Volume 184 THE Number 1 BIOLOGICAL BULLETIN LIBRARY FEB191993 WuOv'S 'I-' 1 ''!. Vi.TSS. FEBRUARY, 1993 Published by the Marine Biological Laboratory al Laooratoiy THE 1 9 1993 BIOLOGICAL BULLETIN PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY Associate Editors PETER A. V. ANDERSON, The Whitney Laboratory, University of Florida DAVID EPEL. Hopkins Marine Station, Stanford University J. MALCOLM SHICK, University of Maine, Orono Editorial Board WILLIAM D. COHEN, Hunter College DAPHNE GAIL FAUTIN, University of Kansas WILLIAM F. GILLY, Hopkins Marine Station, Stanford University ROGER T. HANLON, Marine Biomedical Institute, University of Texas Medical Branch CHARLES B. METZ, Llniversity of Miami K. RANGA RAO, University of West Florida RICHARD STRATHMANN, Friday Harbor Laboratories, University of Washington STEVEN VOGEL, Duke University SARAH ANN WOODIN, University of South Carolina Editor: MICHAEL J. GREENBERG, The Whitney Laboratory, University of Florida Managing Editor PAMELA L. 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Tables, with their headings and footnotes, should be typed on separate sheets, numbered with consecutive Roman numerals, and placed after CONTENTS No. I, FEBRTARY 1993 CELL BIOLOGY Costas, Eduardo, Angeles Aguilera, Sonsoles Gon- zalez-Gil, and Victoria Lopez-Rodas Contact inhibition: also a control for cell prolifer- ation in unicellular algae? DEVELOPMENT AND REPRODUCTION Fenteany. Gabriel, and Daniel E. Morse Specific inhibitors of protein synthesis do not block RNA synthesis or settlement in larvae of a marine gastropod mollusk (Haliati* n//o//v) Freeman, Gary Metamorphosis in the brachiopod Ti >< 'Innlulni: ev- idence for a role of calcium channel function and the dissociation of shell formation from settlement ECOLOGY AND EVOLUTION Curtis, Lawrence A., and Karen M. K. Hubbard Species relationships in a marine gastropod-tre- ntatode ecological system Douillet, Philippe, and Christopher J. Langdon Effects of marine bacteria on the culture of axenic oyster Crassostrea gigas (Thunberg) larvae 15 25 36 Okamura, Beth, and Lita Ann Doolan Patterns of suspension feeding in the freshwater bi vo/oan Plumatella irpens 52 Scheltema, Amelie H. Aplacophora as progenetic aculiferans and the coe- lomate origin of mollusks as the sister taxon of Si- puncula 57 IMMUNOLOGY Rinkevich, B., Y. Saito, and I. L. Weissman A colonial invertebrate species that displays a hi- erarchy of allorecognition responses 79 S.i u .id.*, Tomoo, Jeffrey Zhang, and Edwin L. Cooper Classification and characterization of hemocytes in Sl\t-/a f/ava . 87 PHYSIOLOGY Hidaka. Michio, and Kiwamu Afuso Effects of cations on the volume and elemental composition of nematocysts isolated from acontia of the sea anemone ('.ulliacti* f>nl\j)ii.\ 97 Mangum, Charlotte P. Hemocyanin subunit composition and oxygen binding in two species of the lobster genus Homiinn, and their hybrids 105 No. 2, APRIL 1993 DEVELOPMENT AND REPRODUCTION Abraham, Vivek C., Sunita Gupta, and Richard A. Fluck Ooplasmic segregation in the medaka (On~ias ta- lipes) egg 115 Hamel, Jean-Francois, John H. Himmelman, and Louise Dufresne Gametogenesis and spawning of the sea cucumber Psolus fabririi (Duben and Koren) 125 ECOLOGY, EVOLUTION AND BEHAVIOR Bridges, Todd S. Reproductive investment in four developmental morphs of Streblospw (Polychaeta: Spionidae) .... 144 Emschermann, Peter On Antarctic Entoprocta: nematocyst-like organs in a loxosomatid, adaptive developmental strategies, host specificity, and bipolar occurrence of species 153 Saigusa, Masayuki Control of hatching in an estuarine terrestrial crab. II. Exchange of a cluster of embryos between two females 186 Takeda, Satoshi, and Minoru Murai Asymmetry in male fiddler crabs is related to the basic pattern of claw-waving display 203 PHYSIOLOGY Ellington, W. Ross Studies of intracellular pH regulation in cardiac myocytes from the marine bivalve mollusk, Merce- naria campechiensu 209 CONTENTS Matsushima, O., T. Takahashi, F. Morishita, M. Fujimoto, T. Ikeda, I. Kubota, T. Nose, and W. Miki Two S-Iamidf peptides, AKSGEYRIamide and VSSEYRIamide, isolated from an annelid. McFarland, F. K., and G. Muller-Parker Photosynthesis and retention of zooxanthellae within the aeolid nudibranch Amluhti papillosa . . 216 223 Rees, Bernard B., and Steven C. Hand Biochemical correlates of estivation tolerance in the moiiniainsnailOwi//i-//\(Piilmonata:Oreohelicidae) 230 Wright, Jonathan C., and John Machin Atmospheric water absorption and the water budget of terrestrial isopods (Crustacea, Isopoda. Onisci- dea) 243 No. 3, JUNE 1993 REVIEW McEdward, Larry R., and Daniel A. Janies Life cycle evolution in asteroids: what is a larva.' PHYSIOLOGY 255 DEVELOPMENT AND REPRODUCTION Buckland-Nicks, John Hull cupules of chiton eggs: parachute si rue lures and sperm focusing devices? 269 Bollner, Tomas, and I. A. Meinertzhagen The patterns of bromodeoxyuridine incorporation in the nervous system of a larval ascidian, Ciinin ni- testinalis 277 Harvell, C. Drew, and Richard Helling Experimental induction of localized reproduction in a marine bryozoan 286 Montgomery, Mary K., and Margaret McFall-Ngai Embryonic development of the light organ of the sepiolid squid Euprymna sculopes Berry 296 BIOCHEMISTRY Weis, Virginia M., Mary K. Montgomery, and Mar- garet J. McFall-Ngai Enhanced production of ALDH-like protein in the bacterial light organ of the sepiolid squid Eiijji-yiiniii seolupes 309 Gaus, Gabriele, Karen E. Doble, David A. Price, Mi- chael J. Greenberg, Terry D. Lee, and Barbara-Anne Battelle The sequences of five neuropeptides isolated from Liinulii-, using antisera to FMRFamide 322 Tamura, Shouhei, Takahiko Shimizu, and Susumu Ikegami Endocytosis in adult eel intestine: immunological detection of phagocytic cells in the surface epithe- lium . 330 RESEARCH NOTE Smith, Andrew M., William M. Kier, and Sonke Johnsen The effect of depth on the attachment force of lim- pets 338 VIEWS AND DISCUSSION Rinkevich, Baruch Immunological resorption in Bo/n7/in schlosseri (Tunicata) chimeras is characterized by multilevel hierarchial organization of histocompatibility alleles. A speculative endeavor 342 Index to Volume 184 . 346 the Literature Cited. Figure legends should contain enough in- formation 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. 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Science, Evolution NOT Nature, Land.. Science, N.Y.; Evolution. Lancaster. Pa.) 6. Reprints, page proofs, and charges. Authors receive their first 100 reprints (without covers) free of charge. Additional re- prints may be ordered at time of publication and normally will be delivered about two to three months after the issue date. Authors (or delegates for foreign authors) will receive page proofs of articles shortly before publication. 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 184: 1-5. (February. Contact Inhibition: Also a Control for Cell Proliferation in Unicellular Algae? EDUARDO COSTAS, ANGELES AGUILERA, SONSOLES GONZALEZ-GIL, AND VICTORIA LOPEZ-RODAS Genetica Production Animal, Facnltad de 1'eterinaria. Universidad Complutense, Madrid, Spain Abstract. According to traditional views, the prolifera- tion of unicellular algae is controlled primarily by envi- ronmental conditions. But as in mammalian cells, other biological mechanisms, such as growth factors, cellular aging, and contact inhibition, might also control algal proliferation. Here we ask whether contact inhibition reg- ulates growth in several species of unicellular algae as it does in mammalian cells. Laboratory cultures of the di- noflagellate Prowcentrum lima (Ehrenberg) Dodge show contact inhibition at low cell density, so this would be an autocontrol mechanism of cell proliferation that could also act in natural populations of P. limn. But, Synecho- cystis spp., Phaeodactylum tricornntnm (Bohlin), Skele- tonema costatmn (Greville), and Tetraselmis spp. do not exhibit contact inhibition in laboratory cultures because they are able to grow at high cellular density. Apparently their growth is limited by nutrient depletion or catabolite accumulation instead of contact inhibition. Spirogyra in- signis (Hassall) Kutz, Prorocentrum triestinum Schiller, andAlexandrium tamarense (Hsfiaa) Balech show a com- plex response, as they are able to grow in both low and high cell density medium. These results suggest that con- tact inhibition is more adaptative in benthic unicellular algae. Introduction Environmental conditions (light, nutrients, tempera- ture, and turbulence) are thought to be the main controls of proliferation in unicellular algae. Thus, axenic cultures of algae progressively increase in cell number until division slows due to nutrient depletion, the shadowing of some cells by others, or metabolite accumulation. But other mechanisms could play an important role in autocontrol Received 26 March 1992; accepted 23 October 1992. of algal proliferation. In this respect, endogenous rhythms have been proposed as pacemakers of algal proliferation (reviewed by Edmunds, 1988). Also, mucilage production has been considered a mechanism of biological autocon- trol in unicellular algae (Margalef, 1989). Recently, Wyatt and Reguera (1989) proposed that the onset of phyto- plankton blooms and red tides are due to a mechanism of ecological autocontrol acting at the Gaian level. Several biological mechanisms that control the cell di- vision cycle in mammalian cells have recently been elu- cidated. They are based on growth factors, genes, and gene products that respond to growth factors (Baserga ft ai. 1986; Goustin ft ai, 1986; Cantley et ai. 1991; North, 1991 ). Although these mechanisms have been interpreted as adaptations for regulating cellular proliferation in mul- ticellular organisms, they are common to all eukaryotic cells, even regulating the cleavage of zygotes (Murray and Kirschner, 1989). Recently, we have proven that the cell division cycle in unicellular algae from different phyla (Cyanophyceae, Dinophyceae, Bacillarophyceae, and Chlorophyceae) are regulated by growth factors just as are mammalian cells (Costas and Lopez-Rodas, 199 la; L6- pez-Rodas et ai, 1991). In addition to regulation by growth factors, other mechanisms control the cell proliferation of mammalian cells. For example, some cells are genetically programmed to degenerate and die of old age after a determined number of generations. Also, the unicellular algae Spirogyra in- signis (Conjugatophyceae) undergoes cellular aging as do mammalian cells (Costas and Lopez-Rodas, 1991b). In mammals, another important regulator of cellular proliferation is contact inhibition. Mammalian cells grow in monolayers, colonizing the bottom of culture flasks, but they only increase until their growth is inhibited by contact with neighboring cells. Various mechanisms seem to be involved in this complex phenomenon, from growth E. COSTAS ET AL Table Characteristics ofllie species used Species Phyla Charactenstic Synechocystis spp. Cyanobacteria unicellular. planktonic Prorocenlrum lima Dmophyceae unicellular. (Ehrenberg) Dodge benthic Prorocenlrum Iriestinum Dinophyceae unicellular. Schiller swimming Alexandrium tamarense Dinophyceae unicellular. (Halim) Balech swimming Tetraselmis spp. Praxinophyceae unicellular. swimming Skek'lonema costatuin Bacillarophyceae cenobial (Greville) filamentous. planktonic Pliaeodaclylum tricormitum Bacillarophyceae unicellular. (Bohlin) benthic Spirogyra insignis* Conjugatophyceae cenobial (Hasak) Kutz filamentous. benthic * Spirogyra insignis grows in cenobial filaments anchored to the bottom of the flask by the distal cell. Every cell of the filament can divide (more detail in Costas and Lopez-Rodas, 1991b). factor competence to cell shape changes related to intercell contacts (review Alberts el a!., 1983). This paper attempts to determine whether contact in- hibition can limit the growth of unicellular algae, as is the case in mammalian cells. Several species of unicellular algae from different phyla are analyzed in a combined ecological and evolutionary approach. Materials and Methods Cultures Isolation and culture procedures for the species used were previously described in detail (Costas, 1990; Costas and Lopez-Rodas, 199 la, b, c), so only a brief description is provided here. The characteristics of the eight species employed are summarized in Table I. Freshwater and marine species were grown, respectively, in Petri dishes with 20 ml of WC medium or f/2 medium (Guillard, 1975), at 22.5 0.5C and 80 Mmol nT 2 s~', 12:12 h light-dark cycle. Cultures were treated with 150 mg 1 ' penicillin and 100 mg 1~' streptomycin and were, therefore, axenic. Be- fore the experiments were performed, the cultures were tested for the presence of bacteria using epifluorescence procedures as previously described (Costas, 1990). The possible effects of antibiotics on algal proliferation were obviated, because the antibiotic treatment was applied two months before the experiments took place, so the cultures were grown under axenic conditions. Cultures were maintained by serial transfers of a 500 30 cell inoculum to fresh medium once every day. The cells grew exponentially for 20-30 days, and then the cul- tures showed density-dependent inhibition of growth. We determined that a culture reached saturation when its growth rate approached zero and its cell density reached the maximum. Saturation was easily detected because, growth rates and cell densities were determined daily. The experiments took place three days after the cultures were saturated. Experimental Design Many factors act in the cell density-dependent inhibi- tion of growth. In this investigation, we attempted to an- alyze whether contact inhibition also takes part in this process. Clonal cultures of each species were grown until saturation density was reached, and then the following two experiments were performed. Experiment 1: Cells at saturation density growing in fresh medium. All the cells of each saturated culture were collected (by centrifugation at 1000 rpm for 20 min), and resuspended in the same quantity of fresh medium. In this way we obtained a culture in fresh medium with sat- urated density of cells. Growth rates and cellular densities were measured during the five following days. Five rep- licates were performed for each species. Experiment 2: Cells at low density growing in saturated medium. In the second experiment, the saturated medium, after centrifugation, was filtered through a 0.22 ^m pore filter to produce a completely axenic, saturated medium that was free of cells. In this saturated, cell-free medium, a centrifuged inoculum of the same species growing ex- ponentially, was cultured. Growth rates and cellular den- sities were measured during the five following days. Five replicates were performed for each species. If inhibition of growth by contact inhibition and other factors are mutually exclusive, then contact inhibition of growth can be detected by this system, according to the following logic. If a species exhibits contact inhibition, then it will probably be able to grow in Experiment 2. but it won't be able to proliferate in Experiment 1. On the contrary, if the growth inhibition is due to other factors (nutrient depletion or catabolite accumulation), then it will probably be able to grow in Experiment 1 but not in Experiment 2. But, if other factors (i.e., soluble factors), as well as contact inhibition affect growth, then the simple two possibility choice won't happen. To determine whether contact inhibition is a factor in growth inhibition of those algae that grow in monolayers, the following experiment was performed; i.e.. the same method used to detect contact inhibition in mammalian cells was applied to algae. The cells from half a Petri dish were removed mechanically from each saturated culture CONTACT INHIBITION IN MICROALGAE Table II Growth rates and percentage increase oj cell density in fresh medium at saturation density and in saturated medium at low density Cells at saturation density in fresh medium Cells at low density in saturated medium Exponential growth-rates Saturated growth-rates Growth rates % increase cell density Growth rates cell density Syiicchocystis spp. 0.79 0.01 -0.07 0.006 0.49 0.06" 64 5% -0.04 0.01** -4 i"; Prorocentrum lima 0.38 0.03 0.01 0.01 0.03 0.02** 2 1% 0.39 0.07** 47 2% Prorocentmm triestinitm 0.91 0.05 0.01 0.01 0.29 0.02** 33 1% 0.07 0.02** 7 2% Alexandnum tamarense 0.43 0.03 -0.03 0.02 0.12 + 0.05* 12 5% 0.07 0.02* 7 1% Telraselmis spp. 0.96 0.05 -0.04 0.01 0.87 0.07** 1 38 6% 0.01 0.01** 1 1% Ske/etonema costaturn 1.01 0.07 -0.02 0.03 0.58 0.03** 78 3% -0.07 0.01" -6 r; Phaeodactilum tricomiaum 0.88 0.04 0.02 0.01 0.52 0.01** 68 2% -0.03 0.02** -2 3% Spirnt>yra insignis 0.94 0.05 0.03 0.02 0.18 0.05** 19 4% 0.68 0.05** 97 6% * Statistically no significant differences were found (P > 0.05). ** Statistically significant differences (P < 0.01) were found between growth rates of Exp. 1 and Exp. 2. sample of monolayer species. If contact inhibition exists, the cells on the full side will continue growing into the cell-free half of the dish. Five replicates were performed in each case. A continuous recording by video microscopy helped us to evaluate this experiment. Control of hand/ ing effects Because some dinoflagellates are very sensitive to shear stress, we performed the following two preliminary ex- periments to determine whether manipulation would have detectable effects on the analyzed species. (a) Exponentially growing cells of each species were collected by centrifugation at 1000 rpm for 20 min and resuspended in the same quantity of fresh medium. Their growth rates (5 replicates of each species) were measured during the following five days and compared with the growth rates of uncentrifuged exponentially growing con- trols (5 replicates of each species). ANOVA analysis showed no significant differences (P > 0.05) between growth rates of centrifuged and uncentrifuged cells. Fur- thermore, the number of dead cells was estimated by the yellow cosine exclusion procedure (more details in Costas, 1986; Gonzalez-Chavarri, 1991), and ANOVA showed Table III Growth rates of Prorocentrum lima and Spirogyra insignis after cells were mechanically removed from half a Petri dish Border where Zone where cells cells had been had not been removed removed F no significant differences (P > 0.05) in the rate of cell death between centrifuged and uncentrifuged cells. (b) A similar procedure was employed with saturated cells, and the same results were obtained; (i.e.. there were no significant differences (P > 0.05) between centrifuged and uncentrifuged cells). More details about the proce- dures used to control the effects of handling are set out in Costas ( 1 986) and Gonzalez-Chavarri (1991). Experimental evaluation Once an experiment was initiated for each of the five replicates, both the mean growth rates (during the sub- sequent five days) and the percentage of cell density in- crease (during the subsequent 24 h) were determined. Cell density was estimated as the number of cells per square or cubic centimeter in monolayer or suspension cultures, respectively. The number of cells in each culture was de- termined by counting samples in a hemocylometer. The number of samples counted was determined according to the mean progressive technique (Williams, 1977) to obtain 95% accuracy. Growth rates were calculated as doublings per day: Prorocentrum lima Spirogyra insignis 0.31 0.07 0.47 0.03 0.01 0.01 0.13 0.02 dd' 1 = l/Ln2 Ln(Nt/No)/t, Where Nt = cells at time t; No = cells at time 0; and t = number of days between times t and (more detail in Costas, 1990). Results and Discussion Growth inhibition of saturated cultures of unicellular algae is a complex process, influenced by various factors, such as nutrient depletion, catabolite accumulation, shading effects, and possibly by contact inhibition. Be- cause these factors do not act independently, their inter- E. COSTAS ET AL Figure 1 . Growth of Prorocenlntm lima and Spirogyra insignia when cells were mechanically removed from half a Petn dish. The arrows represent the border produced in the experiment. Only the cells bordering the cell-free zone were able to grow, (a) Saturated P. lima culture at the time of removal, (b) P. lima culture 72 h after the removal. New cells have only proliferated into the open half of the plate, (c) Saturated 5. inaignis culture at the time of removal, (d) 5. insignia culture 72 h after the removal. New cells have only proliferated into Ihe free half of the plate. actions complicate a precise evaluation of the relative im- portance of each. Thus, our experimental design was aimed only at detecting whether contact inhibition takes part in cell dependent inhibition of growth. Table II summarizes the growth rates and the percent- age of cell density increases in both fresh and saturated culture media. Apparently, the dinoflagellate P. lima showed contact inhibition of growth. Both the growth rates and the cell densities of Experiments 1 and 2 were sig- nificantly different (P < 0.0 1 ). P. lima cells were not able to grow at saturation density in fresh medium (Experiment 1 ), but their growth started again in saturated medium when their cell density decreased (Experiment 2). In contrast, Synechocystis spp., Phaeodactilum iricor- nutitm, Skeletonema costatum and Tetraselmis spp. did not exhibit contact inhibition. In all the cases, statistically significant differences (P < 0.01) were detected between both the growth rates and the cell densities of Experiments 1 and 2. Apparently, their growth was limited by nutrient depletion or catabolite accumulation; thus they could proliferate at high cellular density in fresh medium (Ex- periment 1 ), but were not able to grow in saturated me- dium at low cell density (Experiment 2). Contact inhibition of growth may be an important mechanism in Spirogyra insignis. Although this species grew slowly at saturation density in fresh medium (Ex- CONTACT INHIBITION IN MICROALGAE periment 1), its growth was significantly increased (P > 0.01) at low density in saturated medium (Experiment 2). So, in S. insignis. the contact inhibition component seems to prevail because proliferation is faster in a satu- rated medium with low cell density than in fresh medium with high cell density. In Pmwcentrum tricstimun. however, a nutrient de- pendent inhibition or catabolite accumulation seemed to be more important than contact inhibition. P. tricstinum was able to grow in both experiments, although its growth in fresh medium at high cellular density was significantly (P > 0.01) faster than that in saturated medium at low cell density. In Ak'xandnwn tamarcnsc, all of the factors seemed to slow down proliferation. A. tamarcnsc cells were scarcely able to grow in either experiment. The cells of P. lima and 5. insignis were mechanically removed from half a Petri dish, and the resulting growth rates are summarized in Table III. In agreement with pre- vious experiments, the growth of P. lima and S. insignis seemed to be inhibited by a contact inhibition mechanism. In particular, only the cells bordering the cell-free zone were able to grow (Fig. 1 ). This experiment, which employs the traditional method of detecting contact inhibition in mammalian cells (Alberts et a/.. 1983), supports the hy- pothesis that contact inhibition takes place in the growth inhibition of P. lima and S. insignis saturated cultures. Only two of the three benthic species analyzed seemed to exhibit contact inhibition. These results suggest that contact inhibition is a more adaptative mechanism in benthic unicellular algae. Contact inhibition is usually thought of as a mechanism developed by animal cells to limit cell division. The results obtained in these experiments suggest an alternative in- terpretation. The dinoflagellates, which could be consid- ered the earliest group of protist, but which are also far removed from actual eukaryotes (Dodge. 1955; Herzog et ai, 1984; Costas and Goyanes, 1988), have developed contact inhibition, thereby suggesting that such a mech- anism had already been developed by unicellular organ- isms in an early era, probably as an autocontrol mecha- nism regulating natural populations. Nevertheless, contact inhibition has also evolved in the Conjugatophyceae (a recent group of higher algae that are phylogenetically far removed from dinoflagellates), suggesting that such mechanisms may have been developed independently in phylogenetically different groups of unicellular organisms. Acknowledgments Supported by DGICYT grants IN89-0163 and PS89- 0014. Literature Cited Alberts, B., D. Bray, J. Lewis, M. Raff, K. Roberts, and J. D. Watson. 1983. Molecular Biology <>/ I lie Cell. Garland Publishing, New York. Baserga, R., L. Kaczmarek, B. Calabrelta, R. Battini, and S. Ferrari. 1986. Cell cycle genes as potential oncogenes. Pp. 3-12 in Cell Crc/e ami Oncogenes. W. Tanner ami D. Gallwitz. eds. Springer- Verlag. New York. Cantley, L. C., K. R. Auger, C. Carpenter, B. Duckworth, A. Graciani, R. Kapeller, and S. Snlloff. 1 99 1 . Oncogenes and signal transduc- tion. CV//64: 281-302. Costas, E. 1986. Vltraesinictura cromosomica en dinoflagelados. Con- sidcntcioncs fvo/n/m/.v. Ph.D. Thesis. Univ. Santiago de Compostela. 240 pp. Costas, E. 1990. Genetic variability in growth rates of marine dinofla- gellates. Genenea 83: 99-102. Costas, E., and V. J. Goyanes. 1988. Comparative analysis of dinofla- gellate chromosomes and nuclei. Genet. (Lije Sci. Adv.) 7: 15-18. Costas, E., and V. Lopez-Rodas. 1 99 la. On growth factors, cell division cycle and the eukaryotic origin. Endocytobiosis & Cell Res 8: 89- 92. Costas, E., and V. Lopez-Rodas. 1991 b. Persistence of cell division synchrony in S/'/n^vra mvn,vi/s (Gamophyceae): membrane pro- teoglycans transmitting synchronizing information throughout gen- erations. Chronohiol Int 8(2): 85-92. Costas, E., and V. Lopez-Rodas. I99lc. Evidence for an annual rhythm in cell aging in Spin>f>yra m.v/#m.v (Chlorophyceae). Phmilngia 30(6): S97-S99. Dodge, J. D. 1955. Chromosome structure in the dinoflagellates and the problem of the mesokaryotic cell. 2ml. Internal. Con] on Pro- lo-ool. Exc Med Inter Cont-r Ser. No. 91: 39. Edmunds, L. N. 1988. Cellular and molecular hasis <>/ 'biological docks Spnnger-Verlag, New York. 497 pp. Gonzalez-Chavarri, E. 1991. I'roduccion de biomasa a base de 1111- eroalgas v sus a/ilicacioncs en la prutliiecion animal. Ph.D. Thesis. Universidad Complutense. 142 pp. Goustin, A. S., E. B. Leof, G. D. Shipley, and H. L. Moses. 1986. Growth factors and cancer. Cancer Res 46: 1015-1029. Guillard. R. 1975. Culture of phytoplankton for feeding marine in- vertebrates. Pp: 26-60 in Culture of Marine Invertebrate Animals, W. Smith and M. Chanley. eds. Plenum Publ. Co., New York. Herzog, M., S. Boletzky, and M. O. Soyer. 1984. Ultrastructural and biochemical nuclear aspects of eukaryote classification: independent evolution of the dinoflagellates as a sister group of the actual eu- karyotes. Origins of Lite 13: 205-215. Lopez-Rodas, V., M. Navarro, L. De La Campa, E. Gonzalez De Cha- varri, S. Gonzalez-Gil, A. Aguilera, R. Segura, and E. Costas. 1991. Tras las pistas de los primeros mecanismosde control de la division celular: Una aproximacion evolutiva. Pp. 94-108 in Crimo- canccrolngia. F. Chavarria, ed. Fundacion Cientifica A.E.C.C. Ma- drid. Margalef, R. 1989. Condiciones de aparicion de la purga de mar y presiones de seleccion sobre sus componentes. Cuademos da Area de Ciencias Marinas 4: 1 3-20. Murray, A. W., and M. W. Kirschner. 1989. Dominions and clocks: The union of two views of the cell cycle. Science 246: 614-621. North, G. 1991. Starting and stopping. Nature 351: 604-605. Williams, M. 1977. Stereological techniques. Pp. 226 in Practical methods in Electron Microscopy !'<>/ 17 M. Hayat. ed. Elsevier Sci. Publ. Co., New York. Wyatt, T., and B. Reguera. 1 989. ( , Ha alcanzado el cultivo de mejillon en Galicia su masa critica? Cuademos da Area de Ciencias Marinas 4:63-71. Reference: Bio/, Bull. 184: 6-14. (February, 1993) Specific Inhibitors of Protein Synthesis Do Not Block RNA Synthesis or Settlement in Larvae of a Marine Gastropod Mollusk (Haliotis mfescens) GABRIEL FENTEANY 1 AND DANIEL E. MORSE 2 Department of Biological Sciences and the Marine Biotechnology Center, Marine Science Institute. University of California, Santa Barbara, California 93106 Abstract. Antibiotic inhibitors of protein synthesis were tested for their effectiveness in larvae of the red abalone, Haliotis mfescens (gastropod mollusk). Emetine and an- isomycin proved highly effective in this system, while cy- cloheximide, fusidic acid, puromycin. and tetracycline were less effective. Emetine and anisomycin specifically inhibited protein synthesis but not RNA synthesis. The contribution to protein synthesis by chloramphenicol- sensitive prokaryotic contaminants was found to be un- detectable, except following the onset of symptoms of toxicity resulting from prolonged exposure to emetine or anisomycin. The induction of larval settlement and plan- tigrade attachment by 7-aminobutyric acid (GABA), a functional analog of the natural inducer of settlement, occurred even under conditions in which most protein synthesis was inhibited, as expected for a chemosensory system response, whereas subsequent developmental metamorphosis was completely blocked. Because emetine and anisomycin block protein synthesis including the synthesis of new transcription factors but do not block early transcription, treatment of marine invertebrate em- bryos and larvae with these inhibitors can be used to ob- tain a selective enrichment in the mRNA population of "early gene" transcripts induced directly by GABA and other morphogenetic signals, without dilution by new mRNAs, the appearance of which is dependent on the synthesis of new protein transcription factors. Received 5 August 1992; accepted 18 November 1992. Abbreviations: GABA, -j-aminobutync acid; TCA, tnchloroacetic acid; SSC, standard sodium chlonde-sodium citrate buffer. Present address: ' Program in Cell and Developmental Biology, Har- vard Medical School. Boston, Massachusetts 021 15. 2 Author to whom correspondence should be addressed. Introduction Developmentally competent larvae (0.2 mm in diam- eter) of the marine gastropod mollusk Haliotis mfescens (red abalone) are induced to settle from the plankton and begin metamorphosis by oligopeptides and proteins as- sociated with the surfaces of crustose red algae (Morse et al., 1979a, b, 1984; Morse and Morse, 1984), and by functional analogs of these natural inducers, such as 7-aminobutyric acid (GABA), muscimol, and baclofen (Morse et al.. 1979a.b, 1980a. Morse, 1992). These com- pounds apparently bind to chemosensory receptors, with subsequent transduction of the signal mediated by the second messengers cyclic AMP and Ca ++ (Trapido- Rosenthal and Morse, 1986a; Morse, 1992). This trans- duction pathway culminates in an excitatory depolariza- tion that is apparently triggered by the regulated opening of chloride ion channels (Morse et al.. 1980; Baloun and Morse, 1984; Morse, 1990, 1992). The morphogenetic response can be facilitated or amplified by the presence of lysine or lysine analogs (Trapido-Rosenthal and Morse, 1985, 1986b), acting through a separate lysine receptor that, in turn, stimulates a G protein-diacylglycerol signal transduction cascade (Baxter and Morse, 1987, 1992; Wodicka and Morse, 1991; Morse, 1990, 1992). Before we can understand the molecular mechanisms by which these convergent chemosensory pathways regulate larval settlement behavior and the subsequent induction of gene expression controlling cellular differentiation and prolif- eration (cf. Cariolou and Morse, 1988; Groppe and Morse, 1989; Spaulding and Morse, 1991; Degnan and Morse, 1993), we must first determine the requirements for de novo protein synthesis in these processes. Specific inhibitors of protein synthesis, such as cyclo- heximide and puromycin, have proved invaluable for such INHIBITORS OF PROTEIN SYNTHESIS studies in many other systems. Yet these well-known in- hibitors were ineffective with larvae of the red abalone (Haliotis mfesL'cns: gastropod mollusk) developing in sea- water. This finding prompted our search for inhibitors of mRNA translation that would specifically block protein synthesis in abalone larvae in seawater media, while not inhibiting RNA synthesis. The antibiotic protein synthesis inhibitors emetine and anisomycin exhibit the necessary effectiveness and spec- ificity. These compounds do not inhibit RNA synthesis or the induction of settlement and plantigrade attachment of the planktonic abalone larvae, as would be expected if these processes are mediated by a chemosensory system, but they completely block the subsequent metamorphosis which, as expected, is apparently dependent on de novo protein synthesis. These inhibitors should therefore help investigators identify the primary response genes (the transcription of which does not depend on de novo protein synthesis) and messenger RNAs responsible for the in- duction of metamorphosis. Materials and Methods Haliotis rufescens broodstock was collected off the coast of Santa Barbara, California, and production and culti- vation of larvae conducted as previously described (Morse et ai. 1977. 1978, 1979b). Spawning was induced by ex- posing gravid adults to 10 mA/ hydrogen peroxide. Male and female gametes were collected and washed separately, and then mixed to allow fertilization. Embryos and larvae were maintained in 5 ^m-filtered, U.V. -sterilized flowing seawater at 15 1C. Antibiotic inhibitors of protein synthesis were pur- chased from Sigma Chemical Company (St. Louis, Mis- souri), dissolved to make concentrated stock solutions and used fresh on the day of preparation. Tetracycline was purchased as the hydrochloride, fusidic acid as the sodium salt, and puromycin and emetine as the dihydrochlorides. Stock solutions were prepared in 0.22 /urn-filtered distilled water, either alone, or containing the minimum amount of ethanol required to completely solubilize the antibiotic. After addition of antibiotics to experimental samples, no more than 0.2% (v/v) ethanol was present in any seawater sample. Control experiments showed that the presence of 0.3% ethanol had no effect on larval behavior and settle- ment or on the level of [ 3 H]leucine incorporation into TCA-insoluble material in the larvae; higher concentra- tions of ethanol (above ca. 0.75%) and other organic sol- vents induced settlement of the larvae, with a rapidity corresponding to the concentration of solvent (data not shown). Similar results were reported earlier by Penning- ton and Hadfield (1989) for larvae of the nudibranch mollusk Phestilla sibogae. Synthesis of protein and RNA was measured by incor- poration of radioactive amino acid or nucleoside into acid- precipitable macromolecules. For each assay ca. 2000 lar- vae were placed in 10 ml of 5 /urn-filtered, U.V. -sterilized seawater in 40 ml Oakridge tubes. Rifampicin, a specific inhibitor of bacterial RNA synthesis, was added to a final concentration of 2.4 jtA/ in all samples to limit bacterial growth, except where otherwise noted. After incubations in the presence or absence of inhibitors, either L-[4,5- 3 H]leucine (150 Ci/mmol; Amersham Corporation, Ar- lington Heights, Illinois) or [5,6- 3 H]uridine (42 Ci/mmol; Amersham) was added to 0.1 or 0.2 jiCi/ml, as noted in the figure legends. For each treatment at each time point, three larval samples were used. Larvae were kept at 1 5 1 C, except as noted. To end the labeling, nonradioac- tive L-leucine or uridine was added to a final concentra- tion of 0.8 mA/ or 0.4 mA/, respectively. The Oakridge tubes then were centrifuged (16,000 rpm; 4C) for 5 min (Sorvall RC5 or RC5C Superspeed Centrifuges, Clare- mont, California); Sorvall SA-600 or SS-34 fixed angle rotors were used to pellet the larvae. The tubes were placed on ice, the water was drained off, 2 ml of cold 1 X SSC was added, and the larvae were re-suspended and ho- mogenized completely in an ice-cold Dounce homoge- nizer (7 ml Pyrex tissue grinder). One aliquot of 0.5 ml for each sample was removed, placed in a 1.5ml microfuge tube, and frozen for future quantitation of protein. For each sample, 1.5 ml of the homogenate was then placed into another microfuge tube on ice, and 100% (w/v) tri- chloroacetic acid (TCA) was added to yield a final con- centration of 10% (w/v). After acid precipitation for 30 min at 4C, each sample was poured through a 2.4 cm glass microfiber filter (GF/C; Whatman International Ltd., Maidstone, UK), washed three times with 5% TCA, then washed twice with 100% ethanol. The filters were com- pletely dried in an oven at 55C and then placed in liquid scintillation vials; 1 ml of scintillation cocktail (Bio-Safe II; Research Products International, Mount Prospect, IL) was added and radioactivity determined by liquid scin- tillation. Protein was quantitated by the method of Brad- ford ( 1976) according to the protocol of the reagent man- ufacturer (Bio-Rad Protein Assay; Bio-Rad Laboratories, Richmond, California); assays were conducted in triplicate and evaluated relative to a bovine serum albumin standard measured in parallel. The incorporation data presented are the means of triplicate determinations, with error bars representing one standard deviation. Assays of settlement and metamorphosis were con- ducted with larvae in glass scintillation vials (ca. 200 larvae in 10 ml of rifampicin-containing 5 ^m-filtered, U.V.- sterilized seawater) maintained under low illumination and observed with a dissecting microscope. Each treat- ment was conducted in triplicate. Larvae also were placed at a density comparable to that used in the incorporation G. FENTEANY AND D. E. MORSE 150 ^ 100 O O 2 o Q. o o _c 50 100 200 300 400 200 - g 150 - m CD CC 100 200 300 400 [Antibiotic] (\iM) 500 Figure I. Incorporation of ['H]leucine as a function of the concen- tration of antibiotic. After incubating 8-l()-day-old larvae (ca. 2.000 lar- vae/10 ml rifampicin-containing seawater) for 2 h in the presence or absence of blocker at the concentrations indicated, [ 3 H]leucine was added to 0.1 /iCi/ml, and the pulse allowed to proceed for 2 h. Mean control values (representing incorporation in the absence of antibiotic) are dis- placed on the abscissa for clarity. (A) Incorporation in the presence of emetine (diamonds) or anisomycin (rectangles). (B) Incorporation in the presence of cycloheximide (diamonds), puromycin (rectangles), tetra- cycline (triangles), or fusidic acid (circles). Details as described in Materials and Methods. assays (ca. 2000 larvae/ 10 ml seawater), and observed for mortality and other responses to the protein synthesis in- hibitors. Results Inhibition of protein synthesis Larvae of H. rufescens take up exogenous amino acids from seawater, as demonstrated by these and other in- vestigations (Jaeckle and Manahan, 1989), although these larvae are lecithotrophic. Several commonly used inhib- itors of protein synthesis, including cycloheximide, fusidic acid, puromycin, and tetracycline, had little or no inhib- itory effect on the overall incorporation of [ 3 H]leucine into TCA-insoluble material at concentrations that were not toxic to the larvae (Fig. 1). In marked contrast, both emetine and anisomycin proved strongly inhibitory in a concentration-dependent manner. Emetine (9 n.\f) efficiently blocked the incorporation of [ 3 H]leucine into Haliotis larvae under conditions in which 100 nM chloramphenicol (an inhibitor of protein synthesis only in prokaryotes) had no significant effect (Fig. 2A). Identical results were obtained for a range of chloramphenicol concentrations (50-600 n.\f), both in the presence or absence of 2.4 pM rifampicin (an inhibitor of bacterial RNA polymerase). Thus, in the absence of emetine, prokaryotic incorporation of [-'H]leucine was not detectable. Inhibition by emetine was quite rapid; incu- bation for 10 min with 9 fiM emetine prior to addition of radiolabel was sufficient to block incorporation to a level comparable to that produced by an incubation for 2 h (data not shown). The inhibitory effect of a single 200 150 c 100 ' 50 o "I 200 2 o 9- 8 15 c 100 50 Figure 2. Incorporation of [ 3 H]leucine in 7-day-old larvae in the presence or absence of emetine (9 nM) or anisomycin (200 //A/) and chloramphenicol (150 nKf). 7-day-old larvae were used. Pulse-labeling at 24 h following the time of initial addition of emetine was for 2 h (0.1 nCi/ml). (A) Treatments: ( 1) No emetine or chloramphenicol. (2) Chlor- amphenicol added at 2 1 h. (3) Emetine added at h. (4) Emetine added at h and chloramphenicol added at 21 h. (5) Emetine added to a con- centration of 9 ^M at h and the same amount added again at 12 h. (B) Treatments: (1) No anisomycin or chloramphenicol. (2) Chloram- phenicol added at 21 h. (3) Anisomycin added at h. (4) Anisomycin added at h and chloramphenicol added at 2 1 h. (5) Anisomycin added to a concentration of 200 /iA/ at h and the same amount added again at 12 h. INHIBITORS OF PROTEIN SYNTHESIS addition of emetine relaxed with time (Fig. 3A), and a second addition of the same amount of emetine at 12 h reduced incorporation slightly further (Fig. 2A). However, following prolonged incubation in the presence of eme- tine, the addition of chloramphenicol 3 h before labeling led to significantly lower levels of incorporation, partic- ularly at 24 h and 48 h following the addition of emetine (Figs. 2A, 3A). Therefore, some of the apparent relaxation of inhibition by emetine may be due to an increase in the proportion of protein synthesis attributable to contami- nating chloramphenicol-sensitive prokaryotes. This is likely to be the result of bacterial growth on the emetine- treated larvae themselves, as these larvae become weaker, although rifampicin (2.4 (iAf) was present throughout. The inhibitory effect of a single addition of anisomycin (200 pM) persisted longer than that caused by 9 /J.M eme- tine (Figs. 2B, 3B), and a second addition 12 h after the first did not reduce the incorporation of [ 3 H]leucine fur- ther (Fig. 2B). In the presence of anisomycin, the addition of chloramphenicol 3 h before pulse-labeling did not lead to significantly lower levels of incorporation up to 24 h (Figs. 2B, 3B). Much of the inhibition of protein synthesis by a single addition of anisomycin was reversed between 24 and 48 h (Fig. 3B). This late apparent relaxation was blocked by chloramphenicol (Fig. 3B), suggesting that it was due to an increase in prokaryotic incorporation. Effects of emetine and anisomycin on RNA synthesis To test whether emetine affects RNA synthesis, larvae were pulsed with [ 3 H]uridine both in the presence and absence of 10 6 M GABA (added 30 min following the addition of emetine). No significant inhibition of RNA synthesis was observed except at 6 h in the presence of both GABA and 9 nM emetine (Fig. 4A). The presence of emetine (9 n.M) may have a stimulatory effect on the incorporation of ['H]uridine in Haliolis larvae after 12.5 h. A similar experiment showed that the incorpo- ration of [ 3 H]uridine also was not inhibited by the addition of 200 pM anisomycin (added 60 min before addition of GABA; Fig. 4B). Effects of emetine and anisomycin on settlement, metamorphosis, and survival At concentrations sufficient to inhibit most protein synthesis, emetine and anisomycin did not block the initial induction of larval settlement and plantigrade attachment by GABA, although subsequent metamorphosis was completely blocked. Toxicity of these inhibitors was both time- and concentration-dependent. Initial settlement and plantigrade attachment of larvae induced by GABA ( 10~ 6 M and 10~ 3 M) occurred normally in the presence of 9 nM emetine (Fig. 5 A, B). Both in the presence and absence of emetine, larvae ceased their swimming behavior after c 'CD 2 Q. O) 300 200 100 . A 12 18 24 30 36 42 48 = 200 2 o Q. 150 100 50 B 12 18 24 30 Time (h) 36 42 48 Figure 3. Incorporation of ['HJleucme in the presence or absence of emetine (9 pM) or anisomycin (200 n\I) and chioramphenicol (150 nM) as a function of time following addition of emetine. Larvae were pulsed at the times after addition of emetine or anisomycin indicated for 2 h (O.I jiCi/ml). In the chloramphemcol-treated samples, chlor- amphenicol was added 3 h prior to pulse-labeling. Mean values are dis- placed slightly on the abscissa for clarity. (A) 4-day-old larvae were used (6 days old by the end of the experiment in the last 3 sets of samples). No antibiotic (diamonds); emetine (rectangles); emetine plus chioram- phenicol (triangles). (B) 5-day-old larvae were used (7 days old by the end of the experiment in the last 3 sets of samples). No antibiotic (dia- monds); anisomycin (rectangles); anisomycin plus chloramphenicol (tri- angles). addition of GABA, and plantigrade attachment followed. Attached larvae exhibited normal pedal locomotion in the presence of emetine. Abscission of the velum was also observed in the presence of emetine and occurred whether GABA (10~ 6 A/) was present or not, although at 9 /j.M emetine abscission occurred at lower levels when GABA was not present. Abscission was often premature or in- complete, particularly at higher concentrations of emetine. By 6 h after the addition of 10~ 6 M GABA, most of the larvae had settled both in the presence and absence of 9 juM emetine (Fig. 5 A). New shell growth was not observed in the presence of emetine when larvae were induced to settle with 10~ 6 M GABA, although it was observed nor- mally in settled larvae in the absence of the inhibitor by 48 h. Attachment proceeded more rapidly at the higher G. FENTEANY AND D. E. MORSE c '0) "o Q. O5 40 30 20 10 I -o 12 15 18 21 24 -.5 40 2 o Q. O o 30 20 10 B 9 12 15 18 Time (h) 21 24 Figure 4. Incorporation of [ 3 H]uridine in the presence or absence of emetine or anisomycin as a function of time after addition of GABA ( ICT 6 M). The larvae were pulsed for 20 min with radiolabeled nucleoside (0.2 ^Ci/ml) at the times indicated. Mean values slightly displaced on the abscissa for clarity. (A) I0-day-old larvae were used. Where indicated, emetine (9 p.\l) was added 30 min prior to the addition of GABA. No emetine or GABA (diamonds); emetine with no GABA (rectangles): no emetine, plus GABA (triangles); emetine plus GABA (circles). (B) 9-day- old larvae were used. Where indicated, anisomycin (200 nl\f) was added 60 min prior to the addition of GABA. No anisomycin or GABA (dia- monds); anisomycin with no GABA (rectangles): no anisomycin, plus GABA (triangles): anisomycin plus GABA, (circles). concentration of GABA; virtually all of the larvae were attached within 20 min, with no inhibition by emetine (Fig. 5B). Although emetine did not inhibit the initial rate of attachment of the larvae induced by GABA. the larvae failed to maintain their plantigrade attachment (Fig. 5A. B), and progressively more were found on their sides, ap- parently due to the toxic effect of prolonged exposure to emetine. There also was some attachment in the presence of emetine when GABA was absent (Fig. 5A. B). Prolonged exposure of larvae to emetine proved lethal. Even before any mortality was observed, larvae treated with 9 fiM emetine appeared to spend more time on the bottom of the test vial than larvae in control vials. By 36 h after the addition of emetine (9 nAf) both in the presence and absence of GABA (ca. 20 larvae/ml), few larvae were swimming and many appeared dead, while in the control vials lacking GABA, many of the larvae remained swimming and virtually all remained alive. All the larvae were dead by 54 h in the presence of 9 ^M emetine at ca. 20 larvae/ml, and by 72 h at ca. 200 larvae/ ml. Toxicity was progressively accelerated by higher con- centrations, although the initial rate of GABA-induced attachment remained unimpaired below 80 ^/emetine; in the presence of 18 nM and 40 nM emetine, virtually all of the larvae were attached within 20 min following the addition of 1(T 3 M GABA (data not shown). Exposure to 80 pM or 160 fiM emetine produced marked symptoms of toxicity; GABA-induced settlement was reduced, pre- mature abscission of the velum occurred in the presence and absence of GABA. and all of the larvae died within 6-12 h at the low density. Anisomycin appeared to exert a stimulatory effect on the activity of the larvae, particularly on the movement of the cilia. The level of swimming activity of the larvae was markedly greater in the presence of 200 nAI aniso- mycin than in control or emetine-treated vials, even after only 20 min following addition. This effect appeared to partially antagonize the initial GABA-induced attach- ment, with the attached larvae abnormally continuing sustained beating of their swimming cilia and displaying little pedal locomotion; plantigrade larvae often were dis- placed by collision with other swimming larvae, and sometimes began swimming again. The initial rates of settlement and attachment induced by 10 3 M GABA were relatively unaffected by anisomycin, although long- term attachment was reduced (Fig. 5C, D). [The weak settlement-inducing activity of high concentrations of an- isomycin itself (cf. Fig. 5C, D) may explain the biphasic settlement observed in the presence of GABA.] Aniso- mycin also produced concentration-dependent and time- dependent symptoms of toxicity, with complete mortality resulting from prolonged exposure (96 h) of larvae, at high or low density, to 200 ^M concentration. Discussion Inhibition of protein synthesis Emetine and anisomycin were found to be highly ef- fective inhibitors of protein synthesis in Haliotis larvae, whereas cycloheximide, fusidic acid, puromycin, and tet- racycline proved far less effective. Possible reasons for the limited effectiveness of these widely used inhibitors may include their instability or low solubility in seawater, or their inefficient diffusion into the deeper layers of larval tissue. There is some structural evidence supporting the suggestion that membrane permeability may be an im- portant determinant of effectiveness in the marine larval system. Emetine contains four methoxy groups, while an- isomycin contains one methoxy and one acetoxy group, all carbon-linked to cyclic nuclei in both of these anti- INHIBITORS OF PROTEIN SYNTHESIS 11 40 60 Time (h) 20 40 60 Time (h) 80 100 Figure 5. Attachment of larvae in the presence or absence of emetine or anisomycin as a function of time following addition of GABA. Cu 200 were placed in 10 ml rifampicin-treated seawater in triplicate, as described in Materials and Methods. Following a 30-min incubation with or without emetine (A and B) or a 2-h incubation with or without anisomycin (C and D), GABA was added to the samples indicated, and the mean percentage of the larvae showing attachment was scored at the times indicated. Standard deviations for all points, assayed in triplicate, were <4%/. 80 .. -. - \ \ss 1 1 -nr | i [ 20 40 60 80 Percent Metamorphosed Figure 7. (A) Histograms showing the effect of pretreatment at four days of larvae with high K + seawater that either induced partial meta- morphosis or no metamorphosis on the ability of these two categories of larvae to undergo metamorphosis after treatment with a metamor- phosis substrate or high K + seawater at five days. (B) Histograms showing effect of treatment with a metamorphosis substrate or high K* seawater at five days on metamorphosis of larvae that had not been pretreated with high K + seawater at four days. The hatched segment of each bar indicates the proportion of cases that underwent normal metamorphosis. The clear segment ol the bar indicates the percentage of cases that un- derwent partial metamorphosis. The number of cases is at the top of the bar. were not disturbed underwent normal metamorphosis by the next day (six cases). The larvae that were removed from their settlement site did not resettle but underwent partial metamorphosis (four out of six cases). This ex- periment suggests that settlement is necessary for normal metamorphosis. Discussion The role of voltage-dependent Ca 2+ channels in metamorphosis The following lines of evidence indicate that voltage- dependent calcium channels may play a role in meta- morphosis: ( 1 ) Treatment of larvae with high K + seawater which presumably depolarizes the cells of the larva induces metamorphosis and treatment of larvae with high K + in Na + -free seawater is just as effective in inducing meta- morphosis, (2) Treatment of larvae with high K 1 in Ca 2+ - free seawater inhibits metamorphosis, (3) Treatment of larvae with high K + in seawater with elevated Ca 2+ levels or Mg 2+ -free seawater increases the percentage of cases metamorphosing, (4) Treatment of larvae with high K + seawater in the presence of the calcium channel blockers Co 2+ and Nifedipine inhibits metamorphosis. In order to make this work more convincing one would have to dem- onstrate electrophysiologically that target cells are not only depolarized but give an action potential which is typical of voltage-dependent Ca 2+ channels and that Ca 2+ moves into the target cells from the external environment during depolarization. The identities of the target cells where voltage-depen- dent calcium channels function to mediate the meta- morphic stimulus is not known. One possible target cell IONIC CONTROL OF METAMORPHOSIS 23 candidate is a subset of cells in the larval nervous system. There is evidence that the nervous system receives and mediates the metamorphic stimulus in echinoid larvae (Burke, 1983b). Unfortunately, virtually nothing is known about the organization of the nervous system in articulate brachiopod larvae; however, nerve cell processes have been noted in ultrastructural studies done on these larvae for other purposes (Strieker and Reed, 1985a). Another possible set of target cells could be some of the cells that make up the surface epithelium of the larva (e.g., the cells of the distal part of the pedicle lobe). After these cells receive a metamorphic stimulus, it could be transferred to other epithelial cells of the larva by epithelial conduc- tion. There is evidence that epithelial conduction mediates the metamorphic stimulus in hydrozoans (Freeman and Ridgway, 1990). Both substrate and high K + seawater induced meta- morphosis appear to depend on calcium channel function. Substrate induced metamorphosis also depends on the pedicle lobe while high K + seawater induced metamor- phosis does not. The simplest model that accounts for these results is that there is a substrate-induced meta- morphosis receptor at the distal end of the pedicle lobe. When this is activated a metamorphic signal is sent from this site to cells outside of the distal region of the pedicle lobe that must have their putative voltage-dependent cal- cium channels activated in order to spread the metamor- phic stimulus (Fig. 8). When the cells outside the distal region of the pedicle lobe are activated, they also send an inhibitory signal to the cells in the distal region of the pedicle lobe preventing them from responding to substrate mediated metamorphic cues (Fig. 7). The signijii'iiiur <>/ '"partially metamorphosed" larvae The partially metamorphosed larva is most probably the result of an abnormal metamorphic response. This larva is characterized as a larva that forms a protegulum in the absence of mantle reversal and settlement. Because the formation of a protegulum under these conditions probably renders the mantle lobe incapable of reversal and because the mantle lobe does not spread out to occupy a larger area as it does after reversal, this metamorphic response is probably maladaptive. I have made only lim- ited attempts to look for later manifestations of normal metamorphosis in partially metamorphosed larvae. Two partially metamorphosed larvae were fixed and sectioned four days after the initiation of high K + seawater induced metamorphosis. Both of these larvae showed suggestions of cuticle deposition by the pedicle. In order to make this point with certainty, it would be necessary to do a study of these larvae at an electron microscope level of resolu- tion. I did not observe any indication of mouth formation in these partial larvae; however, they may not have been cultured long enough. N PI / !/ i >* i \ \ \ Figure 8. Diagrammatic view of a swimming larva with an apical lobe (AL). mantle lobe (ML), and pedicle lobe (PL). At the distal end of the pedicle lobe there is a postulated center composed of cells ( 1 ) which may use voltage-dependent calcium channels to transduce a substrate mediated metamorphic signal. This center sends a stimulatory meta- morphic signal to other cells in the larva including center (2) which functions via voltage-dependent calcium channels that acts as a secondary metamorphic center. Here this center is shown in the mantle lobe but it could be any place outside of the distal end of the pedicle lobe. The cells of this secondary metamorphic center send a stimulatory meta- morphic signal to other cells of the larva and an inhibitory signal to the cells that transduce the substrate mediated metamorphic signal turning off the metamorphic stimulus from these cells. This model accounts for the experiments described in this paper. A variety of factors probably play a role in generating the partial metamorphosis phenotype. In larvae that have been reared for a number of days in a sterile environment intrinsic maturational changes may occur so that various parts of the metamorphosis signaling pathway or cells that respond to the signaling pathway may be activated. If the postulated distal pedicle lobe substrate receptor cells were activated, an aged larvae may undergo normal metamor- phosis. This happened in a small percentage of cases (Ta- ble II). If cells that are part of the metamorphic pathway that reside outside of the distal region of the pedicle lobe are activated or if cells that will form the protegulum are activated, a larva that shows the partial metamorphosis phenotype would be generated. There is evidence that in some species with a bathy-pelagic life cycle that larvae which do not see an appropriate metamorphic cue in na- ture will metamorphose or partially metamorphose and still continue a pelagic existence (Thorson, 1946; Paine, 1963). The mechanics of mantle lobe reversal during meta- morphosis are not understood. There is a pair of muscles that insert in the mantle lobe and the pedicle lobe that are thought to contract during metamorphosis causing the mantle lobe to flip (Franzen, 1969; Long, 1964). Sub- strate adhesion by the pedicle lobe may be necessary for these muscles to contract or to cause the pedicle lobe to be compressed in an appropriate way as the muscles con- tract so that the mantle lobe is reversed. The production of larvae that show partial metamorphosis following sub- strate detachment could occur because protegulum for- mation is activated even though mantle reversal is inhib- ited. The small number of cases where partial metamor- 24 G. FREEMAN phosis occurs following the culture of larvae in the presence of substrates that induce metamorphosis can be explained in this way. Partially metamorphosed larvae and the conditions where they are formed provide an in- sight into the normal metamorphosis process. Acknowledgments I am grateful to Dr. A. O. D. Willows and the staff of the Friday Harbor Laboratories for their hospitality. I want to thank Sarah Cohen and her diving companions for collecting animals using SCUBA, Dr. Craig Staude for saving animals for me that were collected on dredging trips, and Drs. Alan Kohn and Patricia Morse for letting me use animals that were dredged for class use. I want to thank Judith Lundelius, Bob Goldstein, and Hyla Sweet for their comments on this manuscript. This work was supported by NSF grant DCB-8904333 and a URI re- search leave from The University of Texas. Literature Cited Burke, R. D. 1983a. The induction of metamorphosis of marine in- vertebrate larvae: stimulus and response. Can. J Zoo/. 