to the gut, and nerve roots from each of the six thoracic divisions extending to their corresponding thoracic appendage. The antennular nerve is the major peripheral extension of the nervous system and is composed in pail by afferent fibers that innervate setae on the antennules. The cyprid nervous system is small, containing fewer than 2000 neurons, but is well organized for coordinating a response to settlement cues. Introduction The cyprid (cypris larva) is the final larval stage of the barnacle. Cyprids are specialized for settlement (Anderson, 1994), a behavioral process in which a site is selected for permanent attachment and metamorphosis (Anderson, 1994; Walker, 1995). Cyprid settlement is known to be mediated by specific environmental cues (Clare. 1995; Walker. 1995), but little is known about the mechanisms of cue detection and, in particular, how the detection of cues results in the centrally coordinated motor patterns of settle- ment behavior. Cyprids are highly mobile and bear numerous sense or- Received 22 March 1999; accepted 27 July 1999. * Current address: Department of Biology, Georgia State University. PO Box 4010. Atlanta, Georgia 30302. gans (Walley, 1969). The nauplius eye (= median eye) is present during the cyprid stage and is remodeled into the adult ocelli during metamorphosis (Takenaka et ai, 1993). A pair of compound eyes are also present, which are unique to the cyprid. These develop during the final naupliar stage and are lost during metamorphosis (Walley, 1969; Hallberg and Elofsson, 1983). The compound eyes are closely asso- ciated with a pair of frontal filaments (Walker. 1974), and many setae are located on the antennules (Nott and Foster, 1969; Nott, 1969; Clare and Nott, 1994; Glenner and H0eg, 1995), thoracic appendages (Glenner and H0eg, 1995), cau- dal rami (Walker and Lee, 1976; Glenner and H0eg, 1995), and carapace valves (Walker and Lee, 1976; Jensen et ai. 1994; Glenner and H0eg, 1995). Many of these setae are thought to function as mechano- and chemoreceptors. Pu- tative sensory structures located on the carapace include dermal pits, wheel organs (Elfimov, 1995), and lattice or- gans (Jensen et nl.. 1994; H0eg et ai. 1998). Recently, cilia-type dendritic segments were shown to innervate the lattice organs, suggesting a chemosensory function (H0eg et ul.. 1998). To date, morphological studies of the cyprid have fo- cused primarily on external structures (Elfimov, 1995), and particularly on the antennules because of the role played by these appendages during settlement (Nott and Foster, 1969; Nott. 1969; Moyse et al.. 1995). Fewer details are available on the internal organization of the cyprid. Walley (1969) described the larval development of Semibalanus bal- anoides (previously Balanus balanoides) and outlined the nervous system and major sense organs of both the cyprid and nauplius. Other studies have shown that the antennules (Nott and Foster, 1969), frontal filaments (Kauri, 1961; Walker, 1974), dermal pits (Walker and Lee, 1976), lattice organs (H0eg et ai. 1998), and cement glands (Walker, 1971; Okano et ai, 1996) are innervated, but the nerves 144 CYPRID NEUROANATOMY 145 associated with each of these structures have not been traced back to the central nervous system. The cyprid is well equipped to detect settlement cues, but little is known about the underlying role of the nervous system. Recent studies have suggested that cyprid settle- ment behavior is affected by exposing cyprids to certain neuroactive substances (Clare et ai, 1995; Kon et ai, 1995; Yamamoto et ai. 1995, 1996: Okano et ai. 1996, 1998). Studies aimed at investigating the underlying mechanisms of settlement would benefit from a detailed account of the cyprid nervous system. We report here the results of an anatomical study of the central nervous system and the major sense organs of the cypris larva of B. amphitrite. gained from microdissection, semithin serial sections, and electron microscopy. We find that the central nervous sys- tem is made up of about 2000 neurons and that it contains regionalized neuropils, many of which are linked to periph- eral sense organs. Although the cyprid nervous system is small, it is well organized, which is consistent with the cyprids' need to detect and respond to multiple cues for settlement. Materials and Methods Cyprids used in this study were obtained from a labora- tory culture of Balanus amphitrite (see DeNys et ai, 1995). The selected individuals were between 1 and 3 days old (nauplius-cyprid molt = day 0), were active, and had clear (i.e., non-milky) carapaces, obvious cement glands, and compound eyes. Dissection of the animals provided a useful overview of their structure, including the placement of the antennules and limbs within the bivalved carapace and the gross organization of internal organs. Specimens were placed on a stereomicroscope and dissected using tungsten microscalpels and pins (Conrad et ai, 1993). Individuals were placed in a calcium-free saline (in mmol 1~', 485 NaCl, 13 KC1, 10 MgCU 10 HEPES, pH 7.4) to reduce movement and secured (ventral surface upward) to a sili- con-coated microscope slide using either tungsten pins or a nontoxic, rapid-setting silicon adhesive (Kwik-Sil, World Precision Instruments). A cut along the ventral midline allowed separation of the carapace valves to expose the central nervous system and internal organs of the