61: 1701- 1719. Burke, R. D. I983b. Neural control of metamorphosis in Dendraster excentricus. Biol. Bull. 164: 176-188. Cameron, A., T. Tosteson, and V. llensley. 1989. The control of sea urchin metamorphosis: ionic effects. Develop. Growth Differ. 31: 589- 594. Franzen, A. 1969. On larval development and metamorphosis of Ter- cbralulina Brachiopoda. /<><>/, Bid. Uppsala 38: 155-174. Freeman, G. 1981. The role of polarity in the development of the hy- drozoan planula larva. Rou.\'s Arch. Dev Biol. 190: 168-184. Freeman, G.. and K. B. RidgHa\. 1990. Cellular and intracellular path- ways mediating the metamorphic stimulus in hydrozoan planulae. Rou\'sArch. Dcv. Biol. 199: 63-79. Hille, B. 198-1. Ionic Channels and Excitable Membranes Sinauer As- soc., Sunderland, MA. Long, J. A. 1964. The embryology of three species representing three superfamilies of articulate brachiopoda. Ph.D. Dissertation. University of Washington. Long, J. A., and S. A. Strieker. 1991. Brachiopoda. Pp. 47-84 In Re- production in Marine Invertebrates, I 'ol. 6 Echinoderms ami Lopho- phorales. A. C. Giese, J. S. Pearse. and V. B. Pearse, eds. Boxwood Press. Palo Alto. CA. Morse, D. K. 1990. Recent progress in larval settlement and meta- morphosis: closing the gaps between molecular biology and ecology. Bull. Mar Sci. 46: 465-483. Paine, R. 1963. Ecology of the brachiopod Glottidia pyramidata. Ecol. Monogr. 33: 187-213. Strathman, M. 1987. Reproduction ami Development of Marine I/t- vertehrates oj the Northern Pacific Coast. University of Washington Press, Seattle. WA. Strieker, S. A., and C. G. Reed. 1985a. The ontogeny of shell secretion in Terehratalia transversa (Brachiopoda, Articulata) I. Development of the mantle. ./ Morphol. 183: 233-250. Strieker, S. A., and C. G. Reed. 1985b. The protegulum and juvenile shell of a recent articulate brachiopod: patterns of growth and chemical composition. Lclhaia 18: 295-303. Strieker, S. A., and C. G. Reed. 1985c. Development of the pedicle in the articulate brachiopod Terebratalia transversa ( Brachiopoda, Ter- ebratulida) '/.oomorphology 105: 253-264. Thorson, G. 1946. Reproduction and larval development of Danish marine bottom invertebrates. Medd. Komm. Dan Fisk: Havundersog Ser. Plankton 4: 1-523. White, B. II., and C. S. Nicoll. 1981. Hormonal control of amphibian metamorphosis. Pp. 363-396 in Metamorphosis: A Problem in De- velopmental Biology. L. I. Gilbert and E. Frieden, eds. Plenum, New York. Yool, A. J., S. M. Green, M. Hadfield, R. Jensen, D. Markell, and I). Morse. 1986. Excess potassium induces larval metamorphosis in four marine invertebrate species. Biol. Bull. 170: 255-266. Reference: Biol. Bull 184: 25-35. (February, 1993) Species Relationships in a Marine Gastropod-Trematode Ecological System LAWRENCE A. CURTIS' AND KAREN M. K. HUBBARD* University Parallel, * School of Life Sciences, and College of Marine Studies. University of Delaware. Newark, Delaware 19716 Abstract. Individual snails (Ilyanaxsa obsoleta) on Cape Henlopen, Delaware, frequently are host to one or more trematode species. When different species occupy the same host, interactions might be expected. We investigated five species of parasites to determine whether their existence in different combinations would lead to altered within- host distributions or changed numbers of shed cercariae. Snails (32 samples, total = 379) were collected from June to August, in 1989, and microscopically examined. Par- asite species and stages present in five sections through each snail were recorded. Before examination, 206 of these snails were held in individual chambers in the field. After two high tides (ca. 24 h), the chambers were checked for species and the numbers of cercariae shed. Overall, 22 trematode combinations in single hosts were observed. Analysis revealed that co-occurrence with other species had no significant effects on any trema- tode. Further, analyses of species richness of infecting assemblages over two distinct intervals failed to show that competition is important in determining assem- blage richness. One pair of trematodes (Himasthla quissetensis and Lepocreadium setiferoides) has been reported not to co-occur. We observed co-occurrences, but so few that the apparent conflict between them could not be statistically demonstrated. We suggest that, in this system, parasites are adapted to the host only, they may interact, but they are not adapted to each other. Chances for a parasite to live free from other parasites seem too great for evolved (adapted) relationships to develop. The host, for similar reasons, is probably not adapted to the parasites. Received 14 February 1992: accepted 6 October 1992. 1 Mailing address: Cape Henlopen Laboratory, College of Marine Studies, University of Delaware, Lewes, DE 19958. Introduction For one species to be adapted to another, they must interact in such a manner that one consistently exerts a selective pressure on the other. Species interactions may be thought of as a continuum from local to global. A local interaction (as used here) results in genetic changes in restricted parts of a gene pool (and may result in local ecotypes). On the other hand, if an interaction is global, one species can be a source of biotic selective pressure over the whole operating gene pool of another. Reciprocal genetic changes between species amount to coevolution (Futuyma and Slatkin, 1983). This paper considers the species interactions in a marine gastropod-trematode system. Because the host gastropod has a planktonic larva and the trematodes are dispersed by highly mobile defin- itive hosts, both local and global phenomena must be considered. The interactions between hosts and parasites have been much discussed (see Moore, 1987 for an extensive review), and the levels at which such discussions may be focussed should be distinguished. In this work, two levels are nec- essary. The component community includes all parasite species using a particular host species (population). The infracommunity includes all the parasites in a single host (Esch el ai, 1990). An individual host, harboring a mul- tispecies parasite assemblage, is a biological unit where parasite-parasite as well as host-parasite interactions can occur. There are four basic patterns of evolutionary relation- ships that may be found in any host-parasite system (Fig. la-d). In scheme a, the parasites are adapted to the host (the minimal condition), whereas in scheme b, the host is also adapted to the parasites. Scheme c illustrates the case where the parasites are adapted to the host and to 25 26 L. A. CURTIS AND K. M. K. HUBBARD PARASITE A PARASITE B HOST PARASITE A HOST PARASITE B PARASITE A PARASITE B PARASITE A HOST PARASITE B Figure 1. Four models of possible adaptive relationships among spe- cies in a snail-trematode system. Parasites A and B may coexist in a single host. An arrow from one participant to another indicates that the participant at the origin of the arrow has evolved adaptations to selection pressures coming from the other (i.e.. "PARASITE A - PARASITE B" means A is adapted to B). One-way interactions between parasites (/.('., A adapts to B but not the reverse) are possible, but not figured. each other. Scheme d shows the case where parasites are coevolved with the host and with each other. There should be evidence of an adaptive relationship between species before it is assumed to exist (Williams, 1966). In this work, we have tested for species interactions among trematodes inhabiting the same gastropod host. The goal is to gather evidence to support the elimination of one or more of the above schemes and thereby improve our understanding of host-parasite systems. Studies of trematodes infecting gastropod populations have often revealed patterns of species co-occurrence that suggest interactions (see Rohde, 1981 for references). However, few workers have examined trematode assem- blages in individual gastropods taken from their natural habitat, to determine whether fitness of certain members is consistently affected by co-occurrence with other members (see DeCoursey and Vernberg, 1974). This is largely because multiply-infected hosts are difficult to ob- tain in numbers for study. The prevalence of trematodes in the population of Ilyanassa obsoleta (Prosobranchia, Neogastropoda) on Cape Henlopen, Delaware Bay is high. and a diversity of multiply-infected snails may be ob- tained (Curtis, 1985, 1987, 1990; Curtis and Hubbard, 1990). This allowed us to test for species interactions in a variety of trematode ensembles. Of the nine trematode species in Ilyanassa obsoleta ob- served in Delaware, five are commonly observed in the Cape Henlopen population and figure in this study: ///- masihla quissetensis, Lepocreadium setiferoides, Zoo- gonus rubellus. Aiistmbilharziu variglandis, and Gvnae- cotyla adunca. The snail is the first intermediate host. A variety of second intermediate hosts is used by these spe- cies. Various shorebirds serve as definitive hosts for H. quissetensis. A. variglandis and G. adunca, whereas fish species are used by L. setiferoides and Z. rubellus (see Stunkard, 1983 for life-cycles and taxonomic matters). Any direct species interactions among these parasites must occur in the snail, the only host they all have in common. There is no indication that Ilyanassa obsolete! lose in- fections (Curtis and Hurd, 1983). so the ensembles ob- served in snails probably represent relatively longstanding (period unknown) assemblages. Enduring species assem- blages, proximity in a natural habitat unit, and utilization of similar resources (Smyth and Halton. 1983). suggest that strong interspecific interactions might occur. If competitive interactions are frequent within individ- ual hosts whereby dominant species come to monopolize the host population through time, a pattern should emerge at the component community level. Early on, most snails should have single species infections; as time progresses species accumulate and there should be a preponderance of double and triple infections; and eventually there should be mostly single infections again, as the dominant species evict subordinates (Sousa, 1990). We searched for such a component community pattern among our snails at two time scales, through the summer and over several years. To examine within-snail parasite interactions, we tested individual species to see whether existence in different assemblages had consequences in terms of ( 1 ) alterations of within-snail spatial distributions, (2) complete suppres- sion of cercarial production, and (3) changes in numbers of cercariae released from hosts. Materials and Methods One sandbar (Fig. 2), located near the mouth of the Delaware Bay on Cape Henlopen (75 06'W, 38 471^), was chosen as the source for snails. Certain species of trematodes affect the behavior, distribution and temporal occurrence of Ilyanassa obsoleta on sandbars (Curtis, 1987, 1990). To avoid over-representing snails harboring particular parasite ensembles, we randomly chose collec- tion sites according to the angle and distance from a ref- erence point at the peak of the sandbar (Fig. 2). Samples GASTROPOD-TREMATODE INTERACTIONS 27 "0 10 20 30 40 METERS (ALONG BEACH) NE Figure 2. An elevational contour map of the 1989 sandbar on Cape Henlopen. Delaware where samples ofllyanassa ohsolctu were collected for this study. The 32 randomly selected sample sites are indicated by filled diamonds. The highest point on the map (the sandbar peak at center) is 56 cm above the lowest. were taken between 16 June and 17 August 1989 on both day and night low tides. We wanted many multiply-in- fected snails in the samples, and the 379 snails obtained (Table I) were purposely biased to include them. The snails came from an area where many multiples were likely to be found (e.g., Curtis, 1987), and large snails that were likely to be infected were chosen (Curtis and Hurd, 1983). Usually, two collections of 10 to 13 snails were collected and processed at a time. All the snails were dissected and 206 were also tested for cercarial release. We were interested in revealing gross within-snail dis- placements of individual parasite species by other species or combinations of species. Such displacements would be required if dominant species gradually evicted subordi- nates from the snail. During dissection each snail was removed from its shell and examined in sections to de- termine how individual parasite species, and stages thereof, were distributed within. Heavily parasitized snails are virtual bags of trematodes; they retain no consistent morphological landmarks that are useable as standard points of reference. Consequently, each snail was pinned to a board and cut crosswise into five equal lengths with a razor blade. Section 1 was the most dorsal portion of the snail (the spire), and section 5 was the most ventral (head and foot). The razor was cleaned between cuts and scrupulous care was taken to prevent contamination of one section with material from another. Sections were placed separately in small vials containing 5 ml filtered baywater. Each vial was vigorously shaken 50 times to release the contained trematode stages into the water. A small amount of the water was placed on a slide and examined with the aid of dissecting (32X) and compound (100X) microscopes. We took two samples from each vial. The species and stages of the trematodes were recorded for each section of each snail as follows: parental stages (rediae or sporocysts) and cercariae (PC); cercariae only (C); parental stage without mature cercariae (P); or absent (A). Observed cercariae may have been lib- erated from parental stages during the procedure, but this does not matter as we were only interested in whether formed (mature) cercariae were present. Trematodes were never found in section 5, and after the hundredth snail we stopped examining this section. Table I Trematode infections in Ilyanassa obsoleta collected for this study from a sandbar area (Fig. 2) on Cape Henlopen. Delaware Infecting species n Mean shell height (mm) Range shell height (mm) uninfected 18 21 17-25 singles Hq 74 24 20-27 Ls 25 24 20-27 Zr 29 22 20-26 Av 5 23 22-24 Ga 29 22 18-25 doubles HL 4 23 22-23 HZ 42 24 20-27 HG 10 23 21-25 LZ 8 24 20-28 LA 1 25 LG 32 23 20-26 ZA 1 24 ZG 24 23 20-27 AG 1C) 22 19-24 AD 1 23 GD 1 21 triples HZG 33 24 18-26 LZA 1 28 LZG 22 23 17-26 LAG 4 21 20-24 ZAG 5 23 23-25 Total = 379 Overall = 23 17-28 For each infection, number collected (n), and mean and range of shell heights are given. Snails infected by a single species (singles) are repre- sented by the genus and species initials of the trematode (Hq = Himasllila qitissetensis, Ls = Lepocreadium setiferoides, Zr = Zoogonus rubellits. Av = Austrobilhariia variglandix. Ga = Gynaecotyla adunca). Double and triple infections are represented with the generic initials of the species involved (e.g., a snail infected with H. quissetensis, Z. rubellus. and G adunca goes in the HZG category). Diplostomum nassa (D) occurred only in double infections. Shell height = siphonal canal to apex of shell (e.g., 21 = 20.5 to 21.4 mm). L. A. CURTIS AND R. M. K. HUBBARD The frequencies of parasite presence or absence in the snails were crosstabulated according to the following cri- teria: parasite assemblage (those species infecting the snail); snail section (1 - 4); and the stage of the parasite (sporocysts, rediae, cercariae). Contingency table analyses were employed to test for significant displacements of parasite stages within snails. For each parasite, we used log linear models (Sokal and Rohlf, 1981 ) to calculate the expected frequencies of occurrence of the stages (PC, P, C, or A) in various sections of hosts harboring various trematode ensembles. A saturated log linear model for this kind of analysis includes seven terms: Assemblage; Section; Stage; Assemblage x Section; Assemblage X Stage; Section X Stage; and Assemblage X Section X Stage. The purpose of this analysis is to learn which of these terms are necessary to calculate a set of expected frequencies that do not deviate significantly from the ob- served frequencies. After unnecessary terms are elimi- nated, we are left with the accepted model. The accepted model is expressed in hierarchical form. For example, an Assemblage X Section X Stage hierarchical model would nest all seven terms of the full model; and an Assemblage, Section X Stage model would nest all three one-way terms and the Section X Stage two-way term. If species interactions lead to spatial rearrangements within snails, a 3-way interaction term (i.e.. Assemblage X Section X Stage) would be necessary in the accepted model for any displaced species. For example, suppose species "a" were usually distributed throughout the snail from spire to mantle when it occurred alone, but in the presence of species "b" (i.e., assemblage "ab"), "a" were consistently absent from the spire section. The three-way interaction term would be necessary in the accepted model because absence (A) of species "a" from Section 1 would be a consequence of Assemblage composition. That is, the presence of species "b" in Section 1 would change Stage entries for "a" in Section I to absent (A) from one of the present categories (PC, P, or C). Therefore, a table of expected frequencies that matched observed frequencies could not be calculated without the Assemblage X Section X Stage term in the accepted model. We used cercarial release as a measure of fitness to learn whether parasites were affected by within-host in- teractions with other infecting species. We evaluated cer- carial output from assemblages during one short period, and tested similar assemblages throughout the summer. (An alternative, more manipulation laden, approach would be to follow cercarial output from individual as- semblages over a longer period of time.) Individual snails were confined in chambers in the natural environment for two high tides (ca. 24 h), and the water in which they had been immersed was examined for numbers and spe- cies of cercariae. We used 24-h periods to encompass any daily shedding patterns. The procedure is described in more detail in Curtis and Hubbard (1990). We used a Kruskal-Wallis test (Hollander and Wolfe, 1973) to de- termine, for each species, whether the number of cercariae shed was significantly different when in various co-oc- curring assemblages of parasites. All statistical calculations were done with the software package. Number Cruncher Statistical System, 5X Series. Results A competition model (Sousa, 1990) suggests that trem- atodes might invade a snail population, accumulate in snails, compete, and eventually complete the process by having dominant trematodes evict subordinates. If true, then over the relevant time we should see infecting species richness start low (mostly single infections), increase (mostly doubles and triples), and then decrease again. We looked for such a pattern within two distinct intervals, over the summer (Table II A), and over several years (Table IIB). We divided the sampling period into four two-week intervals; the fourth interval was extended to encompass the 25 snails collected on August 1 7. There were significant changes in richness from one period to the next, but the expected pattern was not seen. In particular, triple infec- tions were quite abundant early in summer and were most abundant in the last sampling period. This would not have been observed if dominant trematodes had defeated sub- ordinates in this period of time. Using size-classes of snails (Table IIB), the interval can be extended from months to years. At the beginning of its third summer, a snail on Cape Henlopen is about 14-15 mm; by the end of that summer, it has grown to about 17-18 mm (Curtis and Hurd, 1983). This means that the smallest snails we collected (17 mm. Table I) were probably in their fourth summer. If 3 mm/summer is used as an estimate of growth for parasitized snails, then the < =22 group in Table IIB is 4-5 yr old; the 23- 25 group is 5-6 yr old; and the 26-28 group is 6-7 yr old. The interval encompassed by Table IIB is about three years using this estimate. Parasitized snails may not grow this rapidly, and the interval is possibly longer. In this years-long interval (size-class range), there were signifi- cant changes in infecting species richness. Note (Table IIB) that single infections were more abundant than triple infections in the youngest snails, but that the proportion of triples increased among older snails. This is not the pattern predicted by the competition model. Occurrence of stages of five trematodes in sections of Ilyanassa obsoleta harboring different assemblages is shown in Table III. Recall that section 1 was dorsal (spire) and section 4 ventral (mantle). In Table IV. models for all five species (except Austrobilliarzia vanglandis) require the one-way Assemblage term because of the widely dif- ferent numbers of snails infected with each assemblage GASTROPOD-TREMATODE INTERACTIONS 29 Table II Tremalode infections in Ilyanassa obsoleta examined during tins work crosstabulated by number of inled IIIR Iremalode species (richness), time of collection in summer 19S9 (A), and si:e (age) of snail (B) Infecting Species Richness (n = 18) % Singles (n = 162) % Doubles (n = 134) % Triples (n = 65) A. Time Collected 16 Jun-29 Jun 30Jun-13Jul 14 Jul-27 Jul 28 Jul-17 Aug B. Size Class (mm) < = 22 23-25 26-28 2-way contingency analyses: Time X richness, Xf,, = 43.69, P < 0.001 Size X richness, X, 2 6) = 25.16, P < 0.001 0.9 15.0 1.1 4.1 10.5 1.0 0.0 39.8 38.7 50.0 42.9 43.8 43.6 32.3 44.3 40.0 30.7 25.5 33.3 36.9 35.5 15.0 6.3 18.2 27.5 12.4 18.5 32.3 113 80 X8 48 153 195 31 Size class ranges are in terms of shell height as in Table I. (see Table III). The frequencies for Lepocreadium setifer- oides can be modeled by taking into account, beyond the Assemblage term, only the one-way Stage term because most of the stage entries are in the PC category. The rest of the models require the Section X Stage term because there was some specificity as to what sections were likely to harbor which stages. This is clearest for Zoogonus ru- bcllus and Gynaecotyla adunca. Stages were often (clearly not always) absent (A) from sections 1 and 4. However, this was not significantly correlated with the assemblage of species infecting the snail. For none of the five species tabulated is an Assemblage X Section X Stage (three- way) interaction term necessary in its accepted hierarchical log linear model (Table IV). That is, co-occurring trem- atodes did not significantly affect the distribution of any of the five species tested. In most snails, parasite stages of all species present occurred throughout. Parasite species interactions could lead to cercarial suppression in a section rather than species eviction. For example, if the presence of species "a" suppressed cercarial production by species "b", the accepted log linear model for species "b" would have to include the Assemblage X Stage term. This would be necessary because, for species "b" in the presence of species "a", the frequency of the PC category would decrease, while the frequency of the P category would increase as compared to other assem- blages involving species "b". Expected values that matched this shift in observed frequencies could not be predicted (modeled) without incorporating the influence of Assemblage on Stage. No species' cercarial production was completely suppressed in this manner (Table I V, lack of Assemblage X Stage terms). The question now becomes: given that cercariae were being produced, was the number released from snails changed as a function of assemblage composition? To answer this, we used data from cercarial release chambers. Prepatent infections (those with no cercariae present) were eliminated from this analysis because their prepatency was not caused by assemblage composition (Table IV, no Assemblage X Stage terms in the accepted models). In- cluding prepatents would add meaningless variability. Absent cercariae are not germane to this analysis if they are not caused by the presence of other species. Table V describes statistically the cercarial output of each of the five species in various assemblages. The magnitude of variability should be noted. Table VI presents the results of Kruskal-Wallis tests that were used to determine whether the assemblage com- position significantly affected the numbers of cercariae released by particular (patent) assemblage members. The results show that although cercarial output (mean rank) did decrease for all species when additional species were present, there was not a significant depression of cercarial output for any one species. Finally, because Himasthla quissetensis and Lepo- creadium setiferoides have not previously been observed together, note that in Table V such a co-occurrence is listed, and that both species shed cercariae concurrently. Four snails contained both H. quissetensis and L. setifer- oides (Table I). Based on observations of a few mature (often moribund) H. quissetensis rediae and cercariae among many L. setiferoides rediae and cercariae, it ap- peared that L. setiferoides was evicting H. quissetensis from the snails. There were not enough of these snails to 30 L. A. CURTIS AND K. M. K.. HUBBARD Table III Spatial distributions of five trematode species (see Table I for parasite abbreviations) within sint>l\- and multiply-infected Ilyanassa obsoleta. Observed frequencies of parasite occurrence thy stage*), in snail sections 1-4 (see text), are given for each species Section Infecting trematodes Species tabulated 1 Stage 2 Stage 3 Stage 4 Stage PC C p A PC C p A PC C p A PC C p A Hq(n = 74) Hq 72 1 1 73 1 73 1 69 2 1 2 HZ(n = 42) Hq 38 2 2 40 2 40 1 1 33 6 2 1 HG(n = 10) Hq 6 3 1 10 9 1 8 1 1 HZG (n = 33) Hq 30 2 1 33 32 1 27 2 4 Ls(n = 25) Ls 25 25 24 1 22 3 LZ (n = 8) Ls 8 8 8 7 1 LG (n = 32) Ls 26 4 1 32 (I 30 2 23 2 3 4 LZG (n = 22) Ls 19 2 1 21 1 (] 21 1 17 5 Zr(n = 29) Zr 27 1 27 2 27 2 17 12 HZ Zr 25 17 37 5 38 4 26 2 14 LZ Zr 4 4 8 7 1 2 6 ZG (n = 24) Zr 22 2 24 23 1 10 14 HZG Zr 20 13 30 3 32 1 17 16 LZG Zr 12 10 21 1 21 1 8 1 13 ZAG (n = 5) Zr 3 2 4 1 5 5 Av (n = 5) Av 4 1 5 4 1 2 3 AG(n = 10) Av 7 3 10 10 2 8 ZAG (n = 5) Av 3 2 5 1 4 5 Ga(n = 24) Ga 25 4 28 1 26 3 16 1 12 HG Ga 6 4 9 1 10 4 1 5 LG Ga 23 1 1 7 29 1 2 29 1 2 16 2 14 ZG Ga 20 4 22 2 24 15 9 AG Ga 9 1 9 1 9 1 8 2 HZG Ga 21 12 28 5 32 1 10 23 ZG Ga 14 8 17 5 21 1 13 9 AG Ga 4 1 5 5 4 1 * Stage abbreviations: PC = parental stage (i.e., sporocysts or rediae) plus cercariae: P = parental stage only; C = cercariae only; and A = all stages absent. Individual species occurred in the context of several different combinations of infecting species (e.g.. Hg occurred alone, in HZ and HG doubles, and in HZG triples). For each species, frequencies are tabulated for each context. The number (n) of snails infected by particular trematode assemblages is indicated. Assemblages found in fewer than four snails are not tabulated. be included in the above log linear or Kruskal-Wallis analyses. Discussion Ilyanassa obsoleta is the only shared host in the life- cycles of these trematode species and is, therefore, the only place they might directly interact. They are tightly packed together in the snail, gather resources in similar ways, and are abundant on Cape Henlopen. Antagonistic interactions between trematode assemblage members have been noted by several investigators (e.g.. Lie el a!.. 1965: Basch el al, 1969; DeCoursey and Vernberg, 1974; Kuris, 1990; Sousa, 1990). On such grounds we anticipated that trematodes co-occurring in /. obsoleta would interact and most likely compete. A between-snail (component com- munity) analysis indicated that competition within snails was not an important determinant of the number of trem- atodes infecting individual snails. Further, regarding within-snail phenomena, no effect of assemblage com- position on any individual species could be discerned sta- tistically. However. Himasthla quissetensis and Lepo- creadiwn setiferoides were seen to co-occur in this study for the first time (Tables I, V), and this observation de- serves special comment. By virtue of their rare co-occur- rence, which eliminated the pair from our statistical anal- yses, these species apparently do interact negatively when they occur in the same snail. Our sample of trematode assemblages from the Cape Henlopen sandflat naturally included only those species combinations that can coexist long enough to be observed by the methods used. These included most of the possible GASTROPOD-TREMATODE INTERACTIONS 31 Table IV Results l log! i near analyses testing the influence of three factors ttrenuitode Assemblage, snail Section, and parasite Stage) on the frequencies of within snail occurrence reported in Table I' Species analyzed Hierarchical log-linear model accepted x : d.o.f. P =* Hq Assemblage, Section 54.21 45 0.163 x Stage Ls Assemblage, Stage 67.33 57 0.165 Zr Assemblage. Section 63.25 90 0.985 X Stage Av Section x Stage 25.79 32 0.773 Ga Assemblage, Section 60.99 105 0.999 x Stage * An insignificant X : (P > 0.05) without the three-way interaction term means that it is unnecessary; no significant displacement occurred. If a trematode's stages (/.<.. sporocysts. rediae. cercariae) were displaced from one snail section to another by the presence of a co-occurring species or combination of species, the accepted model for that trematode would require the three-way interaction term (i.e.. Assemblage X Section > Stage) to calculate expected frequencies without significant deviation from the observed. assemblages and virtually all that might have been ex- pected to occur. Twenty, of the 32 possible for five species analyzed, were actually observed (Table I). Missing as- semblages were the quintuple, the five quadruples, mul- tiples involving the scarce Amtrobilhania variglandis, and two triples involving Himasthla quissetensis and Lepo- creadiwn seiiferoides. A major concern is whether interparasite competitions occur that require considerable time for completion. In the early to middle phases of competition there may be no noticeable effect on any one species. We may have examined most assemblages at a time when coexistence is possible, and erroneously concluded that species do not interact. If such a time-course for competition is involved, how much time is necessary, and was our collection of parasite assemblages (in snails) biased by this? Two pos- sibilities present themselves: competitions could play themselves out over the summer; or over several summers. There was no indication that trematodes assemble in snails, compete, and ultimately evict subordinate species in either the short or the long interval (Table II). To the contrary, species appear to collect in snails as a function of time. Note that older snails, and not either younger group, have the largest proportion of triple infections (Ta- ble IIB). Sousa (1990) looked for a hyperbolic relationship between snail size and infecting species richness and sim- ilarly did not find one. Direct measurements of within-snail species dynamics also indicate no interactions among assemblage members. The occurrence of parasitic stages in different sections of variously infected snails is shown in Table III. No species was excluded from sections of snails because of co-oc- curring species (lack of Assemblage X Section x Stage terms in Table IV). If one species (or combinations of species) leads to gradual eviction of another species from snails, this phenomenon should have been quite common. Neither was cercarial production (from existing parental stages) of any species shut down by co-occurring species (lack of Assemblage X Stage terms in Table IV). Also, there was no indication that cercarial output from hosts (an estimate of fitness) was influenced by co-occurring species. There was no statistically significant reduction of cercarial output of any species as a function of assemblage Table V Descriptive statistics associated with numbers oftremalode cercariae released per hosl (Ilyanassa obsoleta) in 24 h in the field. Information is grouped bv species of cercariae being tabulated (.see Table I for species abbreviations) Infecting Cercariae trematodes tabulated Mean* S.D. Max. Med. Min. Hq(n = 42) Hq 527 709 2739 225 HL(n = 1) Hq 18 18 18 18 HZ(n = 23) Hq 211 344 1428 90 HG (n = 4) Hq 696 1135 2388 177 42 HZG(n = 22) Hq 155 274 1233 60 Ls(n = 18) Ls 319 590 2394 129 HL(n = 1) Ls 567 567 567 567 LZ(n = 2) Ls 130 185 261 131 LG(n = 10) Ls 42 66 165 9 LZG(n = 15) Ls 121 153 483 45 LAG(n = 2) Ls 18 25 36 18 Zr(n = 12) Zr 249 378 1095 15 HZ(n = 20) Zr 68 199 882 1 LZ(n = 2) Zr ZA(n = 1) Zr 1065 1065 1065 1065 ZG (n = 8) Zr 61 67 189 42 HZGln = 19) Zr 47 73 210 6 LZG(n = 13) Zr 79 138 474 21 ZAG (n = 2) Zr 12 8 18 12 6 Av (n = 3) Av ZA (n = 1) Av AC (n = 5) Av 9 16 36 LAG(n = 3) Av 21 16 33 27 3 ZAG (n = 1 ) Av 6 6 6 6 Gain = 14) Ga 222 454 1398 HG(n = 3) Ga 6 10 18 LG(n = 8) Ga 74 168 483 ZG (n = 6) Ga AG(n = 5) Ga 3 5 12 HZG(n = 17) Ga 2 9 36 LZG(n = 13) Ga 27 96 345 LAG (n = 4) Ga 6 12 24 ZAG(n = 1) Ga 18 18 18 18 Only infections that were patent for the species being tabulated are considered (n). For example, there were 33 HZG-infected snails (from Table I): 22 of these were patent for Hq; 19 for Zr; and 17 for Ga. A total of 206 snails were tested for cercarial release. 32 L. A. CURTIS AND K. M. K. HUBBARD Table VI Ri'Htlts ofKruskal-H 'ulli\ tests I'vuliuitmx the null hypothesis for each trematode species (see Table I for species abbreviations), thai the inimher of cercariat! shed was unaffected hv coeMSlhig species Effect of coexisting species on Infecting Mean rank fitness of species (# cercariae) Kruskal- d.o.f. Wallis H P = Hq Hq(n = 42) 52.583 HZ(n = 23) 41.957 HG (n = 4) 53.500 HZG (n = 22) 36.295 3 6.454 0.09 1 Ls Ls(n = 18) 23.972 LG (n = 10) 15.850 LZGIn = 15) 23.733 2 3.186 0.203 Zr Zr(n= 12) 42.417 HZ(n = 20) 29.575 ZG (n = 8) 42.563 HZG(n = 19) 33.763 LZG(n = 13) 41.962 4 5.254 0.262 Av No test Ga Ga(n = 14) 39.536 LG(n = 8) 39.688 ZG (n = 6) 26.500 AG (n = 5) 38.100 HZG(n = 17) 28.471 LZG(n = 13) 33.537 LAG(n = 4) 34.375 6 8.075 0.233 Prepatent infections and trematode assemblages observed fewer than four times were excluded. composition (Table VI). There was much variation, even in single infections (Table V), suggesting that sources of variability other than co-occurring species control cercarial output. DeCoursey and Vernberg (1974) studied assemblages of trematodes infecting Ilyanassa obsoleta in North and South Carolina. At the level of the component commu- nity, they noted that some species co-occur in multiple infections more or less often than would be expected based on the abundance of each in the system. They proposed that such patterns are produced by antagonisms or affin- ities among assemblage members. About 80 snails were dissected, with 30 of these being serially sectioned. The number of snails examined in each assemblage category is not reported. The authors noted "marked overlap in territory and habitat preferences," as we did in this study. Contrary to our conclusion (based on arbitrary snail sec- tions) that the parasites are not displaced, they concluded that some species are displaced from preferred sites (spe- cific snail organs) by other species. Even if small scale displacements (i.e., from organ to organ within our ar- bitrary sections) do occur, they would have to result in reductions in cercarial output (fitness) to have evolution- ary consequences. Cercarial output was not significantly reduced (Table VI). We also note that, if the interest is in adaptation of one parasite to others (Fig. 1 ), then section- ing snails along snail organ boundaries confounds adap- tation to other parasites with adaptation to the host. In the laboratory, DeCoursey and Vernberg ( 1974) also counted the cercariae released from 10 infected snails. Three were infected with Zoogonus lasius ( = rubellus) and five with Lepocreadium setiferoides. The remaining two were doubly infected with these same species. The num- bers of cercariae released in the laboratory by each species of trematode were averaged and compared. When Z. ru- be/Ins and L. setiferoides occurred alone, they each re- leased approximately 3500 cercariae in 24 h. When the species co-occurred, they released 901 and 1477, respec- tively. The authors concluded that L. setiferoides sup- pressed cercarial release by Z rubellus. Data show that cercarial production of both species was lower when they co-occurred. In any case, the number of observations pre- cludes meaningful statistical inference. We are interested in eliminating inoperative models from the four presented in Figure 1. Williams' (1966) dis- tinction between "functions" and "effects" seems useful here. Functions are biological characteristics that are direct products of natural selection (adaptations), whereas effects are characteristics that are a consequence of functions ("side" effects, not directly selected). Holmes (1986) points out that parasitic ". . . interactions should be important [in structuring helminth communities] only when species regularly co-occur at substantial population densities" (p. 203, brackets ours). We note, more specifically, that interactions based on adaptive responses (functions) of one parasite species to another cannot arise unless there is frequent co-occurrence over global gene pools. We cannot imagine how the parasites under study here could have adapted to one another. Definitive hosts (fish and birds) are highly mobile and scatter parasite eggs widely and unevenly. Consequently, spatial distribution of these trematodes within and among host snail popu- lations is patchy (Curtis and Hurd, 1 983; Curtis and Hub- bard, 1990), and there are abundant opportunities for trematode species to exist in isolation. The probability of co-occurrence generation after generation, particularly for specific parasites, is very low. Therefore, evolved parasite- parasite relationships are unlikely in this system. If inter- actions occur, they most likely result from effects, not functions. Our data indicating the lack of interactions among the majority of co-occurring trematodes. and the above considerations, justify eliminating models "c" and "d" (Fig. 1 ). Any evolved features of this system probably stem ultimately from the evolution of parasites to host, or possibly of host to parasites (models "a" and "b". Fig. 1 ). In deciding between models "a" and "b", many of the same arguments apply. Gooch et al. (1972) found that GASTROPOD-TREMATODE INTERACTIONS 33 Ilvanassa obsolete! were electrophoretically homogeneous all along the eastern seaboard, pointing to extensive dis- persal of larvae as the main cause. The planktonic larvae of/, obsoleta would then function analogously to parasite definitive hosts in the dispersal of progeny. Given the het- erogeneity of trematode prevalence in /. obsoleta popu- lations, many snail larvae would settle where parasites are not a frequent environmental challenge. If a snail were to obtain, by mutation, resistance to infection by one or more trematode species, its fitness probably would be en- hanced in parasite-ridden environments such as parts of Cape Henlopen. Yet its progeny would very possibly settle where parasites are infrequent. The mutation, there, would be at best neutral. These considerations suggest that model "a" (Fig. 1) is the operative one the only adaptive re- sponses between species in the /. obsoleta system are most likely those of the parasites to the host. The negative interaction between Hiniasthla quisse- lensis and Lepocreadium setiferoides in the Ilyanassa ob- soleta system deserves comment because it seems to counter the proposition that these parasites are not adapted to each other. A lack of co-occurrence of these abundant species in / obsoleta has been reported (Vern- bergetal., 1969; Curtis. 1985), but the detailed dissection methods used in this study revealed four co-occurrences (Table I). Obviously, miracidia of both species reach the same host, and there is a subsequent eviction (apparently of H. quissetensis). This eviction is important in terms of determining composition of the infra- and component assemblages observed, but is it based on adaptation of one parasite to another? In keeping with the above reasoning that parasite co-occurrence is not globally predictable enough to result in adaptations to other parasites we interpret this negative co-occurrence as based on an effect rather than a function [an exaptation (Gould and Vbra, 1982)] because it results from the way these species have evolved to the host, not to each other. In ecological terms, such a phenomenon is a competitive exclusion. However, in our hypothesis, the exclusion occurs between two spe- cies that are adaptively unaware of each other. If species interactions are an evolutionary force driving the struc- turing of interactive, co-adapted species assemblages, then we should distinguish between function- and effect-based relationships among species. A deeper appreciation of causal relationships in ecological systems will require un- derstanding these relationships. Factors structuring the assemblage of larval trematodes in populations of the California estuarine snail Cerithidea californica have been examined by Sousa (1990) and Kuris (1990). Two direct lines of evidence convinced these au- thors that competitive exclusions were occurring among C. californica trematodes. Sousa (1990) cites personal laboratory observations in which dominant species preyed upon stages of subordinates. Both authors reported trem- atode species replacements in individual snails periodically reexamined for infection by cercarial release. Kuris (1990) constructed a competitive hierarchy among trematode species in infracommunities, which he concluded would produce component community structure. In the Ily- anassa obsoleta system, such cercarial release data would have to be used judiciously because cercariae, even if present, often are not shed (Curtis and Hubbard, 1990). Data are not presented that assess this source of error for the C. californica system. In any event, there is consid- erable heterogeneity in prevalence of trematodes among C. californica populations (Kuris, 1990; Sousa, 1990). Parasite progeny are dispersed by definitive hosts similar to those in the I. obsoleta system, giving species the same opportunities to exist in isolation. This may mean that, whether they interact or not, parasites are not co-adapted in the C. californica system either. Because C. californica has direct development (Sousa, 1990) making popula- tions more insular the host may have the ability to evolve to its parasites. Several authors have examined snail-trematode sys- tems for interactions among parasites infecting the same host individual. Some have emphasized direct micro- scopical observations of antagonisms occurring in fresh- water snails (e.g.. Lie el al, 1965; Basch el al. 1969; Mouahid and Mone, 1990). Based upon such observa- tions, there can be no doubt that antagonisms between trematodes can and do occur, but their frequencies in natural snail populations are less certain. Other authors have emphasized observations of multiple infections in marine (e.g., Kuris, 1990; Sousa, 1990) and freshwater (e.g.. Fernandez and Esch, 199 la, b; Williams and Esch, 1991 (gastropods. In no case are multispecies assemblages reported to be particularly frequent. Such species-rich as- semblages are more frequent and various in the Ilyanassa obsoleta system on Cape Henlopen (Curtis, 1985, 1987, 1990; present study) than in any studied so far (see Cort el al. ( 1937)). The most frequent assemblage observed on Cape Henlopen is Lepocreadium setiferoides with Gy- naecotyla adiinca. and it occurred in only 4.4% of snails (n = 4870) examined by dissection (Curtis, unpub. data). Individual occurrence of each was 16.9 and 20.3%, re- spectively. Thus, even when species can and do co-occur, the probability of co-occurrence is slight. The opportunity to evolve adaptive responses to other particular trematodes seems minimal or nonexistent, which suggests that models "c" and "d" (Fig. 1 ) may be generally inoperative. The best opportunity for trematode-trematode adaptive re- sponses would be in a situation where all the necessary hosts are confined to one habitat, such as in a freshwater pond, as described by Williams and Esch ( 1 99 1 ) and Fer- nandez and Esch (199 la). However, Williams and Esch (1991) and Fernandez and Esch (1991b) conclude that within-snail trematode interactions in their system are in- 34 L. A. CURTIS AND R. M. K HUBBARD frequent and not the factor structuring the infra- and component communities. Can the host be adapted to its parasites? The evolution of a host to several parasites is a problem of "overwhelm- ing complexity" (McLennan and Brooks, 1991). and the issue is not resolvable with the data at hand. Dobson and Merenlender ( 199 1 ) suggest, as we content here, that the probability of such evolutionary responses would depend on host and parasite dispersal abilities, llyanassa obsoleta. because of its widespread dispersal, is unlikely to evolve to its parasites (model "b"), but it is a possibility with a snail in a more insular system, such as a pond. How can the coexistence, in a small habitat unit, of several species with similar resource requirements be ex- plained? This study has provided considerable compar- ative data on the fitness of parasites when they occur in different assemblages. The extensive variation in cercarial output (Table V) is not explainable by looking to presence or absence of other species. Perhaps resources for trem- atodes living in llyanassa obsoleta are somehow not lim- iting. We have suggested that the only adaptations (func- tions) in the system are those of the parasites enabling them to live in the snail (model "a". Fig. 1 ). We offer the following possible explanation. Each of these five trem- todes has evolved to castrate the host snail. Castration of the host stems from a parasite adaptation to channel en- ergy to the parasite that would otherwise go to the support of host gonadal tissue (Baudoin, 1975). llyanassa obsoleta is a long-lived host (7 years or more); the largest (oldest) snails are nearly all parasitized where trematodes are prevalent; and they appear not to lose infections (Curtis and Hurd, 1983). The host must survive the rigors of suc- ceeding winters. A trematode adapted to such a host may have been selected to exact intermediate to minimal damage (besides castration) because it could then "farm" the host for many years (see Minchella et al. (1985) and Gill and Mock (1985) for similar interpretations of host- parasite systems). We propose that, if the trematodes of /. obsoleta operate this way, then they should not singly, or in multiples, drain resources to the extent that they become limiting. In brief, they can coexist if they are all adapted to live well below the level at which the host is stressed. Acknowledgments We would like to thank the Undergraduate Research Office and the School of Life Sciences, University of Del- aware for a Science and Engineering Scholar grant, and two Peter White Fellowships awarded to support K. H.'s undergraduate thesis, from which this paper is adapted. We thank J. Moore for helpful comments on an earlier version. We are also grateful for the efforts and comments of two anonymous reviewers. 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Pp. 4 1 -67 in Parasite Communities Patterns and Processes. G. Esch, A. Bush, and J. Aho. eds. Chapman and Hall, London. Stunkard, H. \V. 1983. The marine cercariae of the Woods Hole, Massachusetts region, a review and a revision. Biol. Bull. 164: 143-162. Vernberg, \V. B., F. J. Vernberg, and F. W. Beckerdite. 1969. Larval trematodes: double infections in the common mud-flat snail. Seienee 167: 1287. Williams, G. C. 1966. Adaptation and Natural Selection. Princeton University Press, Princeton, NJ. Williams, J. A., and G. \V. Esch. 1991. Infra- and component com- munity dynamics in the pulmonate snail Helisoma anceps. with spe- cial emphasis on the hemmnd trematode Ha/ipegus occidualis. J Parasitol. 77: 246-253. Reference: Biol. Bull 184: 36-51. (February, 1993) Effects of Marine Bacteria on the Culture of Axenic Oyster Crassostrea gigas (Thunberg) Larvae PHILIPPE DOUILLET 1 AND CHRISTOPHER J. LANGDON Oregon State University, Department of Fisheries, Hatfield Marine Science Center, Newport, Oregon 97365 Abstract. Bacteria-free oyster larvae ( Crassostrea gigas) were cultured under aseptic conditions; they were fed axenic algae (Isochrysis ga/bana), and the medium was inoculated with isolated strains of marine bacteria. Twenty-one bacterial strains were tested, and most were detrimental to larval survival and growth. However, ad- ditions of strain CA2 consistently enhanced larval survival (21-22%) and growth (16-21%) in comparison with con- trol cultures that were fed only algae. Size-frequency dis- tributions of populations of larvae cultured for 10 days on axenic algae were skewed due to the poor growth of many individuals; whereas size-frequencies from popu- lations of larvae fed axenic algae supplemented with CA2 bacteria were distributed normally. Strain CA2 may therefore make a nutritional contribution to the growth of oyster larvae. /. galbana did not grow under the light intensities used for larval culture; thus the improvement in larval growth cannot be attributed to bacterial en- hancement of algal growth and, consequently, food avail- ability. Naturally occurring microflora from Yaquina Bay, Oregon, depressed survival or growth of larvae-fed live algae. Introduction Bivalve larvae in culture vary substantially in survival and growth (Davis, 1953;Loosanoff, 1954;Walne, 1956a). Between 25% and 50% of the variability in the growth of a single population of mussel larvae (Innes and Haley, 1977), or different populations of larval Crassostrea vir- ginica (Newkirk el al, 1977), are due to genetic factors. A significant proportion of the variability in the survival of C. gigas larvae was similarly attributed to genetic factors Received 3 June 1992; accepted 10 November 1992. ' Present address: The University of Texas at Austin, Marine Science Institute, P.O. Box 1267, Port Aransas. Texas 78373. (Lannan, 1980). Exogenous factors, such as temperature (Loosanoff, 1959), salinity (Bayne, 1965), pH (Calabrese and Davis, 1970), food quantity (Walne, 1965), food quality (Davis, 1953), age of the algal food (Dupuy, 1975), larval concentration (Loosanoff et al., 1953), size of con- tainer (Dupuy, 1975), silt (Davis and Hidu, 1969), exu- dates of unfavorable algal species (Bayne, 1965), water quality (Millar and Scott, 1967) and toxicants (Walne, 1970) have been found to contribute significantly to vari- ability in larval growth. Nonetheless, even different cul- tures of larvae obtained from the same parents and grown under identical conditions of temperature, salinity and ration have been commonly reported to vary in their growth (Bayne, 1983). The role of bacteria as beneficial or harmful agents in the culture of bivalve larvae has been the subject of many investigations, but this role has not been fully evaluated. Thirteen different isolates of marine bacteria did not sup- port the growth of oyster larvae when provided as the sole source of paniculate food (Davis, 1950, 1953). High bac- terial densities in cultures of bivalve larvae are generally considered to be deleterious to the larvae (Walne, 1956a, 1956b, 1958), and even innocuous bacteria in large num- bers have been reported to depress the rate of algal inges- tion (Ukeles and Sweeney, 1969). Some bacterial strains are reportedly able to invade larvae, to produce toxins, or both (Guillard, 1959; Tubiash et al.. 1965; Tubiash et al., 1970; Brown, 1973; Di Salvo, 1978; Nottage and Birkbeck, 1986). In contrast, bacteria have also been im- plicated as a food source for bivalve larvae (Carriker, 1956; Hidu and Tubiash, 1963) or as improving the growth of larvae fed on algae (Martin and Mengus, 1977; Beese, in Prieur et al., 1990). The elimination of microbial contaminants is prereq- uisite to a study of the effects of a bacterial strain on an organism in culture. This approach has been used to study the effects of several bacterial strains on cultures of the 36 BACTERIAL EFFECTS ON OYSTER LARVAE 37 protozoan Amoeba nitrophila (Frosch, 1897 in Luck et al., 1931); the cladoceran Moina macrocopa (Stuart et ai, 1931); and larvae of the clam Mercenaria mercenaria (Guillard, 1959). In the present study, axenic larval Crassostrea gigas, obtained without the use of antibiotics, were used in a series of experiments meant to reveal whether selected strains of marine bacteria can consistently improve the survival and growth of algal-fed oyster larvae. Materials and Methods Maintenance of larvae, bacteria and algae Bacteria-free oyster larvae were obtained according to the method of Langdon ( 1983). Adult oysters Crassostrea gigas were held at 1 8C in a recirculating seawater system for a period of 4 to 6 weeks, depending on the initial reproductive condition of the broodstock. After this con- ditioning period, the oysters were opened and shucked. Using aseptic techniques in a laminar-flow hood, we dis- infected the external surface of the gonads of each oyster with a 1% solution of sodium hypochlorite. A small in- cision was made through the surface of the gonads with a heat-sterilized scalpel, and gametes from each oyster were removed with sterile Pasteur pipettes and transferred to separate sterile flasks containing 0.2 ^m-filtered, au- toclaved seawater (FSSW). Eggs were fertilized by the ad- dition of a few drops of sperm suspension and then were transferred to Erlenmeyer flasks containing FSSW at a density of 1 00 eggs ml ' . Eggs were incubated on an orbital shaker at 25 C for 48 h. When the trocophore larvae had developed into veligers (straight-hinged larvae), subsam- ples of larvae were aseptically withdrawn for axenicity tests, and the remaining larvae were then held at 5C for 5 days. Axenicity of larvae was determined by epifluo- rescence microscopy using 4'6-diamidino-2-phenylindole (DAPI) staining techniques (Porter and Feig, 1980). Sam- ples of larvae were also added to 1/10 recommended con- centration of Difco marine broth 2216 (3.74gr', salinity 30 ppt) and incubated at 25C under aerobic or anaerobic conditions (BBL GasPak Pouch). Larvae from cultures that showed no evidence of microbial contamination from either the epifluorescence test or the 5 day broth incu- bations were considered adequate for experimentation. To confirm that the larvae were axenic, broth incubations were continued for 30 days. Axenic straight-hinged larvae were transferred to 250 ml Erlenmeyer flasks, each con- taining 1 50 ml of FSSW, closed with cotton plugs and capped with aluminum foil. Final larval density was 5 ml" 1 . Growth experiments were then initiated by the addition to the culture flasks of axenic algae and the dif- ferent bacterial strains. Shell lengths of 100 randomly se- lected larvae were measured, either with an optical mi- crometer fitted to a compound microscope, or with an image analysis system (Zeiss Videoplan 2). Strains of marine bacteria were isolated from cultures of algae or oyster larvae at the Whiskey Creek Hatchery in Netarts Bay, Oregon. Other bacteria were isolated, ei- ther from the guts of adult oysters, or from incubations of protein capsules (Langdon, 1989) suspended in unfil- tered seawater. Pure bacterial strains were obtained by the dilution method of Rodina (1972). Strains were grown, at 25C, on marine agar 22 16 or brain heart infusion agar (Difco). Bacteria grown on such solid media for 3 to 5 days were resuspended for 24 h in FSSW; they were then washed by centrifugation at 20,000 X g for 10 min and resuspended in FSSW. Strains were added to larval cultures at concentrations of 10 5 -10 6 cells ml" 1 . Cell concentrations were derived from equations relating spectrophotometric absorbance (600 nm) and bacterial concentration; the latter value was determined by direct count after staining with DAPI (Porter and Feig, 1980). Such equations were developed and used for each strain tested. Axenic Isochrysis galbana Parke (clone ISO) was ob- tained from the Culture Collection of Marine Phyto- plankton (Maine). Algal cultures were grown at 20C in 200 ml f/2 medium (Guillard and Ryther, 1962) illumi- nated by 1000-1500 lux of cool white fluorescent light under a 12 h light/ 12 h dark photoperiod. Algal axenicity was determined as described above for larvae. All glassware was washed in 10% nitric acid, rinsed seven times with distilled water, and baked overnight at 450C. Disodium ethylenediamine-tetraacetate (EDTA) was added at a final concentration of 1 ppm to all seawater to reduce the load of dissolved organic matter (Utting and Helm, 1985). Salinity of seawater after sterilization varied between 28 and 3 1 ppt. Heat sterilization was carried out for 15 min at 121C and 1.06 kg cm 2 pressure. Larvae fed on live algae and bacteria Twenty-one marine bacterial isolates were tested in three culture experiments for their effects on the survival and growth of larvae fed axenic Isochrysis galbana. Ex- periment I included seven microbial isolates from the Whiskey Creek Hatchery (H1-H7) and five isolates from the guts of adult oysters (G1-G5). Control treatments were either larvae fed only algae or starved larvae. In Experiment II, two strains (H6, H7) that improved larval growth in Experiment I were tested along with five strains isolated from the Whiskey Creek Hatchery (H8- H12), one strain isolated from the gut of an adult oyster (G6), and three strains isolated from protein capsules in- cubated in seawater (CA1-CA3). Control treatments in- cluded starved larvae and larvae fed only algae. In third control (SW), cultures of larvae were inoculated at the beginning of the experiment with naturally occurring bacteria present in 5 ml samples of 1 ^m-filtered seawater collected from Yaquina Bay, Oregon. The larvae in the 38 P. DOUILLET AND C. J. LANGDON third control treatment were fed axenic algae every other day. Experiments I and II were carried out with four rep- licates per treatment. Experiment III was designed to retest strains that had enhanced larval survival and growth in Experiment II (H7, CA2). Control treatments similar to those described for Experiment II were included. Experiment III was carried out with eight replicates per treatment. Cultures of bacteria-free oyster larvae (75.5-82 ^m shell length) were inoculated once at the beginning of each ex- periment with bacterial strains. Bacteria-free algal cells, harvested from cultures in exponential growth phase, were added to the larval cultures every two days. The seawater of the larval cultures was not renewed during the culture period. The concentration of algal cells in each larval cul- ture flask was estimated, as follows, before each feeding. A 2-ml sample of the larval culture medium was asepti- cally removed from each flask with a pipet; to prevent removal of larvae, the end of the pipet was covered with a 64 j/m Nitex screen. Algal cells were preserved with formalin, concentrated by centrifugation, and re-sus- pended in 100 jul of 0.2 ^m-nltered seawater. Algal con- centrations in the samples were then determined with a hemocytometer. Fresh algae were then added to larval culture flasks to provide cell concentrations at pre-deter- mined levels. Algal cell concentrations were increased by 15,000 cells ml ', from 40,000 to 100,000 cells ml" 1 over a 10 day culture period. To provide uniform food quality during the experiments, algae from a single culture were added at each feeding period, to all larval cultures receiving an algal diet. Larval culture flasks were placed randomly on orbital shakers in a temperature-controlled room at 25C. The cultures were exposed to a light intensity of 50-70 lux for 12 h each day. No algal growth occurred at this low light intensity. After 10 days of culture, samples of water were aseptically withdrawn from flasks containing starved lar- vae or larvae fed only axenic algae; these samples were analyzed for microbial contamination as described above. The experimental data were analyzed only if these control treatments were bacteria-free at the end of the 10 day culture period. Effects ofCAl bacteria on the growth of algae in tan-al cultures Cells of axenic /. galhana were initially suspended at a concentration of 40,000 ml~' in f/2 medium and then subdivided in sixteen 250 ml Erlenmeyer flasks. CA2 cells were added at 10 5 cells ml ' (final concentration) to eight flasks, while FSSW was added to the other eight flasks to maintain similar initial algal concentrations in all flasks. The final volume of each algal culture was 200 ml. Four algal cultures inoculated with bacteria and four cultures that had received only FSSW were placed in conditions conducive to the growth of I. galbana (1000-1500 lux and 20C); the remaining algal cultures were exposed to the conditions used for larval culture (50-70 lux and 25 C). The algal cultures were incubated on orbital shak- ers for three weeks. Every second day, 10 ml samples were removed aseptically from each algal culture, and algal concentrations determined with a Coulter counter (Mo- del ZB1). Larvae fed on dead algae and bacteria Interactions between strain CA2 and living Isochrvsis galbana that could modify algal food quality were not addressed in the previous experiments. To determine whether bacteria could enhance cultures of larvae fed on non-living diets, live /. galbana were replaced with dead algae. In Experiment IV, known concentrations of axenic /. galbana were frozen at -5C. Freezing and thawing broke the cell walls and membranes of the algal cells. Larvae were fed dead freeze-killed algae (FA) every two days ac- cording to the same protocol used with live algae. One group of larval cultures fed FA was maintained bacteria- free, and two groups were inoculated at the beginning of the experiment with either strain H6 at 10 5 cells ml" 1 (final concentration), or with an inoculum of naturally occurring bacteria (SW). The wild strains were added in 5 ml samples of 1 /jm-nltered seawater collected from Yaquina Bay, Oregon, at a concentration of 10 5 -10 6 cells ml" 1 . Other larval cultures received on alternate days, ei- ther additions of strain H6 (at a final concentration of 10 ? cells ml ') alone, or naturally occurring bacteria (SW) (5 ml of 1 ^m-nltered seawater) alone. Control treatments included starved larvae and larvae fed every second day on live axenic /. galbana. Culture conditions and sample treatments were similar to those of experiments carried out with live algae. Four replicates were tested per treat- ment. Algal cells were also killed by w 'Co-irradiation (5 me- garads) at the Radiation Center at Oregon State Univer- sity. Non-viability of irradiated algae (IA) was evident by the lack of growth of cells in f/2 medium at 20C under 1000-1500 lux of fluorescent light emitted 12 h a day. The irradiation process also destroyed contaminants, as demonstrated by incubations, at 25 C, of irradiated algae in 1/10 diluted marine broth 2216 (3.74 g 1~', salinity of 30 ppt) under either aerobic or anaerobic conditions (BBL GasPak Pouch). The integrity of the irradiated algal cells was verified by microscopic examination. Cell volumes of irradiated and non-irradiated algae from seven different cultures were determined with a Coulter counter (Model ZB1) equipped with a calibrated Coulter channelyser (Model 256). To ensure that IA were acceptable to larvae as a food source, the ingestion rates of larvae fed on either IA or BACTERIAL EFFECTS ON OYSTER LARVAE 39 live /.SW///T.V/.V galbana were compared. Ingestion rates were calculated according to the methods described by Checkley (1980). Larval ingestion rates for live and ''"Co- irradiated algae were compared with a 2 sample t-test, after verifying homocedasticity by Cochran's test for ho- mogeneity of variances at the 0.05 level of probability (Douillet, 1991). In Experiment V, oyster larvae were fed IA every second day according to the methods employed with live algae in Experiments I to III. Three groups of larval cultures were fed IA. One group was maintained bacteria-free, while the two others were inoculated at the beginning of the experiment with strains H7 or CA2. Control treat- ments included starved larvae or larvae fed every two days on live axenic Isocfuysis galbana. Eight replicates were tested per treatment. Larval survival and growth were de- termined as described below. Data collection and analysis At the end of each experiment, the larvae were carefully transferred to scintillation vials containing buffered form- aldehyde (2% final concentration, pH = 8). The larval tissues were stained with rose of Bengal, so that the larvae that were alive could be distinguished from empty shells. The whole larval population in each flask was counted with a dissecting microscope, and the shell lengths of 100 randomly selected larvae were measured, either with an optical micrometer fitted to a compound microscope, or with an image analysis system (Zeiss Videoplan 2). Sur- vival and growth data were transformed to satisfy as- sumptions of ANOVA. Survival data were transformed as: arcsin (square root (percent survival 100 ')) Growth data were transformed as: arcsin (square root ((In L, - In L,,)t ')) where L, is the final mean shell length (^m); L u is the initial mean shell length (^m); and t is the culture period (10 days). These transformations were successful in reducing the heterocedasticity of the survival data but not of the growth data (Cochran's test for heterogeneity of variances, at the 0.05 level of probability). Treatment effects on larval sur- vival were tested with one-way ANOVA. Where significant differences were indicated, Tukey's honestly significant difference test (T-HSD) was applied to determine the sta- tistical significance of differences among individual treat- ments at the 0.05 level of probability. Treatment effects on larval growth were analyzed with the Kruskal-Wallis test (KW). Differences among individual treatments were determined by means of the Games and Howell test (G&H) of equality of means with heterogeneous variances (Sokal and Rohlf, 1981), at the 0.05 level of probability. All tests were performed with the computer program Sta- tistix (NH Analytical Software), except the Games and Howell test which was carried out with the program Biom (Rohlf, 1982). The size-frequency distributions of populations of algae- fed larvae that were bacteria-free were compared with those fed algae supplemented with CA2 bacteria in Ex- periments II and III. Skewness coefficients (gl : Sokal and Rohlf, 1981) of larval populations from each replicate flask were calculated and used to compare larval size fre- quency distributions. A normal size distribution would have a gl coefficient equal to 0. A skewness coefficient higher than indicates that the size distribution is posi- tively skewed (higher proportion of small-sized individ- uals), while a coefficient smaller than indicates negative skewness. After confirmation of homocedasticity of gl values by Cochran's test at the 0.05 probability level, data were analyzed by two-way ANOVA with treatment (algae, algae + CA2) and experiment as factors. As dictated by the results of ANOVA, appropriate multiple comparisons of means were conducted at the 0.05 level of probability using the Student-Newman-Keuls procedure (SNK), controlling for experiment-wide error (Underwood, 198 1 ). Cryopreservation ot bacteria Bacteria have been described as adaptable chimaeras, the metabolic plasticity of which results from widespread transfer of genetic information though plasmids or pro- phages (Sonea. 1988). This strategy for adaptation to changing environments may result, during evolution, in the loss of beneficial characteristics of selected bacterial strains. In order to reduce the possibility of changes in bacterial characteristics between successive experiments, selected strains were cryopreserved at -70C in 10% (V/V) glycerol in sterile 1/10 diluted marine broth 2216. Identification of strain CA2 The identification of bacterial strain CA2 was based on Bergey 's Manual of Systematic Bacteriology (Holt, 1 984). The methodology used for different procedures followed the Manual of Methods of General Bacteriology (Gerhardt et ai, 1981). Exponentially growing cells cultured on ma- rine agar 22 1 6 were used for the following tests performed at the Hatfield Marine Science Center, Newport, Oregon, (a) Cells were Gram stained, (b) Motility was determined by observations of wet mounts with light microscopy, (c) Oxidase activity was determined by spreading CA2 cells with sterile cotton swabs over Pathotec cytochrome oxi- dase test strips (General Diagnostics), which contained a derivative of dimethyl-p-phenylenediamine and -naph- thol. (d) Cultures of CA2 cells were flooded with 3% hy- drogen peroxide for catalase testing, (e) Oxidation and fermentation of glucose was assayed with the modified O-F medium of Leifson (1963). (0 Utilization of inorganic ORIGIN OF BACTERIA ADDED TO LARVAL CULTURES m H rcn c Q on I > cc Z) A 100- r\ 80- / / / 1 60- / f / / / 1 X 7 y 1 ' / / / X / . / . / / ' X 40- J, / ', ^ ; / / / ; A X X X X X 20- / / ' / ^ / / X X 1 X / y / / ^ / / a.. X X M X - r 1 , y / / / / / / / M v X H X V STARVED ALGAE H1 H2 " 2 H4 H5 H6 H7 G1 G2 G3 G4 G5 ALGAE + BACTERIA 155 in 145- E 135- O 125- 1 15- 105- T JL I / STARVED ALGAE HI H2 H3 H4 H5 H6 H7 Gl G2 G3 G4 G5 ALGAE + BACTERIA Figure I. Effects of different bacterial strains on oyster larvae cultured on a diet of axenic Isochrrsis galhana for 10 days. (A) Survival in Experiment I. (B) Survival in Experiment II. (C) Growth in Experiment I. (D) Growth in Experiment II. Bacteria were isolated from the Whiskey Creek Hatchery. Oregon (H). from the guts of adult oysters (G), from incubations of protein capsules in seawater (CA), or were naturally- occurring in 1 nm-nltered seawater (SW). Larval control treatments were starved or fed axenic /. galbana. Results of Tukey's HSD pairwise comparisons and Games and Howell's tests are displayed below the his- tograms of survival and growth, respectively. Squares that occur together on any one of the horizontal lines indicate mean values that are not different at the 0.05% level of significance. 40 BACTERIAL EFFECTS ON OYSTER LARVAE 41 ORIGIN OF BACTERIA ADDED TO LARVAL CULTURES KS Sw I~TI H GZ) C E\S CA 100- B 1 1 I 1 1 oo 80- / / 1 / f X A ;' 6 - > / 1 / 1 ^ 1 X X | / / / / . X v ^ > y / ' / ./ ' ~x V X s^ 40- co i ^ / / / ^ ', / x x x 1 20- X / / '. V, / / / ~x ^ ^ N v \ - i 1 ^ ^ / / / / / >(. X 1 1 1 cTiOv/irn !r.r SW H6 H7 H8 H9 HlO H1 1 HI 2 G6 CA1 CA2 CA3 ALGAE + BACTERIA o CO E X I o I CO 75 150- 125- iOO- 75 I I I STARVED ALGAE SW H6 H7 H8 H9 HlO H11 H12 G6 CA1 CA2 CA3 ALGAE BACTERIA Figure 1. (CiiHtinued) 42 P. DOUILLET AND C. J. LANGDON sources of nitrogen was evaluated by culturing CA2 cells on media prepared with NH 4 C1 or NaNO, (0.5 g 1 '), glucose (0.1 g 1 '), Na 2 HPO 4 (0.1 g 1 '), FePO 4 (0.004 g 1 '(and 1 ml 1 ' off/2 vitamin mix (Guillard and Ryther, 1962). The culture media used as controls were prepared by replacing NaNO, or NH 4 C1 with peptone or tryptone (Difco) at 0.5 g 1 '. (g) Anaerobic growth was determined by transferring CA2 cells either into solid media in Petri dishes, or into 25 ml 1/10 diluted marine broth 2216 (3.74 gT 1 ; salinity 30 ppt) contained in 50 ml Erlenmeyer flasks, placing these cultures in anaerobic GasPak pouches (BBL), and incubating the cells at 20C for up to one month. The following tests were carried out by Dr. Ronald Weiner (University of Maryland at College Park). Meth- odology followed the Manual of Methods for General Bacteriology (Gerhardt et ai, 1 98 1 ). (a) Salt requirements were evaluated by culturing CA2 cells in tryptic soy agar (TSA) prepared at different salt concentrations; NaCl was added at 1% increments up to 10% of the control level. (b) As evidence of anaerobic growth and motility, CA2 cells on a straight needle were used to inoculate a tube containing semisolid tryptic soy broth enriched with 0.8% agar and 1%. NaCl, and the pattern of growth observed. (c) Flagellar staining was carried out by the Leifson method (Gerhardt et ai, 1981). (d) Synthesis of exopoly- saccharides was evaluated by the phenol-sulfuric acid re- action (Gerhardt et ai, 198 1 ). (e) The mole percent gua- nine plus cytosine (mol% G + C) in extracted deoxyri- bonucleic acid (DNA) was determined by the thermal melting (denaturation) methods of Marmur and Doty ( 1962) with a Gilford UV programmable spectrophotom- eter. (f) Antibodies of 20 different bacteria strains be- longing to the Alteromonas/Shewanella group were tested for reaction with exopolysaccharides of CA2 cells, (g) Fatty acid analyses of strain CA2 were carried out for compar- ison with profiles of other marine bacteria by Dr. Fred Singleton (Center for Marine Biotechnology, University of Maryland) and by Dr. Warren L. Landry (Food and Drug Administration, Dallas, Texas). Results Larvae fed on live algae and bacteria Single additions of marine bacterial isolates to oyster larvae cultures significantly affected larval survival (AN- OVA, P< 0.01) and growth (KW, P < 0.01 ) after 10 days of culture in all experiments (Figs. 1, 2). The microbes tested can be divided into categories depending on their effects upon oyster larvae: adverse, neutral, or beneficial. Bacteria belonging to the last category were tested further, and their effects upon oyster larvae were designated as either variable or consistently beneficial. Adverse strains. Strains Gl. G2 and G4 adversely af- fected larval survival (T-HSD, P < 0.05), whereas strains Gl, G2, G4, G5, H8, and H10 adversely affected larval growth (G&H, P < 0.05). Bacteria present in 5 ml aliquots of 1 ^m-filtered seawater depressed larval survival (T- HSD. P < 0.05 ) in Experiment II and larval growth (G&H. P < 0.05) in Experiment III. Neutral strains. A large proportion of the strains (HI, H2. H3. H4. H5. H9. Hll, HI 2, G3, CA1, and CA3) added to cultures of oyster larvae had no significant effect on larval survival (T-HSD, P > 0.05) or growth (G&H, P > 0.05) compared with cultures fed algae alone. ( 'ariable strains. Addition of strains H6 and H7 to larval cultures caused inconsistent improvements of larval growth. For example, larval growth was enhanced (G&H, P < 0.05) in cultures inoculated with strains H6 and H7 in Experiment I. but the enhancement with strain H7 was statistically insignificant in Experiments II and III (G&H, P> 0.05). Moreover, larval growth was depressed (G&H, P < 0.05) when strain H6 was added to larval cultures in Experiment II. Beneficial strains. In both Experiments II and III. larvae grown in cultures inoculated with strain CA2 had a sig- nificantly greater shell length than control larvae fed only axenic algae (G&H, P < 0.05). Larval survival was en- hanced in cultures inoculated with strain H7 and CA2, but this enhancement was statistically significant only in Experiment III (T-HSD, P < 0.05). Size frequency distributions of populations of larvae fed axenic algae were skewed compared to those from cultures fed algae supplemented with CA2 bacteria (Fig. 3; Table 1 ). Analysis of variance indicates a significant interaction between treatment and experimental factors (Table 2). In both Experiments II and III, skewness coef- ficients for populations of larvae fed axenic algae alone were significantly larger (SNK, P < 0.05) that those for populations of larvae fed algae and inoculated with CA2 bacteria. The difference between the skewness coefficients of treatments in Experiment II is larger than that in Ex- periment III, explaining the significant interaction deter- mined by the two-way ANOVA test. Effects ofCA2 bacteria on the growth of algae in larval cultures Cells of Isochrysis galbana, with or without inoculations of CA2 bacteria, did not grow under the conditions used to culture larvae (Fig. 4). The occurrence of CA2 cells in the culture medium had no effect on algal growth under favorable light intensity (1000-1500 lux) and tempera- ture (20C). Larvae fed on dead algae and bacteria Significant differences among treatments in Experi- ments IV and V were determined for larval survival (AN- OVA, P < 0.0 1 ) and growth (KW. P < 0.0 1 ). The survival of larvae cultured on axenic FA or IA alone was signifi- cantly lower (T-HSD, P < 0.05) than that of larvae BACTERIAL EFFECTS ON OYSTER LARVAE 20 STARVED ALGAE ALGAE + BACTERIA O uo + 1 I \ O UJ I in STARVED ALGAE ALGAE + BACTERIA Figure 2. Survival and growth of oyster larvae after 10 days of culture on axenic Isochrysis galbana supplemented with different bacterial strains (Experiment III). Bacteria were isolated from the Whiskey Creek Hatchery, Oregon (H) or from incubations of protein capsules in seawater (CA). Naturally-occurring bacteria present in 1 Aim-filtered seawater (SW) were added in a control treatment. Other control treatments included larvae fed axenic / ga/hana or starved. Results of Tukey's HSD pairwise comparisons and Games and Howell's tests are displayed below the histograms of survival and growth, respectively. Squares that occur together on any one of the horizontal lines indicate mean values that are not different at the 0.05% level of significance. 44 P. DOUILLET AND C. J. LANGDON CO i rr z CO o UJ M LO IT DIETS O O AXENIC ALGAE AI GAF 4- CA2 DIETS O O AXENIC ALGAE ALGAE + CA2 105 140 175 210 245 280 LARVAL SHELL LENGTH Figure 3. Size frequency distributions of larvae cultured for 10 days on a diet oflsuchrysis galhana with or without addition of CA2 cells. Points represent percent larvae for each shell length interval of 30 ^m. Lines used for illustrative purposes only. Data from Experiments II (n = 400) and III (n = 800) for each treatment. cultured on live axenic algae alone (Figs. 5, 6). However, the survival of larvae fed FA or IA was higher (T-HSD, P < 0.05) than that of starved larvae. In contrast, no sig- nificant differences in larval survival were detected be- tween cultures fed live algae and cultures fed FA or IA inoculated with strains H6 and H7, respectively (T-HSD, P> 0.05). Survival of larvae fed every two days on bacteria H6 alone was not significantly different (T-HSD, P > 0.05) from that of larvae fed live algae, and was significantly higher (T-HSD, P < 0.05) than that of starved larvae (Fig. 5). Larvae from cultures inoculated every two days with 5 ml of 1 /jm-filtered seawater (SW) also showed higher survival (T-HSD, P < 0.05) than that of starved larvae. Larvae fed on FA or IA were significantly smaller than larvae fed on live axenic algae (G&H, P < 0.05), and were not different from the size of starved larvae (G&H, P > 0.05) at the end of the experiment (Figs. 5, 6). Additions of single bacterial strains to cultures of larvae fed FA or IA did not improve larval growth compared to larvae fed FA or I A alone (G&H, P > 0.05). In contrast, growth of larvae fed FA inoculated with 5 ml of 1 ^in-filtered sea- water was significantly enhanced (G&H, P < 0.05) com- pared to that of larvae fed FA alone or starved larvae (Fig. 5). Similarly, additions every two days of 5 ml of 1 //m- filtered seawater or strain H6 alone to larval cultures sig- nificantly enhanced the growth of larvae (G&H, P< 0.05) compared to that of starved larvae. The poor growth of larvae fed FA may have been due to the rupture of the freeze-killed algal cells. 60 Co-irradia- tion did not affect the integrity of the algal cells but re- BACTERIAL EFFECTS ON OYSTER LARVAE 45 Table I Skewness coefficients (gl) jrom si:efm/neney ilistrihulianx of populations oj larvae cultured in Experiments II and III Experiment Diet Average skewness of populations 1 S.D. 11 II II! Ill ISO ISO + CA2 ISO ISO + CA2 0.7906 0.21 34 (n = 4) -0.0605 0.2235 (n = 4) 0.3801 0.1720 (n = 8) -0.0466 0.29 10 (n = 8) Larvae were cultured with either axenic Isoclin'sis galbana (ISO) alone or /. galbana plus CA2 bacteria. duced their volume from 44.4 1 .92 ^m 3 to 26.3 0.59 /urn 3 (x 1 SD; n = 7). A high proportion of irradiated cells remained intact while in suspension in seawater, as demonstrated by the small decrease in cell concentration in control flasks, from 59,043 1,1 19 cells ml~ ', to 58,539 1,505 cells mr' (x 1 SD; n = 4) in 105 min. IA cells were ingested by oyster larvae at rates significantly (2 sample t-test, P < 0.01 ) greater than that for live cells. Identification of strain CA2 Strain CA2 was presumptively identified as Altero- nwnas sp. on the basis of the following characteristics: Gram negative rod; aerobic; oxidase positive; requires 250 nA/ salt; motile with polar flagella; exopolysaccharide synthesis; and guanine plus cytosine 43 mol% (T m ). The exopolysaccharides of CA2 bacteria did not react with antibodies to 20 species of A/temmonas. Further- more, both analyses of fatty acids revealed a very unusual fatty acid profile with a high proportion of C-14, C-15 fatty acids (Table 3); this is not characteristic of the genus Alteromonas. However, the fatty acid profile was not sim- ilar to any of the species profiles listed in Dr. Landry's marine library. Therefore, strain CA2 may be an Altero- monas species not typical of the genus. Further characteristics of strain CA2 include yellow pigment production, oxidation and fermentation of glu- cose, but no gas production, and inability to utilize in- organic sources of nitrogen, such as NH 4 C1 or NaNO 3 for growth. Catalase was weakly positive. Discussion Axenic larval Crassostrea gigas were used to determine the effects of additions of single bacterial strains on the survival and growth of larvae cultured with algae. Bacteria can be categorized as adverse, neutral or beneficial, de- pending on their effects upon oyster larvae. Furthermore, bacteria found beneficial in one experiment were reiested in subsequent experiments and could be further catego- rized as either variable or consistently beneficial strains. Additions of strain CA2 to larval cultures consistently enhanced larval survival (21-22%) and growth (16-21%) compared with that of larvae fed on algae alone. The specificity of bacterial strains as food for grazers has frequently been reported (Frosch, 1897 in Luck a ai. 1931; Stuart el al., 1931; Curds and Vandyke, 1966). Fur- thermore, Curds and VanDyke (1966) found that one bacterial strain was either slightly toxic, unfavorable, or favorable depending on the ciliate species tested. In con- trast, a single bacterial strain (PM-4) was found to promote the growth of both shrimp (Penaeus monodon) and crab (Port units tridentatus) larvae (Maeda, 1988; Maeda and Nogami, 1989). Consequently, no generalization about the beneficial effects of specific bacterial strains can be made; i.e., each strain must be tested again with each new target species. Bacteria may be used directly as a food item by oyster larvae (Douillet, 1991). Starved axenic oyster larvae showed poor survival and did not grow after 10 days of culture. In contrast, larvae in cultures inoculated with single bacterial strains or mixtures of naturally-occurring marine bacteria had higher survival rates than starved lar- vae, but lower growth rates than larvae fed on algal diets. Consequently, the bacterial strains tested did not provide all the nutritional requirements for larvae, but appeared, at least, to partially satisfy larval metabolic requirements, as demonstrated by the beneficial effects of bacteria on larval survival and growth. Straight-hinged oyster larvae, fed for 10 min on l4 C-labeled CA2 cells at 1.5 X 10 7 cells ml ' and purged of undigested l4 C-material, retained enough bacterial carbon to meet over 140% of their active carbon metabolic requirements during a 10 min period (Douillet, 1991). Beese(in Prieureta/.. 1990) determined that xenic, starved larval Crassostrea gigas grew 60% in size after seven days of culture, whereas starved axenic larvae did not grow. The ability of starved xenic bivalve larvae to grow has been determined to be greater for larvae Table II Two-way analysis of variance of skewness coefficients (gl)for size frequency distributions of populations of larvae cultured in Experiments II and III Source of variation d.f. Sum of squares Mean squares F-ratio Sig. level Experiment (A) 1 0.31468 0.31468 5.79 0.0259 CA2 addition (B) 1 3.2656 3.2656 60.12 0.0000 Interactions (A*B) 1 0.36039 0.36039 6.63 0.0180 Replicates (C) Residual (A*B*C) 20 1.0864 0.05432 Total 23 5.0271 Larvae were cultured with Isochrysis galbana alone or /. galbana plus CA2 bacteria. 46 P. DOUILLET AND C. J. LANGDON = 3 V -*-- CO DC c o 60 50 40 30 20- 0) 0) - 10-1 1 2 I Slow Fast Flow Figure 1. Ingestion rates (mean number of particles per fecal pellet per colony) of small (S) and large (L) colonies in slow and fast ambient flow (numbers above columns indicate number of colonies sampled). Bars represent two standard errors. SUSPENSION FEEDING IN PLUMATELLA 55 colonies may have relatively greater metabolic demands (possibly they invest more in statoblast or larval produc- tion per unit mass) than small colonies and therefore have a higher propensity to feed. However, the greater increase in the rate of feeding in small colonies with increased flow (by a factor of five) relative to large colonies (in which feeding increased by a factor of 1.8) (see Fig. 1 ) suggests that small colonies respond more strongly to increases in particle flux (or flow). Why small colonies should show such a marked response is not apparent. Perhaps small colonies create stronger ciliary currents in response to an abundance of food (particle flux serving as a cue) as has been observed in marine bryozoans (Best and Thorpe, 1983). Concerted pumping in large colonies may preclude the necessity to create individually stronger feeding cur- rents and may provide for a more constant food supply. Our results contrast with those obtained by Bishop and Bahr (1973) who found that clearance rates of the phy- lactolaemate Lophopodella carteri decreased with colony size. This discrepancy may relate, in part, to differences in colony morphology and growth in the two species, but it also is complicated by comparing feeding studies con- ducted under static and dynamic conditions and in dis- similar volumes of suspension. Lophopodella is a higher phylactolaemate, producing gelatinous, globular colonies with no branching (Wood. 1991 ). Colonies of Lophopodella do not grow indefinitely but undergo fission, the resulting colonies slowly creeping apart. Fission in Lophopodella may result in avoidance of lophophoral feeding interference that occurs as colonies get bigger, hence maximizing filtering efficiency (Bishop and Bahr. 1973; Hughes, 1989). To some extent, our re- sults for feeding in Plumalella support this contention. Plumatella does not undergo fission and its feeding does not decrease with increased colony size. The lack of in- terference in feeding in Plumatella may partly reflect its morphology. Plumatella colonies are tubular and branch- ing and their lophophores are spaced much further apart than those of Lophopodella. However, we also believe it is crucial to consider differing patterns of excurrent flow and food depletion in our experiments and in those of Bishop and Bahr (1973). In Bishop and Bahr's study (1973), Lophopodella col- onies were placed in small vials (diameter = 22 mm) that contained 10 ml of an algal suspension. Thus colonies will have had ample opportunity to resample previously filtered water because the total volume of water was small and because, under conditions of still water, previously filtered water was not carried away. Thus, it is not sur- prising that clearance rates were lower for large colonies. The volume of suspension in our study was large (25 1), and food depletion was not significant. Furthermore, in- corporation of ambient flow meant food-depleted water was carried away from colony surfaces. Conclusion This study indicates that feeding by freshwater bry- ozoans is less constrained by increased flow than it is in marine forms. As suggested above, the relatively large lophophores of phylactolaemates create powerful feeding currents that may be beneficial in both lotic and lentic environments. The complex hydrodynamics characteristic of marine habitats (see Denny, 1988) may ensure delivery of food to the level of small, circular lophophores of ma- rine bryozoans. Furthermore, small, circular lophophores maximize the collective surface area for feeding, and col- onies can benefit from the larger energy surplus associated with small size (Sebens, 1979, 1982; Ryland and Warner, 1986; Hughes, 1989). Thus lophophore size and shape in marine and freshwater bryozoans may reflect different so- lutions to different kinds of problems faced by small, co- lonial suspension feeders in the two sorts of environments. However, the role of phylogenetic constraint in deter- mining lophophore morphology cannot be ruled out (tra- ditional views hold U-shaped lophophores to be primi- tive). Although the majority of freshwater bryozoans possess large, U-shaped lophophores, small, circular lo- phophores are found in the phylactolaemate Fredericella and in the few gymnolaemates that have invaded fresh- water habitats. These exceptions to the rule indicate that the significance of lophophore size and shape in freshwater habitats merits further investigation. Acknowledgments We thank Pauline and David Whittington for their friendly interest and kind permission to collect Plumatella from their pond at Cassington Nurseries and Mark Brown for technical help. This work was submitted in partial fulfillment for the Zoology Honours Degree in the De- partment of Zoology, University of Oxford by L. Doolan. The manuscript has been improved by comments from two reviewers. Literature Cited Best, M. A., and J. P. 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Polypide morphology and feeding behavior in marine ectoprocts. Bull. Mar. Sci. 28: 1-31. Wood, T. S. 1973. Colony development in species of Plumalella and Fredericella (Ectoprocta: Phylactolaemata). Pp. 395-432 in Devel- opment and Function of Animal Colonies through Time. R. S. Board- man, A. Cheetham, and J. Oliver, eds. Dowden. Hutchinson & Ross. Stroudsburg. PA. Wood, T. S. 1989. Ectoproct bryozoans of Ohio. Ohio Biol. Surv Bull \ew Series 8(2): X + 70 pp. Wood, T. S. 1991. Bryozoans. Pp. 481-499 in Ecology and Classifi- cation />/ \orih American Freshwater Invertebrates. J. H. Thorp and A. P. Covich. eds. Academic Press. Inc.. San Diego. CA. Reference: Bin/. Bull 184: 57-78. (February, 1993) Aplacophora as Progenetic Aculiferans and the Coelomate Origin of Mollusks as the Sister Taxon of Sipuncula 1 AMELIE H. SCHELTEMA \Vootls Hole Oceanographic Institution, Woods Hole, Massachusetts 02543 Abstract. Evidence is presented in support of the fol- lowing phylogenetic hypotheses: ( 1 ) Sipuncula are the sis- ter taxon of Mollusca; (2) the two aplacophoran taxa, Neomeniomorpha (= neomenioids) and Chaetodermo- morpha (= chaetoderms), are monophyletic with a com- mon neomenioid-like ancestor, and of the two taxa, Chaetodermomorpha are more derived; (3) Aplacophora and Polyplacophora are sister taxa and form a clade, Acu- lifera; (4) Aculifera are the sister group of the remaining extant mollusks, Conchifera; and (5) Aplacophora are progenetic Aculifera. The evidence is based on homologies of early and late emhryological development, adult morphologies, and molecular analyses. Embryological development in si- punculans and mollusks shows a close relationship be- tween them, and embryological development of the shell separates Aculifera and Conchifera. Adult morphologies indicate: ( 1 ) monophyly of Aplacophora; (2) sister-group relationship between Aplacophora and Polyplacophora; (3) a molluscan plesiomorphy of nonsegmented serial replication of organs; and (4) progenesis in Aplacophora. Molecular evidence supports the embryological and mor- phological relationships between Sipuncula and Mollusca. Mollusca are thus hypothesized to be coelomate Eu- trochozoa, which share an ancestor that probably had se- rial replication of organs. Differences in size and structure of the coelom among Eutrochozoa are hypothesized to have been brought about by changes in the timing and the process of cavitation of the mesodermal bands that arise from cell 4d. Through the process of progenesis Aplacophora retained an ovoid embryological shape and Received 19 August 1992; accepted 25 November 1992. ' Contribution Nos. 8205 from the Woods Hole Oceanographic In- stitution, and 314 from the Smithsonian Marine Station at Link Port. several internal structures that, although they appear to be in a primitive state, are actually secondarily derived as is quadrant D specification during early cleavage. Introduction The uniqueness of Aplacophora among Mollusca lies in their derived vermiform body in combination with an internal organization that appears to reflect a primitive molluscan state, especially the simple ladderlike nervous system, serial musculature, distichous radula (two teeth per row) in its plesiomorphic aplacophoran state, simple digestive system, and epidermis that produces an aculif- erous cuticle. Their evolutionary significance to the phy- lum has long been a matter for conjecture. First came the question of whether Aplacophora were even mollusks, as they lack a number of "typical" characters such as a shell, mantle, and kidneys (e.g., Thiele, 1902; H. Hoffmann, 1929-30), but they have more usually been considered to belong within the phylum because of similarities to chitons in their nervous system ( Amphineura) and spicules (Acu- lifera) (e.g., Spengel. 1881; Heath, 1911). Further discus- sions were concerned with whether aplacophorans were "degraded" or truly "primitive" mollusks (see Hyman, 1967, pp. 68-70 for a historical account). There have been no current arguments which separate Aplacophora from Mollusca since evidence for a close relationship between Aplacophora and Polyplacophora was published by S. Hoffman (1949), but under present discussion is their origin and position within the phylum (Salvini-Plawen, 1972. 1981a, 1985; Scheltema, 1978, 1988), as well as the origin of the phylum Mollusca itself. Mollusca have been argued either to have a noncoelomate origin and to be the sister taxon of the eucoelomate An- nelia-Echiura-Sipuncula (Salvini-Plawen, 1972, 1985 fig. 42), or to be eucoelomates with an ancestor in common 57 58 A. H. SCHELTEMA with other coelomates (Wingstrand, 1985; Scheltema. 1988). In either argument, Aplacophora have been con- sidered stem forms and therefore preceded the Monopla- cophora with serial replication of organs. Hypotheses for a noncoelomate origin rest on the ar- gument that the worm-like Aplacophora with replicated lateroventral musculature evolved from a turbellario- morph ancestor, and that consequently the molluscan coelom is not homologous to that in the Eutrochozoa. A coelomate origin has been hypothesized from annelid- mollusk relationships, including the presence of a cell 4d that gives rise to mesoblasts and consequently a homol- ogous coelom, the presence of a trochophore larva, and serial repetition of body parts. Because the molluscan coelom is small and unsegmented, the idea that annelids and mollusks form a clade with a common segmented ancestor is poorly accepted. The dichotomous choice be- tween either a turbellariomorph or an annelid-like ances- tor for mollusks has dominated recent thinking about molluscan evolution (e.g., Hyman, 1967; Haszprunar, 1992), and the relationship of mollusks to other Eutro- chozoa has not been examined. However, recent molec- ular data discussed below urge reconsideration of mol- luscan relationships to other phyla. Evidence is presented here to support the hypotheses that ( 1 ) Mollusca are eucoelomates with their closest living relatives in Sipuncula, their sister group; (2) Aplacophora and Polyplacophora are sister groups in the subphylum Aculifera (contradicting Scheltema, 1978, 1988); (3) Acu- lifera are the sister group of the remaining living mollusks, Conchifera; (4) the aplacophoran taxa Chaetodermo- morpha (= Caudofoveata, here also called chaetoderms) and Neomeniomorpha (= Solenogastres sensu nomine Salvini-Plawen, here also called neomenioids) are mono- phyletic, sharing a neomenioid-like ancestor; and (5) aplacophorans are progenetic Aculifera. Considered in the discussion is the homology of the eutrochozoan coelom and the evolutionary difference between metamerism, or segmentation as it occurs in the annelids, and serial rep- lication of organs, as found in Neopilina and Vema (Wingstrand, 1985). The term "metamerism" is used here only to denote a segmented coelom; "serial replication" is used to denote the more general case of serial repetition of organs, whether or not by metameres. Evidence that Mollusca are Descended from Coelomates Mollusca have a coelom consisting of gonadal lumina, pericardium, and kidneys, as well as part of the gameto- ducts in Aplacophora. A noncoelomate ancestry calls for the widening of a pericardial space lined by mesoderm as protection for a heart (Salvini-Plawen, 1968a, 1972; not discussed 1985, 1990) and for gonads separate from the pericardium. This development of coelomic spaces would be a molluscan apomorphy. not homologous with annelid or sipunculan coelom. Alternatively, the molluscan peri- cardium can be considered as reduced from a large coe- lomic space homologous to that in other eutrochozoa. The involvement of the pericardial coelom in excretion is unique to mollusks. Ultranltration of blood occurs through podocytes that are present in most molluscan classes including Aplacophora (Andrews, 1988; Reynolds and Morse, 1991). Five independent lines of evidence indicate that re- duction of coelom is the case, and that Mollusca are eu- trochozoan coelomates: ( 1 ) presence of the molluscan cross in mollusks and sipunculans and (2) homology of certain characters in larvae of mollusks and sipunculans indicate that mollusks and sipunculans are sister taxa; (3) a large pericardium among "primitive" mollusks indicates that it is a molluscan plesiomorphy; (4) the embryological development of mesoderm in annelids, mollusks, sipun- culans, and nemertines is similar, and the coelom in the four groups is homologous; and (5) molecular data groups mollusks with other eutrochozoans. Sipunculans as sister taxon of the mollusks An evolutionary relationship between sipunculans and mollusks lies in their early embryological development and in morphological features of sipunculan pelagosphera and molluscan larvae. Molluscan cross. The molluscan cross is found in the embryological development of Gastropoda, Polyplaco- phora, Scaphopoda, and Aplacophora by the end of the 64-cell stage (Verdonk and van den Biggelaar, 1983; Heath, 1899; van Dongen and Geilenkirchen. 1974; Baba, 1951). It is formed by la' 2 -ld 12 cells and their descen- dents, with cells la" 2 -ld" 2 , called peripheral rosette cells, forming the angle between the arms of the cross (Fig. 1 A, B, D, peripheral cells solid black). In Annelida, however, it is cells la" 2 - Id" 2 that form the cross (Fig. IE, cross cells solid black) (Wilson, 1892). In the 64-cell stage of the neomenioid aplacophoran Epimenia vermcosa figured by Baba (1951), a molluscan cross seems apparent from Baba's shading (Fig. ID), although Salvini-Plawen (1985) found "no definite cross formation" in the same source. Manuscript drawings by G. Gustafson of developing Chaetoderma nitidulum eggs likewise show a molluscan cross. In contrast to most mollusks, early cleavage in Pe- lecypoda is asynchronous and bilateral, and no cross is formed; its absence would seem to be an apomorphy. Likewise, development in Cephalopoda seems an apo- morphy of that group, which has telolecithal eggs, early bilateral cleavage, and no molluscan cross. In Sipuncula, a molluscan not an annelid cross is formed, as Rice ( 1975, 1985) has emphasized and refig- ured from Gerould ( 1906), who first described its presence APLACOPHORA: PROGENETIC COELOMATES 59 Figure 1. (A-D) The molluscan cross. (A) Gastropoda (Lyniiiucu siagnalis. after Verdonk and van den Biggelaar. 1983, p. 1 1 1 fig. 3b); (B) Polyplacophora (Stenoplax heathiana. after Heath, 1899. pi. 32, fig. 23); (C) Sipuncula (Golfingia vulgaris. after Gerould, 1 906. p. 99, fig. D, as published in Rice, 1975, p. 99. fig. 17); (D) Aplacophora (Epimenia verrucosa. after Baba, 1951. p. 46, fig. 18). The apical rosette la'"-ld'" is shown in fine, close stippling; arms of the cross la' : - Id' 2 and daughter cells are shown in fine, open stippling; tip cells of cross 2a"-2d" are shown in coarse stippling; peripheral rosette cells la" 2 -ld" : are solid; and trochoblast cells I a 2 - Id 2 are clear. In Epimenia (D), the cleavage stage appears to be earlier than shown in A-C, as the tip cells have not yet separated from 2a' and 2c' (indicated by question marks), and the arms of the cross are not quite straight, similar to an earlier stage in Polyplacophora (Heath. 1899. pi. 32, fig. 17). In B, only one tip cell was discernible in Heath's illustration, and in C tip cells were not indicated in Gerould's original figure. (E) Annelid cross, Polychaeta (Nereis) (after Wilson, 1892, p. 396. diagram II). The apical rosette la 1 "-Id"' is shown in fine, close stippling; peripheral cells la' 2 -ld 12 are shown in fine, open stippling; and the arms of the cross from la" 2 -ld" 2 are solid. in sipunculan development (Fig. 1C). The presence of a molluscan cross during embryological development is understood here to be of phylogenetic importance, and sipunculans and mollusks share a character not found in either annelids or flatworms (Freeman and Lundelius, 1992). Its presence can be considered apomorphic to the embryonic morphology of turbellarians, which lack a cross. Similarities between sipunculan and molluscan lan'ae. Gerould (1906) noticed certain other resemblances to mollusks besides the molluscan cross in the development of sipunculans. In particular, he found similarity between sipunculan pelagosphera and molluscan larvae. The pela- gosphera is unique to sipunculans. It is a swimming larva that metamorphoses from a trochophore stage (Rice, 1975, 1985). Gerould noted the resemblance of the pela- gosphera lip glands to chiton larval pedal glands, and of the pelagosphera buccal organ to the radula sac in chiton larvae (Figs. 2, 3, 4). Pelagosphera larvae can either swim upright with the large metatroch or creep, head-down, along a solid surface. These activities are lost along with the larval head at metamorphosis. Jagersten (1963) first described creeping in living pelagosphera, and he related it to a creeping gastropod. He also noted that the buccal organ (= pharyngeal bulb, Schlundkopf) was used in feeding. Later Jagersten (1972) proposed a possible, but not certain, homology of the pelagosphera lip, which is the creeping surface posterior to the mouth, and the creeping lobe, or foot, between mouth and anus of mol- luscan larvae. Rice (1975, pp. 120-121) described the creeping lo- comotion of pelagosphera as follows: "The larva is able to ... glide along with . . . [the] head flattened against the bottom. Frequently the larvae . . . may crawl in the manner of an inchworm. presumably scraping material from the bottom. The continual eversion of the buccal organ during feeding probably aids in the removal of food from the substratum. This tough muscular organ [covered by cuticle. Rice, 1973] is believed to function in breaking up material into small panicles for feeding. . ." A mucus- like substance from the lip glands is secreted as the animal moves along a natural substratum (Rice, 1981 and pers. comm.). My own recent observations on living pela- gosphera corroborate many of Rice's. Precise descriptions of the protrusible buccal organ and lip gland have been given by Rice (1973). The buccal organ is a muscular sac, ventral and posterior to a cuticle- lined invagination called the buccal groove that lies below the esophagus. The epithelium of the buccal organ is overlain by the cuticle of the ventral side of the buccal groove and is the area first protruded (Fig. 2). Although the precise innervation of the buccal organ was not dem- onstrated, the circumesophageal connectives, which arise from the dorsal cerebral ganglion, are closely associated with the organ. Both the topography and function of the buccal organ and groove are remarkably similar to those of the radular apparatus in mollusks: ventral odontophore = buccal organ; ventral radula sac = buccal groove; and ventral cuticular radula = ventral portion of the cuticular buccal groove. Furthermore, the odontophore and prob- ably the buccal organ are innervated through connectives united with the cerebral ganglion. The homology would be more certain if it were known whether the buccal organ musculature is formed from mesoderm, as is the odon- tophore of mollusks (Raven, 1966), or whether it is myo- epithelial as in archiannelids (Jagersten, 1947; Rice, 1973). The lip gland takes several forms in various pelago- sphera, from a bilobed to a paired or four-lobed body A. H. SCHELTEMA Figure 2. Peiagosphera larvae of Sipuncula. (A) Frontal view of head. Siinmailu* sp. (from Rice, 1981, fig. 4). (B) Entire larva. Aspidosiphon sp. (from Rice, 1981, fig. 6). Numbers as in Figure 3: I huccal gland, 3 pore of lip gland, 4 mouth, 5 lip. which opens either directly, or by way of a ciliated duct or ducts, into the lip pore. In comparison, the anterior pedal gland in larval chitons and in Aplacophora is duct- less (cf.. Figs. 2A, 4B, 6C). Aplacophora, but not chitons, have a central ciliated pit. Similarities in form and function in these three struc- tures lip and foot, lip glands and pedal glands, and buc- cal organ and radula with its sac are striking. Their morphologies are particularly clear in sagittal sections of a pelagosphera and a chiton larva (Figs. 3, 4). There are also similarities in their development, as they all arise from posttrochal ectoderm, with these differences: in si- punculans, the origin of all three structures is stomodeal, whereas in mollusks, the ventral somatic plate, usually from cell 2d, gives rise to the foot and its glands, and only the radula sac is stomodeal (Raven, 1966). In Sipuncula as well, cell 2d gives rise to the somatic plate, which forms the ectoderm of the trunk (Rice, 1976). In mollusks, how- ever, the proximity and functional interdependence of the somatic and stomodeal structures are indicated by the pedal contribution to feeding in veliger larvae. An anterior, medial ciliary tract is formed on the foot by which particles unsuitable for ingestion are rejected (Moor, 1983). Only the head region of the pelagosphera, which is rad- ically altered during metamorphosis to a juvenile sipun- culan. can be compared to the Mollusca. The posterior part of the body with its large coelomic sac, nephridia, mid-dorsal anus, and ventral nerve cord, are already de- finitive adult structures. Evidence from the presence of the molluscan cross and from locomotary and feeding structures that are similar in mollusks and larval sipunculans is sufficiently strong that the two phyla can be considered as sister groups, and mollusks, therefore, as eucoelomates. Of course, if the primitive mode of sipunculan development should prove to be by way of a nonfeeding, lecithotrophic larva, then the similarities between planktotrophic pelagosphera and molluscan larvae would be convergent. However, Rice (1985) most recently considered evolutionary questions of sipunculan larval development and concluded that a yolky egg and short-lived planktotrophic pelagosphera was the primitive mode of development. Other considerations. Two further observations can be made to support arguments for a sipunculan-molluscan sister relationship, one embryological, the other paleon- tological. The first is the embryological development of Echiura (Newby, 1940) compared to that of the sipun- culans. Echiurans have traditionally been linked with si- punculans, both having worm- or sac-like, unsegmented coelomate bodies, but echiurans afford a contrast to si- punculans in their closer relationship to annelids. They have an annelid cross rather than a molluscan cross during early cleavage, and as in annelids, the major ciliary band API ACOPHORA: PROGENETIC COELOMATES 61 Figure 3. Midsagittal section of the pelagosphera larva, Phascolosoma agassi:ii (from Rice, 1973, pi. 5). 1 buccal organ, 2 lip gland, 3 pore of lip gland, 4 mouth, 5 lip, 6 stomach, 7 coelom, 8 esophagus. of older echiuran larvae is the prototroch anterior to the mouth. In sipunculan pelagosphera. the metatroch below the mouth, not the prototroch, is the major swimming organ. Indeed, the region in pelagosphera that forms the head with its locomotory lip, lip gland, and buccal organ, is represented in echiuran larvae by only a few rows of cells between the prototroch and metatroch, and no larval organs are present. If sipunculans are sister taxon of the Mollusca, they must have arisen, like mollusks, early in the evolution of metazoans. One piece of evidence for an early sipunculan history is the mid-Cambrian genus Ottoia from the Bur- gess Shale. Considered priapulids by Conway Morris ( Whittington, 1985) and close to priapulids by Banta and Rice (1976), the genus indicates great diversity of spe- cialized sacciform, coelomate or pseudocoelomate, worm- like animals already in the early Paleozoic. Sipunculans therefore could have a very long, but unobservable and unverified, geologic history. A second piece of evidence is that sipunculans contain hemerythrins, found also only in priapulids, lingulid brachiopods, and some annelids (Curry and Runnegar, 1 990). Because lingulids and prob- ably priapulids and annelids are known from the early Cambrian, the presence of hemerythrins indicates a very long history for all forms having these oxygen transport molecules. Si:e of the pericardium in "primitive" mollusks The pericardium is larger relative to the heart in Apla- cophora, Monoplacophora, and Polyplacophora than it is in Gastropoda, Pelecypoda, and Cephalopoda (Schel- tema, 1973, 1988; Scheltema and Kuzirian, 1991) (Figs. 5, 6A). Ontogenetically, the pericardium is already large before the heart develops from pericardia! epithelium in Aplacophora (Baba, 1938), and in Polyplacophora de- velopment of the pericardium precedes development of the gonad (Hammersten and Runnstrom, 1925). Thus the polarity of pericardia! size is from large to small in Mollusca, and the continued reduction within the phylum is considered to be a derived condition of the Mollusca. B Figure 4. Newly settled larvae of Polyplacophora. (A) Midsagittal section of Acanthochiton discrepans (after Hammarsten and Runnstrom, 1925, fig. E, figure reversed). (B) Ventral view of Stetwplax hcalhiana just after metamorphosis (after Heath. 1899, fig. 59). The opening of the pedal gland (3) lies posterior to the mouth (4); the gland opens through "a series of. . . intercellular channels" rather than a duct (Heath, 1899, p. 631); compare with Figure 6C. 1 radula sac, 2 anterior pedal gland, 3 opening of pedal gland, 4 mouth, 5 foot, 6 larval eye. Structures num- bered 1-5 are homologous to structures with the same numbers in Figs. 2 3. 62 A 9 9 9 Figure 5. Large pericardia! space and heart in primitive molluscs. (A) Aplacophoran, Chaetaderma nitidulum, sagittal section. Gametes pass from the gonads through the pericardium with its large, paired, lateral extensions ("horns") and thence into gametoducts leading to the mantle cavity (from Scheltema. 1973, fig. 2, and Scheltema, 1988, fig. 1 3). (B) Polyplacophoran, Chiton sine/am, cross section (after Wissel, 1904, pi. 24, fig. 49). (C) Monoplacophoran. Neopilina galathea. dorsal view, with paired pericardia! sacs, paired ventricles, and two pairs of auricles (after Lemche and Wingstrand, 1959, from Scheltema, 1988, fig. 1 3). (D) Polyplacophoran, Acanlhopleiira echinata, dorsal view, with two pairs of openings between auricles and ventricle (after Plate, 1898. from Scheltema, 1988. fig. 13). 1 pericardium, 2 ventricle, 3 auricle, 4 opening between auricle and ventricle, 5 auriculoventricular valve, 6 aortal bulb, 7 gonopericardial duct, 8 lateral extension of pericardium, 9 gametoduct. Development ofmesoderm The interpretation that the coelom is reduced in Mol- lusca assumes that the molluscan pericardium is homol- ogous to the coelom in other spiralian coelomates, namely Annelida and Sipuncula. In all three, the coelom is formed from mesoderm that originates from embryonic cell 4d. This cell gives rise to a pair of mesodermal teloblasts, which migrate inward to a ventrolateral position, one on each side of the midline ( Verdonk and van den Biggelaar, 1983; Anderson, 1973; Rice, 1975) and proliferate forward into two lateral mesodermal bands. Mesodermal bands APLACOPHORA: PROGENETIC COELOMATES 63 Figure 6. (A) Cross-section through the pericardium of a neomenioid aplacophoran, Helicoradomenia juani (from Scheltema and Kuzirian. 1991, fig. 5C). (B) Cross-section through the pedal gland and pedal pit of a neomenioid aplacophoran, Ocheyoherpia sp. The voluminous pedal gland occupies most of the head region; the lobes of the gland are in varying stages of secretion. (C) Ciliated pedal pit of Helicoradomenia juani. The pedal gland discharges into the pedal pit, not through distinct ducts, but through numerous channels as described for chitons (Fig. 4B). (D) Secretory epidermal papillae of the neomenioid aplacophoran, Helicoradomenia juani (from Scheltema and Kuzirian, 1991, fig. 2C). (E) Secretory epidermal papillae of the polyplacophoran. Acanthochiton fascicularis (from Fischer el ai, 1980, fig. 3). 1 pedal gland, 2 ciliated pedal pit, 3 dorsal blood sinus, 4 dorsal cecum of midgut, 5 cerebral ganglion, 6 oral cavity, 7 pericardium, 8 auricle, 9 ventricle, 10 ovum. 11 U-shaped gametoduct, 12 copulatory spicule pocket, 13 foot. Asterisks in D and E, cavities of dissolved spicules. 64 A. H. SCHELTEMA are present as well in Nemertini (Turbeville, 1986). In annelids, sipunculans, and nemertines, the coelom is formed by cavitation (schizocoely) of the bands. The coe- lom constitutes the major body cavity in annelids and sipunculans, but in nemertines it forms only vessels for blood circulation (Turbeville, 1986). In mollusks, the me- sodermal bands break up into masses of coelenchyme, from which is formed a solid anlage or pair of anlagen that cavitate to form the pericardium, heart and kidneys (Raven, 1966; Moor, 1983). In some mollusks with paired anlagen, the pericardium begins as paired cavities before becoming united (Raven, 1966). In Neopilina the peri- cardium is still paired (Fig. 5C), and the large pericardia! "horns" in some Aplacophora (Fig. 5A) may reflect an ancestral paired condition. The coelom among the spiralian protostomes described here is interpreted as being homologous because of sim- ilarities in early embryological development. Differences in coelom formation among the four phyla apparently arise from variations in the timing of cavitation after the mesodermal bands have formed; but the differences in process are not considered sufficient to deny homology of the coelom. A single pericardium formed by fusion in mollusks other than Neopilina is thus an apomorphy. Molecular evidence Recent sequencing of 18S ribosomal RNA among 22 classes (not including Aplacophora), in 10 animal phyla, split off acoelomate Platyhelminthes as sister group of the remaining bilaterian taxa, the eucoelomates, which fall into four closely rooted groups (Field ct ai, 1988). The group termed Eutrochozoa (Ghiselin, 1988) includes five analyzed phyla: Mollusca, Annelida. Brachiopoda, Po- gonophora, and Sipuncula. More recently Turbeville el al. (1992) have added Nemertini to the Eutrochozoa, bas- ing their results on 18S rRNA and analyzing two Platy- helminthes, in addition to the single flatworm analyzed by Field el al. (1988). A re-analysis by Lake (1990) of the 1988 data positioned Sipuncula closest to Mollusca and Brachiopoda, with Annelida and Pogonophora as sister groups. The presence of hemerythrins in Brachiopoda, Sipuncula, and some Annelida affords independent sup- port from molecular data for some of the results of Field t't al. (Curry and Runnegar, 1990). The relationships among Sipuncula, Mollusca, and Brachiopoda, however, remain unresolved, and possible synapomorphies of sipunculan and molluscan larval characters were not taken into account by Lake. Although the molecular evidence is still incomplete, it suggests that mollusks have descended from a coelomate ancestor, and that sipunculans are their closest sister group. In proposing that the last common ancestor of the Annelida-Mollusca lineage was hemocoelic and segmented. Lake did not dis- cuss the presence or absence of a coelom. Ghiselin ( 1988) considered the evolution of Mollusca in light of the mo- lecular evidence given in Field et al. (1988), amplifying the data with an analysis of specific nucleotides and a useful history of molluscan phylogenetic hypotheses. Ghiselin favored a segmented, coelomate eutrochozoan ancestor, with loss or reduction of segmentation in the Mollusca. Salvini-Plawen (1990), however, retained a preference for a turbellariomorph molluscan ancestry and refuted the validity of the sequencing by Field et al. (1988) and Ghiselin (1988), because "for some selected, tradi- tionally monophyletic groups [including mollusks] eu- phemistic premises are made" by eliminating some data as convergences. Willmer and Holland (1991) also con- sidered that mollusks had a flatworm origin and suggested that RNA analysis of several Platyhelminthes might show them to be poly- or paraphyletic, but the work of Turbe- ville et al. (1992) indicates that they are monophyletic. Monophyly of Aplacophora A proposed homology of the chaetoderm oral shield with the creeping sole of the archimollusk was the basis for separating the two aplacophoran taxa into two classes (Fig. 7B, C; Fig. 8A) (Salvini-Plawen, 1972, 1985, 1990). This homology was based on the innervation of the oral shield (Salvini-Plawen, 1972), the character of the epi- dermis, and the presumed homology of cuticular struc- tures (Fig. 8C, arrowhead) (S. Hoffman, 1949), but it is not upheld either by light or transmission electron mi- croscopy (Scheltema et al., in press, fig. 9; Tscherkassky. 