cephalon (see Figs. 1, 2). The carapace valves, antennules, and tho- racic appendages were then secured with fine (<10 ju,m diameter) tungsten pins. The secured preparation was trans- ferred to a fixed-stage Olympus BH-2 microscope and viewed and photographed using water immersion objec- tives. Fixation and embedding. Larvae were placed in equal volumes of 0.4 M MgCU and 0.22 jam-filtered seawater (FSW) and gently agitated for up to 3 h. which had the effect of relaxing the carapace adductor muscles and expos- ing the antennules. Specimens were cooled to 4C for 30 thoracic appendages 100u.m Figure 1. The cypris larva of Balumis amphitrite. (A) Light micro- graph of a live cyprid with antennules and thoracic appendages extending from the bivalved carapace. Internal structures visible include a compound eye (CE), translucent oil cells (OC), and a cement gland (CG). Frontal filaments (FFl extend posterior to each antennule. (B) Longitudinal section of the cyprid showing the central nervous system, which consists of a brain and posterior ganglion. The brain connects via paired circumesophageal connectives (not visible in this section I to the posterior ganglion. A single antennular nerve (AnN) and its associated antennular soma cluster (ASC) are visible. The ASC contains the somata of bipolar sensory neurons. Also apparent in this section are densely stained oil cells, the oral cone, esoph- agus, and gut. The compound eyes and cement glands are located lateral to this plane and are not seen (0.5 /urn section; stained with toluidine blue and osmium tetroxide). min, transferred to chilled (4C) fixative consisting of 2.5% glutaraldehyde and 2.0% formalin in FSW (pH 8.2; 950 mosmol). The formalin used was prepared fresh from para- formaldehyde (37% w/v paraformaldehyde in H 2 O). Micro- wave treatment was used to facilitate the penetration of fixative. For microwave fixation, specimens were placed in 20-ml glass vials filled with chilled fixative; the vials were secured in a beaker filled with chilled water, which in turn was placed in a beaker of crushed ice. Microwave treatment continued until the water in the beaker reached a tempera- ture of 37C (typically 50 s). Specimens were then removed from the oven and allowed to cool to ambient temperature; fixation continued overnight. The following day, specimens were rinsed in FSW for 1 h (three changes of 20 min each), post-fixed in 2% osmium tetroxide (in H-.O) for 30 min, 2% uranyl acetate (in H 2 O) for 20 min, dehydrated through an ethanol series, cleared in propylene oxide, exposed to 146 J. H HARRISON AND D. C. SANDEMAN thorax PMC PMC posterior Ventral Figure 2. Schematic drawings of the cypnd nervous system and major organs in longitudinal and horizontal planes. (A-B) The body of the cyprid is organized in two main compartments, the cephalon and the thorax. The bivalve carapace encloses anterior (AMC) and posterior (PMC) mantle cavities about the cephalon and thorax respectively. The brain, compound eyes (CE), median eye (ME), and cement glands (CG) are contained within the cephalon. The antennules extend from the cephalon and bear the adhesive discs and putative chemoreceptive and mechanoreceptive sensilla. The brain connects with the posterior ganglion via circumesophageal connectives (CC). The posterior ganglion and gut are contained within the thorax. Six pairs of thoracic appendages (TA) and a pair of caudal rami (CR) extend from the thorax. (C-D) The orientation of neural structures in the cyprid depends on the relative position of the antennules and thoracic appendages, both of which extend beyond the carapace when the c\pnd either swims or contacts the substratum (C), or can be withdrawn for protection (D). Planes are identified on the basis of the orientation ot the nervous system in (C). Other abbreviations: ASC. antennular soma cluster; OC. oil cell; FF, frontal hlamein. increasing concentrations of Araldite epoxy resin, and Hat- embedded on microscope slides. Flat embedding allowed the orientation of the specimen to be determined using a light microscope. The Araldite was then removed from the slides by cold shock (using liquid nitrogen) and specimens cut from the blocks and remounted on Araldite stubs for sectioning. Lii>!ii microscopy. Twelve animals were serially sec- tioned at cither 0.5 or 1.0 /j,m (six in sagittal plane, three frontal, and three horizontal see Fig. 2 for orientation) with a Reichen -Jung ultramicrotome and diamond histology knife. Sections were transferred to microscope slides and stained with toluidine blue (17r in 6<7r borax, 0.6<7r boric acid, pH 8.3)ormethyleneblue (1% in 0.1% borax, pH 8.0); reconstructions were made from camera lucida drawings and photographs and with the aid of PC-based. Adobe Illustrator software. Electron microscopy. Specimens for transmission elec- tron microscopy were prepared as described above, sec- tioned at 60-70 nm on a Reichert-Jung ultramicrotome using a diamond knife, and viewed and photographed using a Hitachi H-7000 transmission electron microscope. For scanning electron microscopy, anesthetized and fixed ani- mals were washed for 10 min in H^O (three changes of 3 min each) with sonication during the first two steps, dehy- drated, and transferred to acetone for critical-point drying. Dried specimens were mounted on microscope stubs with double-sided carbon tape, then gold coated and photo- graphed on a