1989). The oral shield cuticle is continuous with that of the pharynx and is a lip. and the innervation of the shield is cerebral, lying anterior to that part of the anterior ner- vous system considered "tentacular," and thus part of the head region, by Ivanov ( 1 99 1 ). Accordingly the two apla- cophoran taxa cannot be separated on the basis of the chaetoderm oral shield, although Salvini-Plawen (1990) recently argued that the homology holds because the fore- gut and oral-shield epithelia are different, and the presence of the cuticle is secondary. In a schematic drawing through an oral shield, Salvini-Plawen (1990, fig. 7) showed a sep- aration, the "mantle rim," between the oral shield cuticle and body cuticle, but this separation does not exist in my experience (Scheltema et al., in press, fig. 9B). The ar- gument would be clarified if it were known whether the oral shield is stomadeal in origin. Several synapomorphies suggest that the two aplacoph- oran taxa are monophyletic. The outgroup for comparison is Polyplacophora. The tetraneural nervous system, including the cerebral commissure, lateral and ventral nerve cords, and supra- rectal commissure, is more heavily ganglionated in both neomenioids and chaetoderms than in chitons. The radula APLACOPHORA: PROGENETIC COELOMATES 65 Figure 7. (A-C) Chaetodermomorpha. (A. B) (.'kern 'derma lurnerae. entire animal (antenor to left) and divided oral shield (I'rom Scheltema, 1985, fig. 3L, O. P). (C) Oral shield of SculOfiim megaradulalu.i (cf., Fig. 8A) (from Scheltema, 1988, fig. 6). (D. E) Neomeniomorpha. (D) Dorymenia sp. (E) A new neomenioid genus and species in the family Simrothicllidae. D and E are drawn to the same scale, anterior to left; the midgut and gonad lie between X-X and Y-Y. in its plesiomorphic state in Aplacophora is distichous, that is, only two teeth per row (Scheltema, 1988; Schel- tema el al, 1989), a reduction in number from the doco- glossate chiton radula. Both neomenioids and chaeto- derms have a dorsoterminal sense organ (= dorsocaudal sensory pit), or sometimes several, in the epidermis. It is of unknown function, although homology to the osphra- dium has been conjectured (Spengel, 1881; Haszprunar, 1987). Whether or not this homology is correct, the po- sition of the dorsoterminal sense organ is an autapomor- phy of the Aplacophora, for there is no compelling evi- dence that this position, postulated to be primitive for the molluscan osphradium (Salvini-Plawen, 1985), is other than an apomorphy shared only by neomenioids and chaetoderms. The two aplacophoran taxa share a similar reproductive system unique among mollusks. Paired gonads, some- times fused, open directly into the pericardium, and paired U-shaped gametoducts lead from the posterior end of the pericardium, first anteriorly and then posteriorly, to the mantle cavity (Figs. 5 A, 6 A, 9D). Separate gonaduct openings in species of Phyllomenia (Salvini-Plawen, 1978) are interpreted here as a derived condition of that genus. The mantle cavity in both neomenioids and chaeto- derms is small and posterior, acting as little more than a cloaca. In neomenioids, the groove on either side of the foot-fold can also be considered as reduced mantle grooves (Figs. 6A, 8C). The paired ctenidia in chaetoderms, which fill most of the mantle cavity, is probably a plesiomorphy. with loss in the neomenioids resulting from the space re- quirements of a secondarily more complicated reproduc- tive system, including sometimes very large copulatory spicules. Finally, the worm shape itself is here considered a syn- apomorphy of the Aplacophora. and not separate. Figure 8. (A) Cross-section through the oral shield of a chaetoderm, Scutopus megaradulatus, showing continuity between pharyngeal and oral-shield cuticle. Arrow indicates transition between homogeneous pharyngeal cuticle and more specialized fibrillar oral-shield cuticle with a thickened outer layer (from Schel- tema, 1988, fig. 5). (B) Sagittal section through a neomenioid, liymnomenia sp., showing serial lateroventral musculature. (C) Cross-section through the nonmuscular, heavily ciliated foot of a neomenioid, Helicora- domeniajuani. The arrowhead indicates the nonspiculose cuticle of the mantle cavity extending along each side of the foot groove, which was considered homologous to the chaetoderm oral shield by S. Hoffman (1949). (D) Cross-section through the radula. radula bolsters, and paired, hollow radula vesicles in Helicor- adomenia /lunu (from Scheltema and Kuzinan, 1991. fig. 4D). 1 oral-shield cuticle, 2 pharyngeal cuticle, 3 cuticle of body wall, 4 nerve fibers from precerebral ganglion, 5 ovarian region of hermaphroditic gonad, 6 digestive cells of undifferentiated midgut. 7 copulatory spicule pocket, 8 foot. 9 radula vesicle, 10 radula, 1 1 dorsal cecum of stomach/digestive gland. 66 APLACOPHORA: PROGENETIC COELOMATES 67 Figure 9. Nervous system and reproductive system in a neomenioid, Strophomenia scandens (A, D) and nervous system in a chaetoderm, Limifossor lalpoideus (B, C). (A) Lateral (= pleural, visceral) cord with its ongm in the cerebral ganglion separate from the origin of the ventral (= pedal) cord. Lateral and ventral cords remain separate posteriorly (after Heath, 1904, pi. 27, fig. 2). (B) Anterior end; the lateral and ventral cord have a single origin in the cerebral ganglion (after Heath, 191 1, pi. 10, fig. 8). (C) Posterior end: the ventral cord runs close to the lateral cord and fuses with it. The suprarectal commissure is ganglionated (after Heath, 1905. pi. 43, fig. 18). (D) Posterior end; the lateral and ventral cords are well separated, with the separation maintained throughout. The gonad empties into the pericardium, which is shown in fine stippling. The U-shaped gametoduct, with a many-lobed seminal receptacle, is shown in coarse stippling; it runs from the posterior end of the pericardium to the mantle cavity, indicated by dashed lines (after Heath, 1904, pi. 27, fig. 6). 1 cerebral ganglion, 2 lateral cord, 3 pedal cord, 4 suprarectal ganglion/commissure, 5 buccal ganglion, 6 gonad, 7 pericardium, 8 seminal receptacle, 9 mantle cavity. 68 A. H. SCHELTEMA convergent apomorphies in the two taxa. When this char- acter and those mentioned above are considered together, the Aplacophora clearly emerge as a monophyletic taxon. Chaetodermomorpha, derived Aplacophorans Neomeniomorpha are more similar than Chaetoder- momorpha to the outgroup, the Polyplacophora, in ner- vous system (Fig. 9A, D), form of epidermal papillae (Fig. 6D, E), presence of anterior pedal glands (present only in the larvae of chitons) (Figs. 4, 6B), presence of paired pharyngeal glands, serial lateroventral musculature (Fig. 8B), and inequality of height and width dimensions. Sev- eral autapomorphies indicate that the burrowing Chae- todermomorpha have been derived from a creeping neo- menioid-like ancestor. Criteria for considering a structure to be apomorphic are fusion or elaboration. Changes in the nervous system are pronounced. In chaetoderms the lateral and ventral cord on each side have a single origin from the cerebral ganglion, whereas in nearly all neomenioids lateral and ventral cords have sep- arate origins (Fig. 9A, B). In chaetoderms, the lateral and ventral cords on each side soon run close to each other, finally fusing into a single cord anterior to the suprarectal ganglion (Fig. 9C). In neomenioids, the cords remain apart and are well separated from each other (Fig. 9 A, D). There are few commissures between the ventral cords in chae- toderms, and many in neomenioids. In chaetoderms, the suprarectal commissure and precerebral ganglia are larger and more swollen than in neomenioids. Related to the loss of the ventral cord commissures, chaetoderms have entirely lost the foot and anterior pedal glands. The homology of mucous glands of the oral shield with pedal glands, proposed by S. Hoffman (1949), does not hold in TEM studies (Scheltema el ai. in press. Fig. 9A). In some species of Scut opus and Psilodens, ventral fusion of the mantle is marked by a longitudinal furrow between the spicules (Salvini-Plawen, 1968b; author's unpub. data). The gut of chaetoderms is modified from the simple combined stomach-digestive gland midgut of neomenioids to a separate stomach and blind digestive gland. In its most derived state in Chaetodermatidae, there is a gastric shield and style sac with a mucoid rod (Scheltema, 1978; Salvini-Plawen, 1981b). The serial lateroventral musculature of neomenioids (Fig. 8B) is lost in chaetoderms, although a few vestigial anterior bundles have been reported in a species of Scu- topus (Salvini-Plawen, 1985). Body form in chaetoderms is circular in cross-section; in neomenioids there is usually a small but measurable difference between height and width. The circulatory system is somewhat better denned in chaetoderms than in neomenioids, with anterior and posterior vertical septa defining hemocoelic sinuses, and with an often thick-walled aorta and aortal bulb (Schel- tema, 1973) (Fig. 5A). Finally, the chaetoderm oral shield represents a specialized cuticular structure. The autapomorphies of Chaetodermomorpha all seem to be related to their form of locomotion burrowing in muds and silts and feeding habits, either as carnivores on small benthic organisms, or as detritivores. Autapo- morphies also exist in the Neomeniomorpha, particularly the sensory vestibule and rather complicated reproductive system with accompanying loss of mantle cavity ctenidia, but specializations of the Chaetodermomorpha mark them as the more derived of the two taxa. Relationship of Aplacophora and Polyplacophora Aplacophora and Polyplacophora are here considered to be sister taxa, the Aculifera, on the basis of shared char- acters of nervous system, spicules, and epidermal papillae. An attempt is made to determine the polarities of these characters, and other anatomical similarities are noted. Nervous system Aplacophora, Polyplacophora, and the monoplacoph- oran Tryblidiacea Neopilina and Vema all have a fully developed tetraneury, with paired lateral and pedal nerve cords arising from a cerebral commissure or ganglia and a circumoral or circumesophageal nerve ring. In the monoplacophorans, both cords are joined posteriorly ventral to the rectum, whereas in Aplacophora (Fig. 9) and Polyplacophora, only the lateral cords are joined, and they unite above the rectum in a commissure (chitons) or ganglion (aplacophorans). There is only a single cross- pedal commissure in the monoplacophorans and numer- ous ones in chitons and neomenioid aplacophorans. What is the polarity of these two plans, both of which are plesiomorphic to more specialized nervous systems in other mollusks? Obvious outgroups for comparison, An- nelida, Echiura, Nemertini, and Sipuncula, appear to have a reduced nervous system and offer no clues. In Annelida there is only a paired ventral cord, except for a secondarily derived tetraneury in Amphinomidae (Gustafson. 1930); in Nemertini there is a pair of lateral cords joined either above or below the rectum; in Echiura, there is a single ventral cord; and in sipunculans there is also a single ven- tral cord which is paired in the larval pelagosphera of Phascolosoma agassizii (Rice, 1973). One might surmise that Neopilina, a deep-sea deposit or xenophyophore feeder (Tendal, 1985), is less mobile than either aplacoph- orans or polyplacophorans and has retained a simpler nervous system, and the aplacophoran-polyplacophoran system is more specialized (derived) owing to habitat (chi- tons) or to carnivory (aplacophorans). Of course, a sec- ondary loss and shifting of nerve elements in the mono- placophorans might also be considered, and Wingstrand APLACOPHORA: PROGENETIC COELOMATES 69 (1985) and Salvini-Plawen (1972) suggest that the sub- rectal commissure is an apomorphy. Whichever interpre- tation is correct, one can say that monoplacophoran and aplacophoran-polyplacophoran nervous systems are each apomorphic to some unknown ancestral state, and the suprarectal ganglion or commissure of the Aplacophora- Polyplacophora serves to relate them phylogenetically and set them apart from the Monoplacophora. Spicule formation Spicule formation in aplacophorans and polyplacoph- orans has most recently been investigated by Haas (1981) (Fig. 10). Spicules in both taxa are aragonite and formed extracellularly within an invagination of a single basal cell, which secretes calcium carbonate within a crystalli- zation chamber sealed by neighboring cells (Scheltema a al., in press, fig. 6D). In chitons, megaspines are formed from a proliferation of the basal cell and do not occur in aplacophorans. Spicules of the Aculifera are usually considered to be a plesiomorphic state of calcium carbonate formation within Mollusca, since both spines and shell occur in chi- tons and only spines occur in Aplacophora, both being "primitive" groups in the general sense. However, Mono- placophora, likewise considered primitive, have no spines. The dorsal, calcium-carbonate-secreting epidermis of Mollusca, in combination with a ventral locomotary sur- face, is probably an apomorphy. However, the shell-bear- ing Brachiopoda are rooted with the Mollusca-Annelida group by RNA sequencing (Field el al.. 1988). and some boring Sipuncula have calcium carbonate deposits at the dorsal anterior end of the trunk (Rice, 1969). Further comparative work needs to be done to compare calcium carbonate secretion among the Eutrochozoa before ho- mology can be assumed. It cannot be concluded from outgroup comparison that spicules and shell are homologous structures (and the ar- gument will be made further on that they are not), or that either is the plesiomorphic state. It can be concluded, however, that because of the way in which they are formed, spicules of Aplacophora and Polyplacophora are homol- ogous and can be construed as a synapomorphy. Epidermal papillae The epidermis of both chitons and aplacophorans are liberally supplied with secretory papillae (Fig. 6D, E). In chitons, papillae are homologous with aesthetes (Fischer c/ al.. 1980). Although homology with other conchiferan shell-penetrating structures has been suggested (Salvini- Plawen, 1985), the homology was considered spurious by Wingstrand, who reviewed the literature on the subject (1985, pp. 58-59). The presence of these papillae is COn- Kigure 10. Spicule formation in (A) Aplacophora and (B) Polypla- cophora (after Haas. 1981. figs. 6, 12). The spicule is formed within an invagination of a basal cell which secretes CaCOj. The crystallization chamber is sealed by a nng of neighboring cells, which in Polyplacophora produce a pellicle around the spicule. 1 spicule, 2 neighboring cell, 3 CaCO r secreting basal cell. sidered here to be an apomorphy of the Aplacophora- Polyplacophora. Rciluccd aerial replication Compared to Monoplacophora, there is less serial rep- lication in both Polyplacophora and Aplacophora, but both have greater serial replication than other mollusks. Serial replication appears as regular, lateroventral mus- culature in Neomeniomorpha (Fig. 8B) and as 8-fold rep- etition of muscles and shell plates in chitons. Other anatomical homologies Aplacophorans and polyplacophorans share certain other anatomical structures that are probably homolo- gous, but they may be plesiomorphies of the Mollusca. Dorsal paired gonads, becoming fused during ontogeny in chitons and most Chaetodermomorpha, lie like sacs more or less free above the gut and digestive gland in the dorsal hemocoel. In Neopilina, the gonad is ventral to the digestive system (Lemche and Wingstrand, 1959), and in many other Mollusca the gonad is intermingled closely with lobes of the digestive gland. The circulatory system in both groups is extremely open with posterior paired auricles and a ventricle, a dorsal aorta leading to the head (lacking in many Neomeniomorpha), and open sinuses, the latter more profuse in chitons. Taken together, the above reasons are sufficient for concluding that Aplacophora and Polyplacophora belong together in a single taxon, the Aculifera, which is therefore a clade, and not a grade. 70 A. H. SCHELTEMA Aculifera as the Sister Taxon of the Conchifera Chitons provide evidence that Aculifera are separate from their sister group, the Conchifera. The evidence is based on shell ontogeny, shell structure, and perhaps mo- lecular data. She/I ontogeny In Conchifera, the shell originates within an ectodermal invagination. the shell-field invagination. which is covered by an organic pellicle (Eyster and Morse, 1984) (Fig. 1 1 ). In Aeolidia papillosa, long cytoplasmic processes overlie the pellicle. In chitons, there is no shell field invagination, and shell plate anlagen are deposited within transverse depressions which are sealed, not by a pellicle, but by long, overlapping microvilli that lie beneath a gelatinous mucoid substance (Kniprath, 1980; Haas cl a/., 1980; Haas, 1981; see Scheltema, 1988, for a more complete discussion). Furthermore, in healthy larvae, shell is not deposited as separate granules, as illustrated by Kowalev- sky (1883), but as uninterrupted rods (Kniprath, 1980). This fact conflicts with the hypothesis that chiton shell arose from fused spicules (Salvini-Plawen, 1985. 1990). Shell structure The crystallography of chiton shell has been said to indicate an autapomorphy of chitons by Haas ( 1976), who found that "The . . . c-axis of [the] hypostracum lies in the bisectrix of the crystalline fibers. The whole complex acts crystallographically as a single crystal" (p. 392). If this crystallographic orientation is correct, then no ho- mology exists between polyplacophoran and conchiferan shell. Further differences are a lack of true periostracum in chitons (although Haas [ 1 98 1 ] has demonstrated a thin cuticle overlying the shell plates) and a lack of a nacreous layer (for further discussion see Wingstrand, 1985; and Scheltema, 1988). On the other hand, the shell of the try- blidiacean Monoplacophora does not differ from other primitive conchiferan shells (Lemche and Wingstrand, 1959). Molecular evidence The evolutionary tree of 1 8S rRN A has three branches for three classes of mollusks a nudibranch, two species of clam, and a chiton. This trifurcation of mollusks also appears in Lake's (1990) re-analysis of the data. Further molecular data for all molluscan classes should resolve the branching, but there is a hint of molecular distance between chitons and the two other classes analyzed. Evidence for Progenesis in Aplacophora A vermiform body is a character that could have been added rapidly by a small change in a regulatory gene or Figure 11. Shell deposition in larvae of Conchifera and Aculifera. (A, B) Gastropod, Aolidia papillosa- An organic pellicle (arrowheads) covers the lumen of the shell held invagination; a cytoplasmic extension shown in B seals the edge of the pellicle (after Eyster and Morse, 1984, figs. 1,2; from Scheltema, 1988, fig. 4). (C, D) Polyplacophoran, Ischno- chiton rissoi. The shell plate is first secreted beneath microvilli (stragulum) which are covered by a layer of mucus (C); later (D) the microvillar processes have pulled apart and a cuticle begins to form (after Kniprath. 1980, fig. 5, from Scheltema. 1988, fig. 4). Haas (1981) illustrated a similar process except for showing that cuticle covered the stragulum before CaCO, deposition. 1 shell field invagination, 2 cytoplasmic ex- tension, 3 microvillar process (stragulum), 4 calcium carbonate of shell plate, 5 mucous layer, 6 ?mucous cell, 7 cuticle. in timing of cell assembly early in the ontogeny of an aculiferan mollusk (for mechanisms and examples see Raff and Kaufman, 1983; McKinney and McNamara, 1991). In the embryological development of the chiton Lepido- pleums asellus, swimming larvae are first oval and then become secondarily flattened and sink to the bottom (Christiansen. 1954). Even with development of the foot, chiton larvae remain ovoid for a time (Heath, 1899, Fig. 52; Eernisse, 1988, Fig. 7). One can imagine that larvae of some aculiferan, not necessarily a chiton, might not APLACOPHORA: PROGENETIC COELOMATES 71 have become dorsoventrally flattened through a small change in gene regulation and the worm-like shape arose. The change to a vermiform shape could have occurred either early in the evolution of Mollusca or late. Recent phylogenies presume that a vermiform shape evolved as an early offshoot of the Mollusca, placing Aplacophora closest to the stem form, either as a monophyletic clade (Scheltema, 1988; Wingstrand, 1985), or as two separate clades, with the Chaetodermomorpha evolving first as the sister-group to all extant Mollusca (Salvini-Plawen, 1972, 1985). Serial replication thus was seen to he an apomor- phy. If Aplacophora are closest to the molluscan ancestor, then the imperatives following from that phylogenetic construct fit poorly with the arguments given above, that is: ( 1 ) Aplacophora and Polyplacophora are a clade; (2) shell is not formed by fusion of spicules; and (3) chiton shell is not homologous to conchiferan shell. The question of when serial replication evolved in mollusks becomes critical, for it is either a plesiomorphy of mollusks. or not. Polyplacophora, belonging to Aculifera, have some structures homologous with Monoplacophora, belonging to Conchifera, that are not shared with the Aplacophora (Wingstrand, 1985); radula dentition and radular appa- ratus including musculature; 8-serial pedal retractors; pre- oral unpaired fold, or velum; perhaps the heart with two pairs of atria; and coiled intestine. Wingstrand noted that some of these structures "could be plesiomorphic, i.e., could have been present already in some Aplacophoran ancestors" (1985, p. 74), but considered that the radula and radular apparatus, in particular the paired, hollow radula vesicles, are synapomorphies. It was not then known that paired radular vesicles are also present in some neomenioids (Fig. 8D). Here, structures argued to be apo- morphic by Wingstrand are considered plesiomorphic with exception of the coiled gut, a character widely con- vergent among mollusks. Thus, serial replication is here considered a plesiomorphy of Mollusca. The possibility that a worm shape was acquired by aplacophorans late in aculiferan evolution leads to a wholly different concept of molluscan phylogeny. It calls for progenesis in Aplacophora, wherein nonserial but ple- siomorphic-appearing anatomical characters are retained. The following evidence supports the hypothesis that Aplacophora are progenetic; i.e., that they have retained ancestral juvenile characters in adult form through ac- celeration of sexual maturation (Gould, 1977). ( 1 ) If narrowing of the body by acquisition of a worm shape arose early in aculiferan evolution without proge- nesis, then this process should be reflected somehow in the internal anatomy, and the more elongate (that is, nar- rower) the shape, the more pronounced should the internal changes become. Within the Neomeniomorpha, the least derived aplacophoran taxon, there is little organizational difference between short and elongate species in anterior and posterior ends or in musculature. Elongation of ex- ternal form is accompanied internally by a simple length- ening of the gonad and midgut (Fig. 7D, E). The situation in the more derived chaetoderms differs and does not serve the argument. A comparison can be made to Cryptoplax. a genus of chiton with a derived worm-like shape. In Cryptoplax there are at least four specializations of adult characters: (a) the mantle is very thick relative to internal body di- ameter; (b) there is loss of circulatory pathways; (c) there is loss of shell and shell musculature; and (d) the intestinal tract is remarkably long and complicated, turning back on itself in numerous spirals (Wettstein, 1904; H. Hoff- mann, 1929-30). Furthermore, an analysis of the allo- metric equation defining shape in 408 chiton species in 39 genera indicated great uniformity in allometry, except in the carnivorous Placiphorella and in genera of Cryp- toplacidae (Watters, 1991). Species ofCryptoplacidae, ex- cept those in the most primitive genus, are allometrically similar to each other but have shifted markedly from the allometry of other chitons. Although no allometric studies have been made of neomenioids, the extremes in ver- miformity (Fig. 7D, E) do not predict uniformity. Thus there may be an ontogenetic difference in the evolutionary pathway to a worm-like shape taken by the two aculiferan taxa. Progenesis, an intrinsic process, is hypothesized for Aplacophora, and selection working on structural genes, an extrinsic process, for Cryptoplax. (2) Progenesis results in early reproduction (Gould, 1977). One abundant northwestern Atlantic aplacophoran species living at 2000 m, Prochaetoderma yongei, is known to mature within one year, a remarkably rapid rate, given the ambient temperature (~3C) and in comparison with other cold-water mollusks. P. yongei is interpreted as being an opportunistic species (Scheltema, 1987), but since it is the only aplacophoran for which even part of the life history is known, one cannot be sure that early repro- duction is the usual case in Aplacophora. (3) Progenesis results in a reduced body size (Gould. 1977), but the size of the nearest ancestor to Aplacophora is, of course, unknown. Most neomenioids are usually less than 5 mm long, and one can only infer from the generally larger size of chitons that the first ancestral apla- cophoran was already small. Like some other deep-sea taxa, such as protobranch bivalves (Sanders and Allen. 1973) and isopods (Hessler et ai, 1979), aplacophorans have evolved primarily in the deep sea, where they reach their greatest diversity (Scheltema, 1990). Food is limiting there, and small body size of macrobenthic organisms is the norm (Monniot and Monniot, 1978; Allen, 1983; Soetaert and Heip, 1989). Large neomenioids do exist in the deep sea, but they are usually either specialized (giant Neomenia species: Baba, 1975; Kaiser, 1976) or live in environments where there is high productivity (e.g., high 72 A. H. SCHELTEMA latitudes: Proncomenia sluitcri, Derjugin. 1915, 1928). Large body size in Aplacophora is probably an apo- morphic character because it is found scattered amongst unrelated families, some of which have derived characters such as loss of radula or a thick dermis. (4) Certain structures in Aplacophora are less devel- oped than homologous structures in Polyplacophora or other mollusks. (a) The organic compos : *ion of the cuticle is simpler than in chitons (Beedham ano Trueman, 1968). (b) The radula in its plesiomorphic state in neomenioids has only two teeth per row (distichous), a condition found in the early ontogeny of several gastropods (Kerth, 1983; Scheltema, 1988; Scheltema ct a!., 1989). (c) The apla- cophoran mantle cavity, located ventroposterior to pos- terior, is small, serving as little more than a cloaca (Fig. 7A, D). (d) Both neomenioids and chaetoderms lack kid- neys, (e) The foot is developed only as a ciliated ridge without musculature in neomenioids (Fig. 6A). (f ) Gonads and pericardium are united in aplacophorans, reflecting the early ontogenic state in chitons, where the gonad orig- inates as an anlage of the pericardium (Hammarsten and Runnstrom, 1925) (Figs. 5 A, 6A, 9D). (g) The gut in neo- menioids is simple, with a united stomach and digestive gland; the digestive gland is separate from the stomach in other mollusks. (5) Aplacophora have retained a structure found in chitons only as larvae. The anterior pedal glands are large and specialized in neomenioids (Fig. 6B, C), but are lost soon after metamorphosis in chitons, where they serve only for early postmetamorphic attachment (Heath, 1899) (Fig. 4B). Although progenesis results in primitive-appearing structures, they are actually derived. Therefore, some process within the Aculifera should be primitive in the Polyplacophora but derived in the progenetic Aplacoph- ora. Such seems to be the case in early embryological development. Freeman and Lundelius (1992) have pro- posed that, among the spiralian coelomates Mollusca, Annelida, Sipuncula and Echiura, two mechanisms de- termine which blastomere is specified as the D quadrant. They hypothesized that the primitive mechanism for D quadrant specification is by induction after the fifth cleav- age, when one of the four macromeres has maximum contact with the micromeres. The derived mechanism is by segregation of the cytoplasm into one macromere, which is then specified as the D quadrant; it occurs by the second cleavage. In Polyplacophora, macromeres cleave equally and the D quadrant is specified by induc- tion, the primitive mechanism. But in the cleaving egg of the neomenioid Epimenia, a polar body is formed and therefore macromeres of unequal size; thus the D quadrant is specified by cytoplasmic determinants, the derived mechanism (Baba, 1951; Freeman and Lundelius, 1992). The evidence for progenesis presented here argues for heterochrony in the Aplacophora, but this idea cannot be tested either against fossils, which are unknown, or against a more complete phyletic lineage, as has been done for progenetic meiofaunal forms (Westheide, 1987) and deep- sea tunicates (Monniot and Monniot, 1978). When the early embryological development of aplacophorans is better known, and with further intrataxon comparative studies, the validity of the hypothesis may be clarified. Phytogeny of the Mollusca The phylogeny represented in Figure 12 proposes a coelomate molluscan ancestor with serial replication; two separate evolutionary molluscan lineages, the Conchifera and the Aculifera, based on synapomorphies of differences in CaCO, deposits; and morphologies arising from pro- genesis in the Aplacophora. The molluscan ancestor is considered to have had the following plesiomorphies: (1) extracellular CaCO 3 depo- sition by the dorsal epidermis (Mollusca generally); (2) serial replication, probably originally 8-fold (Monopla- cophora, Polyplacophora, Nautilus, neomenioids, some bivalves); (3) coelom from the 4d cell, paired pericardial cavities (in Monoplacophora, and fused but large in Apla- cophora and Polyplacophora); (4) radula, radular appa- ratus with hollow radula vesicles (Polyplacophora, Monoplacophora. Aplacophora, Fig. 8D); (5) nervous system poorly ganglionated, with cerebral ganglia and commissure, circumenteric ring, and paired lateral and pedal cords with cross-commissures and posterior con- nection (Monoplacophora in part, Aplacophora and Polyplacophora); (6) dorsoventrally flattened, small size (Cambrian Mollusca: Runnegar and Pojeta, 1985; Hasz- prunar, 1992; but note that the Cambrian fossil halkierids and Wiwaxia, perhaps near relatives of mollusks, are cen- timeters in length [Conway Morris, 1985; Conway Morris and Peel, 1990]); (7) dorsal cuticle (Aplacophora, Poly- placophora); (8) ventral ciliated locomotory sole (Mollusca generally); (9) head separate from the locomotory sole and with cerebral ganglia (Mollusca generally); (10) a groove between the dorsal and ventral surfaces, the future mantle cavity (Mollusca generally): (11) pre-oral fold; (12) the presence of podocytes in pericardial tissue (mollusks generally); (13) ductless anterior pedal mucous glands (as a glandular epithelium in Monoplacophora; Lemche and Wingstrand, 1959); (14) a one-way gut with mouth, anus, large digestive gland poorly differentiated from stomach (Neomeniomorpha, Monoplacophora); (15) paired pha- ryngeal diverticula; (16) poorly defined circulatory system; and (17) gonad and pericardium joined at least during ontogeny (Mollusca generally). The phylogeny presented in Figure 12 requires that the original calcium carbonate deposition in mollusks was APLACOPHORA: PROGENETIC COELOMATES 73 ACULIFERA APLACOPHORA CONCHIFERA 0. O tr oc 65 ili < o 39' 23 B -- 22' 39' 18 6-17 [c?| - 5 4 [d?| - 3 2 1 Figure 12. Proposed phylogeny of extant "primitive" Mollusca. (A) Apomorphies of Mollusca: I extracellular CaCO, deposition by dorsal epidermis; 1 eight-fold serial replication; 3 paired coelom, including pericardium; 4 radula; 5 poorly ganglionated tetraneury; 6 small size, dorsoventrally flattened; 7 dorsal cuticle; 8 ventral locomotory sole; 9 head separate from sole; 10 groove between dorsal and ventral surfaces; 1 1 pre-oral fold; 12 nonsegmented pericardium, pencardial tissue with podocytes; 13 ductless anterior pedal gland; 14 poorly differentiated stomach/digestive gland (model: Neopilina); 15 paired pharyngeal di- verticula; 16 poorly denned circulatory system; 17 joined gonad/pen- cardium during early ontogeny. (B) Separation of Conchifera and Acu- lifera: 18 calcareous shell; 19 spicules; 20 epidermal papillae; 21 supra- rectal ganglion/commissure; 22 reduced serial replication and fused pericardium. (C) Separation of Polyplacophora and Aplacophora (24-31 the result of progenesis): 23 eight shell plates; 24 worm shape; 25 reduced foot; 26 reduced mantle cavity; 27 joined gonad/pericardium; 28 kidneys absent; 29 chemically simple cuticle; 30 serial lateroventral musculature; 31 distichous radula; 32 U-shaped gametoducts; 33 gan- glionated nervous system; 34 dorsoterminal sense organ. (D) Separation of Chaetodermomorpha and Neomeniomorpha: 35 ventrally fused cu- ticle, foot lacking; 36 oral shield; 37 fused, reduced nervous system; 38 serial replication absent; 39 stomach separate from digestive gland; 40 large anterior pedal gland; 41 elaborated reproductive system; 42 ctenidia absent. * = convergent morphologies; c? = presence of ctenidia ques- tionable; d? = radula questionably docoglossate. neither as spicules nor as shell. CaCO, was first deposited, perhaps, as granules within a dorsal cuticle, which was thereby stiffened. Such a reinforced cuticle could act as the antagonist to the dorsoventral pedal musculature. During chiton ontogeny, the pedal musculature develops earlier than the shell plates (Hammarsten and Runnstrom, 1925). One can speculate from this fact that, perhaps, the various forms of shell and spicules among mollusks have resulted from selection for different modes of locomotion in various habitats, rather than selection just for protec- tion. In terms of CaCO 3 secretion among phyla, the impor- tant synapomorphy for mollusks, which sets them off from other spiralian coelomates, is the locomotary sole in com- bination with a cuticle- and CaCO r secreting dorsal epi- dermis. Certain rock-boring sipunculans also secrete CaCOj dorsally, forming a plug for their tubes (Rice, 1969), and Brachiopoda, which fall in with spiralian coe- lomates in molecular analysis, also have calcium carbon- ate shells. However, animals in neither of these phyla have the combination of dorsally produced CaCO, and a ven- tral locomotary surface unique to mollusks. It is hypothesized that after, or as, Conchifera diverged from the stem line, the mantle deepened and gills devel- oped. Serial replication was retained in Monoplacophora but lost in the rest of the Conchifera, except for serial pedal musculature in some taxa and the renal system in cephalopods. Aculifera may have evolved either at the same time as Conchifera or later. By the Upper Cambrian or Lower Ordovician, the serial shell plates of Polypla- cophora had evolved (Runnegar and Pojeta, 1985). This event was preceded by the loss of serial replication other than lateroventral muscles and perhaps by an increase in size. In a separate evolutionary event of progenesis, the Aplacophora evolved with probable reduction in size, further loss of serial replication, loss of nephridia, retention of gonad-pericardial connection, and acquisition of a worm shape with concomitant reduction of the foot. Chaetodermomorpha were derived from the neomenioid- like stem with complete loss of foot, reduction and fusion of the nervous system, and specializations of the gut. This hypothesized phylogeny does not call for an evo- lutionary process in which CaCO, deposits, or the cells that produce them, become fused. Furthermore, it should allow some of the Early Cambrian sclerite-bearing forms now coming to light, such as the shell-bearing, articulated halkieriid described recently from the Lower Cambrian of Greenland (Conway Morris and Peel, 1990), to find their place in relation to the extant Mollusca. In this phylogeny, the Monoplacophora with clear serial replication are not evolved after Aplacophora, and mol- luscan serial replication is considered to be a plesiomor- phy. As Wingstrand (1985) pointed out, it is difficult to imagine that serial replication evolved after the shell. The 74 A. H. SCHELTEMA careful and original anatomical analysis of Wingstrand showing close affinities of the monoplacophoran Trybli- diacea and Polyplacophora are upheld here as retained plesiomorphies of the common ancestor. Whether Pru- vot's neomenioid larva with its supposed seven rows of spicules actually exists does not change the argument (see Salvini-Plawen, 1972, 1981a, 1985; Scheltema, 1988 for discussions and figures of the larva). Manuscript drawings of Chaetoderma nitidithtm larvae made by G. Gustafson show eight rows of spicules for this taxon as well. If further observations on aplacophoran development prove that serial rows of spicules do exist, the larva still would not necessarily reflect progressive evolution from spicules to fused shell plate formation, but more likely would indicate a breakdown of plate formation similar to the breakdown of larval chiton shell plates caused experimentally by Kniprath (1980) (See also Scheltema, 1988). Age of the Aplacophora If known fossils reflect the actual time of evolutionary events, then the evolution of Polyplacophora late in the Cambrian (Runnegar and Pojeta, 1985) from a continuing line of aculiferous creatures was probable, with increased size and muscles being the determinants of shell plates rather than vice versa (see Hammarsten and Runnstrom, 1925, p. 276, for ontogenetic development of muscle be- fore shell). Aplacophora, with their highly derived shape and paedomorphic internal organization, give information about the primitive conditions of mollusks without being themselves primitive. A Late Cambrian-Early Ordovician origin from an aculiferan form with a developed mantle groove and posterior mantle cavity is postulated for Apla- cophora, with the 8-fold dorsoventral muscles rearranged in neomenioids into a series of indeterminate number. Cautionary Notes on Convergences Digestive system The molluscan gut appears to have evolved similar morphologies more than once (Fig. 12, no. 39). Evidence for convergence lies in presence of the style sac and gastric shield, found in a number of molluscan classes. In the aplacophoran family Chaetodermatidae, one of the most derived of the chaetoderm groups based on radula mor- phology (Scheltema, 1972, 1981), the gut is the most complicated among chaetoderms. with a gastric shield and a mucoid rod in a style sac (Scheltema, 1978; Salvini- Plawen, 1981b). The polarity of a less to a more compli- cated gut configuration within the chaetoderms is clear (Scheltema, 1981). Thus, the presence of a style sac and gastric shield is convergent among Mollusca. Metamerism Reduction of serial replication (Fig. 12, no. 22) is hy- pothesized for several molluscan classes Cephalopoda, Bivalvia, Polyplacophora. and Aplacophora. The evidence from morphology, ontogeny, and molecular analysis seems not to favor the hypothesis that replication origi- nated in annelids. If the altogether unsegmented Sipuncula are sister taxon of the mollusks, then arguments that the molluscan coelom is the result of a reduced annelid-like segmented coelom are not convincing. Evidence presented here could be interpreted in three ways (Fig. 13, s'-s 4 ). (1) A nonsegmented ancestor that had serial replication of organs and a coelom lies at the base of the lineage giving rise to Eutrochozoa (s 1 ). (2) The eutrochozoan ancestor had no serial replication, which O z O 0) Q _l UJ 9* 6-8 o 2. 10 11 9* S3 1-5 Figure 13. Phylogenetic relationship among Sipuncula, Mollusca. and Annelida. 1 spiral cleavage; 2 paired coelom originating from two teloblasts derived from 4d; 3 trochophore larva (?); 4 tetraneury; 5 ciliated creeping sole; 6 molluscan cross; 7 ventral, cuticular, pharyngeal (sto- madeal), protrusihle invagmation and attendant musculature; 8 anterior pedal gland; 9 fused nerve cords; 10 reduced coelom; 1 1 loss of creeping sole, s = serial replication: s 1 symplesiomorphic for all three taxa, but lost in Sipuncula; s 2 symplesiomorphic for Sipuncula and Mollusca, but lost in Sipuncula, and convergent with s 1 as metamerism in annelids; s 3 metamerism plesiomorphic for Annelida, convergent with either s 2 or s 4 ; s 4 plesiomorphic for Mollusca, convergent with s 1 . * = convergent morphologies. APLACOPHORA: PROGENET1C COELOMATES 75 later arose de novo twice: once in the stem form leading to mollusks and sipunculans (s 2 ). which was lost in the latter, and secondly in the ancestral annelid as metamer- ism (s 3 ). (3) Molluscan 8-fold serial replication (s 4 ) evolved after the stem form that gave rise to Sipuncula and Mol- lusca; and annelidan metamery (s 3 ) [as in (2)], arose as an unrelated evolutionary event. The first interpretation is perhaps closest to what may actually have occurred and seems the most parsimonious explanation. Differences in the coelom among eutrochozoan groups can be related to locomotion, a theme emphasized cor- rectly, I believe by Salvini-Plawen (e.g., 1972, 1985). Locomotion among Eutrochozoa is most rapid in annelids and mollusks. Serial pedal musculature is related to a creeping locomotion and is the most conservative serial structure in mollusks, present as a plesiomorphy in Monoplacophora. Polyplacophora, Aplacophora, and (much reduced) Pelecypoda and perhaps the neritid Gas- tropoda. In Annelida, coelom and muscle have combined in the perfection of a hydraulic locomotion (Clark, 1964). Perhaps, then, a re-examination of the relationship of muscles and coelom during ontogeny would be a useful exercise in providing insights into understanding the de- velopment of metamerism in Eutrochozoa. For instance, in at least some Annelida, ectodermal segmentation of the three anterior segments precedes segmentation of me- soderm (Anderson, 1973, pp. 36-37). Radula Wingstrand (1985) gave a detailed description of the radular apparatus in Polyplacophora and Monoplacoph- ora, demonstrating their great similarity, especially the docoglossate radula and radula vesicles. There are three possibilities: such a radula is a molluscan plesiomorphy; it is an apomorphy of Polyplacophora and Monopla- cophora; or it is convergent. Evidence from Aplacophora and ontogeny of some Gastropoda suggests that the plesiomorphic radula in mollusks was distichous (Keith, 1983; Scheltema el ai, 1989). An outgroup for comparison is the Cambrian sclerite-bearing Wiwaxia (Conway Morris, 1985) with two or three rows of teeth which appear much like the ple- siomorphic radula in Aplacophora. The phylogenetic po- sition of Wiwaxia. however, remains enigmatic, consid- ered either to be close to mollusks (Conway Morris, 1985) or to be an annelid (Butterfield, 1990). If the plesiomor- phic radula is distichous, then the docoglossate radula is convergent in Polyplacophora, Monoplacophora, and patellacean Gastropoda. If the docoglossate radula is a molluscan plesiomorphy, it is difficult to imagine how it functioned in a small Cambrian mollusk and what evo- lutionary steps would be necessary to account for all other molluscan radulae. The strongest evidence given by Wingstrand (1985) for monophyly of polyplacophorans and conchiferans is presence of a pair of hollow, presumably liquid-filled rad- ula vesicles found at that time only in Polyplacophora and Monoplacophora. None had been reported in Apla- cophora. However, a re-examination of the neomenioid Helicoradomenia juani and other species in the genus, which have a plesiomorphic aplacophoran radula, has led me to conjecture that paired, elongate, hollow vesicles present in this genus are a homolog to the radula vesicles in Polyplacophora and Monoplacophora (Fig. 8D). Therefore these vesicles are a molluscan plesiomorphy. However, further study of the aplacophoran radula and its apparatus is needed. Larval forms The phylogenetic significance of larval forms in Spiralia is not addressed here. There is still no agreement on whether a pelagic organism gave rise to benthic forms U'.t,'.. Nielsen and Norrevang, 1985), or vice versa, and whether the trochophore larva arose once or several times (Ivanova-Kazas, 1985a, b, for careful discussions). Within Mollusca. Salvini-Plawen ( 1972, 1985) regarded the peri- calymma larva, which lacks purely larval organs except the swimming test and is found only in aplacophorans and protobranch bivalves, as the ancestral type. The questions are left here as unresolved and not affecting the arguments for homology of early cell fate among Eutro- chozoa, although my preference is indicated by use of the latter term. Classification of Extant Molluscan Classes With shell and spicules considered as synapomorphies for Conchifera and Aculifera, respectively, the following classification of extant Mollusca emerges: Phylum Mollusca Subphylum Conchifera Class Monoplacophora Class Bivalvia Class Gastropoda Class Scaphopoda Class Cephalopoda Subphylum Aculifera Class Polyplacophora Class Aplacophora Subclass Neomeniomorpha Subclass Chaetodermomorpha This arrangement is similar to that already proposed in the last century with little knowledge of the soft anat- omy of Monoplacophora. Garstang (1896) considered the 76 A. H. SCHELTEMA Aplacophora as "degraded" from an ancestral chiton-like form, but although he later stressed the importance of paedomorphosis in evolution, he did not see it as per- taining to Aplacophora. It is curious that a classification based on what are here inferred to be synapomorphies and on progenesis should be much the same as classifi- cations of a hundred years ago. Conclusions The hypotheses, arguments, and pieces of evidence presented here lead to the conclusions that Mollusca ( 1 ) are eucolomates with an ancestry in common with spiral- ian trochozoans; (2) are related to Annelida, but not as closely as they are to Sipuncula: (3) have a reduced coelom which was never segmented; (4) are not directly descended from an aplacophoran-like or turbellariomorph prede- cessor; and (5) are descended from an ancestor with serial replication. Acknowledgments The idea that aplacophorans may have evolved through progenesis originally came from David R. Lindberg. I have benefitted from critical discussions with Dave and with Carole S. Hickman, Bruce Runnegar, Claus Nielsen, Tom Waller, Douglas Eernisse, and Bertil Akesson, all of whom also steered me towards relevant literature. Doug Eernisse and Bruce Runnegar read an earlier version of this paper as well. Thanks are also due to Gerhard Haszprunar, who read the manuscript in its present form. Two reviewers most helpfully suggested literature that I had overlooked. I have tried to keep the phylogeny presented here as straightforward as criticisms and discussions suggested, and hopefully there is not too much "story telling." My gratitude goes to each of my critics. Mary E. Rice opened the way for an understanding of the Sipuncula-Mollusca relationships presented here, and the visit with her at the Smithsonian Marine Station at Link Port, Ft. Pierce, Florida, afforded the opportunity to work with both living pelagosphera larvae and apla- cophorans. I thank her deeply, and for prints of the splen- did photographs of pelagosphera. I thank Franz P. Fischer for providing me with a copy of his photograph of polyplacophoran epidermis, and Claus Nielsen for a copy of G. Gustafson's original draw- ings of Chaetoderma nitidulum larvae. As always. I gratefully acknowledge helpful discussions with Rudolf Scheltema, who has provided me space and who has always taken an energetic interest in my work. The following credits for previously published illustra- tions are acknowledged: figures 5A, C, D, 7C, 8A, and 1 1 from American Malacological Bulletin 6 (1988): 57-68, figs. 4, 5, 6, 13; figure 2A, B from American Zoologist 21 (1981): 605-619, figs. 4, 6; figure 3 from Smithsonian Contributions to Zoology 132 ( 1973): pi. 5; figures 6A, D, and 8D from Ve liger 34 (1991): 195-203, figs. 2C, 4D, and 5C; figure 6E from Zoomorphologie 94 (1980): 1 2 1 - 1 3 1 , fig. 3; figure 7 A, B from Biological Bulletin 169 (1985): 484-529, fig. 3L, O, P. Note added in proof: Two papers have just been published that have direct bearing on the ideas presented here. (1) Bengston S. 1992. The cap-shaped Cambrian fossil Maikhanella and the relationship between coelosclentophorants and molluscs. Lethaia 25: 401-420. 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Division chronology and development of the cell pattern in Dentalium 3)'3C> /o i _ ] 45(138)*- 3uu% JTTT 1 ' 62 (081)* 63% " ' i D47( 17 VS 10) " - 065(14 VS 8) 53(061)* 194% 041 (38VS 27) 073 16 VS 13) 68 (073)*233/ 093 ( 16 VS 13) L-l 2O r- CM fi 9 CM 3 f 8 =) d 100 _*_ j CO c f ~ _i ie K-) CO O ID -| d 51 g 2 f T i/i > S OJ QD 31 CO, _J r 1 ~ ^ d 16 l j5 CD i* 6 i P in o O Q c g J-T 3 49~1 o 5?^ o -i si 5 Co 9 CD ^ d 5] Figure 2. a. A hierarchy in the resorption between tour eolonies of generation II (refer to Fig. 1). The arrowheads point to the inferior partner. Numbers printed along the arrows refer to, respectively: days for complete resorption, zooid ratio (in parentheses, calculated as: the number of zooids in the inferior/superior partners on the day of fusion), percent increase or decrease of zooids in the superior partner from the day of fusion until a complete resorption of the inferior partner. The letter D refers to a case where a disconnection between the partners in a specific chimera occurs. In that case, the numbers along the arrow indicate: days from fusion to disconnection, number of zooids of the left colony on the day effusion vs. the number of zooids of the right colony (in parentheses). Disconnection between the partners within a Botryllus chimera is one of the variations in the outcome to chimera formation, resulting from unsuccessful fusion (Rinkevich and Weissman, 1989), reciprocal resorption (Rinkevich and Weissman, 1987a, 1989), or from a retreat growth phenomenon (Rinkevich and Weissman, 1988). These physiological-genetic-morphological parameters may lead to early separation between the partners before a complete resorption of the inferior partners in a chimera is obtained (Rinkevich and Weissman, 1988, 1989). The hierarchial tendency in the resorption phenomenon is, in most of the cases, already observed before separation between the candidates cancels this reaction. However, we did not count disconnection even when figuring hierarchy. In each such case, at least one additional chimera, where full resorption was accomplished, is assayed. It should be noted, however, that the incompleled results of disconnections are always in agreement with the results where resorption is completed. Subclone sizes may alter the direction of chimera resorption. However, this occurs only when ALLORECOGNITION RESPONSES IN COLONIAL INVERTEBRATE 85 the subordinate partner is much larger than the winner. All subclones used in the present study were matched to pairs with zooid ratios, below that may reverse the direction of resorption. b. Hierarchy in the resorption within the self-crossed offspring of generation III (the two left schemes) and within the detined-cross offspring of generation IV (refer to Fig. 1). The letter M refers to a case where the chimera dies. In that case, the numbers along the arrow indicate: days from fusion until the death of the chimera, number of zooids of the left and the right partners, respectively, on the day of fusion (in parentheses). A dashed arrow with an arrowhead points to a case where the direction of resorption is evident; however, the chimera either died or the partners disconnected before the resorption was completed. Additional subclones for doing new chimeras were absent; therefore, the hierarchy in resorption was not fully determined, c. Hierarchy in the resorption between generation II colonies and the self-crossed offspring of generation III. A dashed arrow without an arrowhead indicates a case where hierarchy is not evident before interactions of the partners in a specific chimera were interrupted by chimera mortality or disconnection. In those cases, no more chimeras were done because of the lack of additional subclones. d. 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(February, 1993) Classification and Characterization of Hemocytes in Styela clava TOMOO SAWADA 1 , JEFFREY ZHANG : , AND EDWIN L. COOPER Department of Anatomy and Cell Biology. School of Medicine, University of California, Los Angeles. California 90024 Abstract. Viable hemocytes of the common tunicate Styela clava are classified into four groups designated as eosinophilic granulocytes, basophilic granulocytes, hyaline cells and lymphocyte-like cells. Eosinophilic granulocytes, actively amoeboid, have large refractive granules that stain with neutral red. Basophilic granulocytes do not stain with neutral red and formed couplets or triplets. Hyaline cells, which often contain phagosomes. have electron-dense small vesicles recognizable only by electron microscopy. Hemoblasts have a characteristic large nucleolus which is visible by light microscopy. Eosinophilic granulocytes and hyaline cells actively ingest yeast particles in vitro. This classification simplifies former ones by correlating electron microscopy, with light microscopy, and viable with fixed hemocytes. Clearly viable tunicate hemocytes can be identified by simple methods. We have provided clear and more accurate descriptions which will lessen the con- troversy often associated with assigning hemocyte func- tions in immunodefense responses both in vivo and in vitro. Introduction The classification of tunicate hemocytes remains con- fused, not withstanding Wright's attempt ( 1 98 1 ) to devise useful categories. Recent progress in tunicate biology, however, requires a precise correlation between various cellular functions and particular types of hemocytes. Styela clava. especially, has been used in investigations of immunological responses including those associated with hemocytes: allogeneic reactions (Raftos and Cooper. 1991); cytotoxic reactions (Kelly et ai. 1992a); humoral Received 20 May 1992; accepted 9 November 1992. Present address: ' Department of Anatomy, Yamaguchi University School of Medicine, Ube-city, 755 Japan. 2 Undergraduate under the SRP program at UCLA. opsonin (Kelly et ai. 1992b, 1993a, b, in press); and the production of cytokines (Beck et ai. 1989; Raftos et ai. 1991). Humoral lectins (Yokozawa el ai. 1986; Harada- Azumi et ai. 1987). antibacterial substances (Azumi et ai. 1990), and a metallo-protease (Azumi el ai. 1991) were studied in another species, Halocynthia roretzi. Although the classification of Styela clava hemocytes began early (Ohue, 1936) and the site of hemopoiesis is described (Ermak. 1975. 1976), the literature includes de- scriptive morphologies with a plethora of terms, but rel- atively little experimental information uniting structure with function. Previous analyses of hemocytes failed to correlate age, season, and cell behavior in a systematic way, and these variables were not related to the various techniques used for examining them (e.g.. staining and fixation versus observation of live cells). Recent molecular and cytological studies focusing on the hemocytes and immune system of Stye/a will reveal a more precise picture of the functional contribution of individual effector cells. But this development depends on a thorough and con- sistent classification of the hemocytes. To establish an acceptable and predictable classification scheme, we examined hemocytes from Stye/a clava and correlated the morphological and behavioral character- istics of living hemocytes, and compared appearance of viable cells with those analyzed by light and electron mi- croscopy. Our work offers a strategy for classifying hem- ocytes in any invertebrate, especially tunicates which are becoming increasingly more important as we decipher the nature of effector cell activity during immune re- sponses. Materials and Methods Hcmocvtes Hemocytes were harvested by severing the stolons of Styela clava after rinsing the outside with 70% ethanol. 87 T. SAWADA ET AL Exuding hemolymph was collected into 0.5 M NaCl (NaCl-solution, pH 7.0 by 0.01 N NaOH) in polystyrene tubes; this prevented the nonspecific coagulation of he- mocytes and allowed individual hemocytes to be observed. Hemolymph was mixed with the NaCl solution one to one in final volume. Staining Hemolymph or hemocyte suspensions in NaCl-solution were loaded onto glass slides. After 10 min, adhering hemocytes were fixed for 15 min and stained with he- matoxylin and eosin (H&E). Cold ethanol, cold methanol or 4% paraformaldehyde (0. 1 M sodium cacodylate buffer, pH 7.0) were used as fixatives, and the morphological preservation was compared. For vital staining, neutral red (NR, 0.01% in final concentration) was added to hemocyte suspensions: 1 5-30 min later, the hemocytes were loaded onto glass slides and observed. Correlation of NR-staining with H&E-staining We photographed NR-stained hemocytes adhering on glass slides, then fixed them for regular light microscopy without moving the slides, and photographed them again under phase-contrast microscopy. After H&E-staining, we found exactly the same cells as in the former two pho- tographs (NR-staining and phase-contrast) to compare their appearance. Transmission electron microscopy (TEM) Hemocytes in the hemolymph and inside pharyngeal tissue were examined by TEM. Hemolymph collected into polystyrene tubes was centrifuged (400 X g for 5 min) and the pellet fixed. Pieces of pharynx (about 1.5 mm square) were dissected and fixed. Specimens were pre- fixed in a mixture of 2% glutaraldehyde and 2% parafor- maldehyde (0.75 M sucrose, 0.2 M sodium cacodylate buffer, pH 7.0), then post-fixed with 1% osmium tetroxide in the same buffer. The specimens were dehydrated in ethanol series and embedded in Medcast (Ted Pella, Red- ding, CA). Propylene oxide was used to infiltrate the resin. Autonomous fluorescence of viable hemocytes Hemocytes suspended in NaCl-solution were loaded on glass slides and observed with a Nikon EFD2 fluores- cence microscope with blue (420-490 nm)-and ultraviolet (330-380 nm)-illumination. Composition of hemocytes Different hemocyte types were counted by light mi- croscopy after H&E or NR-staining. and also by TEM. A sample of hemocytes was taken from 6 animals, and five to ten different viewing fields (1 10-130 cells in total) from each sample were examined in light microscopy with a 100X objective lens. Five pharyngeal pieces, one each from 5 animals (one TEM-section for each piece), and a hemocyte-pellet from one animal were examined by TEM. About one hundred cells were examined on each section. Phagocytic activity against yeast particles Saccharomyces cerevisiae (baker's yeast, type II; Sigma Chemicals, St. Louis, MO) was stained with Congo red and suspended in artificial seawater (approximately 1 X 10 8 particles/ml), according to Kelly et ai (1993a). Hemocyte suspensions in NaCl-solution (100 p\) were loaded on cover slips, and yeast particle suspension (100 ^1) was added 5 min later. The hemocytes were incubated for 30 min. After the cover slips were gently rinsed to remove excess yeast particles, 0.01% neutral-red solution was added. Hemocyte types were identified by NR-stain- ing. Types of hemocytes which phagocytized yeast par- ticles were identified. Results Light microscopy of hemocytes Most hemocytes adhered to glass slides, and some of them exhibited amoeboid movement within 5 min. How- ever, many small, transparent cells did not adhere well enough to resist water movement caused by pressure on the cover slip. By phase contrast microscopy, four different types were observed (Table !):(!) hyaline cells, which ex- hibited significant extensions ( 1 5-20 j/m in diameter); (2) round cells (basophilic granulocytes, 6-10 ^m in diam- eter), which contained highly refractive small granules and often formed couplets or triplets; (3) amoeboid cells (eo- sinophilic granulocytes; 8-15 /jm in diameter), which contained large granules and exhibited more active amoeboid movement than the other types; and (4) small spherical cells that did not spread (hemoblasts; 4-6 ^m in diameter), which contained a small amount of cyto- plasm and had a nucleolus clearly visible by light mi- croscopy. The nuclei of hemocytes other than hemoblasts were not visible unless they were spread flat on a glass slide. Phase contrast microscopy was not sufficient to distin- guish all eosinophilic and basophilic granulocytes with certainty. The eosinophilic granulocytes often contained granules as small as those of basophilic granulocytes, and when they were not moving they were just as round as basophilic granulocytes. We observed large cells that had a hyaline cytoplasm lacking visible granules, but they did contain pigmented or non-pigmented large vacuoles. These cells were also identifiable as hyaline cells because they spread wide and flat. The spreading of hyaline cells was rapid once it began, and these cells did not exhibit active amoeboid movement after they spread. HEMOCYTES OF TUNICATE Table I Classification and sonic characteristics o/'Styela clava licmocvies 89 Size 8 ! 5 ^m 610 f*m 15-20 JOT 4-6 j/m H&E-staining Orange Purple Very weak purple or Purple NR-staining Orange or red-violet pink Negative or orange at vacuoles Granules in LM Many retractile Many small G refractile Granules in TEM Not uniform heterogeneous Uniform spherical Small vesicles _ electron dense Adhesion to glass + + ii Phagocytosis + + + + + + Other characteristics Active amoeboid movement Forming couplets or Widely spread (cell Nucleolus visible in LM * Blue fluorecence in red-violet cells aggregates fusion?) Previous classifications Compartment signet-ring vesiculated Finely granular Hyaline signet-ring? Hyaline lymphocyte-like morula coarsely granular amoebocyte hemoblasts Under UV-illumination (330-380 nm). NR-staining and some characteristics of viable cells NR mainly stained the cytoplasmic granules of amoe- boid cells (Fig. 1 ). Hyaline cells were usually not stained except for some cases in which small or large cytoplasmic vacuoles were stained (Fig. IE, F). Two groups of amoe- boid cells were positively stained by NR (eosinophilic granulocytes), but the intensity differed. One was stained a dense red- violet, whereas the other was orange (Fig. 1 ). Both types of cells contained 5-20 large cytoplasmic granules, and they were morula-shaped before starting amoeboid movement. Other granular cells were usually round, unstained by NR, and contained highly refractive small granules (Fig. 1; basophilic granulocytes). Most of the hemoblasts were not stained (Fig. 1 ). but a few some- times stained faintly orange. Following staining with NR. hemocytes were easily distinguished and their characteristic behaviors examined. Both types of NR-positive granulocytes (eosinophilic granulocytes) were active in amoeboid movement, ex- tending many spine-like pseudopodia. NR-negative gran- ular cells (basophilic granulocytes) were less active in amoeboid movement, but extended long pseudopodia. These cells were often observed as couplets (Fig. ID), triplets or small aggregates composed only of this type of cell, and they did not separate once they came in contact. Occasionally, these cells spread flat after 30-60 min in- cubation. H&E-staining We used three different methods to observe hemocytes with H&E-staining after fixation. Ethanol and methanol significantly modified hemocyte morphology, so only paraformaldehyde fixation was utilized. We observed the same cells with NR-staining and H&E-staining. Two NR-positive amoeboid cells (red-violet and orange cells) were stained intensely red or pink. Both cells con- tained various sizes of cytoplasmic granules that stained with eosin, so both cells were classified as eosinophilic granulocytes. The appearance of the cytoplasmic granules was altered by fixation, especially by ethanol and meth- anol. Cells that were fixed with these agents appeared as vacuolated cells, granular cells, compartment cells or sig- net-ring cells. NR-negative amoeboid cells that contain small refrac- tive granules were stained purple with H&E and were des- ignated as basophilic granulocytes. Their cytoplasmic granules were no longer evident after fixation, and cyto- plasmic staining was relatively weak after they spread on slides. However, the nuclei of basophilic granulocytes were smaller and more dense than those of hyaline cells. The cytoplasm of hyaline cells was very thin after spreading so H&E stained them only weakly purple with some orange-stained cytoplasmic vesicles. Phagocytic vacuoles in some of them were stained red. Some hyaline cells contained large cytoplasmic vacuoles and thus ap- peared as signet-rings. Also their nuclei became larger as they spread. A few encapsulations of several small cells were ob- served (small encapsulation; Fig. 2). In addition, small numbers of multinuclear cells were observed in H&E- staining (Fig. 2). These cells spread flat and contained several large nuclei and small vesicles stained with eosin. Finally, hemoblasts stained purple, and their nucleoli be- came unclear after fixation. TEM oj hemocytes In centrifuged pellets of hemolymph, we observed five different hemocyte types (Figs. 3, 4): (1) large cells 90 T. SAWADA ET AL bg hy Figure 1. Living hemocytes on glass slides after NR-staining. (A) Eosinophilic granulocytes included two groups of granulocytes that stained in different colors (o = orange and r = red-violet). The sizes of the cytoplasmic granules are variable in each hemocyte. (B) Neither hemoblasts (hb) and basophilic granulocytes (bg) were stained. Nucleoli were evident in hemoblasts. (C) Basophilic granulocytes (bg) contained many refractive granules which were smaller than those of eosinophilic granulocytes (o = orange cells). (D) A couplet of basophilic granulocytes (bg): these were frequently observed. (E) Hyaline cells (hy) spread wide and flat on the glass slide to form a thin cytoplasmic sheet. (F)Some hyaline cells contained granules (arrow) that stained with NR. x 1250 (10-12 ^m in diameter: hyaline cells) containing signifi- cant amounts of endoplasmic reticulum (ER); (2) small spherical cells (5-6 /urn in diameter: hemoblasts) with little cytoplasm and large nuclei; (3-5) three different granu- locytes (6-12 nm in diameter: basophilic and eosinophilic granulocytes) containing abundant cytoplasmic granules. These five types also constituted the entire hemocyte pop- ulation within the pharyngeal tissue. The large cells contained numerous rough-surfaced and smooth-surfaced ER and also small vesicles (0.1 ^m in HEMOCYTES OF TUNICATE 91 A , Figure 2. Fixed hemocytes on glass slides with H&E-staining. (A) Multinuclear cells with seven nuclei spread wide and flat. Vesicular structures in the cytoplasm were slightly stained. (B) Small encapsulation (arrow) containing 3-7 small cells; these were sometimes observed in the hemolymph. xl 180 diameter) of high electron density (Fig. 3). A few of these cells contained large vacuoles or phagosomes. Their nuclei often had nucleoli and coarse and uniform euchromatin, although heterochromatin was sometimes observed. These cells corresponded to hyaline cells on the basis of size, phagosomes, and the absence of large cytoplasmic gran- ules. The small cells with little cytoplasm contained mito- chondria and small amounts of ER (Fig. 4C). Their nuclei, with characteristic large nucleoli, were usually larger than those of other hemocytes. Chromatin was uniformly dis- tributed and slightly more dense in comparison with hya- line cells. These cells corresponded to hemoblasts in cell size, i.e., little cytoplasm and characteristically large nu- cleoli. The three different granulocytes (temporarily designated as type 1, 2 and 3 granulocytes according to TEM) had the same nuclear pattern (usually with dense heterochro- matin at the periphery and sometimes small nucleoli) but differed in their cytoplasmic granules. Type 1 granulocytes (6-10 yum in diameter) contained electron-dense and spherical granules with a diameter range of 0.2-0.5 ^m (Fig. 4A). These cells correspond to basophilic granulo- cytes on the basis of size, the sizes of their cytoplasmic granules (they had smallest granules among granulocytes), and their frequency in the hemolymph. Type 2 gmnit/o- cytes (8-10 /urn in diameter) contained irregular-shaped granules that varied in size (0.1 -1.3 /urn in diameter). The granules contained homogeneous material of intermediate electron-density (Fig. 4B). Type 3 granulocytes ( 8- 1 2 ^m in diameter) had cytoplasmic granules that were also ir- regularly-shaped and remarkably varied in size (0.1-1.5 nm in diameter). These granules were composed of het- erogeneous materials central spheres with high electron density and surrounding material of intermediate electron density (Fig. 4B). Type 2 and 3 granulocytes corresponded to eosinophilic granulocytes on the basis of size, the ir- regular shape of their cytoplasmic granules, and their fre- quency of occurrence. Hemocyte composition We examined percentages of the various hemocytes in hemolymph by counting each type after NR- and H&E- staining and TEM (Table II). The order of dominance for each type was the same in all cases, but the exact values were somewhat different. The most abundant cells were eosinophilic granulocytes (46.3% in NR-staining); second were the basophilic granulocytes (21.0%); hyaline cells were third ( 18.5%-): and the smallest population was that of the hemoblasts (14.1%). The percentage of eosinophilic cells in H&E-staining (68.5%) was about the same as the sum of type 2 and 3 granulocytes in the hemocyte pellets observed by TEM (67.8%), but it was larger than the sum of orange- and red-violet cells in NR-staining (46.3%). Many fewer he- moblasts were found in both H&E-staining (2.3%.) and TEM (2.0%) than in NR-staining (14.1%). The proportion of hyaline cells ranged from 5.5 to 18.5%, even after the percentages of multinuclear cells and phagocytosis were added. Multinuclear cells (1.2% in H&E-staining) were not found in NR-staining or TEM of pharyngeal tissue. A utonomous fluorescence oj hemocytes Blue fluorescence was observed in certain granulocytes under ultraviolet-illumination. NR-staining of those flu- orescent hemocytes, in the same field of view, revealed that the autonomous fluorescence was from eosinophilic granulocytes which stained in red-violet (Table I). Under blue-illumination, no hemocytes exhibited autonomous fluorescence. Phagocytosis Four hemocyte types i.e., hyaline cells, eosinophilic granulocytes (including red-violet and orange cells in 92 T. SAWADA ET AL m t!+^& .. 'JQratfSH * ' : * *. '?& . Figure 3. Transmission electron microscopy of hyaline cells and a hinuclear cell. (A) Hyaline cell (h) in the centrifuged pellet, with heterochromatin at the nuclear periphery. (B) A binuclear cell in the centrifuged pellet. (C) Hyaline cell (h) in pharyngeal tissue; the nucleus has a large nucleolus and uniform euchromatin. All cells (A, B, C) contained electron-dense small vesicles, numerous vesicular structures, and endoplasmic reticulum. Bar = 1 jim. NR-staining). and basophilic cells ingested yeast parti- cles. Among them, hyaline cells and eosinophilic granu- locytes had significantly higher activity than basophilic granulocytes (Table III). In the cell population that had ingested yeast particles, hyaline cells (36-42%) were fewer than eosinophilic granulocytes (5 1-68%), as shown in Ta- ble IA. However, phagocytic activity was higher in hyaline cells, because the phagocytic ratios were higher in hyaline cells (32-78%) than in eosinophilic granulocytes (13- 35%), as shown in Table IB. Many hyaline cells engulfed 2-5 yeast particles, whereas most eosinophilic granulo- cytes incorporated only one particle. Discussion Classification of hemocytes Hemocytes from many species of tunicates have been classified by both light and electron microscopy (Ohue, 1936; George, 1939;Endean, 1960; Andrew, 1961, 1962; Overton, 1966; Smith, 1970; Botte and Scippa, 1977; Figure 4. Transmission electron microscopy of hemocytes in the centnt'uge pellet of hemolymph. (A) Type 1 granular cells ( 1 = basophilic granulocytes) containing relatively uniform and spherical granules. (B) Both type 2 (2) and 3 (3) granular cells (eosinophilic granulocytes) containing irregularly shaped granules. The granules of type 3 cells contain electron dense cores. (C) Hemoblast (hb) with little cytoplasm and without cytoplasmic granules, except for mitochondria and vesicles. The relatively large nucleus contains characteristic large nucleolus. Bar = 1 ^m. 93 94 lli'inncvtc composition examined under different conditions T. SAWADA ET AL Table II Hemocyte types Viable cells (NR-staining) Fixed cells (H&E-staining) EM (pellet) EM (pharynx) Eosinophilic Orange 68.5 5.6% 38.9% 15.8 7.5% granulocytes 16.5 5.0% Red-violet 28.9 32.8 11.0 29.8 8.5 Basophilic 21.0 5.1 21.1 4.4 2 1 .0 21.3 7.0 granulocytes Hyaline cells 18.5 7.9 5.5 2.9 8.0 16.8 3.8 Hemoblasts 14.1 3.2 2.3 2.2 2.0 8.5 6.0 Multinuclear cells 0.0 1.2 1.2 0.2 0.0 Cells of 0.1 0.2 1.4 1.2 1.1 2.3 1.6 phagocytosis No. of individuals 6 6 1 5 examined average S.D. Milanesi and Bunghel, 1978; Fuke. 1979, 1980: Rowley, 1981, 1982; Mukai et ai, 1990), and in morphological terms, such as vacuolated or granular cells, hyaline cells, hemoblasts or lymphocytes (Wright, 198 1 ). or by functions (Freeman, 1964; Fuke, 1980; Fujimoto and Watanabe, 1976; Burighel et ai, 1976; Rowley, 1983; Azumi et al, 1 990, 1 99 1 ; Raftos et al. . 1 990; Raftos and Cooper, 1 99 1 ). However, the inapplicability of these classifications from one species to the next, and the lack of correspondence between different methods (e.g., light versus electron mi- croscopy) have produced confusion. Hemocytes of S. clava have been classified into morula cells, compartment cells, signet-ring cells, granular amoe- bocytes, hyaline cells and lymphocyte-like cells (Ohue, 1936; Wright, 1981). But, among fresh and living hemo- cytes, we observed no signet-ring cells nor any cells with a stable, morula shape. Instead, there were granulocytes that frequently changed their appearance during amoe- boid movement. They appeared morula-like when they rounded up, and could be compartment cells or granular amoebocytes after they had become extended and flat- tened. Fixation, especially with ethanol or methanol, modified hemocyte morphology significantly, and some of the eosinophilic granulocytes and hyaline cells became signet-ring in shape. Therefore, we adopted two cautious guidelines. First, we avoided using such terms as morula, compartment, or signet-ring. Second, we employed no Wright- or Giemsa staining because they require methanol as the fixative. Instead, we preferred to use formaldehyde fixation and H&E-staining. We identified five different hemocyte types by vital NR- staining and TEM, and four types by H&E-staining of fixed cells. We estimated that the granules of the orange cells in NR-staining contain less dense material, and so correspond to type 2 granulocytes in TEM; similarly red- violet cells in NR-staining correspond to type 3 granu- locytes in TEM. The difference between type 2 and 3 cells, or between orange and red-violet cells, is not significant enough to separate them into two cell types. Moreover, both the orange and red-violet cells evidently correspond to eosinophilic granulocytes in H&E-staining. These two granulocytes appear to be similar in amoeboid movement and phagocytic activity. Therefore, we classified both of them into the same group as eosinophilic granulocytes. We suggest that type 2 granulocytes (orange cells) are an earlier stage in cell differentiation than type 3 granulocytes (red-violet cells). The correspondences between the light microscopical and TEM images of basophilic granulocytes (type 1 gran- ulocytes in TEM), hyaline cells, and hemoblasts were clear on the basis of their morphological characteristics and their frequencies of appearance. Multinuclear cells were classified as hyaline cells for the following reasons: (1) morphologically multinuclear cells are in all other respects similar to hyaline cells; (2) they sometimes contain large, eosinophilic vacuoles that we assume to be phagosomes; (3) the morphology and behavior of hyaline cells are quite similar to phagocytes type 1 (pl-ceQs)ofHalocynthiaroretzi(H. rorelii), which evidently fuse together and form multinuclear cell sheets (Sawada el al., 1991). But, we have no strong evidence for cell fusion between the hyaline cells of S. clava. More- over, the frequency of multinuclear cells in fresh hemo- lymph is not clear, because they could be identified only after spreading on glass. Both of these points require fur- ther investigation. In this study, therefore, we have identified four hemo- cyte types in S clava. ( 1 ) Eosinophilic granulocytes con- tain several refractive vacuoles that appeared red in neutral red vital stain, red by H&E. and exhibit active amoeboid HEMOCVTES OF TUNICATE 95 Table III Phagocytosis / yeast particles by hemocytes from three Jil/erent individuals (A) Composition of hemocytes which ingested yeast particles Eosinophilic granulocytes Total Basophilic Hyaline cells Animals (red-violet)* 1 (orange)* 1 granulocytes cells Hemoblasts examined a* 2 29.2% 22.1% 6.2% 42.5% 0.0% 113 b 41.0 11.0 10.0 38.0 0.0 100 c 41.6 16.8 5.0 36.6 0.0 nil (B) Phagocytosis against yeast particles within each kcinocyte type Hemocyte types Animals Ingesting cells Non-ingesting cells Total cells examined eosinophilic granulocytes a* 2 19.4% 80.6% 108 (red-violet) b 30.8 69.2 52 c 12.9 87.1 101 (orange) a 16.4 83.6 110 b ND* 3 ND ND c 1.6 98.4 61 basophilic granulocytes a 7.3 92.7 124 b 3.8 96.2 53 c 0.0 100 57 hyaline cells a 78.0 22.0 100 b 63.6 36.4 22 c 32.8 67.2 61 *' Two sub-populations of eosinophilic granulocytes different in colors of NR-staimng are indicated in parenthesis. * : Animals (a. b. c) in Table A correspond to the animals in Table B. * 3 No data. movement and phagocytosis. (2) Basophilic granulocytes contain numerous small granules that do not stain with neutral red, are purple in H&E, and form specific aggre- gations with the same cell type. (3) Hyaline cells contain fine electron-dense granules in TEM, occasionally contain phagosomes that stain red with neutral red and H&E, and extend into thin circular sheets on glass. (4) Hemoblasts possessed little cytoplasm, large nucleoli visible by light microscopy, but adhere only weakly to glass. Possible cor- respondence between former classifications are shown in Table I. Functions and characteristic behavior of each hetnocyte type Phagocytosis, as is well known, is a ubiquitous and im- portant immuno-defense response found throughout the animal kingdom. Hyaline cells exhibited the highest phagocytic activity, and some of them engulfed more than five yeast particles. Eosinophilic granulocytes were less active than hyaline cells, but they accounted for the largest population because of their abundance and active motility. Hyaline cells were the most likely candidates for ef- fecting encapsulation of larger particles by their ability to spread and form flat sheets and to fuse together into larger multinuclear sheets. Hemoblasts have been referred to as lymphocyte-like cells (Wright, 198 1 ) and as proliferative stem cells (Ermak, 1976). We also observed the charac- teristically large nucleolus also in viable cells and con- firmed their equivalents by light (Wright, 1 98 1 ) and elec- tron microscopy (Ermak, 1976). Motility was also an important and definitive, behav- ioral characteristic. Only eosinophilic granulocytes ex- hibited active movement. In contrast, the basophilic granulocytes did not separate after once contacting others, which resulted in the formation of couplets or triplets. This behavior continues when augmented, resulting in small aggregates. Similar behavior was also observed on gl -cells of//, roretii (Sawada et ai, 1991), and we suggest the presence of common granulocytes that can form spe- cific aggregates within the same cell type. Correspondence to the hemocytes in other tunicate species Hemocyte types found in many species have been cat- egorized into several groups by Wright (1981). However, the hemocytes of a single category often include several different types. In addition, certain hemocytes of one spe- cies are apparently absent in other species. It would not be instructive to compare only morphological aspects of hemo- cytes, and only under a single condition, such as in paraffin 96 T. SAW A DA ET AL sections. Observations of living hemocytes, under different conditions and stained with simple dye, coupled with func- tional analysis, e.g.. of phagocytosis, would be more useful. In such a manner, we compared the hemocytes of St vela clava and Halocvntlria rorctzi which have also been clas- sified in the living state (Sawada et ai, 1991 ), and found interesting correspondences between types. Hyaline cells and basophilic granulocytes were similar to the pi -cells and gl -cells of Halocynthia roretzi. respectively, in mor- phological and behavioral aspects. Hemoblasts, as the candidate for hematopoietic stem cells, may correspond to the ly-cells of Halocynthia roretzi, but their function as the stem cells has not been established in either species. Eosinophilic granulocytes seemed to be similar to the v3- and v4-cells of Halocynthia roretzi in that refractive vacuoles occupy most of the cell volume, and active amoeboid movement and addphilic staining occur. But eosinophilic granulocytes ofStyela clava were evidently more phagocytic. The correspondence between these species of at least two to three cell types may be consistent with their phylogeny. Acknowledgments We thank Sharon Sampogna and Monica Eiserling for technical help in light and electron microscopy. This study was supported by the National Science Foundation (Grant #DCB 90 05061). Literature Cited Andrew. VV. 1961. Phase microscope studies of living blood-cells of the tunicates under normal and experimental conditions, with a description of a new type of motile cell appendage. Q. .1. Micr. Sci. 102: 89-105. Andrew, VV. 1962. Cells of the blood and coelomic fluids of tunicates and echinoderms. Am. Zoo/. 2: 285-297. Azumi, K., H. Yokozawa, and S. Ishii. 1990. Halocyammes: novel anti- microbial tetrapeptide-like substances isolated from the hemocytes of the solitary ascidian. Halocynthia roretzi Biochemistry 29: 159-165. Azumi, K., H. Yokozawa, and S. Ishii. 1991. Lipopolysacchande in- duces release of a metallo-protease from hemocytes of the ascidian, Halocynthia roretzi. Dev. Comp. Immune/ 15: 1-7. Beck, G., G. R. Vasta, J. J. Marchalonis, and G. S. Habicht. 1989. 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The characterization of granular amoebocytes and their possible roles in the asexual reproduction of the polystyelid ascidian, Poly:oa vesiciiliphora. J. Morphol 150: 623-638. Fuke, M. T. 1979. Studies on the coelorruc cells of some Japanese ascidians. Bull. Mar. Biol. Sin. Asamushi, Tohoku University 16: 143-159. Fuke, M. T. 1980. "Contact reactions" between xenogeneic or allo- geneic coelomic cells of solitary ascidians. Biol. Bull. 158: 304-315. George, VV. C. 1939. A comparative study of the blood of the tunicates. Quart. J Micr Sci. 81: 391-431. Harada-Azumi, K., II. Yokozawa, and S. Ishii. 1987. N-acetyl-galac- tosamine-specific lectin, a novel lectin in the hemolymph of the ascidian Halocynthia roretzi: Isolation, characterization and com- parison with galactose-specific lectin. Com. Bioclwm. Physiol 88B: 375-381. Kelly, K. L., E. L. Cooper, and D. A. Raftos. 1992a. In vitro allogeneic cytotoxicity in the solitary urochordates. J. Exp. Zool. 262: 202-208. Kelly, K. L., E. L. Cooper, and D. A. Raftos. 1993a. A humoral opsonin from the solitary urochordate Styela clava. Dev Comp. Imnumol. 17: (in press). Kelly, K. L., E. L. Cooper, and D. A. Raftos. 1992b. Purification and characterization of a humoral opsonin from the Styela clava. Comp. Biochem. Physiol 103B: 749-753. Kelly, K. L, E. L. Cooper, and D. A. Raftos. 1993b. Cytokine-like activities of a humoral opsonin from Styela clava. Zool. Sci. (in press). Milanesi, C., and P. Burighel. 1978. Blood cell ultrastructure of the ascidian Bolryllux schlosseri. I. Hemoblast, granulocytes macrophage. morula cell and nephrocyte. Acta Zool 59: 135-147. Mukai, H., K. Hashimoto, and H. VV atanabe. 1990. Tunic cords, glom- erulocytes, and eosinophilic bodies in a styelid ascidian. Polyandro- carpa misakiensis. J. Morphol. 206: 197-210. Ohue, T. 1936. On the coelomic corpuscles in the body fluid of some invertebrates. III. The histology of the blood of some Japanese as- cidians. Sci. Rep Tohoku L'niv. 11: 191-206. Overton, J. 1966. The fine structure of blood cells in the ascidian Per- ophora viriilix. J. Morph. 119: 305-326. Raftos, D. A., D. L. Stillman, and E. L. Cooper. 1990. //; vitro culture of tissue from the tunicate Styela clava. In Vitro Cell Dev Biol 26: 962-970. Raftos, D. A., and E. L. Cooper. 1991. Proliferation of lymphocyte- like cells from the solitary tunicate. Styela clava. in response to al- logeneic stimuli. J. Exp. Zool. 260: 391-400. Raftos, D. A, E. L. Cooper, G. S. Habicht, and G. Beck. 1991 . Invertebrate cytokines Tunicate cell proliferation stimulated by an interleukin-1- like molecule. Proc. Nat. Acad. Sci. USA 88: 9518-9522. Rowley, A. F. 1981. The blood cells of the sea squirt, dona intestinalis: morphology, differential counts and in vitro phagocytic activity. J. Invertebr. Palhol. 37:91-200. Rowley, A. F. 1982. Ultrastructural and cytochemical studies on the blood cells of the sea squirt, dona intestinalis. I. Stem cells and amoebocytes. 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(February, 1993) Effects of Cations on the Volume and Elemental Composition of Nematocysts Isolated from Acontia of the Sea Anemone Calliactis polypus MICHIO HIDAKA AND KIWAMU AFUSO Department of Biology, University of the Ryiikyus, Nishihara Okinawa, 903-01 Japan Abstract. The hypothesis that exchange of intracapsular divalent cations with Na + in seawater increases the inter- nal osmotic pressure during discharge of nematocysts of marine cnidarians was tested by examining effects of ex- ternally applied cations on the volume and elemental composition of nematocysts isolated from acontia of the sea anemone Calliactis polypus. The volume of isolated nematocysts increased with increasing concentrations of cations if the cation was monovalent but appeared to de- crease if the cation was divalent. Ca 2+ reduced the internal osmotic pressure of the nematocysts more efficiently than Mg 2+ . X-ray microanalysis of nematocysts incubated in 1 M solutions of various salts showed that Ca 2 + in isolated nematocysts was only partially replaced, if at all, by ex- ternally applied Na + and Mg 2+ while most Mg :+ was re- placed by Na + and Ca 2+ . The present results suggest that exchange of intracapsular divalent cations with external monovalent cations increases the internal osmotic pres- sure, and that selective binding of Ca 2+ to polyanions in the capsule decreases it. Whether the increase in the in- ternal osmotic pressure caused by the cation exchange is large enough to trigger discharge remains to be investi- gated. Introduction Lubbock and his colleagues proposed that loss of Ca 2+ from a nematocyst increases the osmotic pressure of the intracapsular fluid and thus causes discharge of the ne- matocyst (Lubbock and Amos, 1981; Lubbock el ai, 1981; Gupta and Hall, 1984). They proposed that poly- peptides in undischarged nematocysts are crosslinked by Ca 2+ to form polypeptide chains and that the release of Received 14 May 1992; accepted 20 October 1992. calcium from the nematocyst dissociates the polypeptide chains, thereby increasing the number of osmotically ac- tive molecules. Because of this report, Ca 2i has been con- sidered to play a major role in nematocyst discharge. Recently Weber (1989) demonstrated that naturally occurring cations of Hydra nematocysts can be replaced by externally applied cations. Nematocysts loaded with other cations generally retain discharge capabilities. Gerke d t/l. ( 199 1 ) found that in situ nematocysts of Hydra con- tained high concentrations of potassium (K) instead of calcium (Ca). These observations suggest that Ca 2+ is not indispensable for the discharge of certain kinds of ne- matocysts. Weber (1989) proposed that Hydra nematocysts can be considered as Donnan-equilibrium dominated osmotic systems and that cations associated with polyanions in the capsule, rather than polyanions themselves, contribute to high intracapsular osmotic pressure. Because Hydra nematocysts contain high concentrations of K (Gerke et a/., 1991 ) and are surrounded by a membrane that might serve as a diffusion barrier against ions of low molecular weight (Lubbock et a/., 1981), nematocysts of Hydra might be in equilibrium with high concentrations of K + . If such nematocysts are exposed to freshwater as a result of exocytosis, the osmotic pressure difference across the capsule wall would increase, leading to the discharge of the nematocysts. Indeed, isolated Hydra nematocysts im- mersed in concentrated NaCl or KC1 solutions swell up to 1 1 5% of the original volume and tend to discharge when the external concentration of the salts is lowered (Weber, 1989). The above process, however, may not account for the discharge of nematocysts of marine cnidarians, because nematocysts of marine cnidarians must discharge in sea- water, which contains high concentrations of salts. X-ray 97 98 M. HIDAKA AND K.. AFUSO microanalysis of frozen sections of various marine cni- darians show that the predominant cation of nematocysts /// situ is either Ca :+ , Mg : + , or K* (Tardent el a/.. 1990). If nematocysts of marine cnidarians also behave as Don- nan-equilibrium dominated systems, exchange of intra- capsular cations with cations in seawater will occur when nematocysts are exposed to seawater as a result of exo- cytosis. In Ca- or Mg-containing nematocysts, the ex- change of divalent cations in the capsule with monovalent cations such as Na + in seawater might increase the internal osmotic pressure, since one divalent cation is replaced by two monovalent cations to maintain electroneutrality. If the increase in the internal osmotic pressure is large enough, the nematocysts would discharge. The purpose of the present study is to examine the hypothesis of divalent-monovalent cation exchange. Un- discharged nematocysts isolated from various cnidarians contain high concentrations of Ca and Mg (Weber et a!., 1987; Mariscal, 1988; Hidaka, 1993). These isolated ne- matocysts provide a useful model for studying the re- sponses of Ca- and/or Mg-containing nematocysts to var- ious cations. We determined the effects of mono- and divalent cations on the volume of nematocysts isolated from acontia of the sea anemone Calliactis polypus. We also studied whether Ca 2+ and Mg 2+ found in the isolated nematocysts could be replaced by externally applied cat- ions as in Hydra nematocysts (Weber, 1989). Materials and Methods Specimens of Calliactis polypus on the shells of hermit crabs belonging to the genus Dardanus. were collected from the reef around the Okinawa island, and maintained in an aquarium supplied with a subgravel filter. The her- mit crabs were fed with chopped Tapes every 2-4 days. The anemones were used as a source of acontial nema- tocysts 1-3 days after feeding. Acontial filaments were obtained by prodding the sea anemone with blunt-tipped forceps. Undischarged basitrichous isorhiza nematocysts were isolated either in artificial seawater (ASW) or in distilled water (DW), since nematocysts isolated in ASW and those isolated in DW display different discharge capabilities (Hidaka and Mariscal, 1988). A piece of acontium was placed in a drop of ASW or DW on a glass slide. The glass slide was treated with a drop of a 0.1% solution of poly- 1-lysine (Sigma; approx. mol. wt. 90,000) in distilled water for 10 min in a wet chamber prior to use (Mazia et ai, 1975). Two strips of thin adhesive tape (Scotch 3M) were placed on both sides of the drop to make a narrow space between the glass slide and a cover slip, and to make it easy to replace the solution in this space. When nema- tocysts were isolated in ASW, the acontium was squashed under a cover slip. Only a small percentage (about 5%) of nematocysts discharged during this procedure, and most of them only partially discharged eversion of the tubule stopped halfway. Cellular debris and partially discharged nematocysts were removed by washing the squashed acontium with a few drops of ASW. Most of the nema- tocysts isolated in this manner from acontia ofCalliaclis tricolor discharged when immersed in 5 mAf EGTA (Hi- daka and Mariscal, 1988), suggesting that the isolated ne- matocysts are functional. When nematocysts were isolated in DW, the acontium was immersed in a drop of DW for 5 min and then the remaining acontium was removed. The extruded undischarged nematocysts were allowed to settle onto the glass slide for 10 min. Then, the isolated nematocysts were washed with more than five drops of ASW or DW to remove unattached nematocysts. Photomicrographs of nematocysts were taken in ASW or DW using a plan objective lens (X 100). Then, test so- lutions were applied by perfusing the nematocysts with at least eight drops of each test solution (Hidaka and Mar- iscal, 1988). Nematocysts isolated in ASW were treated with decreasing concentrations of salt solutions, that is, 1000, 100, 10, 1, and mAf solutions. Nematocysts iso- lated in DW were treated with increasing concentrations of salt solutions. Nematocysts were immersed in each test solution for 10 min, because changes in the volume of isolated nematocysts in solutions with or without Ca 2+ were completed within 10 min (Hidaka, 1992). After 10 min a pair of photomicrographs of the nematocysts was taken for each test solution. The length and diameter of nematocysts capsules were measured to the nearest 0.1 mm (corresponding to 0.05 /*m) on two photomicrograph prints using calipers. The average value was used for each capsule. The volume was calculated assuming that the capsule was an ellipsoid. The volume of nematocysts im- mersed in test solutions was normalized by the original volume of the nematocysts in ASW or DW, and expressed as relative volume. The original volume (mean SD) was 1 17.7 18.8 Mm 1 (n = 37) in ASW-isolated nematocysts and 100.7 10.1 M' (n = 37) in DW-isolated nema- tocysts. For each salt solution, three or four experiments were performed and at least seven nematocysts were mea- sured. The significance of regression of the relative volume of nematocysts on log (salt concentration) was tested in each salt solution. When the regression was not significant, the relative volume at 1 M salt concentration was com- pared with that at 1 mAf using Duncan's multiple range test. The difference in the relative volume of nematocysts was tested among pairs of cations at 1 M salt concentration using the multiple range test. For substitution experiments, nematocysts were isolated by immersing 20 acontia in 5 ml of DW for 5-10 min. Remaining acontial tissues were removed by filtering the nematocyst suspension through 60 ^m nylon mesh. Ali- quots (0.8 ml) of the filtrate were placed in each of six EFFECTS OF CATIONS ON NEMATOCYSTS 99 microtubes and centrifuged at 1940 X g for 5 min. One ml of each test solution was added to the pellet. Nema- tocysts were resuspended in the test solutions and allowed to stand for 10 min. Test solutions were ASW and 1 M solutions of NaCl, KG, CaCl : , MgCl 2 , and SrCl 2 . Next, the nematocysts were washed in DW by centrifuging at 1940 X g for 5 min and by resuspending the nematocysts in 1 ml of DW. The nematocysts were washed in DW and collected by centrifugation three times. Finally, the nematocysts were resuspended in 1 50 n\ of DW. Aliquots (20 n\) of the nematocyst suspension were placed on meshes with formval or collodion membranes that had been coated with carbon and treated with poly-1-lysine. The nematocysts were allowed to settle on the membrane for 1 h, then air-dried after the remaining solution was soaked up with a piece of filter paper. Specimens were observed under a scanning transmission electron micro- scope (JEOL JEM-2000EX) equipped with an energy dis- persive spectrometer (TN 42 U). X-ray spectra were ac- quired at an acceleration voltage of 100 kV. Semiquan- titative elemental analyses were performed using an application software (Noran Instruments Inc. SMTP) on 4-6 nematocysts for each test solution. The software, which was designed for standardless semiquantitative analysis of metallurgical thin films, removes background and integrates peak areas. The peak intensities were con- verted to ratios of element concentrations by multiplying by calculated K-factors. Correction for absorption was not made. The ASW contained (in mAI): NaCl, 480; KC1, 10; CaCl 2 , 10; MgCl 2 , 26; MgSO 4 , 29; and was adjusted to pH 8.0 with 10 mM HEPES. All the salt solutions and DW were buffered to pH 8.0 with 10 mM HEPES, and all the experiments were done at room temperature (23-26C). Results Nematocysts isolated from acontia of Calliaclis polypus in ASW swelled in concentrated solutions of monovalent cations (Fig. 1 ). There was a significant positive regression between the volume of nematocysts and the concentration of Na + and K + (regression analysis, P < 0.05). Though there was no significant regression between the volume of nematocysts and the concentration of divalent cations, the volume of nematocysts was significantly smaller in 1 M MgCl 2 and SrCl 2 than in 1 mM solutions (Duncan's multiple range test, P < 0.01). At 1 M concentration, nematocysts immersed in divalent cations were signifi- cantly smaller than those immersed in monovalent cations (Duncan's multiple range test, P < 0.01). When nema- tocysts that had been immersed in various salt solutions were immersed in a buffer solution without added salts, there was no significant difference in the mean volume 120 110 g 100 JS 90 0> oc 80 10 100 1000 Concentration of salts (mM) Figure 1. Effects of cations of various concentrations on the volume of nematocysts isolated from acontia of Calliaclis polypus in ASW. Ne- matocysts isolated in ASW were immersed successively in salt solutions of decreasing concentrations. The salt solutions examined were NaCl (). KCI (), CaCN (O), SrCl : (A), and MgCl 2 (O). The volume of ne- matocysts in each solution is expressed as a percentage of the original volume of nematocysts in ASW. Vertical bars represent standard devia- tions; some SD bars are omitted for clarity. (one-way ANOVA, P > 0.25). Thus the volume of the nematocysts increased with increasing concentration of cations if the cation was monovalent, but decreased if the cation was Mg :+ or Sr + . The volume of the nematocysts was smaller in 1 A/CaCl 2 than in 1 A/MgQ 2 (P < 0.01). The volumetric behavior of nematocysts isolated in DW was almost the same as that of nematocysts isolated in ASW (Fig. 2). When the concentration of external K + was increased, the volume of isolated nematocysts increased (regression analysis, P < 0.05). Though regression between the volume of nematocysts and concentration of Na + was not significant, nematocysts immersed in 1 M NaCl were larger than those immersed in 1 mAf NaCl (Duncan's multiple range test, P < 0.05). Nematocysts immersed in 1 M Cad: and SrCl 2 were smaller than those immersed in 1 mM solutions (P < 0.01). Nematocysts immersed in 1 M CaCl 2 or SrCl 2 were smaller than those immersed in 1 A/MgCl 2 (/ > <0.01). A scanning electron micrograph of a nematocyst sample prepared for X-ray microanalysis is shown in Figure 3. X-ray spectra of nematocysts were different depending on the incubation solutions (Fig. 4). The major elements of nematocysts incubated in ASW were Ca and Mg in ad- dition to sulfur (S), though a small Na-peak was present (Fig. 4A). When nematocysts were incubated in 1 M NaCl, the Na-peak increased, the Ca-peak remained high but the Mg-peak disappeared (Fig. 4B). When nematocysts were immersed in 1 M KCI, a small K-peak appeared, but the peaks of the other elements were not affected (Fig. 100 M. HIDAKA AND K. AFUSO 120 r - 110 0) 100 o > 0) 2 90 4) DC 80 10 100 1000 Concentration of salts (mM) Figure 2. Effects of cations of various concentrations on the volume of nematocysts isolated from acontia of Calliactis polypus in DW. Ne- matocysts isolated in DW were immersed successively in salt solutions of increasing concentrations. The symbols are the same as in Figure 1. The volume of nematocysts in each solution is expressed as a percentage of the original volume of the nematocysts in DW. The verlical bars represent standard deviations; some SD bars are omitted for clarity. 4C). Nematocysts incubated in 1 A/ CaCl 2 showed large Ca- and small Na-peaks in addition to the S-peak (Fig. 4D). When nematocysts were incubated in 1 M MgCl 2 , the Mg-peak increased (Fig. 4E). Nematocysts incubated in 1 A/ SrG 2 showed large Sr- and small Ca-peaks in ad- dition to the S-peak (Fig. 4F). When discharged nema- tocysts were analyzed, peaks of metals were absent re- gardless of the incubation solutions. Table I shows the relative abundance of metal cations in undischarged nematocysts that were isolated in DW and then incubated in various salt solutions. Ca accounted for about 50% of the metals in nematocysts immersed in ASW or 1 A/ MgCl 2 and more than 50% in nematocysts immersed in 1 A/ NaCl or KC1. Ca was replaced substan- tially only by strontium (Sr). Most of the Mg disappeared when nematocysts were immersed in 1 A/ NaCl, CaCl 2 , and SrCl : . Only a small amount of K was present in ne- matocysts incubated in 1 A/ KC1. Discussion Weber (1989) studied the volumetric behavior of iso- lated stenoteles of Hydra under different ionic conditions. He showed that nematocysts immersed in 1 A/ solutions of various salts swell when the concentration of salts is lowered, regardless of whether the cations are monovalent or divalent. The volumetric behavior of isolated nema- tocysts of the sea anemone Calliactis polypus was different from that of Hydra nematocysts. Calliactis nematocysts appeared to shrink in concentrated solutions of divalent cations as in Hydra nematocysts, but swelled in concen- trated solutions of monovalent cations. Thus the volu- metric behaviors of the marine anemone nematocysts and the freshwater Hydra nematocysts are different in solu- tions of monovalent cations. Weber (1989) showed that the volumetric behavior of Hydra nematocysts immersed in salt solutions of various concentrations can be accounted for by a Donnan-equi- librium model. The Donnan potential generates an asym- metrical distribution of ions across the capsule wall. Ac- cording to Weber's simulation studies, the difference in total ion concentration between the inside and outside of the capsule increases as the external salt concentration is lowered from 3 M to 0. 1-0.01 A/. When the external salt concentration is further lowered, the osmolarity difference drops due to protonation of polyanions, unless the exter- nal pH is high. The volume of Calliactis nematocysts, however, decreased as the external concentration of monovalent cations was lowered from 1 to A/. Thus the volumetric response of the sea anemone nematocysts to monovalent cations cannot be accounted for by the simple Donnan-equilibrium model. Weber ( 1989) showed that naturally occurring cations in Hydra nematocysts can be replaced by externally ap- plied cations. If this is true for nematocysts of marine cnidarians, cations contained in the isolated capsule might be replaced by external cations when nematocysts are im- mersed in various salt solutions. Isolated Calliactis ne- matocysts contained predominantly Ca 2+ and Mg :+ (Hi- daka, 1993). IfCa 2+ and Mg 2+ in the isolated nematocysts are replaced by monovalent cations, the internal osmotic pressure would increase as one divalent cation is replaced by the two monovalent cations required to maintain elec- troneutrality. The swelling of the sea anemone nemato- Figure 3. Scanning electron micrograph of isolated nematocysts used for X-ray microanalysis. The white spot represents the site irradiated with an electron beam during the acquisition of spectra. These nema- tocysts were isolated in DW and incubated in 1 A/ MgCl : for 10 mm. I I I I ( IS in (A [ IONS ON NEMATOCYSTS 101 flSM- 1 o.d.d C- 1 od-glutamic acid)s are the major constituents of nematocysts in Hydra (Hydrozoa, Cnidana). ./. Binl. Chcm 265: 9664-9669. Weber, J. 1991. A novel kind of polyanions as principal components of cnidarian nematocysts. Cnnip. Biochem. Physiol. 98A: 285-291. Weber, J.. M. Klug, and P. lardent. 1987. Detection of high concen- trations of Mg and Ca in the nematocysts of various cnidanans. E.\- pcricntia43: 1022-1025. Vanagita, T. M. 1959. Physiological mechanisms of nematocyst re- sponses in sea-anemone II. Effects of electrolyte ions upon the isolated cnidae. / Fac. Sci. Univ. Tokyo Sect. II' 8: 381-400. Reference: Biol. Bull 184: 105-113. (February. 1993) Hemocyanin Subunit Composition and Oxygen Binding in Two Species of the Lobster Genus Homarus and Their Hybrids CHARLOTTE P. MANGUM Bodega Marine Laboratory. University of California. P. O. Box 247. Bodega Bay, California 94923 [ Abstract. The monomeric subunit composition and O : binding properties of the hemocyanins (Hcs) of Homarus americamts. H. gammarus and their hybrids are very similar, though not identical. //. americanus He has six major electrophoretically separable polypeptide chains; H. gammarus He has four major and two minor chains; and the hybrid He has four major and one minor chain. Four chains co-migrate in all three groups, and the fifth chain in the hybrid co-migrates with a fifth chain in H. gammarus. Thus, qualitatively, the hybrid He is more like that of//, gammarus than H. americanus. a similarity reflected in respiratory properties. Although the O : affinity of the hybrid hemocyanin appears to lie intermediate be- tween that of the two parent hemocyanins at 25C, in fact it is significantly different from that of//, americanus but not H. gammarus. The cooperativity of the hybrid He also differs significantly from that of //. americanus but not H. gammarus He. The distinctive properties of H. americanus hemocyanin at 25C are believed to be due to either or both of two chains: a unique and also invariant chain in H. americanus. and one that is present in H. gammarus and the hybrids but not in H. ameri- canus. H. americanus He also appears to be slightly less sensitive to the allosteric modulator L-lactate. No differ- ence in CaCl 2 sensitivity was found. At lower temperatures respiratory properties are indistinguishable. In adult H. americanus that had been held under identical conditions for long periods, variation in subunit pattern was not en- tirely absent, but it was smaller than that found in natural populations of other species. No differences in O 2 binding at 25C were found in morphs differing qualitatively in Received 3 June 1992; accepted 8 October 1992. Permanent address: 'Department of Biology, College of William & Mary, Williamsburg, VA 23185-8795. one chain and quantitatively in two others. No effect of a combination of rearing temperature and diet was found on the He subunit composition of juveniles. Introduction The arthropod hemocyanins (Hcs) are multiples of hexamers built of 70-80 kDa polypeptide chains. Often the 2 X 6-mers predominate in the bloods of adult decapod crustaceans, including the lobster Homarus americanus (Olson et ul.. 1988). The number of different monomers is usually large, with a dozen or more found in several species of Uca (Sullivan et ai. 1983: Callicott and Man- gum, 1992; Mangum, 1992 and unpub. data). The mono- mers have been grouped into one of four categories on the basis of their electrophoretic mobilities, immunolog- ical reactions and roles in oligomer assembly (Markl, 1986). Within a species, the monomeric heterogeneity also plays a functional role in respiratory adaptation during the adult stage (Mason et ai, 1983; Mangum and Rainer, 1988; deFur et ai. 1990; Mangum et ai. 1991). By com- paring morphs, the functional differences have been at- tributed to particular electrophoretic bands (Mangum and Rainer, 1988; Mangum et ai, 1991; Mangum, 1992). The role of He subunit composition in bringing about functional differences between species is less clear. A sur- vey of forty-two species of various degrees of taxonomic relatedness suggests a high degree of specificity (Reese and Mangum, 1992). More intensive investigation of the Hcs of seven species of the genus Uca. which are extremely polymorphic as well as heterogeneous, supports the in- ference of species specificity (Callicott and Mangum, 1992; Mangum, 1992; C. P. Mangum, unpub. data). In every case, including sibling species such as U. panacea and pugilator, even low frequency He morphs of a species can be readily distinguished from those of another. Functional 105 106 C. P. MANGUM properties can also differ in sibling congeners with different latitudinal ranges. In comparisons of congeners that are less closely related, however, functional properties are more clearly related to environmental factors than to phylogenetic allinity or subunit composition (Reese and Mangum, 1992). In the only two species in which the effect of laboratory acclimation has been examined, the subunit phenotype of an adult individual is not fixed (Mason ct a/.. 1983; deFur el a!.. 1990; Callicott and Mangum, 1992). In Cal- lincctes sapidus. moreover, the variation both in the lab- oratory and in nature can be related to environmental factors such as salinity and hypoxia (Mangum, 1990; Pihl ct ai. 199 1;C. P. Mangum and S. P. Baden, unpub. obs.). Thus the members of the highly polymorphic samples of natural populations may have been acclimated to different environmental (or nutritional) conditions. Here I report data for the monomeric subunit com- position and oxygen binding of the Hcs of the adults of two species of lobsters in the genus Homarus and the hybrid progeny of their spontaneous matings. The two parent species had been brought from their native Atlantic habitats to the Bodega Marine Laboratory, where they were held under identical conditions for periods far longer than the species investigated previously. They are known to be highly homozygous at 41 loci and, at most loci, the allozymic phenotypes of the two are either extremely sim- ilar or identical. It is believed that the two speciated al- lopatrically when isolated for the first time during the Pleistocene (Hedgecockrt al.. 1977). In one species, I also examined the He subunit composition of juveniles which had been reared on either of two diets, and at different temperatures. Materials and Methods The sample All available adults, a total of 36. were examined; they were large (28-42 cm from rostrum to tail), intermolt individuals. Two (one of each sex) belong to Homarus gammarus (Linnaeus), formerly known as H. vulgaris: they were collected near lona, Scotland in 1975. They are the sole survivors of the larger sample characterized by Hedgecock et al. in 1977. Twenty-five adults ( 16 females, 9 males) are members of Homarus americanus H. Milne Edwards. All but one were caught on various dates in 1988-92 in waters surrounding Martha's Vineyard, Mas- sachusetts, and had been held in the mariculture facility at the Bodega Marine Laboratory for periods ranging from three months ( 1 individual) to more than three years (3 individuals). One individual of H. americanus (age > 6 years) was born in the Bodega Marine Laboratory. Nine hybrid adults (7 fertile females, 2 infertile males) were progeny of spontaneous matings between //. gammarus and americanus. Most were produced in 1983 by a female H. gammarus and a male H. americanus: one, of un- known parentage, was born in 1978. All adults had been fed the same diet of surf fish and shrimp, and had been held under identical photoperiods in the running seawater system of the Bodega Marine Laboratory. A seven year compilation of data ( 1 985-9 1 ) indicates that the water temperature ranges from 10 to 15C, and usually varies within about two degrees; over the four month period of sampling the salinity varied from 32.5-33.5%o, which is typical. The He subunit composition of 14 juvenile //. amer- icanus (8-10 cm length, both sexes), which had hatched in the Bodega Marine Laboratory 20-22 months earlier, was also examined. Half of these animals, which were their natural color, had been fed since stage IV a diet of brine shrimp, fish and crabs, and had been held at the seawater system temperature. The other half, phenotypic albinos, had been fed a diet based on casein; for the past year they had been held at room temperature (ca., 23C). The diets, rearing conditions and molt history of animals such as these were described in detail by Baum ( 1990). Preparation oj material and electrophoresis Blood was taken from the base of the last leg and serum expressed from the clot in a tissue grinder. After centrif- ugation an aliquot of the material was dissociated to its monomers by dilution with 0.01 mol I" 1 EDTA + 0.05 mol r 1 Tris (pH 8.9), to reduce light scattering; He con- centration was estimated from the absorbance of disso- ciated material at 338 nm (Bausch & Lomb Spectronic 2000 spectrophotometer), using the extinction coefficient reported by Nickerson and van Holde (1971). An addi- tional aliquot was diluted (1:10 or 1:30, depending on concentration) with the dissociating buffer for electro- phoresis, and the remainder frozen for future use. Ab- sorbance of the material from several individuals, detailed below, was compared at 280 and 338 nm. PAGE electrophoresis of native monomers was carried out at constant current according to Hames and Rickwood (1985). Following determination of the He phenotype in each individual, the variants among H. americanus were examined several times in adjacent lanes on the same gels. Representatives of each of the three groups were also compared many times on the same gels. Finally, gels were overloaded with six times the usual amount of material, the presence of Cu was determined according to Bruyn- inckx et al. (1978), and then the gels were stained as usual with Coomassie Blue. Oxygen binding On the basis of the PAGE, particular individuals were selected for a second bleeding, performed within a week HEMOCVANIN-O, BINDING AND SUBUNIT COMPOSITION IN LOBSTERS 107 of the final phenotype determination. Serum was dialyzed overnight, against seawater for most of the measurements or against a Tris maleate buffered salt for the experiments on inorganic ion sensitivity. Oxygen binding was deter- mined within a few days, using the cell respiration method (Mangum and Lykkeboe, 1979). Data analysis Bohr plots of the values for P 50 (oxygen affinity) were described by regression lines and their 95% confidence intervals compared. Mean values for n 50 (cooperativity) and He concentration were compared by Student's /-test. The data for CK binding as a function of [Cad:] were analyzed similarly. However, the nonlinearity of the re- sponse of Psn to [NaCl] and [Na^SOj] precluded statistical analysis. Results Hemocyanin concentration Adults of Homarus americanus had significantly (P = .02) higher levels of He [6. 1 1 (0.41 S.E.) g 100ml '] than the hybrids [4.18 (0.70 S.E.) g 100 ml" 1 ]. The values for the two members of//, gammarus (2.75 and 4.84 g 100 mr 1 ) also fall below the 95% confidence interval around the mean of the H. americanus sample. In the // americanus data there is no clear trend with length of time in the laboratory, suggesting that the nutritional state of the animals was good. The juveniles of this species had considerably lower He concentrations [1.02 (0.15) g 100 mr 1 ]. which were unrelated to diet. Monomeric subunit composition The two adult Homarus gammarus had identical He phenotypes, which were also the same as that of one of the two individuals examined two years earlier (C. P. Mangum, unpub. obs.). Four high density (or major) and two intermediate density (or minor) electrophoretic bands separated by charge (Fig. 1 ). All six were positive for Cu. The 25 adults of//, americanus exhibited very similar but not identical He phenotypes (Fig. 1 ). As many as eight bands separated on the lower third of the gels, four of which had co-migrants in H. gammarus (Fig. 1 ). The two most anodic bands ( 1 and 2) were always present in trace quantities, if at all. Material at their position appeared to quench the fluorescence of bathocuproine sulfonate, in- dicating the presence of Cu. However, it was not possible to ascertain the site of the quenching more precisely, only one of the two may contain Cu. These bands had no co- migrants in H. gammarus or the hybrids. In H. americanus bands 3-8 could reach high concen- trations. The gels on which the best separation was ob- tained exhibited less density in the middle of the material designated as bands 3 and 4, suggesting the presence of two chains that are similar in charge and extremely dif- ficult to resolve. Moreover, the leading edge of this ma- terial clearly co-migrated with bands 2 in the hybrid and 3 in H. gammarus. whereas the trailing edge clearly lagged behind. Thus I assigned two numbers (3 and 4) to this position of the H. americanus material, even though the separation was not great enough to photograph. In ad- dition, I was not able to decide whether the trailing ma- terial was present in all 25 individuals. The quantities of chains 6 and 7 in //. americanus are similar to the cor- responding ones in //. gammarus, but chains 4 and 8 always occurred in higher levels in H. americanus than H. gammarus (Fig. 1 ). In the early PAGE, band 5 in H. americanus did not appear to be sharply delineated at its leading and trailing edges. It was the only high density band that clearly varied qualitatively as well as quantitatively, ranging from absent (3 adults) to low concentration (5) to high concentration (17). Since I suspected that this band might not be a He chain, I examined the ratio of the absorbance at the protein peak (280 nm) and the active site (338 nm). According to this index, however, the total Cu content of H. amer- icanus samples containing maximal levels of band 5 did not clearly differ from those that lacked it; nor did it differ from the samples from //. gammarus and the hybrids. none of which contained co-migratory material. In ad- dition, on subsequent gels band 5 was as sharply delineated as the rest (Fig. 1). Bands 6 and 8 varied quantitatively, though the mag- nitude was not great. They decreased concomitantly to intermediate levels in two of the 25 animals; band 8 was intermediate in two additional individuals in which band 6 remained maximal. Band 7 appeared to be absent in a single individual in which 6 and 8 were maximal. This female, from which larvae had hatched two months ear- lier, had been in the laboratory for only three months. All other individuals had maximal levels of these three chains. Although I did not investigate material held at tem- peratures above freezing, there was no correlation between phenotype and age of frozen preparations; this has been true in my experience with Hcs from all species examined thus far. In the present case the same banding pattern was observed before and after four months. Finally, material prepared on a second occasion from four of the same individuals three months after the first bleeding showed no change in phenotype. The hybrids, all adults, exhibited a single phenotype which did not vary quantitatively or qualitatively. With the exception of the higher levels of band 2, it resembles the phenotype of H. gammarus more than that of H. americanus (Fig. 1 ). The hybrid He has four major chains and one minor chain, all of which correspond in mobility 108 C. P. MANGUM 1 2 H. americanus hybrid H. gammarus 1 i a 3 4 m 5 6 Figure 1. Banding patterns of the dissociated hemocyanins of the two parent species of Homarus and their hybrids, and a diagram illustrating the correspondence (arrows) of band positions. The anode is at the top. In the gel for // americanus, each pair of lanes shows a sample from a different individual in higher (left) and lower (right) concentrations. The lanes on the far right were overloaded to show the anodic material (numbered I and 2) that occurs in trace quantities. The cathodic triplet of bands in this species is more clearly shown in lanes that were not overloaded. The middle panel shows // gammarus ( 1 individual) and the hybrid He ( 1 individual) in alternating lanes. to one of the six chains of H. gammarus. The hybrids differ from both parents, but from H. gammarus only in the absence of the most anodic band (H. gammarus band 1 ) and the higher levels of hybrid band 2. They differ from H. americanus in the absence of its three most anodic bands (H. americanus bands 1-3), in the absence of H. americanus band 5, in the consistently lower quantity of their most cathodic chain (hybrid band 5) and in the pres- ence of a distinctive band 1. As in all other species I have examined (e.g., Mangum et a!., 1985; Mangum, 1992), the phenotypes of males and females in each of the three groups were indistinguishable no sex specific material was present. HEMOCYANIN-O, BINDING AND SUBUNIT COMPOSITION IN LOBSTERS 109 The juvenile H. americanus were indistinguishable from the adults. Six of the seven members of each dietary- thermal group had the maximum number of bands. One in each group lacked chain 5. Chains 6-8 were invariably present in maximal concentrations. Oxygen binding First, the intraspecific variation in //. americanus was examined. One adult lacked band 5 and also had minimal (= intermediate) levels of chains 6 and 8; at 25C, however, the oxygen binding properties of its He (stripped of organic co-factors) were indistinguishable from those of another individual containing maximal amounts of all eight bands. Therefore the data have been combined for presentation. The coefficient of determination (r) for the regression line describing P 50 in Figure 2 is 0.962, further affirming the absence of a perceptible effect of phenotype. The single individual with low quantities of band 7 had been sacri- ficed at the time the O 2 binding measurements were per- formed. Second, the Hcs of the two parent species were com- pared. At all but the lowest pH investigated. He O : affinity at 25 C is significantly lower in H. gammarus than H. americanus, though the difference is fairly small (Fig. 2). Third, the hybrid He was compared with each of the par- ent Hcs. Whereas the data for the hybrid He appear to be intermediate between those for the two parent species, the difference from //. gammarus is not significant even in the middle of the pH range investigated. In contrast, the difference between the hybrid and //. americanus is significant throughout most of the pH range exam- ined (>7. 2). The mean value for cooperativity is somewhat smaller (P = .001) in H. americanus (3.24 .11 S.E.) than //. gammarus and the hybrids (3.95 .13), which do not differ from one another (P = .15). Thus the respiratory properties of the hybrid He are also more like those of//. gammarus than H. americanus. At lower temperatures, the significant differences dis- appear completely. Ninety-five percent confidence inter- vals around regression lines fit to the O : affinity data in Figure 3 overlap fully throughout the pH range investi- gated. Often this is true because the numerical values di- minish and are thus more difficult to distinguish, but in this example there is not even an apparent trend. Mean values for cooperativity do not differ (P = .2-. 8). As a result, //. americanus He is less temperature sensitive than the other two Hcs, though only in the 15-25C range. For that range, the apparent heat of oxygenation (AH) is only 2.4 kcal mor' for H. americanus He (pH 7.6), whereas the value for the hybrid He is 5.6, and the value for H. gammarus He is -6.6. For the range 5-15C the value of AH for all three Hcs is -9.4 kcal mol~' (same pH). //. americanus He is slightly less sensitive to the allo- steric effector L-lactate (Fig. 4) than H. gammarus He; once again, the sensitivity of the hybrid He appears to be intermediate. At pH 7.6 the addition of 10 mmol 1~' lac- tate changes log P 50 of H. americanus He by 0.166, H. gammarus He by 0.232, and the hybrid He by 0.203. However, O ; affinity in H. gammarus and the hybrids is so 40 20 10 8 o . 5 - A A c ' o * 7.2 7.6 8.0 7.2 7.6 8.0 Figure 2. Oxygen binding at 25C of Homarus americanus (closed circles, solid lines), H. gammarus (open circles, dotted lines) and their hybrid (triangles, dashed lines) hemocyanins. The curves are fitted regression lines 95% confidence intervals. 0.05 mol T' Tris maleate buffered seawater. Material obtained from two individuals of H americanus was used (see text), whereas H gammarus and the hybrids are represented by a single individual. 110 C. P. MANGUM S 4 15 C 5 C ;-.** , i , i A 00 o in Q_ 70 SO 30 10 7 5 o, 7.0 7.5 8.0 7.0 7.5 PH 8.0 Figure 3. Oxygen binding al 1 5 and 5C of//, americanus (closed circles), H gammarus (open circles) and their hybrid (triangles) hemocyanins. The regression lines and confidence intervals were omitted for clarity. 0.05 mol 1~' Tns maleate buffered seawater. Origin of material as in Figure 2. not quite significantly different in the presence of lactate, even in the middle of the pH range. In the presence of lactate, O : affinity of //. gammarits He remains signifi- cantly lower than that of H. americanus throughout the pH range investigated. In contrast, the hybrid He has a significantly lower O 2 affinity than that of H. americanus He only at high pH. In all three groups cooperativity is significantly diminished in the presence of lactate. The mean value drops from 3.2 to 2.86 .05 S.E. (P = .05) for H americanus He, from almost 4 to 3.06 .24 (P = .05) for H. gammarus He, and from almost 4 to 2.95 .29 (P = .002) for the hybrid He. The sensitivity of the three Hcs to CaCh is indistin- guishable (Fig. 5). Regression lines and their 95% confi- dence intervals overlap fully throughout the concentration range investigated. Mean values for cooperativity do not differ (P = .50-.75). NaCl clearly raises He O ; affinity and lowers cooper- ativity of//, americanus He (Fig. 5). Once again, however, the different morphs were indistinguishable, and the data were combined for presentation. In contrast to the allo- steric effect of Ca 2+ . the relationship between P 50 and NaCl is nonlinear on logarithmic coordinates. I used high con- centrations of Na2SO 4 , prepared from the decahydrate. too 70 50 30 o n 10 L 7 5 7.2 7.6 8.0 7.2 7.6 8.0 7.2 7.6 B.O Figure 4. Lactate sensitivity of//, americanus (left panel: closed circles, solid regression lines 95% confidence intervals), H. gammarus (right panel: open circles, dotted lines) and their hybrid (middle panel: triangles, dashed lines) hemocyanins. Origin of material as in Figure 2. Control curves reproduced in each panel from Figure 2. 25C, 0.05 mol I" 1 Tris maleate buttered seawater. HEMOCYANIN-O, BINDING AND SUBUNIT COMPOSITION IN LOBSTERS 111 o in 5.0 4.5 4.0 O* * * 3.5 j 3.0 7 S f 8 o * A * i i i o 7 i | O g 5 - o. e 4 3 ~ o 2 i l l I l i 1.8 - 0.0 0.5 1 .0 1.5 log free [CaCI ] 2.0 1.5 1 .0 o o o 8 0.0 0.5 1.0 1.5 2.0 2.5 3.0 log free [NaCI] or [Na 2 SOj Figure 5. Inorganic ion sensitivities of lobster hemocyanins. The units of free ion concentrations (antilogs) are mmol l~'. Left panels: H amcricanus (closed circles, solid lines). //. gammarus (open circles, dashes) and their hybrids (triangles, dots). 0.05 mol 1~' Tns maleate buffer (pH 7.7) + O.I mol T 1 NaCI. Right panels: the response of H americanus He to NaCI (closed circles) and Na : SO 4 (open circles). 0.05 mol 1' Tris maleate + 0.01 mol 1~' CaCl : . 25C. Ongin of matenal as in Figure 2. to examine specificity. The response differed very little from that of NaCI (Fig. 5), and the apparent difference may lie within the error of preparing an accurate solution of a highly hydrated salt (especially at a marine labora- tory). Discussion The essentially non-specific sensitivity of Homarus americanus He to NaCI is further evidence that the in- organic ion responses of the crustacean Hcs are not all alike. The response of this He differs from that of portunid crab Hcs (Truchot. 1975; Mason el al, 1983), which are insensitive to NaCI, but resembles that of penaid shrimp Hcs (Brouwer et al.. 1978; Mangum and Burnett. 1985). From a physiological point of view, however, NaCI sen- sitivity is unlikely to be important in H. americanus, a stenohaline species. According to Hedgecock et ill. ( 1977 and pers. comm.), the genetic distance between the two parent species of Homarus. though significant, is so small that the numer- ical value is closer to expectation for subspecies than spe- cies. Thus it is of particular interest that the present find- ings support the inference of species specificity of He sub- unit composition (Reese and Mangum, 1992). Although H. gammarus is monomorphic for the common H. amer- icanus allele at 30 allozymic loci, neither of the two parent Hcs in the present sample could be confused with the other. As in the sibling species of Uca (Mangum, 1992 and unpub. obs.), this inference is true in spite of intra- specific variation. In H. americanus. band 3 is both di- agnostic of the species and, at least in the present sample, invariant. Material that co-migrates with chains 1 and 2 of H. gammarus is clearly absent from H. americanus. Furthermore, the hybrid He is structurally distinct from either parent. The present findings also support the inference of little interspecific genetic distance. Even though they are not identical, the two parent Hcs are more similar than any of the ca. 50 Hcs we have examined thus far, with the exception of Menippe adina and M. mercenaria Hcs (Reese 1989). Like the lobsters, these two sibling species of stone crabs are also believed to have speciated recently, and they also hybridize spontaneously (Bert, 1986). In both structural and functional properties the hybrid He resembles that of one parent more than the other. It has only one less chain than H. gammarus He but several fewer than H. americanus He. The electrophoretic be- 112 C. P. MANGUM havior of each of the five hybrid chains is identical to that of some one of the // gammanis chains, whereas hybrid chain 1 has no co-migrant in H. americanus. These re- lationships are reflected in the O : binding of the Hcs in a complete saline, though only at high temperature. In stage IV through adult H. americanus, SDS PAGE separates three He chains (Olson el ai. 1988; Olson and McDowell, 1989). As is often the case (e.g., Sullivan et al., 1983), additional bands are revealed when the sepa- ration is made by charge. In neither juveniles nor adults of this species can the He be categorized as strictly monomorphic at the level of quaternary structure, despite prolonged acclimation of the donors. Moreover, in juveniles the variation is distinctive of neither the stage nor the thermal-nutritional history. In both stages, however, the variation is much smaller than in samples of natural populations of several species of brachyuran crabs (Mangum, 1990, 1992; Callicott and Mangum, 1992). This generalization is true of respiratory properties of the adults as well. Although the sample size is much smaller in the present investigation, the inference remains unchanged when the comparison is made with, for example, the 14-20 individuals of Callinectes sapidus investigated by Mangum el al. in 1991. I emphasize that the small amount of He variation found here may not accurately represent natural popu- lations of H. americanus (much less H. gammanis). Nor is it clear that the lack of variation results from prolonged acclimation rather than limited genetic diversity, as found at other loci (Tracey et al., 1975; Hedgecock el al., 1977). However, I note that the inference of allozymic similarity in the species was made from samples of populations on either side of Cape Cod but not Cape Hatteras, the greater geographic barrier (e.g., Friedrich, 1973; National Geo- graphic Society, 1985). The present findings suggest that, given the common acclimation, the differences observed both within H. americanus, and between this species and the other two groups, represent a fixed condition in an adult individual. Although only intermolt animals were investigated here, the finding of no change with molt stage in Callinectes sapidus (Mangum et al.. 1985) has recently been con- firmed in H. americanus (N. B. Terwilliger, pers. comm.). More important in the present context, the virtual identity of most respiratory properties of the three Hcs appears to reflect the notable similarity of the electrophoretic phe- notypes. Conversely, it is reasonable to suggest that the slightly higher O 2 affinity and lower cooperativity of H. americanus He at high temperature are due to the chains that are unique to one of the three groups. Perhaps the most likely candidate is chain 2 in //. gammanis (= 1 in the hybrids), which is absent in H. americanus. However, the possibility that band 3 is invariant as well as unique to //. americanus cannot be excluded. Bands 1 and 2 are never present in H. americanus in more than trace quan- tities, and morphs containing or lacking chain 5 did not differ in O 2 binding. The latter inference would be un- warranted only if the effect of chain 5 was exactly com- pensated by an equal and opposite effect of chains 6 and 8, which were also variables in the comparison. Acknowledgments Supported by NSF DCB 88-16172 (Physiological Pro- cesses). I am extremely grateful to the University of Cal- ifornia for a Research Fellowship and to the Bodega Ma- rine Laboratory for its unfailing hospitality. Literature Cited |{:iiiin. N. A. 1990. Studies on the role of dietary protein and lecithin in molting and cholesterol transport in juvenile lobsters, Homanis sp. M. A. Thesis. Sonoma State University, Rohnert Park. Cal.. 65 pp. Bert, T. M. 1986. Speciation in western Atlantic stone crabs (genus MenippeY the role of geologic processes and climatic events in the formation and distribution of species. Mar. Biol. 93: 157-170. Brouwer, M., C. Bonaventura, and J. Bonaventura. 1978. Analysis of the effect of three different allosteric ligands on oxygen binding by hemocyanin of the shrimp Pcnaeus setiferus. Biochem. 17: 2148- 2154. Bruyninckx, W. J., S. Gutteridge, and H. S. Mason. 1978. Detection of copper on polyacrylamide gels. Analyt. Biochem. 89: 174-177. Callicott, K. A., and C. P. Mangum. 1992. Phenotypic variation and lability of the subunit composition of the hemocyanin of I'ca pugi- lulor. J Exp. Afar. Biol. Ecol. (in press). deFur, P. L., C. P. Mangum. and J. E. Reese. 1990. Respiratory re- sponses of the blue crab Callinectes sapidus to longterm hypoxia. Biol. Bull 178:46-54. Friedrich, H. 1973. Marine Biology. University of Washington Press. United Kingdom. 474 pp. Hames, B. D., and D. Rickwood. 1985. Gel Electrophoresis of Proteins. 1RL Press, Oxford. 290 pp. Hedgecock, D., K. Nelson, J. Simons, and R. Shleser. 1977. Genie similarity of American and European species of the lobster genus Homanis. Biol. Bull. 152:41-50. Mangum, C. P. 1990. Inducible O, carriers in the crustaceans. Pp. 92- 103 in Animal Nutrition and Transport Processes. 2. Transport, Res- piration and Excretion. Comparative and Environmental Aspects. J.-P. Truchot and B. Lalou, eds. Karger, Basel, Switzerland. Mangum, C. P. 1992. Structural and functional polymorphism of the hemocyanin O 2 transport system of the sand fiddler crab. L'ca pug- ilalor. J. Exp. Mar. Biol. Ecoi (in press). Mangum, C. P., and L. E. Burnett. 1986. The CO 2 sensitivity of the hemocyanins and its relationship to Cl sensitivity. Biol. Bull. 171: 248-263. Mangum, C. P., and G. Lykkeboe. 1979. The influence of inorganic ions and pH on the oxygenation properties of the blood in the gas- tropod mollusc Busycon canaliculalum. J Exp. Zoo/. 207: 417-430. Mangum, C. P., and J. S. Rainer. 1988. The relationship between sub- unit composition and oxygen binding of blue crab hemocyanin. Biol. Bull 174: 77-82. Mangum, C. P., J. Greaves, and J. S. Rainer. 1991. Oligomer com- position and oxygen binding of the hemocyanin of the blue crab Callinectes sapidus. Biol Bull 181:453-458. Mangum, C. P., B. A. McMahon, P. L. deFur, and M. I. VVheatly. 1985. Gas exchange, acid-base balance and the oxygen supply to HEMOCYANIN-O: BINDING AND SUBUNIT COMPOSITION IN LOBSTERS 1 13 tissues during a molt of the blue crab. Callincctcx sapidiis Rathbun. J. Crn.it. Bioi 5:207-215. Mason, R. P., C. P. Mangum, and G. Godette. 1983. The inllucnce of inorganic ions and acclimation salinity on hemocyanin-oxygen binding in the blue crab Callmeaes sapidiis. Biol. Bull. 164: 104-123. Markl. J. 1986. Evolution and function of structurally diverse subunits in the respiratory protein hemocyanin from arthropods. Biol. Bull. 171: 90-115. National Geographic Society. 1985. .-I/An <>/ An///; America Wash- ington, DC. Pp. 66-67. Nickerson. K. \\ ., and K. E. van ilolde. 1971. A comparison of mol- luscan and arthropod hemocyanin. I. Circular dichroism and ab- sorption spectra. Comp Biochcin P/IVMO! 39B: 855-872. Olson, K., N. B. I erwilliger, and J. McDowell Capuz/.o. 1988. Structure of hemocyanin in larval and adult lobsters. Am /.mil 28: 47A. Olson, K. S., and J. McDowell. 1989. Structure and function of he- mocvamn in American lobsters. Am /.mil. 29: 20A. Pihl, I.., S. P. Baden, and R. J. Dia/. 1991. Effects of periodic hypoxia on distribution of demersal fish and crustaceans. Mar Binl 108: 349-360. Reese, J. E. 1989. Structure and function of crustacean hemocy- anins. MA Thesis. College of William and Mary, Williamsburg. VA. 75 pp. Sullinin, B., L. Pennell, B. Hutchison, and R. Mulchings. 1983. Genetics and evolution of the hemocyanin multigene-I. Genetic variability in L'ca pugilator from Beaufort, NC. Comp. Biochem. Physiol 76B: 615-618. Tracey, M. L., K. Nelson, D. lledgecock, R. A. Shleser, and M. I,. Pressick. 1975. Biochemical genetics of lobsters: Genetic variation and the structure of American lobsters (Homanis americanus) pop- ulations. ./ Fish. Res Board Can 32: 2091-2101. Truchot. J.-P. 1975. Factors controlling the in vitro and in vivo oxygen affinity of the hemocyanin of the crab, Carcinus meanas. Resp. Phvswl 24: 173-189. CONTENTS CELL BIOLOGY Costas, Eduardo, Angeles Aguilera, Sonsoles Gon- zalez-Gil, and Victoria Lopez-Rodas Contact inhibition: also a control for cell prolifer- ation in unicellular algae? DEVELOPMENT AND REPRODUCTION Fenteany, Gabriel, and Daniel E. Morse Specific inhibitors of protein synthesis do not block RNA synthesis or settlement in larvae of a marine gastropod mollusk (Haliotis rujescens) 6 Freeman, Gary Metamorphosis in the brachiopod Terebratalia: ev- idence for a role of calcium channel function and the dissociation of shell formation from settlement 15 ECOLOGY AND EVOLUTION Curtis, Lawrence A., and Karen M. K. Hubbard Species relationships in a marine gastropod-tre- matode ecological system 25 Douillet, Philippe, and Christopher J. Langdon Effects of marine bacteria on the culture of axenic oyster Crassostrea gigas (Thunberg) larvae 36 Okamura, Beth, and Lita Ann Doolan Patterns of suspension feeding in the freshwater bryozoan Pltimatella repens 52 Scheltema, Amelie H. Aplacophora as progenetic aculiferans and the coe- lomate origin of mollusks as the sister taxon of Si- puncula 57 IMMUNOLOGY Rinkevich, H., Y. Saito, and I. L. Weissman A colonial invertebrate species that displays a hi- erarchy of allorecognition responses 79 Sawada, Tomoo, Jeffrey Zhang, and Edwin L. Cooper Classification and characterization of hemocytes in Stvela dava . 87 PHYSIOLOGY Hidaka, Michio, and Kiwamu Afuso Effects of cations on the volume and elemental composition of nematocysts isolated from acontia of the sea anemone Calliactis polypus 97 Mangum, Charlotte P. Hemocyanin subunit composition and oxygen binding in two species of the lobster genus Homarus and their hybrids 105 Volume 184 THE Number 2 BIOLOGICAL BULLETIN APRIL, 1993 Published by the Marine Biological Laboratory 1993 LATE SUMMER COURSES AT THE MBL History of Biology: Human Genetics in the Twentieth Century (AUGUST 1- AUGUST 11, 1993) APPLICATION DEADLINE: MAY 21, 1993 Open to students from a wide variety of backgrounds and ranks who share an interest in the history and philosophy of human genetics and eugenics. This course will focus on the history of human genetics in the United Slates, Great Britain, France, Germany and Russia in the twentieth century. Themes will include clinical and eugenic aspects of human genetic studies, the history of efforts to control human evolution, ethical questions arising from present as well as past attempts at such control, and the social construction of scientific knowledge. 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Authors (or delegates for foreign authors) will receive page proofs of articles shortly before publication. 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: Bioi Bull 184: I 15-124. (April, 1993) Ooplasmic Segregation in the Medaka (Oryzias latipes) Egg VIVEK C. ABRAHAM, SUNITA GUPTA. AND RICHARD A. FLUCK Department of Biology, Franklin and Marshall College, P.O. Box 3003, Lancaster, Pennsylvania 1 7604-3003 Abstract. Using time-lapse video microscopy, we found that ooplasmic inclusions in the fertilized medaka egg dis- played two types of movement during ooplasmic segre- gation. The first manifested itself as the movement of many inclusions (diameter = 1.5-1 1 ^m) toward the an- imal pole at about 2.2 pm min~'; this type of movement appeared to be streaming. The second type of movement was faster (about 44 //m min ') and saltatory; inclusions displaying this type of movement were smaller (diameter < 1.0 /urn) and moved toward the vegetal pole. The move- ment of oil droplets toward the vegetal pole of the egg may represent a third type of motion. All these movements began only after a strong contraction of the ooplasm to- ward the animal pole, which at 25C began 10-12 min after fertilization and <3 min after formation of the second polar body. In eggs treated with microtubule poisons colchicine, colcemid, or nocodazole oil droplets did not move to- ward the vegetal pole, saltatory motion toward the vegetal pole was absent, and the growth of the blastodisc was slowed. Eggs treated with 0-lumicolchicine, an inactive derivative of colchicine, showed normal movements. Colchicine, while not inhibiting formation of the second polar body, did inhibit pronuclear migration. These results suggest that microtubules are involved in the movement of some ooplasmic inclusions, including oil droplets, to- ward the vegetal pole; the movement of ooplasmic inclu- sions toward the animal pole; and pronuclear migration. Introduction Eggs of many animal species display a remarkable va- riety of movements soon after they are fertilized. Many of these movements, known collectively as ooplasmic Received 18 May 1992; accepted 25 January 1993. segregation, are important for the rearrangement of egg cytoplasm during the minutes and hours following fertil- ization. In some animals, amphibians and ascidians for example, these movements lead to cytoplasmic localiza- tion of morphogenetic determinants, which are subse- quently segregated to specific cells during cleavage and ultimately affect gene expression in the cells that incor- porate them (Reverberi, 1971; Davidson, 1976; Illmensee et al.. 1976; Jeffery, 1984; Speksnijder el ai, 1990a). In contrast to a relatively detailed understanding of oo- plasmic segregation in eggs of ascidians, annelids, and amphibians (Vacquier, 1981), relatively little is known about it in fish eggs. Except for Roosen-Runge's (1938) classic study in which time-lapse cinemicrography was used to monitor ooplasmic segregation in the zebrafish egg (Brachydanio rerio), there have been no published reports of the use of time-lapse microscopy to monitor segregation in a fish egg. Given the increasing use of fish embryos as model systems in the study of development (Kimmel, 1989; Powers, 1989; Kimmel et al.. 1990; Schindler, 1991), it is important to examine segregation in this group of organisms more closely. Microtubules are required for ooplasmic movements in several taxa of animals, including amphibians (Wak- ahara, 1989; Houliston and Elinson, 1991; Peter et al., 1991), ascidians (Zalokar, 1974; Sawada, 1988; Sawada and Schatten, 1989), and annelids (Eckberg, 1981; Shi- mizu, 1982; Astrow et al., 1989). Microtubule poisons drugs that block either assembly or disassembly of mi- crotubules have been useful tools in these studies (Za- lokar, 1974; Eckberg, 1981; Shimizu, 1982; Astrow et al., 1989; Sawada and Schatten, 1989), in which a role for microtubules is presumed when a particular movement is inhibited by one or more of these poisons. Because these poisons can have cytotoxic effects unrelated to their effects on microtubules, many studies have compared the 115 116 V. C. ABRAHAM ET AL effects of more than one such class of these poisons and. also, have used as controls chemically similar derivatives that have low affinity for tubulin, the protein subunit of microtubules. For example, /i-lumicolchicine, a derivative of colchicine (Wilson and Friedkin, 1967), can be used as a control for colchicine (Sabnis, 1981; Achler et al., 1989; Peter el al.. 1991). We have studied ooplasmic segregation in the egg of the medaka (Oryzias talipes). This large (diameter = 1.2 mm) clear egg, with its thin peripheral layer of ooplasm surrounding a central yolk vacuole, permits microscopic study of both the gross movements of ooplasm as well as the movement of ooplasmic inclusions. The objectives of the present study were ( 1 ) to describe the movements of ooplasmic inclusions, and (2) to monitor the effects on these movements of three drugs that block microtubule assembly (Wilson et al.. 1974; Dustin, 1984, ch. 5; Bray. 1992, p. 207). A preliminary account of these findings has been pub- lished (Abraham and Fluck, 1991). Materials and Methods We removed gonads from breeding medaka (Yama- moto, 1967; Kirchen and West, 1976; Fluck, 1978) and placed them in a balanced saline solution (BSS; 1 1 1 mM Nad; 5.37 mM KC1; 1.0 mM CaCl 2 ; 0.6 mM MgSO 4 ; HEPES, pH 7.3). Eggs were removed from the ovary, and the long chorionic fibers at the vegetal pole were removed with scissors. Eggs were fertilized in BSS (Yamamoto, 1967) and transferred to a microscope slide on which a cover glass was supported by four pillars of petroleum jelly. The cover glass was then pressed gently against the chorion to flatten a small region of the egg near its equator. Such flattening facilitated optical studies and also enabled us to roll the egg to achieve the desired orientation. All procedures were performed at room temperature (23- 26C in most experiments); in this temperature range, the first cell division begins after about 70 min. Because the rate of development varies inversely with temperature, we have reported the timing of events not only as "minutes after fertilization" but also as "normalized time" (t n ), where t n = 1.0 is the time at which cytokinesis begins. We monitored the movements of ooplasmic inclusions (or parcels) with time-lapse video microscopy, using a Ni- kon Optiphot or Diaphot microscope equipped with phase-contrast optics and connected through a Dage/MTI camera to a Panasonic NV-8050 time-lapse video cassette recorder. Using a 40X phase-contrast objective lens, we usually focused on a patch of ooplasm near the equator of the just-fertilized egg; with the Optiphot, the field of view was approximately 140 ^m X 200 /urn (total mag- nification = 882X) and with the Diaphot it was approx- imately 225 ^m X 325 nm (total magnification = 542X). We measured the diameters of inclusions on the screen of the video monitor and corrected for scale. To measure the speed and direction of movement of the inclusions, we placed a transparent plastic sheet over the video mon- itor during playback and mapped the paths of randomly chosen inclusions at regular time intervals; the length of the time interval chosen, usually either 20 s or 40 s, de- pended on the average speed of the inclusions at the time. To measure the thickness of the blastodisc, we viewed it in profile from the side, measured its thickness along the animal-vegetal axis, and corrected for scale. To mea- sure the volume of the blastodisc, we viewed it in profile from the side and used an image analysis program (Mi- crocomp Planar Morphometry, Southern Micro Instru- ments, Atlanta, Georgia) to measure three parameters (area, centroid x and projected .\) of one-half of the blas- todisc, after drawing a line that bisected the image along the animal-vegetal axis. We then calculated its volume, using the following equation: volume = (2?r) (area) (cen- troid .v-projected .\). The validity of this method was es- tablished by measuring standard objects. To measure the thickness of ooplasm elsewhere on the egg, we measured its thickness en face in the Z axis of the objective lens, using the presence of inclusions as a marker for ooplasm. We used two methods to monitor the timing of the second meiotic division. In the first, we fixed eggs in 3.7% formaldehyde in BSS at regular intervals after fertilization. After rinsing away the fixative and staining the nuclei with Hoechst 33258 (10 ^g ml 1 in BSS containing 1% Triton X-100), we examined the eggs with epifluorescence optics. In the second method, we microinjected Hoechst 33258 (100 Mg ml' 1 , dissolved in 50 mM K 2 SO 4 and 10 mM HEPES, pH 7.2) into unfertilized eggs, injecting ap- proximately 1.5 nl into the ooplasm at about 45 latitude from the animal pole. The method for microinjection was similar to that used by Ruck et al. (1991), except we used a high pressure microinjection system (Narashige IM-200). The injection process parthenogenetically activated the eggs, while Hoechst 33258 stained the maternal nuclear DNA. After placing these eggs between a coverglass and slide, we recorded movements of the ooplasmic inclusions, using a SIT camera coupled to the VCR, and monitored the second meiotic division by examining the eggs at reg- ular intervals with epifluorescence optics. Room temper- ature was 19.5C in this latter series of experiments. Microtubule poisons Stock solutions of colchicine (1 mM in BSS), /8-lumi- colchicine ( 1 mM in BSS), colcemid (0.35 mM in BSS), and nocodazole (2 mg ml' 1 ) in DMSO were diluted into BSS to make working solutions. Working solutions of no- codazole also contained 1% DMSO, which had no ap- OOPLASMIC SEGREGATION IN MEDAK.A 17 parent effect on the eggs. In preliminary experiments, we monitored the effects of several concentrations of each drug on the movement of oil droplets during ooplasmic segregation and found the minimum effective concentra- tions that disrupted their normal movement to be 100 nAf colchicine, 0.35 pAI colcemid, and 0.17 nM nocod- azole; we used these concentrations in subsequent exper- iments. Eggs were generally incubated with the drugs for 1 h before fertilization and then fertilized in the same medium; however, in some experiments, eggs were in- cubated with the drugs for 1.5 h or 2 h before they were fertilized. In each experiment, we monitored one egg with time-lapse video microscopy and monitored an additional 15-20 eggs with a stereomicroscope. To monitor the effect of colchicine on formation of the second polar body and migration of the pronuclei, control eggs (nine eggs from two females) and eggs treated with 100 fiM colchicine (eight eggs from two females) were fixed in 3.7% formaldehyde in BSS at t n = 0.45. After washing away the fixative, the eggs were stained with Hoechst 33258 and examined with epifluorescence optics. Chemicals Colcemid. colchicine, Hoechst 33258, /3-lumicolchi- cine, and nocodazole were obtained from Sigma (St. Louis, Missouri) and formaldehyde from Electron Mi- croscopy Sciences (Fort Washington, Pennsylvania). Results An early sign of egg activation was the cortical granule reaction, which spread as a wave from the animal pole to the vegetal pole in about 90 sec at 26C. a result consistent with the time reported by Gilkey et al. (1978). After the cortical granule reaction, the ooplasm became relatively transparent (Fig. 1 A), and several types of inclusions could be seen in it (Fig. 2). One class of inclusions were oil droplets, which with phase-contrast optics appeared as white spheres with diameters from < 1.0- 100 nm. That these spheres were oil droplets was confirmed by staining them with a lipophilic fluorescent dye, nile red (data not shown). About 1 min after the beginning of the cortical granule reaction, there was a strong contraction of the ooplasm. marked by the movement of all ooplasmic in- clusions toward the animal pole; this fertilization con- traction lasted about 1.5 min and thus was over within 2.5 min after fertilization, times also consistent with Gilkey el al. (1978). Our detailed study of the movement of ooplasmic inclusions began after the fertilization con- traction. At 10- 12 min after fertilization (at t n = 0.16at25C), a second contraction occurred (Fig. 3), in which all oo- plasmic inclusions, including oil droplets, again moved toward the animal pole. After this second contraction. most inclusions continued to move toward the animal pole; however, oil droplets (Fig. 1C-F) and some smaller inclusions began to move toward the vegetal pole. Ac- cumulation of ooplasm at the animal pole and the move- ment of oil droplets and other inclusions toward the veg- etal pole proceeded simultaneously for = 70 min, at which time the blastodisc underwent its first division (Fig. IF). By this time, there were fewer, larger oil droplets, a result of their fusion with each other during their movement toward the vegetal pole. The timing of the second meiotic division was approx- imated by examining fixed eggs and was confirmed by injecting Hoechst 33258 into live eggs. At 1 9. 5 C the sec- ond polar body formed by 13.8 3.3 min (X S.D.. n = 4; t n = 0. 1 2) after activation, and the second contraction began about 3 min later at 16.7 1.2 min (X S.D., n = 6; t n = 0. 15; Fig. 2). In all cases, polar body formation preceded the second contraction. In the following three sections, we describe (1) the streaming movement of inclusions toward the animal pole, (2) the saltatory movement of inclusions toward the vegetal pole, and (3) the movement of oil droplets toward the vegetal pole. The data presented in Figures 4 and 5 were collected from a single egg at t n = 0.43. The move- ments seen in this egg were confirmed in 41 other eggs studied between the second contraction and the first cell division, and an additional 15 eggs were used to obtain the data summarized in Figure 2. Streaming Essentially all the inclusions in Figure 2 appeared to be streaming toward the animal pole. The diameters of these inclusions were in the range 1.5-1 1 /urn, and they appeared to be distributed throughout the depth of the ooplasm. By "streaming" we mean that the movements of the individual inclusions did not appear to be inde- pendent of each other; in other words, all inclusions moved at nearly the same speed and in the same direction. The motion of three such inclusions, moving at about 1 .5 ^m min ', is summarized in Fig. 4A. Though this speed was typical of streaming motion (2.2 0.8 ^m min~', X S.D., n = 31 inclusions from 5 eggs), the speed some- times increased to as high as 8.2 ^m min" 1 for periods lasting up to 10 min and sometimes decreased to near zero for periods lasting up to 12 min. Saltatory movement The circled inclusion in Figure 2 is one that by its size, shape, and appearance (phase-dark) would be expected to exhibit saltatory movement. The number of inclusions showing such movement was usually not more than three per microscopic field. These inclusions were in the same optical section as those showing streaming movement 118 V. C. ABRAHAM ET AL. Figure I. Ooplasmic segregation in the medaka egg. (A) t n = 0.07. The just-fertilized egg consists of a chorion covered with hairs, a large yolk vacuole, and a thin peripheral layer of ooplasm between the yolk membrane and plasma membrane. Oil droplets are present throughout the ooplasm. and a thin blastodisc is visible at the animal pole (AP). (B) t n = 0.25. The thickness of the blastodisc has increased, but oil droplet movement toward the vegetal pole has not yet begun. (C) t n = 0.53. The thickness of the blastodisc has increased even more, and oil droplets have begun to move toward the vegetal pole. (D) t n = 0.69. A biconvex blastodisc has formed, and many oil droplets have left the animal hemisphere. (E) t n = 0.81. The blastodisc has become plano-convex, and oil droplet movement continues. (F) t n = 1.00 (about 70 min at 23C). The blastodisc has begun to undergo cytokinesis, and most of the oil droplets have formed a crude cap over the vegetal hemisphere. Scale bar. 500 ^m. toward the animal pole. The movement of such inclu- sions (Fig. 4B) differed from those that streamed in the following ways: ( 1 ) Their motion was intermittent, hence the designation "saltatory." An inclusion showing sal- tatory motion typically moved at a constant rate for 1 5- 120 sec, paused for 5-20 sec and then began moving again. (2) Their velocity (44.4 13.8 pm min ', X S.D., n = 17 inclusions from 9 eggs) was about 20-fold higher than that of streaming inclusions. (3) They moved toward the vegetal pole, not the animal pole. (4) Whereas streaming inclusions appeared to move directly toward the animal pole, the paths of these inclusions, though generally directed toward the vegetal pole, were more zig-zagged. Movement of oil droplets Immediately after the second contraction, oil droplets, like saltatory inclusions, began to move toward the vegetal pole (Fig. 1B-E). Unlike saltatory inclusions, however, oil droplets appeared to move directly toward the vegetal pole. Moreover, oil droplets moved more slowly than sal- OOPLASMIC SEGREGATION IN MEDAKA 19 Figure 2. Phase-contrast image of a typical microscopic field near the equator of a fertilized egg at t n = 0.43. Ooplasmic inclusions include some that streamed toward the animal pole (arrowheads), oil droplets of various sizes (arrows), and inclusions that moved saltatorily toward the vegetal pole (encircled parcel). The out-of-focus image of chorionic hairs distorts the image in places (outlined by dashed lines). Scale bar, 50 /jm. Ef/ccls of microtubule poisons Colchicine, colcemid, and nocodazole had the same effects on the eggs, while eggs treated with /i-lumicolchi- cine behaved as untreated (control) eggs. No effect of these poisons was apparent until after the second contraction, even in eggs that were incubated in the microtubule poi- sons for 1 .5 h or 2 h before fertilization: The cortical gran- ule reaction (in 96% of the drug-treated eggs vs. 97% of the controls), the fertilization contraction, elevation of the fertilization membrane, and the second contraction occurred normally in these eggs. However, these drugs had dramatic effects on the sub- sequent movement of ooplasmic inclusions toward the poles of the egg. The most obvious effect was on the oil droplets, which floated to the top of the egg instead of moving toward the vegetal pole (Fig. 6B). Moreover, sal- tatory motion toward the vegetal pole was absent from drug-treated eggs. All three poisons also slowed the rate of growth of the blastodisc (Figs. 6B; 7). The volume of the blastodisc of control eggs at t n 0.85-1.0 was 21.3 + 4.0 nl (X S.D.. n = 7), while that in eggs treated with microtubule poisons was 1 1 .6 2.6 nl (n = 12). Moreover, in poisoned eggs the blastodisc did not undergo the changes in shape seen in control eggs from meniscus to biconvex to planoconvex (Fig. 1); instead the blastodisc appeared only to enlarge while maintaining its meniscus form. The microtubule poisons also caused a decrease in the velocity of streaming inclusions [2.2 0.2 |/m min ' (X S.E.M.. n = 3 eggs) versus 2.9 0.5 (n = 3 eggs) in control eggs]. When we looked at the direction of move- ment of inclusions during a 10-min period, we found that tatory inclusions (17.0 5.7 /urn min ', X S.D., n = 1 5 droplets in 3 eggs), and their speed varied more than that of saltatory inclusions during a given stretch in which they were moving continuously (Fig. 4C). Second contraction complex We monitored this contraction by observing either oil droplet movement at low magnification (at which the en- tire egg could be seen simultaneously) or the movements of inclusions at higher magnification. In all 18 eggs (from 8 females) in which we analyzed the second contraction, it was composed of at least two components: ( 1 ) a move- ment of ooplasmic inclusions, including oil droplets, to- ward the vegetal pole, and (2) a subsequent pronounced movement of the inclusions toward the animal pole (Fig. 5A, B). In 5 of the 18 eggs we observed, the movement toward the vegetal pole was preceded by a weaker move- ment toward the animal pole (data not shown). 200 ! 150 D O m o 100 50 U.O 0.2 0-4 0.6 0.8 1.0 1.2 Proportion of Time to First Cleavage Figure 3. Change in thickness of the blastodisc during ooplasmic segregation. Data from 1 5 eggs, grown at 14C-24C, were used to con- struct this figure; shown are X S.D. Arrows mark the times of occurrence of the second meiotic division (M) and the beginning and end of the second contraction (C). The thickness of the blastodisc increased from = 40 Mm in the just-fertilized egg (t n = 0.05) to =160 ^m at t n = 0.7, by which time the blastodisc was biconvex (Fig. ID). The decrease in the thickness at t n > 0.7 was caused by a change in the shape of the blastodisc from biconvex to plano-convex before the first mitotic division (Fig. ID, E). 120 V. C. ABRAHAM ET AL 100 200 400 600 Elapsed Time (sec) 800 80- 60- 40- 20- 100 200 Elapsed Time (sec) 300 40- 100 200 300 Elapsed Time (sec) 400 Figure 4. Graphic summary of the movements of ooplasmic inclusions. The movements of five inclusions in a single egg are shown, beginning at t n = 0.4. (A) Streaming of inclusions toward the animal pole. Throughout most of this 1 1+ minute period, the speed of the inclusions was & 1.5 Mm min~'. (B) Saltatory motion of an inclusion toward the vegetal pole. Movement was intermittent, i.e.. the inclusion sometimes moved rapidly (a -* b) and sometimes paused (c). Occasionally such inclusions reversed their direction (d). The velocity of the inclusion between t ~ 28 s and t = 200 s was ^25.7 ^m mirT 1 . (C) Movement of an oil droplet toward the vegetal pole. The motion summarized here is that of a small oil droplet (diam. =6 ^m). The speed of the droplet was about 30 Mm min~' at 50-100 s and about 9 Mm min~' at 125- 225s. whereas inclusions in control eggs moved in essentially the same direction (3.7 10.6 departure from the an- imal-vegetal axis, X S.D., n = 30 inclusions; note the small standard deviation), inclusions in poisoned eggs varied substantially in their direction of movement (10.9 55.7, X S.D., n = 52 inclusions; note the large stan- dard deviation). Hoechst 33258 stained three bodies in control eggs fixed at t n = 0.45. One was inferred to be the second polar body (Fig. 8A) on the basis of its protrusion from the surface of the egg, its size, and the presence of a halo of membrane ruffles around it (Brummett et a/.. 1985). The other two, the male and female pronuclei, were about 5 jim from each other and 51.8 10.2 ^m (X S.D., n = 9) from the polar body (Fig. 8B, C). As in control eggs, Hoechst 33258 stained three bodies in eggs treated with 100 pM colchicine and fixed at t n = 0.45, one of which was the second polar body (Fig. 8D). However, in contrast to the 30 Is 20 ss 3 0) E 2? 3 0) o- -10 4 6 Elapsed Time (min) Figure 5. The second contraction. (A) The "tracks" of five inclusions during the second contraction in an egg growing at 2lC. The circles represent the positions of the inclusions at 20 sec intervals, beginning at "B" (6 mm after fertilization; t n = 0.06) and ending about 9.5 min lateral "E" (at t n = 0.15). The parcels first moved toward the vegetal pole ("down" in this figure: ), and then reversed their direction at "R" and began to move toward the animal pole (o-o). Note that the movement toward the animal pole was rapid at first and then slowed. Scale bar, 5.65 Mm. (B) Graphic summary of the movement of parcels #1-3 in Figure 5A. The parcels moved hardly at all for 2 min, began to move toward the vegetal pole (down in this figure) after about 3 min (at t n = 0.09), and then reversed their direction after 5 min (> t n = 0.1 1) and began to move toward the animal pole (up in this figure). 10 OOPLASMIC SEGREGATION IN MEDAKA 121 Figure 6. Effects of nocodazole on ooplasmic segregation. (A) Control egg, t n = 0.84. A large blastodisc has formed at the animal pole, and oil droplets have formed a cap over the vegetal hemisphere. (B) Egg treated with 0.17 nM nocodazole. t n = 0.80. The blastodisc is smaller than in the control egg, and most of the oil droplets, instead of moving toward the vegetal pole, have floated to the top of the egg (i.e.. toward the viewer, whose perspective is from above the egg) and coalesced into one large droplet there. Scale bar, 250 Mm. situation in control eggs, in which the male and female pronuclei were within 5 ^m of each other, the male and female pronuclei in colchicine-treated eggs were far apart (132.2 54.8 urn; X S.D., n = 8; Fig. 8E). Discussion All three microtubule poisons used in this study col- chicine, colcemid. and nocodazole affected the move- o m ^ a> c JC u 175 150 125 100 75 50 25 00 0.2 0.4 06 0.8 1.0 1.2 Proportion of Time to First Cleavage Figure 7. Effect of microtubule poisons on the growth of the blas- todisc. The thickness of the blastodisc along the animal-vegetal axis was measured in untreated eggs ( . four eggs) and in eggs treated with 100 iiM fj-lumicolchicine ( D , three eggs), 100 pM colchicine ( , four eggs), 0.35 nM colcemid ( O . three eggs), or 0.17 tiM nocodazole ( A . five eggs). Shown are the mean values; over all the treatments and times, the standard deviation averaged 12% of the mean. Colcemid, colchicine. and nocodazole all slowed the growth of the blas- todisc. while eggs treated with J-lulmicolchicine behaved as untreated eggs. The volume of the blastodisc in control eggs_and poisoned eggs at t n = 0.85- 1 .0 was 2 1 .3 4.0 nl and 1 1 .6 2.6 nl (X SD), respectively. ment of ooplasmic inclusions during segregation in the medaka egg. That the poisons acted specifically as micro- tubule poisons is a reasonable inference because ( 1 ) their effective concentrations were similar to those used in pre- vious studies (Zalokar, 1974; Eckberg, 1981; Shimizu, 1982; Astrow el ai. 1989; Sawada and Schatten, 1989); (2) poisons from two classes of microtubule poisons (Bray, 1992, p. 207) had similar effects on the eggs; and (3) 100 nM /3-lumicolchicine had no apparent effect on the eggs. The results of the present study differ from those of Katow (1983), who reported that the blastodisc formed normally in zebrafish eggs treated with colchicine. This difference could be due to the lower concentration of colchicine used in the earlier study (2.5 nAl vs. 100 nM in the present study) or to a lower permeability of the zebrafish egg to colchicine. It is possible (but unlikely, we believe) that microtubules are not required for ooplasmic segregation in the zebrafish egg. Saltatory movement similar to that observed in the present study is often associated with microtubules (Hay- den el ai. 1983; Brady and Pfister, 1991). Similarities include the intermittent nature of the movement, its in- hibition by microtubule poisons, and the speed of moving particles (Hamaguchi et ai. 1986; Shimizu el ai, 1991). These similarities suggest that in the medaka egg some ooplasmic inclusions move toward the vegetal pole via microtubules oriented approximately along the animal- vegetal axis. The normal movement of oil droplets was also affected by the poisons, suggesting that microtubules are also in- volved in the movement of these droplets. Such an in- 122 V. C. ABRAHAM ET AL Figure 8. Formation of second polar body and pronuclear migration. Untreated eggs (A-C) and eggs treated with 100 n.M colchicine (D-E) were fixed at t n = 0.45, subsequently stained with Hoechst 33258, and viewed with either phase contrast (A. D) or epifluorescence (B, C, E) optics. The second polar body (arrowhead) could be seen near the animal pole in both untreated eggs (A) and in eggs treated with 100 ^M colchicine (D). Of the three fluorescent bodies present in this region of the egg, one corresponded to the polar body (arrowhead in B, E), and the other two were the male and female pronuclei (C, E). In the untreated egg shown here, the polar body and pronuclei were recorded in separate photographs (B, C) because their focal planes were separated by about 25 jim. The two pronuclei were close to each other and were about 37 ^m from the polar body. In eggs treated with colchicine, the pronuclei were much farther apart (E). Scale bars. A, D. 10 ^m; B, C, E, 50 urn. volvement would seem to require the presence of a unit membrane at the surface of the oil droplets to provide a site of attachment for a kinesin-like molecule. Whether such a membrane is present around these droplets is not known. In other types of cells, unit membranes are present around some lipid droplets but not others (Wake, 1974; Nedergard and Lindberg, 1982). An alternative expla- nation for the effect of these poisons on oil droplet move- ment is that in control eggs a dynamic network of micro- tubules holds the oil droplets in place; in the presence of these microtubule poisons, such a dynamic network would eventually disappear as disassembly continues in the ab- sence of assembly. This question will require further study. The movement of ooplasm toward the animal pole in fish embryos has been previously described as streaming (Roosen-Runge, 1938; Beams et ai. 1985) or bulk How (Gilkey, 1981); our results confirm these reports. Micro- tubule poisons slowed both the movement of inclusions toward the animal pole and the growth of the blastodisc, but they did not inhibit either process entirely, suggesting that more than one mechanism is responsible for these phenomena. In ascidians (Sawada and Osanai, 1981, 1984, 1985; Jetfery, 1984; Bates and Jeffery, 1988) and an oligochaete (Shimizu, 1982, 1984), actin microfila- ments form a cortical network that contracts toward one pole of the egg, pulling with it both cortical and subcortical components of the ooplasm. F-actin is present in the cor- tex and subcortex (Beams et a/.. 1985; Wolenski and Hart. 1987; Chang, 1991) of the zebrafish egg, and an acto- myosin-like ATPase has been identified in cortical prep- arations offish eggs (Jorgensen, 1972). Moreover, cyto- chalasins (Katow, 1983; Ivanenkov cl ai. 1987; Fluck, unpub.) and DNase I (Ivanenkov et ai. 1987) inhibit for- mation of the blastodisc in fish embryos. Thus, both mi- crotubules and microfilaments may be involved in the movement of ooplasm and its inclusions toward the an- imal pole in the medaka egg. Calcium ion may both trigger and organize such a con- traction in the medaka egg just as it does in ascidian egg (Jeffery, 1982;Sardetrfa/.. 1 986; Speksnijder 1.9 mm). To prevent the loss of tubule contents during embedding, the tubule sections were cut well beyond the segment se- lected for sectioning. For each individual, six 5 ^m-mi- crotome sections were cut from both the small and large tubules. These sections were first placed on gelatin coated slides (the gelatin was heated to 42 C) and then transferred REPRODUCTIVE CYCLE OF PSOLUS 1-ABR1C11 127 Figure 1. Psolus fabricii. (A) Dissected male showing the respirator, tree (R), intestine (I), body wall (W), aquapharyngeal bulb (A), cloacal muscles (C), longitudinal muscles (L). and testis (G). (B) Photograph of the mouth region showing the feeding podia, one of which is in the mouth and the others extended, and papillae surrounding the gonopore (arrow). Photographs of a testis (C) and an ovary (D) just after spawning showing the large tubules (L), small tubules (S). and tubules with swellings containing residual gametes (RG). The horizontal bars in photographs C and D represent 50 mm. to an oven at 37C for 1 h. This technique usually pre- vented the breaking of the fragile tubules and the loss of gametes. The slides were stained with cosine and hema- toxylin, as described by Galigher and Kozloff( 1971), and good resolution of the various cell types was achieved. A second series of slides was stained with the periodic acid- SchifT(PAS) reaction (Humason, 1981) to identify poly- saccharides (glycogen). Gonadal development was classified into five stages (post-spawning, recovery, growth, advanced growth, and mature stage) that were adapted from the earlier studies of holothurians (Tanaka, 1958; Costelloe, 1985; Cameron and Fankboner, 1986). For each male, we made 15 ran- dom measurements of the thickness of the gonadal tubule wall from slides of both small and large tubules. Only intact areas were used in this assay. For each female and for both small and large tubules, we determined the di- ameters of 200 relatively unbroken oocytes that showed a well-centered germinal vesicle. Size of the gonad at sexual maturity A sample of 132 individuals was collected on 4 May 1988. For each of these individuals, we measured the go- nadal index and made histological sections to determine whether mature gametes were present. We also deter- mined the total number of tubules in each individual, as well as the length of 1 5 randomly selected tubules, and the length of the intestine. Environmental factors Continuous temperature measurements at the study site at Anse a Robitaille were made during most of our study using a Peabody Ryan thermograph placed at 10 m in depth. Data on day length and minimum daily sun- shine were obtained from the weather station at the Que- bec Airport (Environment Canada, Atmospheric Envi- ronment Service). Data on freshwater run-off were pro- vided by Environment Canada (Climatologic Services) by 128 J.-F. HAMEL ET AL using the combined discharges from the Montmorency, Bastiscan, Saint-Anne and Chaudiere rivers. Phytoplankton cells abundance control the spawning of a number of marine invertebrates in the Estuary, but regular phytoplankton measurements could not be made during our study. As an indirect signal of the spring phy- toplankton bloom, we determined, in 1989. the time of spawning in the green sea urchin Strongylocentrotus droe- bachiensis. This species spawns when the adults detect the rapid growth of phytoplankton during the spring bloom (Himmelman, 1981; Starr, 1 990; Starr elal, 1990, 1992). Thus, from March to August, when spawning was anticipated, gonadal indices (percentage gonadal mass) were determined for 15 adult urchins (4.0-6.5 cm in di- ameter) of both sexes at each sampling date. We used two approaches to examine seasonal changes in the intestinal contents of Psolus fabricii. First, for each date, the contents of the first centimeter of the intestine of each of the 30 individuals were suspended in 5 ml of 10% formalin, and the various types of undecomposed organisms present in a 1 ml subsample were identified and counted with a hemacytometer. Large cells were ex- amined under white illumination, and the presence of small phytoplankton cells was determined by the fluo- rometric method of Yentsch and Menzel ( 1963). Second, the contents of the following 30 g portion of the digestive tract was emptied into a Petrie dish, examined with a binocular scope, and the proportion of living phytoplank- ton cells (green in color) to non living materials (decom- posed cells and inorganic materials) was estimated. The phytoplankton cells in the first centimeter of the intestine seemed virtually undigested (green in color and intact). Buoyancy ofoocytes Forty oocytes were collected from five mature females (measuring 25-34 g in dry body wall mass) collected on 14 July 1990. The oocytes were placed in natural seawater to provoke breakdown of the germinal vesicle. The oocytes were then placed in a 500 ml graduated cylinder (5 cm in diameter) at 7-8C and the rate of up- ward vertical movement was recorded (Fig. 8). This movement was taken as a measure of buoyancy. 250n 200- _0j 2 150- .2 1001 50- Male* b I 101 * JD a 73 5H G Ol O Male Juvenile 200 i Size range selected 150- Male X 0.05). In some very large males and females, the terminal ends of some tubules were necrotic, and at times these ends were found floating free in the coelomic fluid. The size of the gonad at sexual maturity was determined from the 132 individuals collected on 4 May 1988 (Fig. 2C). Gonadal tubules were present in every individual examined that had a dry body wall mass of at least 1.2 g REPRODUCTIVE CYCLE OF PSOLL'S FABRICll 129 Figure 3. Psolus lahncn Light micrographs of sections of gonads of juveniles. (A) Immature gonad (from an individual weighing <5.5 g in dry body wall mass) showing the germinal epithelium (GE) and an absence of identifiable precursor cells for two germinal tubules (GT); (B) Immature female (between 5.6- 10 g) with oogonia (O). primary oocytes (PR), but an absence of more advanced stages; (C) Immature male (between 5.6-10 g) showing the proliferation zone (PZ) and the lumen containing only a few spermatozoa (SP); (D) Young female ( = 10 g) showing the germinal epithelium (GE), primary oocytes (PO) and a few vitellogenic oocytes (V); (E) Young male ( = 10 g) showing two distinct germinal tubules (GT) and numerous spermatozoa (SP) in the lumen. The horizontal bar in photograph A represents 800 ^m and applies to all of the photographs. (equivalent to =0.7 cm in distance mouth-anus). The relative size of the gonads increased sharply as the dry body wall mass rose from 3 to 10 g (Fig. 2C). The size of testis and ovary overlapped greatly up to a body wall mass of = 1 5 g, but the testis was generally larger than the ovary for larger individuals (Kruskal-Wallis, analysis of variance, P < 0.01, followed by a non-parametric multiple-range test, P < 0.05; Sokal and Rolph, 1981). The variation in gonadal size was relatively small between 20 and 34 g (5.5 to 6.1 cm); beyond that range the relative gonadal size dropped. Histological preparations showed that only undiffer- entiated precursor cells are present along the germinal epithelium of individuals weighing <5.5 g( = 3.2 cm) (Fig. 3A). The sex of larger individuals could be identified by the presence of oogonia and young oocytes in females, and spermatogonic stages in males (Fig. 3B, C). Beginning at 6.5-7.9 g (4.0-4.6 cm), the sexes were readily recognized 130 J.-F. HAMEL ET AL from gonadal smears (Fig. 2C), although histological ex- amination showed that only individuals weighing >10 g (=4.8 cm) contained mature gametes with the same mor- phology and reaction to PAS and hematoxylin and cosine as for very large individuals (Fig. 3D, E). Immature gonads were cream in color, whereas the mature testis was pink, and the mature ovary reddish brown (individuals > 10 g). Thus, Psolus fabricii starts to produce mature ga- metes at 10 g, but the gonads only attain a plateau in size at 15-20 g (Fig. 2C). Individuals weighing 25 to 34 g showed no variation due to body size and were used in following the gonadal index cycle. No significant departure from a sex ratio of 1 : 1 was observed in any of the samples, and the ratio for all of the samples together was 595 males to 607 females (df = 14, X 2 : = 1.44, P > 0.05). No external differences were observed between the sexes, and hermaphrodites were not encountered. Seasonal changes in body component indices Throughout the study, the mean gonadal index of males was more than twice that of females (Kruskal-Wallis, P < 0.01), and on no date did the maximum value for any given female attain the minimum observed for the males (Fig. 4). Nevertheless, the two sexes showed parallel sea- sonal cycles in gonadal size. The index for males and fe- males dropped significantly between 1 4 May and 1 2 June 1988 (Kruskal-Wallis, P < 0.01 ), suggesting the release of gametes. The gonads showed no further significant change in size until the end of the following winter (in March 1989 for males and April 1989 for females), when a sig- nificant growth was evident (Kruskall-Wallis, P < 0.01). Both testicular and ovarian indices attained a peak in mid July 1989 and then dropped abruptly by 5 August 1989, suggesting a second spawning. The diameter of the germinal tubules showed a similar pattern, although the annual cycle was more pronounced (Fig. 4). In both years, the mean diameter attained a max- imum just before spawning (higher in 1989 than in 1988) and dropped precipitously during spawning (Kruskal- Wallis, P < 0.01). The decrease was by =24% in 1988 compared with =80% in 1989. The diameter of any given tubule was relatively uniform, except after spawning when some tubules had swollen sections containing unspawned gametes. The mean diameter of male tubules was consis- tently larger than that of females during May through August (Kruskal-Wallis, P < 0.0 1 ), but not during autumn and winter (Kruskal-Wallis, P > 0.05). In spite of the distinct seasonal pattern in mean tubule size, extremes in tubule size were always evident, and every gonad con- tained tubules ranging from small to large. The 1989 spawning was also observed directly by per- sons diving in our study site on 22 July (Normand Piche Male Female 10 M J JASOND 1988 F M A M J I A 1989 Figure 4. Psolus fabricii. Seasonal changes in the mean gonadal, intestinal and respiratory tree indices and in the diameter of the germinal tubules for males and females from May 1988 to August 1989. Vertical lines indicate the 95% confidence intervals. and Andrea Cantin, pers. comm.). Numerous females were seen releasing eggs. Spawning was probably wide- spread and massive in the lower St. Lawrence Estuary because on 25 and 26 July 1989 other divers observed an abundance of Psolus fahricii oocytes and embryos throughout the first 3 m of the water column over a long section (>5 km) of the southern side of the Estuary near Rimouski (Lucie Bosse, Institut Maurice-Lamontagne, pers. comm.). Variations in the intestinal index were largely attrib- utable to changes in the intestinal contents: the mass of the wall of the first centimeter of the intestine varied by <2% throughout the study (and no significant seasonal REPRODUCTIVE CYCLE OF PSOLUS l-'AHRK'll 131 changes were detected. Kruskal-Wallis, P > 0.05), whereas the mass of its contents varied by = 37% (Kruskal-Wallis. P < 0.0 1 ). The intestinal index showed intermediate values during the summer of 1988, a minimum between De- cember 1988 to March 1989, and then a sharp increase to a maximum in July 1989. The maximum attained in 1989 was much greater than in 1988 (Fig. 4). The respi- ratory tree decreased during the winter of 1 988-89, started to growth in April 1989, and reached a peak in mid July 1989 (Fig. 4). Female reproductive cycle Oogenesis. The development of gametes in Psolusfa- bricii was transversal, starting at the surface of the germinal epithelium and progressing towards the lumen of the tu- bule. In addition, it proceeded in a relatively uniform fashion along all surfaces of any tubule. Along the surface of the germinal epithelium, oogonia occurred in groups at numerous points, whereas primary oocytes (<100 /urn) were dispersed. The small oocytes (<250 /um) were sur- rounded by follicular cells which persisted until spawning. The germinal vesicle is central and also persisted until spawning. The small oocytes have a PAS-negative baso- philic cytoplasm which becomes slightly PAS-positive upon attaining 300 ^m (indicating the beginning of gly- cogen accumulation), and increasingly positive as vitel- logenesis progressed. The morphology and histological staining indicated that the oocytes were mature at =800 um, although they could attain up to 1 400 ^m in diameter. Nutritive phagocytes were associated with the tubules that had released gametes. For both males and females, the tubule wall became more and more PAS-positive after spawning until January-February, and then progressively PAS-negative until the following spawning period. The following five stages of oogenic development were used to quantify the seasonal oogenic changes (Fig. 5). ( 1) Post-spawning (Fig. 5A). The gonadal tubule wall is thin and extremely convoluted. Although some residual or unspawned oocytes. measuring 400-800 ^m, remained in the tubules, the majority of oocytes measured <300 /im and are generally PAS-negative. Striking elongated empty areas are seen in the tubules, suggesting the passage of oocytes along the length of the tubule during spawning. Nutritive phagocytes begin to appear and are always inside of the follicular cells that surround the residual oocytes. The follicular cells around the residual oocytes were de- generated. (2) Recovery (Fig. 5B, C). The gonadal tubule wall is very thick. The germinal epithelium is convoluted, and beds of small oocytes (<200 /urn in diameter) are present along the epithelium. Nutritive phagocytes are closely as- sociated with nearly all of the residual oocytes and the follicular cells are poorly denned. (3) Growth (Fig. 5D). The thickness of the tubule wall reaches its maximum. Along the surface of the germinal epithelium, many small oocytes (<200 /urn, PAS-negative) and some previtellogenic oocytes (300-600 um, PAS-pos- itive) are present and nutritive phagocytes are virtually absent. (4) Advanced growth (Fig. 5E). The tubule wall is thinner, and the diameter of the tubules is increased. In the lumen of the tubules, well-defined follicular cells are associated with large PAS-positive previtellogenic (400- 600 ^m) and vitellogenic (>600 nm, PAS-positive) oo- cytes. The vitellogenic oocytes are reddish orange. Nu- merous small oocytes (<400 um) are present along the germinal epithelium. (5) Mature (Fig. 5F). The tubules are highly dilated, their walls thin and not convoluted, and they are almost completely filled with mature oocytes (>800 /urn). Each oocyte contains one to four nucleoli and a well-defined germinal vesicle, which occupies 30-50% of the surface of the oocyte in the histological preparations. Immature oocytes are virtually absent. Seasonal changes in the oogenesis. Advanced oogenic stages (advanced growth and mature stages) predominate in the large tubules, and earlier stages (post-spawning, re- covery and growth stages) in the small tubules (Fig. 6). Nevertheless, both categories of tubules showed a seasonal pattern that is correlated with the gonadal index cycle. In the large tubules, the post-spawning and recovery stages are almost always absent, and the major evidence of the June 1988 and July 1989 spawnings was the decrease in the mature stage. In contrast, the mature stage in the small tubules is rare before spawning and absent in other pe- riods, and the major evidence of spawning was a sharp increase in the post-spawning stage (from to 80%-). These observations suggest that the release of mature oocytes during spawning transforms large fecund tubules into small tubules in post-spawning condition. This change coincides with a sharp drop in the mean diameter of tu- bules (Fig. 4). Following spawning there is a period of inactivity until mid January; then oocyte development resumes. In small tubules, a progressive increase in the frequency of the growth stage occurred between January and July 1989 coincident with a decrease, first in the post- spawning stage, and then in the recovery stage. Mean- while, the large tubules show an increase in the advanced growth and mature stages (Fig. 6). Si:e of oocytes. The seasonal pattern in the size structure of oocytes varies markedly between large and small tubules (Fig. 7). In large tubules, a striking change in the oocyte population occurred during the 1988 spawning. Prior to spawning, most oocytes measured >800 /urn, whereas after spawning in June 1988, 500-700 /urn oocytes predomi- nated. Following this, the oocyte population in the large tubules was stable. Then in January 1989, renewed oocyte 132 ... m ^gggsli* \ Figure 5. Psolusfabrk'ii Light micrographs of ovarian sections illustrating the oogenic cycle. (A) Portion of a post-spawning ovary showing the germinal epithelium (GE). residual oocytes (RO). and a channel created by the expulsion ol eggs during spawning (C); (B) Early recovery stage showing primary oocytes (PO), mature oocytes (M), and nutritive phagocytes (P) surrounded by follicular cells. (This section was across a swelling containing residual gametes in a spent tubule); (C) Late recovery' stage showing an abundance of nutritive phagocytes (P); (D) Growth stage showing sites of oogonial proliferation (OP), primary oocytes (PO), and mature oocytes (M); (E) Advanced-growth stage showing an abundance of both vitellogenic oocytes (V) and mature oocytes (M) with nucleoli (N); (F) Mature stage showing large mature oocytes (M) containing the germinal vesicle (GV) and surrounded by follicular cells (F). The bar in photograph A represents 800 Mm and applies to photographs B, C. D and E, whereas the bar in photograph F represents 400 urn. growth was evident, and the predominant mode of oocytes attained a peak of 1 100 to 1300 ^m in mid July 1989. These mature oocytes largely disappeared during the July 1989 spawning, and on 5 August the modal oocyte class was again 500-700 /urn. Although the loss of large oocytes (>800 /urn) during spawning was expected, the presence of a strong cohort of intermediate oogenic stages (500- 700 /urn) in the large tubules after spawning seemed at first surprising. This was because spawning transformed the large tubules into small tubules. Thus, the small tu- bules in June 1988, which were characterized by a strong mode of oocytes measuring <300 j*m, were probably those which had just spawned. They contained residual oocytes that were being attacked by nutritive phagocytes, whereas Large tubules 100 REPRODUCTIVE CYCLE OF PSOLl'S FABR1CI1 Female Small tubules T 133 Post-spawning Small tubules Male Large tubules M J A S O N D J 1988 F M A M 1989 J A Figure 6. Pxolus tahncii. Relative frequency of different gametogenic stages (as defined in the Materials and Methods section) in small and large, female and male, tubules for the period from May 1988 to August 1989. at the same time, the large tubules contained an abun- dance of intermediate stages, and nutritive phagocytes were rare. Most oocytes in the small tubules in mid May 1988 measured 400-600 ^m, whereas those in the large tubules in June 1988 measured 600-800 /urn. The simi- u c 0> cr 0) Large tubules T 500 1000 1500 500 1000 1500 Oocytes diameter (|im) Figure 7. Psolus fabncn. Oocyte diameter distributions for small and large tubules for the period from April 1988 to August 1989. For each distribution the vertical axis is from to 60% and the mean oocyte diameter is indicated by an arrow. larity in size distributions for these two samples suggested the transition from small to large tubules during spawning. Following spawning, until mid December 1988, the oocyte 134 J.-F. HAMEL KT II distributions for the small tubules showed little change. However, a marked increase in the total number of cells per surface of germinal tubule was noted in January 1989. Thereafter, oocyte size and number progressively in- creased in the small tubules until 20 July 1989 when the size structure was virtually identical to that of the small tubules prior to spawning in 1988. The sharp reduction in >800 ^m oocytes in the large tubules during the 1989 spawning closely followed the changes in the large tubules during the 1988 spawning. Buoyancy o/ Oocyles. Thirty three of the oocytes showed a positive floatability which clearly increased with di- ameter (Spearman rank correlation coefficient, r = 0.67, df: 32, P < 0.01). This indicated that 1.2 mm oocytes would move upward at a rate of 20-30 mm min~'. The other four oocytes showed a slightly negative floatability, and we suspect that they were damaged (possibly the egg membrane was not intact) (Fig. 8). These observations indicate that spawned eggs will move to the surface of the water column. This agrees with the abundance of devel- oping Psolus fabricii embryos near the surface, as observed by divers during the 1989 spawning. Male reproductive cycle Spermiogenesis. The following five stages of spermio- genesis are used to quantify the seasonal changes in the small and large tubules (Fig. 9). ( 1 ) Post-spawning (Fig. 9A). The thickness of the tubule wall is at its minimum. In the sections, we observed elon- gated empty areas along the length of the tubules, sug- gesting the passage of gametes during spawning. A few residual spermatozoa are present, and no proliferating zone (containing spermatogonia, spermatocytes and spermatids) was present. (2) Recovery. The tubule wall is extremely thick and highly convoluted. The tubules contain small quantities of spermatozoa and scattered nutritive phagocytes. (3) Growth (Fig. 9B, C). The gonadal tubule wall is beginning to decrease in thickness but is still convoluted. Spermatogonia are abundant along the surface of the ger- minal epithelium. Progressing towards the lumen, there is a layer of spermatocytes, one of spermatids, and finally a small number of spermatozoa in the lumen. (4) Advanced growth (Fig. 9D, E). The tubule wall is thinner and slightly convoluted, and the lumen is filled with spermatozoa. (5) Mature (Fig. 9F). The tubules are stretched to their maximum diameter and completely filled with sperma- tozoa. The tubule wall is nearly smooth, and earlier sper- matogenetic stages are absent. Psolus fabricii spermatozoa are flagellated with a round head measuring 5-6 /urn. Microscopic observation of a sperm suspension in seawater, just prior to spawning, re- vealed a low motility of the spermatozoa. .5 g- I o 3 pa 35- 30- 25- 20 - 15- 10 - 5 - 0- -5- -10 0.3 0.6 0.9 1.2 Diameter (mm) Figure 8. Pxoltis Jahricn. Relation of buoyancy to diameter for oo- cytes dissected for mature females and activated by being placed in sea- water. The regression line is based only on oocytes with positive buoyancy. Seasonal changes in spermatogenesis. In May and June 1988 and again in July and August 1989, advanced stages (advanced growth and mature stages) were found in >85% of the large tubules, whereas earlier stages (post-spawning, recovery and growth stages) predominated in the small tubules (Fig. 6). The most striking evidence of the spawn- ings in June 1988 and August 1989 was the abrupt ap- pearance of post-spawning stages in the small tubules. The tubules classified as large after spawning were char- acterized by an abundance of early spermatogenetic stages and few spermatozoa. These observations suggested, first, that the release of spermatozoa from the large tubules during spawning diminish their size, so that after spawning they were considered as small tubules. Moreover, the tu- bules that were selected as large were those that had re- cently attained a diameter of 1.9 mm (the lower limit for large tubules). Thus a pattern parallel to that observed for the females was found. The post-spawning stage, found only in the small tubules, disappeared by late summer and was replaced largely by the recovery and growth stages. Subsequently, the advanced growth and mature stages be- came more common and attained a peak a few month prior to spawning. Thickness of the gonadal luhit/e wall. In males, the dis- tributions for gonadal tubule wall thickness were virtually always skewed rather than being symmetrical (Fig. 10). Further, all size classes up to 140-160 ^m were present in both small and large tubules throughout the study, ex- cept for four dates near the time of spawning, when the largest classes were absent. In both the small and large tubules, changes in the distributions followed an annual pattern. Just before spawning in May 1988, the mean thickness was 20-40 nm; after spawning it decreased REPRODUCTIVE CYCLE OF PSOLUS 1-'ABR1CI1 135 Figure 9. Psolus fabricii. Light micrographs of testicular sections illustrating the spermatogenic cycle. (A) Post-spawning testis showing the germinal epithelium (GE) and channels where sperm passed during spawning (C); (B) Growth stage showing the highly convoluted germinal epithelium (GE) and the proliferation zone (PZ); (C) Growth stage showing the germinal epithelium, spermatogonia (SG), spermatocytes (SC) spermatids (ST) and spermatozoa (SP) in successive layers progressing towards the lumen; (D) Early advanced- growth stage showing the proliferating zone (PZ) and spermatozoa (SP): (E) Late advanced-growth stage showing the thin gonadal tubule wall and an abundance of spermatozoa (SP); (F) Mature stage showing the thin tubule wall (TW), absence of the proliferation zone, and great numbers of spermatozoa (SP) in the lumen. The bar in photograph A represents 800 ^m and applies to photographs B, D, E and F. whereas the bar in photograph C represents 300 urn. slightly. Subsequently, the thickness of the tubule wall progressively increased, although the pattern varied de- pending on tubule size. Thus, the large tubules grew more rapidly and attained a peak (=120 ^irn) in November 1988, whereas the small tubules did not attain a peak (=140 nm) until February 1989. Subsequently, the size of the modal size class again decreased to 20-40 /um fol- lowing the 1989 spawning (Fig. 10). Environmental factors Temperature. The mid May to mid June spawning, in 1988, coincided with the spring warming period, and temperatures attained about 5C at the time of spawning (Fig. 11). However, temperatures fluctuated markedly during this period. An increase from 4 to 6C was ob- served between 20 and 23 May 1988, and a drop of 6.7 136 J.-F. HAMEL ET AL. Small tubules Large tubules 40 80 120 160 40 80 120 160 Gonadal tubule wall thickness (|im) Figure 10. Psolusfabricii Frequency distributions! 10 nm size classes) of the thickness of the gonadal tubule wall for small and large tubules for males collected from May 1988 to August 1989. The arrows indicate the mean thickness for each sampling date. to 4.4C between 5 and 6 June. These variations were due to the semidurnal tides in the Estuary (Demers el al. 1986). During 1988. the maximum temperature was reached in mid July, and the autumnal decrease began in late August. In 1989, warming began in March, attained a peak in late June, and then varied around 6C until the end of the study (2-4C daily fluctuations were frequent). The accelerated gonadal growth, which began in March or April, occurred at about the time that the vernal warming began. There was no spawning, either during the warming phase or during marked temperature variations in May and June; rather, spawning occurred in late July when temperatures were more stable. Thus, although spawning occurred at about the same temperature in the two years (5-6C), the point in the temperature cycle was quite dif- ferent (Fig. 1 1). Photoperiod. In our study, the renewal of gametogenesis in January coincided with the period when day length and daily bright sunshine were beginning to increase, and the gonadal peak was attained at the photoperiod maxi- mum (Fig. 11). Although Psolusfabricii spawned near the photoperiod maximum in both years, the 1988 spawning occurred at the beginning of this maximum and the 1989 spawning one month later, when photoperiod was just beginning to decline. Freshwater run-off' and the predicted timing of the phy- toplankton bloom. The period during which freshwater run-off decreased in the Estuary was much later in 1989 (July) than in 1988 (late May) (Fig. 11). Nevertheless, spawning in both years coincided with this event, sug- gesting a relationship between the two. 1989 was an ex- ceptional year in that the run-off was markedly greater and more delayed than in the five previous years. It was also unusual in that spawning of the green sea urchin Strongylocentrotus droebachiensis, a signal of the phyto- plankton increase, was much later than in the previous years. The urchin spawned abruptly between 14 July and 5 August, exactly the same period during which Psolus fabricii spawned (Fig. 1 1 ). In contrast, when urchins were studied in the Estuary in the previous years, spawning occurred prior to mid June (Starr, 1990). Intestinal contents. The intestine of adult Psolusfabricii contains two types of materials: ( 1 ) non living particles and (2) phytoplanktonic cells; the major species of plank- ton are the diatoms Thalassiosira sp., Coscinodiscus sp., Chaetoceros sp., and Skeletonema sp. (Fig. 1 1). During the autumn and winter, only 20-50% of the contents were phytoplanktonic cells, and this increased during the spring, attaining virtually 100% in the summer (Fig. 1 1 ). A marked seasonal pattern was evident for the diatoms present in the intestines. Most cells in the first samples in May and June 1988 were Coscinodiscus sp., Thalassiosira sp., and Skeletonema sp., suggesting a diatom bloom at REPRODUCTIVE CYCLE OF PSOLL'S 1-'ABRIC11 137 41 t- 3 "% u 5 3' 1 ns D w- 1.9 mm, a diameter sufficient to be classified as large tubules. During the autumn, the oocyte distributions in the tubules classified as large, remain virtually static, indicating a pe- riod of inactivity; then in January, oocytes growth and tubule enlargement resumes. This growth continues until the following summer when most oocytes measure >800 ^m and the tubules attain their maximum diameter (Fig. 4). Finally, the release of these large oocytes during spawning results in a drop in the size of the tubules. From the time of spawning until the following January, nutritive phagocytes are active in destroying the residual oocytes (Fig. 5). Our study also indicates a prolonged spermatogenesis. As with oogenesis, it begins with the production of pre- cursor cells in the small tubules in mid winter, although a prior accumulation of reserves in these tubules is indi- cated by the thickening of the gonadal tubule wall during the previous autumn (Fig. 10). In late winter and spring, as the thickness of the germinal wall decreases, spermato- gonia. spermatocytes, and spermatids progressively ac- cumulate in the tubules (Figs. 9, 10). Since these tubules contain only small amounts of spermatozoa, they prob- ably do not participate in spawning. During and after spawning they progressively attain the size of large tubules. The major change after spawning in what are now the large tubules is the thickening of the gonadal tubule wall and this peaks in February (Fig. 10). At about the same time, the production of spermatozoa increases, and this amplifies until a peak just prior to spawning (Fig. 6). Fi- nally, with the release of sperm during spawning, these large tubules become small; after spawning nutritive phagocytes become abundant. This is the first report of the testicular cycle taking longer than a year in holothu- rians. Although the examination of large and small tubules indicates that gametogenesis is prolonged, studies with radioactive markers are needed to determine its duration precisely. Our observations suggest that the production of the majority of gametes begins in winter and terminates 15-18 months later (two summers later). In some tubules, however, this process may be much longer or, at times, shorter. That gametogenesis in Psolus fabricii generally takes more than a year was revealed from the separate histo- logical studies of small and large tubules. Smiley and Clo- ney (1985) and Smiley (1988) examined the gonads of female Sticliopus califomicus collected in different seasons. Their observations of three size groups of tubules, with the most advanced stages of oogenesis only being present in the largest tubules, similarly led them to conclude that oogenesis was a long process. In contrast to P. fabricii. the various sized ovarian tubules of S. califomicus are not intermixed. Rather they are arranged in order, the large fecund tubules being located posteriorly. Smiley and Clo- ney (1985) report that the large tubules are completely reabsorbed once the oocytes are released. Based on these observations, they propose that the tubules are produced at the anterior of the gonad and migrate posteriorly as they increase in size and state of development. Reabsorp- tion does not occur in P. fabricii, since a new group of oocytes is evident in the fecund tubules at the time of spawning and persists in spent tubules during the autumn when residual gametes are being phagocytised. Thus, the pattern of tubule and gamete production in S. califomicus contrasts markedly with that in P. fabricii. Resorption of tubules has also been noted in Mesothuria intestinalis (Theel, 1 90 1 ) and Ypsilothuria talisman! (Tyler and Gage, 1983), but probably does not occur in S. japonicus (Ta- naka, 1958) and three species of sea cucumber examined byConand(1981). REPRODUCTIVE CYCLE OF PSOLL'S FABRICII 139 Gametogenesis in another holothurian, Aslia lefrevrei. in the same family as Psolus fabricii (Dendrochirotida), follows still another pattern. The tubules are of uniform width, and gametogenesis follows an annual pattern that is highly synchronized amongst the tubules (Costelloe, 1985). For example, during numerous periods in the year, all of the tubules are at the same stage of gametogenetic development. As in P. fabricii. the tubules are not reab- sorbed after spawning, the growth continues for a sub- sequent year, oocytes (<200 /^m) appearing prior to spawning. The above observations indicate that the pat- tern of gametogenesis varies markedly even within closely related holothurian species. Future studies should there- fore consider the pattern of production of the tubules as well as the gametogenesis within the tubules. Control of gametogenesis The active phase of gametogenesis in Psolus fabricii begins in January and continues until spawning. In small tubules of both males and females, early gametogenetic stages proliferate. And in the large tubules, oocyte growth increases in females, and more advanced spermatogenic stages are produced in males. This renewed gametogen- esis occurs when water temperatures are near freezing (= -1C, Therriault, 1973; Ouellet-Larose, 1973), and an increase does not occur until several months later (March-April). Further, food conditions are minimal as evidenced by the near absence of phytoplankton cells in the intestines. The first increase in phytoplanktonic cells in the intestine is in mid March (Fig. 1 1 ). The only notable environmental change during the mid-winter renewal of gametogenesis is the return to increasing photoperiod. In fact, virtually the entire gametogenetic period coincides with the period of increasing day length and daily bright sunshine, and peak maturity is attained at the maximal photoperiod. These observations suggest that an increasing photoperiod controls gametogenesis. Photoperiod has been experimentally shown to control gametogenesis in numerous taxa including echinoderms (Pearse et al, 1986; McClintock et al., 1990). P. fabricii occurs where annual changes in temperature and food availability are pro- nounced, certainly more so than the habitats of other echinoderms that have been used to study the photoperiod control of gametogenesis. Nevertheless, the activation of gametogenesis well before the increase of temperature and food from their annual minima suggests the potential im- portance of photoperiod for P. fabricii. The increased growth in tubule diameter and gonadal mass observed in March or April (Fig. 4) suggests a second point that may be controlled by environmental factors. At this time, photoperiod has been increasing for several months, and the most notable environmental change is the first increase of phytoplanktonic cells in the intestines (Fig. 11). Phytoplankton are probably not abundant at this time, but abundant enough that the feeding mecha- nism of Psolus fabricii can filter cells from the water col- umn, contributing to gonadal production. An influence of food availability on gonad development is also sug- gested by numerous studies on other invertebrates (Sastry and Blake, 1971; Gimazane, 1972; Bayne, 1975). The vernal warming is another potential environmental change at this time, and our temperature record, which began in mid April in 1989, indicates that warming had occurred in April. Role of nutritive phagocytes and the gonadal tubule wall in supporting gametogenesis The appearance of nutritive phagocytes and their role in eliminating residual gametes is well documented in studies of holothurians (Tanaka. 1958; Costelloe, 1985; Smiley and Cloney, 1985) and other echinoderms (Lieb- man, 1950; Holland and Giese, 1965; Fenaux, 1972). In echinoids, nutritive phagocytes have been shown to transform reserves to dissolved compounds which are later used for gamete production (Holland and Giese, 1965). This may occur in Psolus fabricii, but since the phagocytes disappear before the active gametogenic period, these substances would have to be stored for their eventual use in gametogenesis. In Psolus fabricii, the gonadal tubule wall thickens dur- ing the period of gametogenic inactivity, from autumn to mid winter (Fig. 10). This growth, coincident with the autumnal decrease in food availability, falling tempera- tures, and short photoperiod, appears to be a priority in the use of energetic reserves of the animal at this time. In a variety of echinoderms. gametogenesis is similarly pre- ceded by a thickening of the tubule wall, and this is thought to represent an accumulation of reserves for ga- metogenesis (Pearse, 1969; Conor, 1973). Costelloe (1985) suggests that certain increases in gonadal size in the sea cucumber Aslia lefrevrei are due to the storage of materials in the tubule wall, but P. fabricii does not show an increase in gonadal mass as the tubule wall thickened. The earlier growth of the tubular wall in the large tubules, compared with the small tubules, suggests that resources are chan- neled preferentially to the large tubules. This could be because the later stages of gametogenesis in the large tu- bules require more resources than the earlier stages in the small tubules (Fig. 10). In the small tubules, the massive proliferation of the earlier gametogenetic stages during January and February precedes the thinning of the wall that begins in March. This suggests that the proliferation does not require large amounts of reserves. External spawning cues A massive loss of gametes over a short period strongly suggests that spawning is controlled by external factors 140 J.-F. HAMEL ET AL. (Himmelman, 1981; Giese and Kanatani, 1987; Starr, 1990; Starr el al.. 1990, 1992). In both years of our study, the gonadal indices, measurements of tubule diameter, and histological observations indicated an abrupt spawn- ing between two successive sampling dates (a 4-weeks in- terval in 1988, and 2 weeks in 1989). Temperature more than any factor has been suggested as a spawning signal in invertebrates (Orton, 19 14; Brown, 1984; Bricelij et al.. 1987), but we did not observe a consistent relationship between temperature and spawning in Psolus fabricii. Similar conclusions have been reported for other holo- thurians (Costelloe, 1985; Cameron and Fankboner. 1986). The only study suggesting that temperature might control spawning in holothurians is that of Tanaka (1958). That spawning time of P. fabricii varies between years suggests that spawning is not controlled by photoperiod. Our data suggests that spawning in Psolus fabricii may be signaled by the phytoplankton increase. Therriault and Levasseur( 1985, 1986) demonstrate that the phytoplank- ton bloom in the Estuary is always delayed relative to that of the Gulf of St. Lawrence because of freshwater run-off: the bloom only develops after the surface layer has sta- bilized, which occurs when the spring run-off drops. In both 1988 and 1989, spawning in P. fabricii coincided with the predicted onset of the bloom, based in turn on the decrease in freshwater run-off (Fig. 1 1). Such a syn- chrony is further indicated by the coincidence of the spawning dates of P. fabricii with those of the green sea urchin, whose spawning is triggered by phytoplankton (Himmelman, 1975; Starr et al.. 1990, 1992). Cameron and Fankboner ( 1986) indicated that phytoplankton may also initiate spawning in Stichopus calif ornicus. They noted that spawning individuals in the field were almost only observed after periods of bright sunshine (>5 h d-1 for >4 d), and further that phytoplankton was abundant during some spawnings. The spawning in 1989 resulted in the release of a larger amount of gametes than in 1988 (Fig. 4). Possibly more gametes attained maturity in 1989 because of the delay in spawning. We suggest this because the mean tubule diameter attained a higher value in 1989 than in 1988, and the difference was largely due to the growth that oc- curred during June and July (Fig. 4). The longer period before spawning may have permitted the completion of gametogenesis in additional tubules, tubules that might otherwise not have matured until the following year. In addition, the gonadal index and tubule diameter did not fall as low in 1988 as in 1989. This again suggests that fewer gametes were mature when the spawning cue was detected in 1988. This discontinuation of gamete pro- duction after the early 1988 spawning suggests that phys- iological mechanisms prevent further gamete maturation and secondary spawnings once spawning has occurred. For the urchin, for which phytoplankton has been shown to be the spawning cue, spawning in the laboratory in- creases with plankton abundance (Starr el al.. 1990). If this is true for Psolus fabricii, a more intense phytoplank- ton bloom in 1989 might account for the more massive spawning in that year. The greater mass of intestinal con- tents of P. fabricii in 1989 suggests there was a more in- tense bloom in that year (Fig. 1 1). Plankton ic stages of Psolus fabricii Holothurians with small eggs usually have a larval stage, whereas species with large eggs usually develop directly into juveniles (Tanaka, 1958; Rutherford, 1973; Green, 1978; Tyler and Billett, 1988). Psolus fabricii has excep- tionally large eggs, sometimes attaining 1400 ^m in di- ameter, and lacks a larval phase (pers. obs.). Nevertheless, the juvenile stage is pelagic. Probably, as Tyler and Billett ( 1987) indicate forelasipodid holothurians, the abundant nutritive reserves in the egg account for the high degree of floatability of the pelagic stage. Warmer temperatures near the surface may enhance the rate of development, and in addition the pelagic juveniles may further benefit from increased food resources, either in the form of dis- solved substances or planktonic cells. The feeding podia are well developed around the mouth of the pelagic ju- veniles of P. fabricii (pers. obs.). which suggests that they are capable of feeding on suspended particles. Feeding Some holothurians feed on organic material at the wa- ter-sediment interphase (Hyman, 1955; Reese, 1966; Fer- guson, 1969) whereas others feed on planktonic particles (MacGinitie and MacGinitie, 1949; Brumbaugh, 1965). Psolus fabricii is a highly selective feeder. For example, although numerous phytoplankton and zooplankton spe- cies are common in the region where we collected P. fa- bricii (Cote, 1972; Cardinal and Lafleur, 1977; Fortier et al.. 1978; Maranda and Lacroix, 1983; Therriault and Levasseur, 1985, 1986), the intestines contained almost exclusively four species of diatoms. The proportion of these items decreases in abundance in the intestine as productivity drops in late autumn and winter and is re- placed by nonliving matter. P. chitonoides (Fish, 1967) and Cucumaria elongata (Fankboner, 1978) similarly feed primarily on suspended living particles. That dendrochi- rotes are most abundant in temperate and subtropical waters, and rare in tropical areas and at great depths (Pawson. 1966; Hansen, 1975; Lawrence, 1987), suggests that they require the abundance of small living particles such as found in shallow water northern areas (Lawrence, 1987). Nonliving matter or detritus has been suggested to be an important source of food in the diet of suspension feeders (Baier, 1935; Newell. 1965: Kirby-Smith, 1976) and could provide nutritional resources for P. fabricii REPRODUCTIVE CYCLE OF PSOLUS FABRIC/1 during the winter. The long intestine of dendrochirotides may be an adaptation for digesting vegetal matter (Law- rence, 1987). P.fabricii has a remarkably long intestine relative to its body size (intestinal length = -1.68 + 4.52 dry body wall mass; r = 0.95, n = 37). For example, an adult measuring 6.1 cm in distance mouth-anus, (34 g) has a 1 52 cm intestine. This unusually long intestine may be an adaptation to its diet of diatoms which are protected by siliceous frustules. Acknowledgments We are greatly indebted to N. Piche for his help in collecting the samples and for the underwater photographs ofPsolusfabricii. The aid of S. Paradis, M. Claereboudt, A. Duval, E. Bourget, A. Cantin, A. Cardinal, H. Gu- derley. L.-P. Hamel, O. Hamel. B. Laganiere, and A. Tremblay at various points in the project is also gratefully acknowledged. Thanks are also due to A. Pusterla (De- partement de Pathologic, Universite Laval) and A. J. Col- let (Departement d'Anatomie, Universite Laval) and the Departement d'Oceanographie (Universite du Quebec a Rimouski) for the histological preparations. The first au- thor was supported by a FCAR scholarship and the re- search was supported by NSERC funding to J. H. H. and L. D. Literature Cited Atwood, D. G. 197.1. Ultrastructure of the gonad wall of the sea cu- cumber Lep/osynapta clarki. Z Zclltorscli 141: 319-330. Atwood, D. G. 1974. Fine structure of the spermatogonia. spermatocytes and spermatids of the sea-cucumber (Echinodermata: Holothuro- idea). Can. J /.ool 52: 1389-1396. Atwood, D. G., and F.-S. Chia. 1974. Fine structure of an unusual spermatozoan of a brooding sea cucumber. Ciiaiinaria lithrica. Can J. Zoo/. 52: 519-523. Baier, C. R. 1935. 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S. shruhso/ii (D) and S. benedicti (L) invested about 10 X more C and N in each offspring and 30% more C and N in each brood than did the morphs with planktotrophic development. C and N concentration dug per unit volume) was signifi- cantly greater in S. benedicti (L) than in the other morphs. though no general relationship with embryo size was ev- ident. The C:N ratio of offspring did not differ among the four morphs. Comparisons of estimated lifetime repro- ductive investment made by the two developmental types of S. benedicti indicated that lecithotrophic development involved twice the C and N investment in reproduction. Positive, significant regressions were evident between em- bryo C and N content and embryo volume at the inter- morph level. Significant intra-morph regressions were ev- ident in all morphs but Streblospio n. sp. (P). However, the large amount of variation unaccounted for by embryo volume calls into question the use of embryo size as a predictor of parental investment in offspring. Introduction Three modes of development, strongly correlated with egg size and fecundity (Thorson, 1946, 1950), are recog- nized among marine invertebrates. Planktotrophic de- velopment is descriptive of the production of a relatively Received 13 March 1992; accepted 25 January 1993. * Current address: USAE Waterways Experiment Station, WES-ES- F, 3909 Halls Ferry Road. Vicksburg. Mississippi 39180. large number of larvae, developed from small eggs, which acquire the necessary energy for growth by feeding on particulate matter during planktonic life. Lecithotrophic larvae are produced from fewer, but larger, eggs. These larvae do not feed on particulate matter but subsist, at least in part, on the energy supplied by the mother in the form of yolk during oogenesis. During direct development, offspring complete their development without a plank- tonic phase, usually within the mother or an egg mass; the energy for development is supplied by the mother (Thorson, 1950; Grahame and Branch, 1985). The adaptive significance of development mode has received extensive consideration. Selection pressures such as predation, starvation, and dispersal have been proposed for development mode evolution (Thorson, 1950; Chia, 1974; Strathmann, 1985). Quantitative modeling ap- proaches have been employed in an effort to identify the selection pressures and processes of importance in life history and development mode evolution in marine in- vertebrates (Vance, 1973a, b; Christiansen and Fenchel, 1979;Caswell, 1981; Grant, 1983). One fundamental assumption of the models of Vance ( 1973a, b) and Christiansen and Fenchel ( 1979) concerns the relationship between an egg's size and its energy con- tent. It is assumed that a positive correlation exists between an egg's measured or estimated size and the investment in material or energy that egg represents. This seemingly reasonable assumption enables the models to make use of the large amount of data on egg size and development mode available for marine invertebrates. Interspecific comparisons of egg size and organic con- tent including a broad range of taxa have shown the ex- pected positive relationship to exist (Strathmann and Vedder, 1977). However, the use of interspecies compar- isons to justify the assumed relationship between egg size and organic content appears invalid. McEdward and Car- 144 REPRODUCTIVE INVESTMENT IN STREBLOSPIO 145 son (1987) and McEdward and Coulter (1987) have pointed out that for models describing evolutionary pro- cesses, the relevant level of variation to examine occurs within a single species. For the species of asteroids studied by McEdward, Carson and Coulter, egg size was found to be a poor predictor of organic content when examined intraspecifically due to the large amount of variation in organic content unaccounted for by egg size. Likewise, Qian (1991) found that egg size was not correlated with egg energy content within three populations of the poly- chaete Capitella sp. Variation in egg organic composition may also make egg size an unreliable predictor of an egg's energetic value (Turner and Lawrence, 1979). To date, the relationship between egg size and egg or- ganic content has been examined in only a small number of taxa (mostly echinoderms). One of the questions ad- dressed in this paper concerns the extent to which the pattern identified in echinoderms is present in other taxa, specifically spionid polychaetes. Inter- and intraspecific comparisons of reproductive and per offspring investment in organisms with different development modes have proved to be useful in identi- fying the ecological consequences of development mode as well as the potential evolutionary forces shaping these patterns (Menge, 1974; Perron, 1986; Levin cl nl.. 1991). The spionid polychaete genus Strchlospio. which exhibits developmental variation both within and between species (Levin, 1984; Cazaux, 1985), offers a highly suitable sys- tem for examining questions concerning egg size and egg organic content and the consequences of development mode. Six reproductive variants are known for Strchlos- pio, comprising three or more species (Levin, 1984; Ca- zaux, 1985; Rice, 1991; Levin and Eckelbarger, pers. comm.). To assess the nature of reproductive costs associated with development mode patterns in Streblospio, both re- productive expenditure per offspring and per brood were examined in four reproductive variants. These variants are similar in body size and ecology but are distinguishable in a number of reproductive characteristics (Dean. 1965; Levin, 1984; Cazaux, 1985). Two reproductive morphs of 5. benedict i have been identified from the Atlantic coast of the United States (Levin, 1984; Levin ct til.. 1991). Females with planktotrophic development produce a large number (100-600) of small eggs (70-90 urn dia.) which develop into planktotrophic larvae. Lecithotrophic morphs of this species produce a smaller number (10- 100) of large eggs (100-200 nm dia.) and lecithotrophic larvae (Levin, 1984). Streblospio n. sp. from the Gulf of Mexico also has planktotrophic development and pro- duces a large number (100-700) of small eggs (60-70 ^m dia.) (Levin, 1984; Rice and Levin, in prep.). S. shrubsolii with direct development from near-shore habitats in France produces a very small number (20-50) of large eggs (200-230 ^m dia.) which develop directly into crawl away juveniles (Cazaux, 1985). Materials and Methods Animals used during this study were obtained from three sources. S. hcncc/icli with both planktotrophic and lecithotrophic development were initially collected from intertidal salt marsh habitats in Bogue Sound, North Car- olina. Worms with lecithotrophic development repre- sented second generation laboratory animals whose par- ents were collected in September, 1990 from natural pop- ulations at Fivers Island near Duke Marine Lab in Beaufort, North Carolina. Individuals with planktotrophic development were collected at Tar Landing Bay, North Carolina in June 1991. Individuals of Strchlospio n. sp. were settled in the lab by S. Rice from plankton samples made in the Hillsborough River, Tampa Bay, Florida in July 1991. Individuals of 5. shrub.wlii were taken from laboratory cultures established by L. Levin in 1986 from samples collected by C. Cazaux in Arcachon, France. Males and females of each variant were incubated in pairs at 20C in culture dishes of sieved marsh sediment and 34-36%o seawater. according to the techniques out- lined in Levin and Creed (1986). To make observations of multiple broods from the same female it was necessary to collect recently fertilized embryos rather than eggs. Embryos are brooded on the dorsal surface in all repro- ductive variants and are more accessible than maturing eggs which occur within the body coelom. Embryos were always collected within 24 h of fertilization and were composed of between one (zygotes) and approximately 250 cells. The developmental stage of sampled embryos, estimated from the number of blastomeres present, was used as a covariate in data analyses. Once embryos were separated from females they were collected by pipet, counted, and placed in a dish of filtered (0.45 jum) seawater. Two perpendicular (maximum length and width) measures of embryo diameter were made with a compound microscope and ocular micrometer for ap- proximately 20 embryos within a brood. Embryo volume was calculated using the mean radius and the formula ^Trr 1 since the embryos were more spherical than prolate or oblate in form. Entire broods, produced by a single female, were col- lected for C and N analysis by depositing all the embryos onto a small square of previously combusted Whatman GF/F (glass-fiber) filter paper. A minimum of approxi- mately 1 5 embryos of S. shrubsolii (direct developer) or S. benedicti (lecithotrophic) and 200 embryos of S. hene- dicti (planktotrophic) or Streblospio n. sp. (plankto- trophic) were needed to meet the detection limits of the analysis (2-3 ^g of C or N). Broods smaller than these minimum sizes were not analyzed. Samples were dried 146 T. S. BRIDGHS Siimnuirv <>l the reproductive cluu\i< /i Table I <>t the lour developmental inrph\ <ig N/Brood 7.77** 1.63 14 8.50' 2.80 90 6.75" > ">i 1 5.14" 1.55 20 3. 86 8.99 0.0001 Embryo Volume (fil X 111-') 4.67" 0.78 24 3.08" 0.44 100 0.495' 0.057 6 0.366" 0.041 20 3. 103 1395. 0.0001 Mg C/Embryo 1.09' 0.25 24 0.85" 0.14 100 O.I08 C 0.026 6 0.091" 0.031 20 3, 103 725.4 0.000 1 Mg N/Embryo 0.229" 0.048 19 0.174" 0.026 90 0.023 C 0.0033 1 0.017" 0.0051 20 3, 86 949.0 0.0001 C cone. (>ig//jl) 233.3 s 34.50 24 279.7" 51.54 100 217.2" 48.02 6 248.9' 79.7 2(1 3. 102 12.60 0.0001 N cone. (/ig/MD 48.67' 5.57 19 57.62" 10.19 90 45.72" 4.67 1 47.47' 12.62 20 3. 85 1 3.58 0.0001 C;N Ratio 4.83' 0.54 19 4.88" 0.28 90 4.97" 0.69 1 5.17" 0.48 20 3. 85 2.40 0.0767 Data on embryo volume was obtained by calculating volume (4/3nr'l using a mean radius determined from two perpendicular estimates of diameter. C and N data were obtained from elemental analysis of entire broods of early embryos. Degrees of freedom (df). F. and P values are listed for the ANOVA results for each character. Superscripted letters denote those values within a row which are significant!) different in an oven at 50C for approximately 24 h, then stored in a vacuum desiccator prior to analysis. Appropriate blank samples, without eggs, were prepared to distinguish background C and N values associated with the collection technique. The amount of C and N in each brood was determined by use of a Carlo Erba Elemental Analyzer (model E.A. 1 108). All statistical analyses of data were performed with SAS (version 5.18). All data were log transformed to remove heteroscedasticity and normalize distributions. When sig- nificant differences (P < 0.05) were found among the four reproductive variants, an n-posteriori Least Significant Difference (LSD) test was performed on means of the four reproductive types (a = 0.05). A multiple linear regression model was used to examine the relationship between em- bryo volume and C and N content. Given the repeated measures structure of the data, two covariates were in- cluded in the model to distinguish between among-female and within-female variation. Covariate 1 (cov 1 ), repre- senting mean values of embryo volume for each female, allowed the relationship between either C or N content and embryo volume to be examined. Covariate 2 (cov 2) was formed by subtracting the mean embryo volume of all broods produced by a female during the experiment from the mean embryo volume of each individual brood. These deviations permitted testing for a relationship be- tween embryo C and N content and volume for multiple broods from a given female. Results Reproductive ami offspring investment Per brood measures of fecundity in S 1 . shrubsolii (D) and S. benedicti (L) were significantly lower than 5". bene- dicti (?) and Strehlospio n. sp. (P), where (L), (P), and (D) designate lecithotrophic. planktotrophic and direct de- velopment, respectively (Table I). The differences in fe- cundity among reproductive morphs were accompanied by differences in embryo volume. S. xlirubsolii (D) em- bryos, which were the largest of the four types (4.67 X 10~ 3 Ml), were 12.7 X the volume of Streblospio n. sp. (P) em- bryos, 9.4 X the volume of S. benedict i (P) embryos, and 1.5 X the volume of embryos of S. benedicti (L) (Table I). Embryo volume increased with developmental stage (P = 0.0021). Comparisons of the C and N investment made to in- dividual offspring produced by each reproductive type re- vealed differences similar to those found for embryo vol- ume (Table I). 5". shrubsolii (D) made the largest average investment in each offspring ( 1 .09 /jg C, 0.229 /^g N) fol- lowed by 5. benedicti (L) (0.851 ^g C, 0.174 /ig N), S. benedicti (P) (0.108 ^g C, 0.023 ng N), and Streblospio n. sp. (0.091 M gC, 0.017 jug N). In terms of C and N, the lecithotrophic and direct de- veloper made a greater material investment in each brood than did the morphs with planktotrophic development (Table I). Significant differences were present in ^g C per brood between S 1 . shrubsolii (D) and both planktotrophic developers, and in /ug C and N per brood between 5. bene- dicti (L) and both planktotrophic morphs (Table I). Even though the planktotrophic morphs produced much larger numbers of embryos, the lecithotrophic and direct de- velopers were found to have made a 30% greater C and N investment in each brood. The C:N ratio of brooded offspring was similar among the reproductive variants, ranging from 4.83 in S. shrub- solii (D) to 5.17 in Streblospio n. sp. (P) (Table I). The C:N ratio of embryos decreased with developmental stage (P = 0.01 12). S. benedicti (L) exhibited significantly greater C and N concentration (^g C and ^g N per ^1) than the other re- REPRODUCTIVE INVESTMENT IN STRKBLOSHO 147 A. CARBON CONTENT VS. EMBRYO SIZE I w o tm O.OOO O.O01 0.002 O.O03 O.O04 0.005 O.OO6 EMBRYO VOLUME ( n 1) B . NITROGEN CONTENT VS. EMBRYO SIZE 0.41 1 U 0.3 O.OOO 0.001 O.O02 0.003 O.OO4 0.005 O.OO6 EMBRYO VOLUME ( n 1) Figure 1. Scatter plots describing the relationship between jig C/ embryo (A) and ng N/embryo (B) and embryo volume for all reproductive morphs. Both regressions are highly significant (P < 0.0001 ). productive types (Table I). Both C (P = 0.0306) and N (P = 0.0794) concentrations decreased with developmen- tal stage. Embryo C and N content versus eiuhryo volume Significant, positive correlations were found between jig C per embryo and embryo volume (r = 0.87, P < 0.0001) and ^g N per embryo and embryo volume (r = 0.89, P < 0.0001 ) across reproductive variants ofStre- blospio (Fig. 1). The regression model incorporating cov 1 and cov 2 used in analyzing the relationship between Mg C (or jig N) per embryo and embryo volume explained 95% of the C variation and 97% of the N variation. A strong relationship was evident between C and N content and embryo volume across reproductive types as indicated by the significance of cov 1 in the models (C ANCOVA: F U0 2 = 82.34; P < 0.0001; N ANCOVA: F U85 = 56.38; P < 0.0001). However, the relationships between jug C arid N per embryo and embryo volume within each re- productive type were not identical. The significance of reproductive type (C ANCOVA: F 3 ., 2 == 646.84; P < 0.0001; N ANCOVA: F X85 = 823.51; P < 0.0001) in the models indicated that differences existed among the four morphs in the nature of the regressions, specifically the y-intercept. No differences could be detected in the slope of the lines among the four types as seen by the lack of significance in the cov 1 X type interaction in both models (C ANCOVA: F, ,,, : = 1.85: P = 0.1478; N AN- COVA: F 3 . 85 = 2.29; P = 0.0884). Cov 2 was significant only in the case of the N model (C ANCOVA: F, lo: = 1.98; P =0.1626; N ANCOVA: F,, 85 == 4.37; P = 0.0395). indicating that the relationship between N content and embryo volume could be detected with data from individual females sampled more than once. Differences were evident among the four variants in the strength of the relationship between embryo C and N content and embryo volume within each morph. Signif- icant, positive correlations existed between jig C and ^g N per embryo and embryo volume for S. shrubsolii (D), S. benedict i (L). and 5. benedict i (P). but not for Stre- blospio n. sp. (P) (Table II. Figs. 2. 3). The specific regres- sion parameters for each of the relationships are listed in Table II. The amount of variation accounted for by the regressions, and therefore the strength of the relationship, was highest for S. shrubsolii (D) (71%- for N content and 66% for C content). A much smaller amount of variation was accounted for by the regressions for 5. benedicli (L) Table II Slope and v-intcrcepl estimate* lor the regression equation* of ng C und ng N/embrvo ver\u* embrvo volume tor Streblospio dill morphsi and each morph separately Variants y-intercept SE Slope SE P ,ug C/Embryo versus Embryo Volume Slrcblospio (all morphs) 0.0431 0.0213 0.247 0.00713 0.0001 5. shrubsolii (D) -0.102 0.187 0.256 0.0395 0.0001 S benedicli (L) 0.489 0.0950 0.118 0.0305 0.0002 5. benedicli (P) 0.00382 0.0414 0.210 0.0831 0.0187 S. n sp. (P) -0.00732 0.0627 0.269 0.170 0.1306 fig N/Embryo versus Embryo Volume Sireblospio (all morphs) 0.00812 0.00431 0.0513 0.00147 0.0001 S. shnibsolii (D) -0.0276 0.0406 0.0548 0.00855 0.0001 S. benedict HL} 0.112 0.0187 0.0203 0.00607 0.0012 S. benedicli (P) 0.00271 0.00421 0.0402 0.00847 0.0001 S. n sp. (P) -0.000504 0.00995 0.0489 0.027 0.0869 Estimates and standard errors (SE) are listed for the slope and y-in- tercept of the overall regression for Streblospio. including all morphs. as well as the specific regressions for each morph. P denotes the significance for each relationship. 148 T. S. BRIDGES S. shruhsolii (D) O.OO2 O.OO3 O.OO4 O.OO5 0.006 EMBRYO VOLUME ( M 1) Streblospio n. sp. (P) O.lb- t"= .12 p. 13 0.14 00 0.12 o 0.10 e 6 O.O8 O 0.06 o 00 O.OO03 O.OOO4 O.OO05 EMBRYO VOLUME ( n 1) S. benedicti (L) 1.6 1.4 I- i 10 P O 0.8 tic 3. 0.6 0.4 OOOO O.O01 O.O02 O.OO3 O.OO4 0.005 EMBRYO VOLUME ( M 1) S. benedicti (P) OOOO4 O.OO05 O.OO06 O.OO07 EMBRYO VOLUME ( n 1) Figure 2. Scatter plots describing the relationship between ng C/embryo and embryo volume for each reproductive morph of Strehlospio. (11% for C and 1 3% for N) and 5. benedicti ( P) ( 2 1 % for Cand 51% for N). A more meaningful estimate of the strength of the three significant regressions can be made by examining confi- dence limits for predicted embryo C and N content. Pre- dictions of embryo C and N content using each morph's regression parameters and a single value of embryo size (each morph's mean) produced the following predicted values (95% confidence limits): S. shrubsolii (D), C = 1.10/ug0.31,N = 0.228 /ug 0.058; S. benedicti (L). C = 0.851 0.268, N = 0.174 0.050; S. benedicti (P), C = 0.108 0.050, N = 0.023 0.005. These confidence intervals envelop a large portion of the range of actual values for embryo C and N content found in each of these morphs, between 53%- and 99%. The large amount of variation about these regressions, which results in such large confidence intervals, makes it difficult, if not im- possible, to make significantly different predictions of embryo C or N content from embryo volume within each morph. Discussion Offspring invest men! The negative relationship between offspring size and number described for many marine invertebrate taxa (Thorson, 1946, 1950; Emlet et al.. 1987) including poly- chaetes (Hermans, 1979; Levin et al., 1991), was also found in this study (Table I). This tradeoff can be ex- plained by assuming there to be a finite and limited amount of energy available for reproduction (Vance, 1973a; Smith and Fretwell, 1974; Stearns. 1976), an as- sumption more easily justified among closely related spe- cies which accumulate and apportion nutrients in a similar fashion. Levin et al. (1991) observed a negative genetic correlation between fecundity and egg size in S. benedicti reared in the lab, suggesting that evolutionary forces may influence this tradeoff. The potential evolutionary forces driving differences in per offspring investment and development mode are of particular interest. One of the key preadaptations allowing for the evolution of direct from indirect development may be the evolution of a large yolk-filled egg ( Wray and Raff. 1991). However, experimental embryology has demon- strated that in species developing directly, development can proceed normally at half the egg size, in a size range similar to forms with indirect development (Okazaki and Dan, 1954; Henry and Raff, 1990; Wray and Raff, 1991). If direct development or lecithotrophy could be ac- complished in Slreblospio with only a five-fold increase over planktotrophy in per offspring investment (instead of the 10-fold increase in investment reported here), and the remaining C and N was allocated to increased fecun- dity, the resulting fecundity benefit could make such a REPRODUCTIVE INVESTMENT IN STREBLOSPIO 149 S. shrubsolii (D) S. benedicti (L) O.O03 OOO4 OO05 0.006 EMBRYO VOLUME ( n 1) Streblospio n. sp. (P) O.OOO 0.001 O.O02 0.003 O.O04 O.O05 EMBRYO VOLUME ( M 1) S. benedicti (P) 0.03O 0.025 , ! =.16 ps.OQ o g 0.020 Id g- 0.015 O 8 0.010 O O.OO03 O.OOO4 O.OOO5 EMBRYO VOLUME ( ji 1) OOOO4 OOOO5 OOOO8 O.O007 EMBRYO VOLUME ( n 1) Figure 3. Scatter plots describing the relationship between ^g N/embryo and embryo volume for each reproductive morph of Streblospio. strategy adaptive (Table I). However, greater per offspring investment in S. shrubsolii (D) and S. benedicti (L), in addition to developmental changes, also produces a larger offspring. S. shrubsolii (D) produces a 1000 /um long crawl away juvenile (Cazaux. 1985). Larvae of S. benedicti (L) are released and settle at half the size of 5. shrubsolii (D) (about 550-650 ^m) (Levin, 1984). Planktotrophic larvae ofS. benedicti and Streblospio n. sp. are released at about 250-350 ^m in length and appear to settle at a size com- parable to or smaller than S 1 . benedicti with lecithotrophic development (Levin, 1984). Selection for increased offspring size may have been an important factor in development mode evolution in Stre- blospio. A shift toward larger offspring size in Streblospio offspring with a planktonic phase may be adaptive in the face of size-selective planktonic predation (Kerfoot. 1977; Greene, 1985; Rumrill et at., 1985; Pennington el a/.. 1986). The presumed predator avoidance behavior of some planktotrophic spionid polychaete larvae, including S. benedicti (P), that increase their effective size by flaring long swimming setae, is consistent with the importance of size-selective predation in this species. Larger size at settlement in 5 1 . benedicti (L) and at release from the fe- male in S. shrubsolii (D) may also benefit offspring subject to negative interactions with permanent meiofauna or macrofauna by accelerating passage through vulnerable size ranges (Bell and Coull, 1980; Watzin, 1983, 1986). Juvenile 5. benedicti are sensitive to interactions with macrofauna (McCann and Levin, 1989). Levin and Hug- gett (1990) reported a larval and juvenile survivorship advantage in S. benedicti (L) (relative to 5. benedicti (P)) during a field study of populations of 5. benedicti with lecithotrophic and planktotrophic development. Offspring composition Even though morphological distinctions are evident in yolk granules of S. benedicti with lecithotrophic and planktotrophic development, differences in gross measures of organic composition were not evident in this study (Eckelbarger. 1980, 1986). Offspring C:N ratios of the four reproductive types could not be distinguished statistically, suggesting that the relative proportion of protein to non- nitrogen containing compounds is the same among the four morphs (Table I). Turner and Lawrence (1979) also found that organic composition did not change with egg size in the echinoderms they studied. Lawrence et al. ( 1984) concluded, due to the compositional similarity of eggs of different sizes and development modes, that the significance of larger eggs was not to accommodate dif- ferences in the energetic demands of development, but to create a larger offspring. One would expect to see a higher proportion of lipid in larger eggs if the change in devel- opment involved a greater energetic demand (Lawrence et al., 1984). Increased per offspring investment in Stre- blospio may have similar importance, i.e., the production of larger offspring. 150 T. S. BRIDGES C and N concentration (j/g//ul) was similar among the embryos of three of the four Strcblospio reproductive morphs, and no consistent irend with embryo size was noted (Table I). Qian a id Chia (1992) found that egg energy concentrali >as similar in lecithotrophic and planktotrophic C 'upiiella sp. Strathmann and Vedder (1977) reported that organic matter per unit volume de- creased with egg size in echinoderms with feeding larvae. Such a trend has not been observed in echinoderms with larger eggs, including pelagic lecithotrophs (Turner and Lawrence, 1979; McEdward and Chia. 1991). Energy concentration does appear to be significantly greater in eggs of echinoderms with nonfeeding larvae than those with feeding larvae (Emlet el a/., 1987; McEdward, pers. comm.); this observation is consistent with data presented by Needham (1963). Thus, important fundamental dif- ferences may exist among the eggs of echinoderms with different developmental modes. More data are required before such trends can be discerned for polychaetes. Reproductive investment The lecithotrophic and direct developers made greater material investments in each brood than either plankto- trophic developer. In addition to investing more C and N in each offspring, S. benedicti (L) was also found to have invested 33% more C and 26% more N in each brood than did 5. benedict i (P). However, these values are min- imum estimates of the difference in reproductive invest- ment since S. benedict i (P) produced more broods that were too small to be analyzed for their C and N content. Lifetime investment levels can be estimated by combining data on per offspring investment made in this study with lifetime fecundity data made by Levin el til. (1987), where worms were raised under the same experimental condi- tions. Using these data, S. benedicti (P) (1324.32 eggs/ lifetime) would have a calculated lifetime reproductive investment level of 143.03 jug C and 30.46 /ng N, and S. henedicti (L) (336.6 eggs/lifetime) would have invested 286.45 jug C and 58.57 /jg N. Based on these calculated values, 5". benedicti (L) makes a two-fold higher investment in reproduction than S. benedict i (P). These estimates do not technically represent reproductive effort since repro- ductive effort is defined as the proportion of resources devoted to reproduction (Havenhand and Todd. 1989). However, the similarity of these two morphs in size as well as ecology (Levin et ai. 1987; Levin and Huggett. 1990). would suggest that such estimates may represent a first approximation of reproductive effort, though some caution is warranted (Grahame, 1982). Efforts at deter- mining which reproductive pattern, planktotrophy or lec- ithotrophy, is more energetically expensive have yielded equivocal results (Grahame and Branch, 1985; Strath- mann. 1985). Differences in apportionment of energy to growth and development in S. benedicti with planktotrophic and leci- thotrophic development may partially account for the difference in reproductive investment. S. benedicti with planktotrophic development reaches sexual maturity (first reproduction) earlier and at a larger size than the lecitho- trophic morph, indicating that growth and developmental rates are accelerated in planktotrophs compared to leci- thotrophs (Levin et ai. 1987; Levin et ai. 1991). The importance of accelerated growth and development in planktotrophic 5. benedicti is further suggested by de- mographic analyses of the two developmental morphs. Similarity in estimated population growth rates (X) in the two morphs were the result of a balance between a larval and juvenile survivorship advantage in lecithotrophs and increased fecundity in early adult stages in planktotrophs (Levin el ai. 1987; Levin and Huggett, 1990). Given the effect of age at first reproduction and early fecundity on population growth rates (Stearns, 1976). females with planktotrophic development may be investing in future offspring both through energy committed to eggs directly and through enhanced early growth and development. The evolutionary shift from planktotrophy to lecithotro- phy may involve not only changes in offspring size and investment, but also age and size at maturity in S. bene- dicti. Embryo si:e versus C and N content Significant, positive relationships have been found be- tween egg size and organic content using data from a number of species in this study (Fig. 1) as well as others (Strathmann and Vedder, 1977: Turner and Lawrence, 1979; McEdward and Chia, 199 1 ). In general, the strength of this relationship when examined at the interspecific level, as reflected by r values, appears to be high (present study; McEdward and Chia, 1 99 1 ). However, large errors in prediction may result when using regression equations formulated with interspecific data to predict values of per offspring investment from intraspecific and intra-morph data on embryo size (Bridges, 1992). The strength of intra- morph relationships between embryo C and N content and embryo volume ranged from 5. shrubsolii (D), where the regressions accounted for 66% of the variation in C and 71% of the variation in N to Streblospio n. sp. (P), where significant relationships could not be detected (Figs. 2, 3). Even in the three morphs where significant regres- sions were evident, the size of 95% confidence intervals on predicted values of C and N content would preclude making significantly different predictions of C and N con- tent from embryos of different size within developmental morphs. Observations in this study of lecithotrophic and planktotrophic polychaetes are similar to those in echi- noderms with lecithotrophic development where variation REPRODUCTIVE INVESTMENT IN STREBLOSP1O 151 among species in the nature and strength of the relation- ship between egg size and organic content has been found (McEdward and Carson, 1987; McEdward and Coulter. 1 987; McEdward and Chia, 1 99 1 ). Given that egg or em- bryo size accounts for minimal variation in organic con- tent within species, considerable caution should be taken in presuming egg or embryo size as an accurate measure of per offspring investment. Acknowledgments I would like to thank G. Plaia and N. Blair for providing assistance and advice with elemental analysis and C. Brownie for help with the statistics. Cultures ofStrehloxpio were graciously supplied by L. Levin, S. Rice, and C. Cazaux. I would also like to thank F. Gould, J. Garlich, D. Checkley, D. Wolcott, and three anonymous reviewers for their comments on earlier versions of this manuscript. Special thanks must go to L. Levin for her conscientious advising and critical examination of earlier versions of this paper. This project was supported in part by funds from EPA grant R8 1-72-52-0 10 to L. Levin. Literature Cited Bell, S. S.. and B. C. Coull. 198(1. Experimental evidence for a model of juvenile maerofauna-meiofauna interactions. Pp. 179-192 in Marine Benlluc Dynamics, K.. R. Tenore and B. C. Coull, eds. Uni- versity of South Carolina Press, Columbia. SC. Bridges, I. S. 1992. 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Larval settlement into marine soft-sediment systems: interactions with the meiofauna. / Exp. Mar. Biol. Ecol. 98: 65- 113. Wray, G. A., and R. A. Raff. 1991. The evolution of developmental strategies in marine invertebrates. Trends Ecol. Evoi 6: 45-50. Reference: Bail. Bull 184: 153-1X5. (April. 1993) On Antarctic Entoprocta: Nematocyst-like Organs in a Loxosomatid, Adaptive Developmental Strategies, Host Specificity, and Bipolar Occurrence of Species* PETER EMSCHERMANN Fakulliit fiir Biologic tier Universitat Freiburg i.Br.. Biologic fiir Mediiiner, Schdnilestrafie 1. 7800 Freiburg i.Br. BRD Abstract. In the southern Weddell Sea and the Brans- field Strait a total of eight species of entoprocts were found: four Loxosomatidae. originally known to be common in the Northern Polar Sea and the Atlantic sector of the sub- arctic region (Loxosomella antedonis Mortensen. 1911, L. compressa Nielsen and Ryland, 1961, L. varians Niel- sen, 1964, and L. antarctica Franzen, 1973); three new species of loxosomatids (L. brochobola spec, nov., L. seir- yoini spec, nov., and L. tonsoria spec, nov.); and one single colonial entoproct Barentsia discreta (Busk, 1886) which is distributed worldwide. Loxosomella brachystipes, de- scribed by Franzen in 1973 from South Georgia, is shown to be synonymous with L. varians Nielsen, 1964. The microscopic investigation of the above species revealed several morphological characters, previously unknown, that add to our knowledge of the Entoprocta in general, and also help in characterizing species. The first of these novel characters, observed in L. brochobola spec, nov., are extruding organs similar to cnidarian spirocysts. This is the first description of such organs in entoprocts. Lox- osomella antarctica is capable of calyx regeneration and thereby becomes the only solitary entoproct known to have such a regeneration capacity. Finally, the formation of special resting buds in Barentsia discreta is described. The range of morphological variation of these species, the question of host specificity in the Loxosomatidae, and the bipolar occurrence of some of these species is discussed. Introduction Reports on Antarctic Entoprocta are scarce. Until 1973 only five colonial forms had been recorded from the Received 31 August 1992; accepted 25 January 1993. * These investigations have been supported by the Deutsche Forsch- ungsgemeinschaft. Southern Ocean, predominantly from the subantarctic region: Pedicellina anstralis Ridley, 1881 was reported from the Magellan Strait, at the Patagonian coast and the Falkland Islands (Islas Malvinas); Barentsia capitata Cal- vet, 1904, and Barcnisin variahilix Calvet, 1904 were re- ported from South Georgia and the Falkland Islands; and Barentsia aggrexakt 1 Johnston and Angel, 1940 from Macquarie, Heard, Marion, and the Kerguelen Islands. These latter three species are probably synonymous. Fi- nally, Barentsia discreta (Busk, 1886), common circum- antarctically in subantarctic latitudes (Johnston and An- gel. 1940: Rogick, 1956; Waters. 1904) as well as on the Antarctic shelf itself, was reported in the Commonwealth Bay (Johnston and Angel. 1940) and from the northern- most tip of the Antarctic Peninsula (Franzen, 1973). In 1973, Franzen augmented these reports with observations of older samples from the 1901-03 Swedish Antarctic ex- pedition. He added Pedicel/ inn ccrmia (Pallas, 1774) and four Loxosomatidae to the list of entoprocts from Ant- arctic waters: Loxosomella compressa Nielsen and Ry- land, 1961 var. antarctica; Loxosomella mwmanica (Ni- lus, 1909); Loxosomella antarctica spec, nov.; and Loxosomella brachystipes spec. nov. There have been no more recent studies of the Antarctic entoproctan fauna. During the Antarctic summer 1989-90, in the course of a survey of the Antarctic benthos supported by the Deutsche Forschungsgemeinschaft, the entoproctan fauna of the Weddell Sea and the Bransfield Strait were inves- tigated aboard the German research vessels PFS POLAR- ' A sample of barentsiid colonies from the Californian coast near Santa Cruz, sent me by Kerstin Wasson (University of California, Santa Cruz), proved to consist not only of colonies ofBiirentsia ramosa. being common there, but also of Barentsia aggregata J. and A., which had previously been believed to occur only in the subantarctic region. 153 154 P. EMSCHERMANN STERN and FFS METEOR. Benthic samples were taken in the Weddell Sea at 26 locations along the shelf from its northeastern most edge down to the base of the Ant- arctic Peninsula. Forty-five hauls were taken with both an Agassiz trawl and an epibenthic sledge (Fig. 1. Table 1 ). Except at three stations in the 600- 1 100 m depth range, most samples were taken between 100 and 500 m (see Table I and Fig. 1 ). The benthic fauna, especially the as- sociations of ciliary-feeders, was generally richer, both in abundance and species diversity, in the eastern part of the Weddell Sea where the steep slope is more exposed to the Weddell Sea current than the fauna of the western Weddell Sea where bottoms are less sloped and less ex- posed to the current. In the Bransneld Strait a total of 22 hauls were evalu- ated, nine from a quadrangular dredge and 1 3 from a Van Veen Grab, taken at a depth range between 120 and 400 m at 1 5 locations between Elephant Island to the north and Adelaide Island to the south. (Fig. 2, Table II). Altogether, eight entoproctan species were found. Four of them occurred in both areas and one species was found only in the Weddell Sea. Three new species were described: two from the Weddell Sea and one from the Bransfield Strait. As a general trend, the abundance and population den- sity of Entoprocta in the Bransneld Strait was much higher than in the Weddell Sea presumably because of the higher primary production and consequently higher nu- trient supply in this area. Of special zoogeographical in- terest is that four of the species found to be common in the Antarctic region also occur in the North Atlantic and the Arctic Polar Sea, but seem to be absent from the mid- Atlantic coasts. Sampling Methods and Treatment of Samples The trawling times of the sampling gear varied between about 30 and 90 min, according to the bottom structure and ice conditions. To obtain undamaged living samples for laboratory observations and culture experiments, the hauls were immediately subject to rough presorting. Ap- propriate growth substrates for entoprocts, such as bryo- zoan and hydroid colonies, bivalve shells, small sponges and stones, as well as potential entoproctan hosts such as errant and sedentary polychaetes with their tubes, sipun- culids. echiurans, priapulids, ophiuroids and. occasionally, crinoids, were collected as soon as possible and placed in separate plastic tubs with running fresh seawater of outside -70- SO' -40' -20' 10' 65 >OLAR CIRCLE WEDDELL SEA / ff\ 75 ANTARCTICA < PFS POLARSTERN CRUISE ANT Vlll-5 STATIONS OF AQASSIZ TRAWL AND EPIBENTHrC SLEDGE -50' -30' -20' -10' Figure 1. Station map of the POLARSTERN cruise ANT VIII-5 in the Weddell Sea. ON ANTARCTIC ENTOPROCTA 155 Table I Station l.M / Pularxicrn-Cntiae AKT 1 7/7-5 in the II cddcll Sen Station Position Depth (m) Date Gear Bottom Predominant fauna 16-346 571.08 W 11,77 360-320 29/12/89 A St Sponges. Brso/oa -399 S 72,86 W 19.30 380-390 30/12/89 A St, Sf Sponges. Bryozoa. Ascidia -403 S 76,94 W 49.81 220-250 06/01/90 A Ch. G Bryozoa, Holothuna -405 S 76,52 W 52.63 380-390 07/01/90 A S Ascidia, Pennatulids, S 76.53 W 52,72 Gorgonia, Sponges: S 76,52 W 52,78 Bryozoa -407 S 75.46 W 27,02 240-250 12/01/90 E, A Sf Sponges, Holothuna -41 1 S 74,54 W 25,75 520-530 14/01/90 E, A S, St Cnnoids. Holothuna, Nemerteans. Prawns -421 575,21 W 27,80 430-400 17/01/90 E, A S. G Pennat., Crinoids, Echin., Ophmr.. Holothur.. Prawns, Fishes -423 S 74,84 W 27,56 460-470 17/01/90 A, E S. St Sponges. Pennat.. Ophiur., Prawns -434 S 73,69 W 21,75 260-270 20/01/90 E. A Cb. St Bryoz., Holoth., Prawns -437 S 72,84 S 19.40 390-420 21/01/90 E Cb, St Bryoz., Ophiur.. Amphip. -454 571.08 W 11.69 210-280 26/01/90 E. A Sf Sponges. Gorgon.. Fishes -456 S 71.25 \V 21,01 200-250 26/01/90 A. E Sf Sponges. Ascid., Amphip., Fishes -459 S 70,69 W 11.19 350-390 28/01/90 E, A G. St, Cb Sponges, Pennat., Crinoids, Amphipods -468 S 74,74 W 26.36 480-460 09/02/90 E. A S. G Pennal.. Amphip.. Prawns. Fishes -470 S 74.28 W 34.09 1050-960 10/02/90 E. A Cb Stylasterids. Ophiur.. Ascid.. Sponges. Polychaetes -475 S 76.85 W 49.45 280 1 3/02/90 E. A S Sponges, Echin., Ascid., Cnn. -477 S 76.45 W 53.15 430-450 14/02/90 A, E S Pennat.. Echin.. Prawns -479 S 75.68 W 56.72 340-360 14/02/90 E. A S. Sf Crin., Ophiur.. Prawns, Pantop. -481 S 74,71 W61.14 640-620 15/02/90 A. E St Ophiur.. Amphip.. Prawns -484 S 75,28 W 55.98 450-440 16/02/90 E, A S Pennatul., Ascid., Ophiur., Polychaetes. Brachiopods -486 S 76,50 W 52,15 340-330 1 7/02/90 E Cb Spong., Bryoz., Pennat., Ascid. -489 S 73,68 W 23.13 980-990 21/02/90 A St Ophiuroids. Prawns -490 S 73,70 W 22,66 630-610 21/02/90 E Sf Cnn.. Hololhur., Echin.. Prawns -491 S 73,69 W 22,42 390-370 21/02/90 E S. G Cnnoids, Pantopods, Amphip. -492 S 73,69 W 21,74 250 21/02/90 A Cb Cnn., Bryo/.. Spong., Ophiur. -496 S 70,63; W 08.09 80 27/02/90 A. E Cb Bryozoa. Holothuria Benthos stations: Gear, sediments, and predominant fauna Gear: A, Agassiz trawl: E. epibenthic sledge; Bottoms: Cb. calcareous bryozoan shells: G. sand and coarse gravel; S. sandy silt and mud: Sf. felt ot sponge needles; St. larger stones and rocks. temperature (-1 to +0.5C). The presorted substrates were subsequently checked for entoprocts under the dis- section microscope. About half of the zooids of each spe- cies found were kept alive, whereas the rest of them were preserved, some after narcotization, some without such a pretreatment. Narcotization and fixation A 4% formaldehyde solution in seawater proved to be the best fixation medium, yielding usable results even for electron microscopical purposes. For fast narcotization, especially when specimens were treated while still on their host, the gradual addition of an isosmotic solution of MgSO 4 gave acceptable results. But the local anesthetic amylocaine-hydrochloride [Stovaine R Rhone Poulenc: 1- (dimethylamino)-2-methyl-2-butanol-benzoate hydro- chloride] was more effective, particularly for small samples or single specimens in a small amount of water. By far the best results were obtained with a two-step narcotiza- tion: 8-10 crystals of amylocaine-hydrochloride were gradually added to a small sample of specimens in about 2 ml of seawater until the animals were completely ex- panded and showed no reaction to mechanical stimulation (about 10 min). Subsequently, some crystals of MgSOj were added. After 5 min the sample could be fixed by the addition of 0.2 ml of 4% formaldehyde. Treatment of living samples Living samples for later culture experiments and ob- servations aboard were kept in 5-10 1 aquaria under run- ning seawater at 0-0. 5C on their original substrates or hosts. They were fed by the moderate addition of fresh nanoplankton samples, chiefly diatoms. Only the colonial Barentsia discreta could be cultured successfully and brought home to the laboratory still alive. None of the solitary loxosomatids could be kept alive and actively budding for more than three to four weeks, even when left on their original hosts. Measurements and sketches Measurements of a representative number of living specimens (30-50 if possible) from every locality were 156 P EMSCHERMANN FFS METEOR CRUISE XI-4 STA'RONS OF VAN VEEN GRAB AND JUAURANGULAR DREDGE Figure 2. Station map of the Meteor cruise XI-4 in the Bransfield Strait. taken aboard and. later on, compared with those of ran- dom samples of preserved specimens. These groups of measurements were not significantly different. Freehand sketches were made of living specimens. When ship con- ditions allowed, micrographs of living specimens were taken through the dissection microscope. Higher mag- nification micrographs were made from preserved mate- rial in the home laboratory. Some Remarks on Species Determination and the Description of New Species in Entoprocta Entoprocta in general, and most Loxosomatidae in particular, have a scarcity of reliable species characters. The majority of morphological parameters, such as size, number of tentacles, body proportions, shape of stomach, and even conspicuous structures like cuticular pores, and spines, and body appendices, exhibit great intraspecific variability, and there is often overlap between species. Because of this deficiency of reliable morphological fea- tures, attempts have often been made to use the host or the locality of an entoproct as an aid for identifying its species. But neither the number of true species and their variation, nor their geographical distribution and possible spectrum of hosts, are sufficiently well-known to be useful in species identification. A rigorous biological species characterization by demonstration of their genetic isolation has not been possible for the great majority of entoproct species. Therefore, any species determination, especially any description of new species, founded on the evaluation of a few morphological characteristics, should be based on an intimate knowledge of all comparable species and. if possible, a comparison of the specimens in question with the type material of all similar species or. at least, with definitively identified samples of the latter. The description of a new species is not of value in itself; the demonstration of the real distribution range of a species is much more important. Any description of specimens new to an area should be illustrated with precise drawings in frontal and lateral view, and, if possible, in the contracted as well as the expanded state. Additional micrographs are often very helpful. Proof samples or types should be preserved both in contracted and expanded state. Because of insufficient description and unsatisfactory preservation of type ma- terial, not one Loxosomatidae described by Harmer (1915) from the Siboga samples can be reidentified. ON ANTARCTIC ENTOPROCTA I a Mr II Sun inn List 2 Meitw-Cnnac \l-4 in the Bransfield Strait 157 Station Position Depth (m) Date Gear Bottom 08-90 561.25 W 55.05 125 29/01/90 VG S 14-90 562.53 W 54.15 400 29/12/90 VG. D St 21-90 561.00 W 56.00 337-426 30/12/90 VG S 27-90 561.75 W 57.89 340 01/01/90 VG S 28-90 S 62.09 W 57.64 286-383 01/01/90 VG S 31-90 562.99 W 56.99 80 02/01/90 VG. D St. S 39-90 5 63.42 W 59.86 155 03/01/90 VG, D St 50-90 S 62.25 W 60.57 167-147 05/01/90 VG, D S 64-90a 564.15 W 63.55 135-150 08/01/40 VG, D St 66-90 S 64.47 W64.77 356 08/01/40 VG, D St, S 76-90 S 65.06 W 66.98 220 10/01/90 D St 77-90 S 65.39 W66.18 330-370 10/01/90 VG St 78-90 565.91 W 66.85 75 11/01/90 D St 87-90 S 66.57 W 68.57 450 12/01/90 VG, D S. St 96-90 S 62.77 W 60.90 150 16/01/90 VG. D S, St Benthos stations: gear and sediments Gear: VG. Van Veen grab; D, rectangular dredge; Bottom: Cb, calcareous bryozoan shells; G, sand and coarse gravel, S, silt and mud; St, felt of sponge needles; St, larger stones and rocks. Because of our limited knowledge of the intraspecific variability and geographic distribution of most entoproc- tan species, every describer of a new entoproctan species should be wary that his new species may turn out to be synonymous with a species long known, even if all pre- cautions have been taken. The following descriptions of new species must be seen in this light. Description and Discussion of Species Loxosomella brochobola spec. nov. Holotype. Collected by the author on 20 January 1990 at the type locality, stat. 16-434 ANT VIII-5 (73.69S; 21.75W) at a depth of 260-270 m from sandy and rocky bottoms with abundant calcareous Bryozoa; the entoproctan species was growing exclusively on the inner, abfrontal surface of tube-shaped Porcllu malouinensis colonies (Bry- ozoa). Syntypes. Deposited in the British Museum of Natural History, London (No. 1992.12.14.1) and the Zoologisk Museum, Kebenhavn. Name. From Greek: fipoXof-snare and I3a\\tiv-dis- charge, referring to the sticky threads that can be ejected by nematocyst-like extrusive organs a unique character of this species. Description. This is a tall Loxosomella species, about 1 300 pm in length, the individuals resemble at first glance a Pedicellina zooid (Figs. 3a-c; 4). The bulgy goblet- shaped calyx is sharply delineated from the long, slender, and highly motile stalk. The large tentacular crown with 14-20 slim tentacles is oriented straight up. In the ex- panded state, the calyx is slightly laterally depressed, but is nearly globular when contracted. The rectum bulges out between the aboral pair of tentacles, and the anus opens immediately anterior to the aboral ends of the horseshoe-shaped periatrial ciliary rim (Fig. 4f, i). The peduncle is slender, cylindrical and one and a half to twice as long as the calyx. About halfway down there is usually a slight "waist." The basal attachment area is narrower than the average diameter of the peduncle and animals are not fixed very strongly to their substratum, but can be removed easily without damage. Remnants of the foot gland normally persist as a plug of globular cells in the base of the stalk. As often observed in loxosomatids, the perikarya of the peduncular epithelial cells are arranged in longitudinal rows between the muscle strands. The stomach is voluminous and globular with wide lateral pouches bulging out at either side (Fig. 4k). The longitudinal musculature of the oral side consists of a dense layer of fibers running downwards from the oral and orolateral calyx wall to nearly the base of the peduncle, while at the aboral side and laterally in the upper portion of the stalk, only 2-3 single strands at either side are de- veloped. Basalwards these aboral muscle fibers increase in number, thus forming together with the oral fibers a closed muscular tube in the lower portion of the pe- duncle. Living specimens in normal expanded posture have the peduncle slightly curved, the aboral side of the calyx in- clined downwards, the oral side up (Fig. 40. Seen from above in this position, four large whitish blue, opaque blister-like structures are conspicuously visible at either side between the bases of each of the second and third, as well as the third and fourth, oral tentacle. Upon irri- tation, or sometimes spontaneously, 300-400 nm long delicate, helically twisted threads can be ejected from these enigmatic organs (Figs. 3d-h; 4b-d, n-g), which resemble 158 ON ANTARCTIC ENTOPROCTA 159 cnidarian spiro- or nematocysts. The sticky threads are as long, or somewhat longer than the tentacles, and remain anchored with their proximal ends in the epithelial cells from which they originate, floating with their distal ends, outside the tentacular crown. At higher magnification these extrusive organs each consist of an enlarged barrel- shaped and plurinuclear epithelial capsule, about 80 ^m long and 45 /j.m in diameter. In the unexploded state, it is filled with an invaginated highly coiled tubule, roughly square in cross section. The nuclei, generally four, are situated basally in a narrow area of marginal plasma. When ejected, the evaginated tubule, about 3 jum in diameter, has an X-shaped cross section and is cov- ered by a thin mucous coat. Loxosomella brochobola is the only entoproct known to have such extrusive organs. The function of these organs is obscure, but defense seems unlikely. Possibly these extrusive threads are con- nected with a specialized method of feeding: their ar- rangement around the mouth supports such a presump- tion. The extrusive threads could act as a kind of "fly paper" in a marine biotope poor in suspended matter. They would collect small particles attached to the sub- stratum and inaccessible to the ciliary feeding apparatus, and from time to time would be swallowed together with any adhering material. But this kind of activity has, so far, not been observed. From their genesis, the extruding organs are surely true kamptozoan organs, not "kleptocnides" somehow ac- quired from hydroids growing in their immediate vicinity, such as Halecium. Moreover, in both their overall struc- ture and extrusion mechanism, they are distinct from similar cnidarian organs. Unlike the tubules of cnidarian nematocysts, they do not evaginate by turning inside out, like a glove finger, but rather they are ejected simply by the unfolding of the curled introverted thread through a rupture of the extruding capsule at its proximal tip. (An ultrastructural investigation of these peculiar organs is in progress and will be published separately.) Buds, normally two to three on either side, develop orolaterally, level with the basal half of the stomach. They are fixed to the parent, not by the aboral tip of the foot, but by a junctional zone situated basally at the aboral side of the calyx, as is known from Loxosomella kefersteini (Figs. 3a, d: 4a-e, 1). The long, sickle-shaped glandular foot of the bud points upwards. The main body of the foot gland is situated just below the stomach. From there. a narrow glandular groove bordered by large secreting cells runs all along the foot to its aboral tip. After the bud has detached from the parent, the foot does not degenerate totally, but develops into the basal portion of the adult peduncle. The aboral tip of the foot becomes the attach- ment site for settling on the substrate. The upper portion of the adult stalk above the "slight waist" consequently develops by stretching the zone between the calyx base and the proximal part of the foot. Gonads in different stages of development were ob- served in nearly all specimens examined: Immature and mature testes were developed only in undetached buds and newly settled specimens, while mature ovaries were found exclusively in larger animals, usually with 1 -2 eggs on either side. The testes are positioned laterally to the stomach; the ovaries lie more distally, in the space between stomach, esophagus and the atrial bottom. In a few spec- imens unhatched larvae lacking eyespots were observed (Fig. 4m). Measurements. Total length: 1200 ^m (994-1350 Mm); length of calyx: 380 Mm (260-493 Mm); length of stalk: 800 Mm (423-978 Mm): width of calyx: 335 Mm (239-408 Mm); thickness of calyx: 400 Mm (245-554 Mm); diameter of stalk: 90 Mm (65-1 14 Mm); number of tentacles: 18 ( 12-20). in buds: 12. Hahiuit and distribution. Though its bryozoan host is abundant all over the Weddell Sea, Loxosomella brocho- bola has been found at only two locations in the eastern Weddell Sea (stations ANT-VII1-5 16-396; 16-434; 16- 491; and 16-492). L. hroc/ioho/a grows exclusively on the inner, abfrontal surface of the tube-shaped colonies of Porella malouinensis (Bryozoa) which is sometimes as- sociated with young colonies of an undetermined species of Halecium (Hydroida). At the type locality L. brochobola was growing in small groups of 20-30 specimens/cm 2 , specially on younger host colonies settled only sparsely by other epizoans. Discussion of the species. The loxosomatid described above is the only entoproct known to possess nematocyst- like organs. These may be homologous to and derived from pearl-like glandular cells or cell complexes, which have been found to be more or less regularly scattered around the margin of the tentacular crown in a number of loxosomatids. These organs alone are a striking species character. To date only four other loxosomatids are known which show the budding pattern described above: Loxo- somella kefersteini Claparede, 1867, L. pseudocompressa Konno, 1977, L. annulata Harmer, 1915, and L. mepse du Bois-Raymond-Marcus, 1957. In its general appear- Figure 3. Loxosomella brochobola spec. nov. a-c: living specimens in abfrontal (a) and lateral view (b, c); d: contracted specimen with large bud and ejected sticky threads: e: ejected sticky thread; f: extrusion organ in Nomarski contrast, the coiled ejectable tubule is visible (bar 10 Mm); g: the same organ, two of four nuclei are visible at the left side (bar: 10 Mm); h: part of an ejected tubule [bar in all micrographs 100 Mm unless otherwise indicated). 160 P. EMSCHERMANN Figure 4. l.i>M>somclla hrnchohola spec. nov. a: contracted zooid from fixed sample; b-e: two expanded zooids (b/c; d/e), in lateral-frontal, and latcral-ablrontal view, respectively, with buds and. partly, ejected extrusion organ; f: living expanded zooid in normal posture; g, h: bending movements of a zooid after irritation; i: contracted tentacular crown seen from above, the rectum bulging out between the aboral tentacles; k: calyx seen from the aboral side; lateral pouches of the stomach are visible; I: newly detached bud. the navel being visible at the abtrontal side of the calyx base; m: young larva before hatching; n-q: extrusive organs in different stages of ejecting the sticky thread (from preserved specimens). ance, only the latter shows some similarities to the Weddell Sea specimens; but the smaller size and average number of tentacles of L. niepse. the shape of its stomach not being trilobed. the shorter foot of its buds not being sickle- shaped, and most of all, the lack of the conspicuous ex- trusive organs clearly distinguishes this species from the Weddell Sea specimens. In conclusion, Loxosomella bro- c/ioho/a is a reliable new species. ON ANTARCTIC ENTOPROCTA 161 Loxosomella seiryoini spec. n'S; 6445'W) at a depth of 320 m from a stony and muddy bottom, growing in small numbers ( 10 specimens) dorsally on the anterior segments and gills of an ampharetid polychaete (ct Glyphanoslomum \pcc.: Fig. 7a, b). Name. From Latin: tonsorius-shaver, because of the gibbous appearance of the calyx, which in lateral profile resembles an old Norelco 4 ' electric shaver (e.g.. Fig. 50. Description. A medium-sized species, 600-800 ^m in length, with a characteristic gibbous calyx, a short and thin peduncle of about 0.5-0.7 times the calyx length. The comparatively large tentacular crown with 8 short and stout tentacles faces towards the oral side (Figs. 5f- i; 7c-0. The calyx is slightly laterally depressed (width/thickness ratio 0.8). Below the stomach, the calyx constricts abruptly into the thin peduncle, which tapers somewhat towards its base and terminates in a small attachment area. The latter consists of a small epithelial invagination repre- senting a remnant of the genuine foot-gland (Figs. 5k; 7h, i). The animals are not fixed very firmly to their substra- tum and can be easily removed without damage; they fall off easily after fixation. The stomach is almost globular. and as a result of the humpbacked calyx, the rectum is an unusually long tube. The longitudinal musculature consists of only a few muscle strands: frontally and at either side 2 to 3 fibers each run from the calyx wall down to the base of the peduncle. Buds develop orolaterally in line with the upper half of the stomach. Only two of the specimens had de- veloped very young buds, however, since these did not show any trace of a foot-gland, nothing is known of its structure. Mature gonads (Fig. 7g, were present in all specimens; the majority contained ovaries with 3-4 eggs in different maturation stages. In one single case, testes filled with sperms were observed and in another specimen, the rem- nants of degenerating testes were visible below the ovaries. This indicates a protandric hermaphroditism also in this species. \h-ii\urcnn-ni\ Total length: 600 M (366-795 MHI); length of calyx: 370 Mm (223-461 M'TI); length of peduncle: 197 Mm (95-350); width of calyx: 200 M"! ( I I 1-240 Mm): thickness of calyx: 243 Mm (223-254 MTU), diameter of peduncle: 95 //m (above) to 64 urn (below); number of tentacles: 8. Habitat ami distribution. Only 10 specimens of this loxosomatid were found on the gills and the dorsal side of the first segments of an ampharetid polychaete from silty and rocky bottom, west of Anvers Island at a depth of 320 m. Discussion o/ /he species. Since a small foot-gland is present even in adult specimens (Figs. 5k: 7h, i) a rem- nant of a presumably larger gland in the buds the above specimens belong to the genus Loxosomella. The rudi- mentary attachment gland seems to remain active throughout life. Any conspicuous circular muscle fibers in the peduncle base, which would indicate a sucker-like function of the basal glandular pit, as is characteristic for the genus Loxosonui. are lacking. The body shape is quite distinctive: no other loxosomatid so far described has such a gibbous calyx with the expanded tentacular crown facing exactly towards the front. The species characterization is based on only a few individuals; additional examination of the foot-gland structure in older buds, as well as an investigation of the variation range of this species from more numerous samples is highly desirable. The data available at present suggest that the specimens described above constitute a new species. While nothing is known so far about the distribution of the above new species beyond their type localities, the species described below seem to be distributed not only in the whole Atlantic sector of the Antarctic and subant- arctic sea, but also in the arctic and subarctic region of the northern hemisphere. Loxosomella antarctica Franzen 1973 Material. Collected by the author in the Weddell Sea at stations ANT VIII-5/ 16-396, 16-411, 16-42 Land 16-434, growing on the brittle star Ophiuroli'pis gelida as well as on the aphroditid polychaete Laetmonice pmducta at stations 16-411 and 16-489. In the Bransneld Strait the species has been found by Dr. U. Wirth at the stations Met. XI-4/31- 90. 39-90. and 64-90 growing on the same hosts with an apparent preference for Opluurolcpis xi'liila The original description of this species given by Franzen ( 1973) was based on preserved specimens from samples Figure 5. LoxiiMimclla .wirvnini spec. nov. a-e: contracted preserved specimens in frontal view (a), and lateral view (b); c: specimen with large bud; d: contracted specimen with a small bud; e: stalk of the latter with basally visible remnants of the foot-gland (arrow); f-k: Loxosonu'llu liimmriii spec, nov., preserved specimens in lateral and frontal view; i: with a small bud; k: stalk of i with basal remnant of the foot-gland (arrow) [bar 100 Mm]. Figure 6. l.i>.\osomella seirvonini spec. nov. a-g: different zooids from the rear end of (Jolfingia mar- garitaci'a. a: living specimen with the conspicuous peritentacular collar; b and c: preserved specimens; d: contracted specimen with large bud; e: specimen in semiexpanded state, in abfrontal view; f and g: specimens with larvae in their brood pouches in lateral and frontal view, respectively; h-k: different specimens from the introvert of the host, k with a robust "lorica" out of detritus particles covering the abfrontal part of calyx and stalk. In the foot plates of all specimens remnants of the foot-gland are visible. 164 ON ANTARCTIC ENTOPROCTA 165 Figure 7. LoxmmnclUi tansoria spec. nov. a: host polychaete Glyphanostomum xpcc.; h: head region of the latter with loxosomatids settling on the cirri and the prostomium: c-e: preserved expanded specimen with mature ovaries, in lateral, frontal, and ahfrontal view, respectively; f and g: preserved specimens in lateral view with mature ovary (0 and mature testes (g); h-i: basal tip of the stalk with remnants of the foot- gland. 166 P. EMSCHFRMANN of the 1902 Swedish Antarctic Expedition; these samples were dredged west of the northernmost tip of the Antarctic Peninsula. Franzen's description will be supplemented by my recent observations on living specimens. Description. Loxosoinclla anlarclica is a tall species, up to 2 mm in length, with a high goblet-shaped calyx, almost lyriform when seen from the oral side, and nearly circular in cross section. Only in a strongly contracted state is it at times somewhat flattened (Figs. 8a-f; 9a-g). In the ex- panded state, the large tentacular crown, generally with 12 slender tentacles (only 10 in newly detached buds), is inclined to the oral side at an angle of about 45; when contracted it faces more or less orally. The peduncle of large budding specimens varies in length from -h to 3 times the calyx length. Basally, below a conspicuous con- striction, it terminates in an enlarged foot-plate. The stomach is variable in shape, voluminous and globular to inversely triangular, but in contracted speci- mens, transversely oval. The cuticle is comparatively ro- bust; in younger specimens it is smooth, but in older in- dividuals, especially in the basal portion of their peduncle, broadly wrinkled. The body musculature is well devel- oped. Longitudinal fibers run upwards from the pedun- cular base, fanning out into the calyx where, at either side of the esophagus and intestine, they insert into the frontal and aboral body walls. The muscular layer, compact at the oral side, thins out towards the aboral side into loose bundles of single fibers. Depending on the nutritional conditions, 1-3 buds ap- pear at either side (Fig. 90, developing anterolaterally in line with the middle of the stomach. The glandular foot of the bud has a long posterior extension and only a knob- like frontal protuberance (Figs. 8e: 9h). This is one of the striking differences between this species and the similar- looking Loxosomella antcdonis (Fig. 14e), which is diffi- cult to distinguish from younger specimens of L. untarc- tica. But in the buds of the former, the foot is inversely T-shaped, extending to a conspicuous anterior as well as a posterior, process. Immature and mature gonads are present in most specimens testes exclusively in buds and newly detached specimens, and ovaries only in older zooids. So far the Weddell Sea and Bransfield Strait samples agree quite well with Franzen's description and illustra- tions. But the variability in the ecological conditions of the Weddell Sea, and, consequently, in the size and body shape of the Weddell Sea specimens, is much higher than in the type samples. While the average size of Weddell Sea specimens is about 1000 /urn (600-1750 /urn), the Bransfield Strait samples average 1500 ^m (640-2100 ^m), and both are smaller than Franzen's specimens. The calyx of the latter is, in most cases, distinctly marked off from the peduncle, but in some Weddell Sea populations it transforms gradually into the stalk. In these samples, the stalk usually tapers towards its base to about half of its original diameter (Figs. 9i-l; lOc. d). while in Franzen's samples, the stalk was cylindrical throughout its length. According to Franzen, Loxosomella cintarctica lacks any lateral sensory papillae. But in two Weddell Sea popula- tions (stat. 16-422 and 16-439), in a number of specimens growing on Ophiurolepis gelicla. very small sensory pa- pillae were present on either side of the calyx, in line with the second pair of oral tentacles (Figs. 9i-l; lOc, d). Usually these delicate "sensory spots" are only visible under higher microscopical magnification as pointed cuticular protru- sions equipped with 1 to 3 stiff cilia (Fig. lOc, inset) that protrude from an intraepithelial cluster of sensory cells. I have never found such sensory organs in buds and young individuals. Most remarkable is the ability of L. antarctica to shed and regenerate a calyx (Figs. 1 1: 12) a regener- ative capacity unique to this species amongst loxosoma- tids. .Mi'iiMtivincnl* \\'cddcll Sea \i>d.-inien.f Total length: 1000 Mm (595- 1750 Mm); length of calyx: 400 nm (380-636 Mm); length of peduncle: 680 MHI (240- 1 1 30 /im): width of calyx: 3 1 5 Mm (208-4 14 Mm): thick- ness of calyx: 317 ^m ( 178-477 Mm); diameter of peduncle: 133 Mm (85-180 Mm); diameter of peduncle in specimens with tapering pe- duncle: above 155 Mm 1 127-180 Mm), hasally 104 jim (87-135 Mm); number of tentacles: 12 (10-12). Bransfield Strait specimens: Total length: 1524 M m (636-2142 Mm): length of calyx: 490 Mm (318-625 Mm); length of peduncle: 1074 Mm (318-1525 Mm): width of calyx: 280 Mm (143-357 Mm); thickness of calyx: 290 Mm (159-318 Mm): diameter of peduncle: 133 Mm (95-220 Mm); number of tentacles: 12 (10-12). Hahiun anil distribution. In the Weddell Sea. L. ant- arctica has been found repeatedly at depths ranging from 100 to 400 m. growing in moderate numbers on the oral disc and the arms of the brittle star Ophiurolepis gelida (Fig. 8a), and, occasionally, in small numbers, on the dor- salmost fine setae (Fig. lOa. b) of the polychaete Laet- monice proditcta ( Aphroditidae). In the Bransfield Strait, Loxosoinclla antarctica is the most common loxosomatid. and grows on the same hosts; but it exhibits a conspicuous preference for the brittle star. Usually the ventral body surface and the arms of this host, as well as the dorsal side of the disc, are occupied by crowded populations of the loxosomatid at a density of about 4-6 individuals per mm : . Be\ond its Antarctic occurrence, the same species possibly has a second area of distribution in the Arctic Polar Sea. In the collections of the British Museum, a small sample of a loxosomatide (no. 31.7.3.1.70) was deposited which was collected from an Epizoanthus arborescens colony near Bear Island (Greenland). Although the preservation state of these specimens is not the best, and they are identified by Mortensen himself as L.\\mclla anleili>ni.\. it is evident that they lack bilateral sensory papillae, one of the striking species characters of the latter species (if. p. #). Therefore, these Arctic specimens may also belong to Loxosomella anlarclica Franzen. 1973. Especially with respect to the bipolar distribution of a number of entoprocts, a critical review of museum samples, as well as some new investigations in arctic waters, would be desirable. Discussion of the species and the possibility of hybrid- ization. Specimens from different hosts usually did not differ significantly, but samples from the Weddell Sea and Figure 8. Loxosomella antarctiai. a: Zooids settling tightly on an Ophiumli'pis arm (scale 1 mm); b and c: living specimens in frontal and lateral view, respectively; d and e: young bud and newly detached bud; f: specimen from Franzen's type sample (bar 100 ^m). 167 Figure 9. Lo.WM'iiwIlii iinlanlica a-c: expanded and contracted specimens from the Bransfield Strait; a: expanded specimen from Ophiurolepis (horn life); b-c: preserved contracted specimens from Laetmoiucc: d-g: expanded and contracted specimens from the Weddell Sea (from life): d and e: from Ophiurolepis; f and g: from Laelmonice; h; newly detached bud; i-1: contracted Weddell Sea specimens from Ophiurolepis with tiny sensory spots (m) at either side. 168 Figure 10. Loxosomclla anlarclica. a and b: Preserved Weddell Sea specimens from the setae of Laet- monice products; c and d: expanded and contracted Weddell Sea specimens from Ophiurolepis gelida with minute lateral sensory spots (arrows, inset) [bar 100 urn]. 169 170 P. EMSCHERMANN the Bransiield Strait differ markedly in their average sizes. This may result from the conspicuous differences in the nutritional conditions in these regions; primary produc- tion, predominantly consisting of diatoms, is much richer in the Bransfield Strait than in the Weddell Sea. A discontinuous presence of lateral sensory spots, which was observed in some rare cases in Weddell Sea specimens of L. antarclica, has been reported likewise for Loxoso- mella clavijonnis and L. phascolosomata (Vogt, 1876); but the latter observations are not well established. Since all other characters of such Weddell Sea specimens with small lateral sensory papillae were within the normal range of variation of L. antarctiai. and since such specimens were always found mixed with a majority of "normal" antarctica-zooids, they are considered to belong to the same species. Of course, such cases could also be produced by hybridization between L. antarctica and another spe- cies, such as L. antalonis. that is equipped with lateral sensory papillae. At the one location in the Weddell Sea (stat. 16-369), where both of these species, /.. antarctica and L. antalonis. occurred, they settled on different hosts and only in small numbers: L. antarctica on Ophiurolepis and L. antcdonis on Lac'tmonicc. Where Loxosomella antarctica occurred abundantly on both the ophiurid and (in smaller numbers) the polychaete, L. antedonis seemed to be generally absent. It was exactly under these conditions, amidst a majority of "normal" Antarctica-zooids, that zooids with tiny lat- eral sensory papillae were detected. These findings are strongly suggestive of hybridization between these species, especially since, within the abun- dant L. antarclica populations of the Bransfield Strait where L. antedonis appeared to be generally lacking, no specimens with sensory papillae were detected. In fact, if hybridization between Loxosomella antarctica and Lox- osomella antedonis is possible at all, then small dissemi- nated populations of L. antedonis may well be absorbed by hybridization with the antarctica populations wherever the latter species is dominating. A sporadic appearance of antedonis characters in such hybridized populations must be expected. Both species can maintain themselves unhybridized only in macrobiotopes where they live as small populations that are spatially separated, e.g.. on dif- ferent hosts. Regeneration. Loxosomella antarclica is the only sol- itary entoproct. for which the ability to regenerate a calyx has been demonstrated (Figs. 11; 12). Usually in Loxo- somatidae the regenerative capacity is limited to the repair of single injured tentacles. In abundantly growing populations of Loxosomella antarctica on Ophiurolepis from the Bransfield Strait, amidst great numbers of large active zooids, sporadic headless (no calyx) stalks were found. These stalks were still intact and were actively twisting and bending. At their headless apical end, they were sealed with a cuticular cap. Figure 1 1 . L.t>xo\omclla anlurclica. stages of calkyx regeneration, a: headless stalk in total view; b: apical region of a regenerating stalk with adhering remnants of the calyx cuticle; c-g: apical portions of several stalks in different regeneration stages: the beginning invagination and formation of the primary atrial vesicle (c, d), the initial gut formation (e), and differentiation of esophagus, stomach, intestine, and rectum (f). and (g) the atrial opening, newly broken through, and formation of the hrst oral tentacles (drawn alter preserved samples). and sometimes remnants of the cuticle of the shed calyx were still present. The basal portion of these peduncles is. in general, sharply delineated from the apical part by a different structure of the cuticle; basally, it is roughly wrinkled and coated by detritus particles, while apically it is thinner, smooth, and translucent (Figs. 1 la, b; 12a, c). The same was observed in the stalks of many large active zooids presumably an indication of successive growth periods. Under the microscope, headless peduncles were ob- served in different stages of calyx regeneration. The ear- liest, least differentiated stages, have a thickened body wall epithelium throughout. At their distal ends they are con- tracted by the fibers of the well-developed longitudinal musculature, the remaining wound of the shed calyx being sealed off by a plug of epithelial cells and covered by a newly secreted cuticular cap (Figs, llb-d; 12a, b). The innermost strands of the muscular layer are partly dis- integrating, and the body cavity is filled with voluminous parenchyma cells containing many granules and vesicles, presumably storage proteins from phagocytized muscle cells. This picture resembles the muscular joints of bar- entsiid stalks when transforming into resting buds. It can be inferred from the different stages observed that the subsequent regeneration of a calyx proceeds in the same way as in colonial entoprocts (Figs. 1 Ib-g; 12b, d, e): ( 1 ) A primary atrial vesicle is formed by an apical invagination of body wall cells. (2) Gut and atrial floor ON ANTARCTIC ENTOPROCTA 171 Figure 12. Loxmcimella anlarcnca. calyx regenerating stalks; a and b: heavily cuticularized stalk with germinating tip; a primary atrial vesicle has formed (arrowhead); b-d: formation of the gut, theatrial opening not yet broken through; c and e: in lateral view; d: in frontal view; A - atrial cavity; I - intestine; R - "anlage" of the rectum; S - stomach; arrows - residual storage cells (bar 100 #im). 172 P. EMSCHERMANN differentiate out of a basal cluster of the invaginated cells. (3) After the atrial opening has been broken through, the tentacles develop along the atrial rim beginning at the oral side. The size of the headless peduncles, and their obviously successive stages of differentiation, preclude the possibility that they could merely be young metamorphosing spec- imens or old zooids in the course of degeneration. The former would be expected to be considerably smaller and in any case devoid of adhering apical cuticular remnants (Fig. 1 Ib), whereas the atrium of the latter would never be completely closed (Figs, llf: 12c-e). Whether this calyx regeneration takes place as a con- sequence of external injury to the calyces, or is due to a periodic transformation of peduncles into resting buds under unfavorable environmental conditions, is presently indeterminable. All other loxosomatids examined in this respect under normal temperatures have an individual life span of hardly more than 6-10 weeks. During this time, depending on the nutritive supply, they continuously develop and release buds. Simultaneously, they pass through a short protandric male phase and a subsequent longer female phase. After having released about 10-20 larvae, the zooids degenerate. The larvae require at least about a week for metamorphosis. The presence of apparently older basal portions and younger distal parts of the peduncles in larger zooids in- dicates a life span being extended over several growth pe- riods. Such an increased ability to regenerate is obviously an adaptation to extremely short growth periods dimin- ishing the chance of sexual reproduction. Under such cir- cumstances an extension of the life span by an optional inactive resting phase is advantageous. Loxosomella antedonis Mortensen 1911 Material. Collected by the author in the Weddell Sea at stations ANT VIU-5, 16-396 and 16-405 in depths of 300-400 m growing in moderate numbers on the dorsalmost tine setae of Laetmonice producta (Poly- chaeta. Aphroditidae). The species was originally described by Mortensen (1911) from the northeast coast of Greenland, growing on the cirri of the feather star Antedon prolixa, and has been redescribed by Ryland and Austin (1960) from set- tlement panels off Swansea. I found it again in 1964, growing abundantly for a brief period on rocks and other solid substrates at the rocky shore around Helgoland. The species is very similar to young specimens of Loxosomella antarctica. but does not attain the length of the latter. Description. The Antarctic specimens are 700 to max- imally 1200 //m long, the slender, almost cylindrical pe- duncle being as long, or 1.5 times as long, as the calyx (Figs. 13a-d; 14a-e). The calyx, seen from the oral side, is inversely triangular in outline and slightly depressed in the oral-anal axis. In the expanded state, the calyx trans- forms gradually into the stalk. The large tentacular crown. with 12-16 tentacles, when expanded, is conspicuously inclined to the oral side. In the contracted state the calyx is racket-shaped, flattened, with the lophophore facing frontally. The stomach is oval to inversely triangular with somewhat projecting lateral lobes. The peduncle, which bears longitudinal musculature that is not as strongly de- veloped as in /.. aniarclica. terminates in an enlarged ad- hesive disc. As a striking character, this species possesses at either side of the calyx, just beneath the lophophore and level with the stomach roof, a prominent, non-retractile sensory papilla, about 20-30 ^/m long, with a tuft of stiff bristles (Figs. 13b-d; 14b-d). Buds develop orolaterally in line with the upper half of the stomach. In contrast to those of most other loxosomatids, they have a very distinct T- shaped foot with a long anterior and posterior process, the peduncle inserting in the middle (Fig. 14e). Sometimes several fine sensory bristles are visible at the anterior tip of the foot. Measurements. Total length: 900 /im (690-1450 ^m): length of calyx: 42(1 urn (336-548 jum): length of peduncle: 508 ^m (361-651 urn): width of calyx: 265 ^m (233-308 Mm); thickness of calyx: 185 /jm (169-189 fimY, diameter of peduncle: 107 ^m (93-1 17 /jm): number of tentacles: 14-16: length of sensory papillae: 20-30 ^m. Habitat anil distribution. In Antarctic waters, the spe- cies has been found only in the Weddell Sea. growing exclusively on Laetmonice producta. West of the Antarctic Peninsula it seems to be absent. The actual distribution of Loxosomella antedonis probably consists of the arctic and subarctic region, where it settles on various living as well as dead substrates, showing no host specificity. Additional remarks to the species. The Weddell Sea specimens agree quite well with Mortensen's original de- scription of Loxosomella antedonis, as well as with Ry- land's and my own specimens from the Irish- and the North Sea. Though the original type specimens have been lost, the conspicuous lateral sensory organs, mentioned and figured by Mortensen, present a striking species char- acter. A sample of specimens without such papillae, de- posited in the British Museum (cf. p. #) and identified by the late Mortensen as Loxosmella antedonis. is definitely different from this species. Loxosomella compressa Nielsen and Ryland 1961 Synonym. Loxosomella compressa var. antarctica Franzen. 1 973. Material. This species was abundant at depths from 100 to 400 m at almost all stations in the Weddell Sea. but was absent at depths greater than 500 m. In the Branstield Strait it was found at one single location (Met. Xl-4/39-90). at a depth of 160 m. generally growing on the dorsal setae of a great variety of polynoid polychaetes. Loxosomella compressa, first described by Nielsen and Ryland from the Norwegian coast, growing on the no- topodial setae of several polynoids, turned out to be the most common entoproct in the Weddell Sea. In this area, it apparently prefers the same hosts as in its northern area of distribution. h TV\wmclla >m/r,v,vpn\ Ehlers. 1890; Robertson, 1900; Barentsia unlarclica Johnston and Angel. 1940: Barentsia discreta Annandale, 1915; O'Donoghue, 1920: Emschermann. 1985: Franzen, 1973; Harmer, 1915; Hutchins. 1945: Johnston and Angel 1940: Kirkpatrick 1888; Konno 1971; Marcus. 1922. 1937. 1953; Mature. 1957; Mukai andMakioka, 1980; Okada and Mawatari. 1938;Osburn. 1912. 1914, 1932, 1944, 1953;Rogick, l956:Toriumi. 1949. 1951: Vigeland. 1937/ 38; Waters. 1904; Barcmsia gracilis Norman. 1907/10; Barentsia in- termedia Johnston and Angel. 1940: Barent\ia miMikicnxis Oka. 1895; liarcntxiii tiniula Verrill, 1900. Material. In the Weddell Sea. small colonies were found on diverse sol d substrates from three locations (ANT VIII-5/ 16-396 and 16-456. ami additionally in older samples collected at 76 36,0'S; 30 33.3'W). In the Branstield Strait, samples were found at all stations, except 66- 90. in a depth range of 80 to 400 m. Description. Living colonies of Barentsia discreta (Fig. 19) can immediately be recognized macroscopically by the vivid bending and twisting movements of the tall. 4- 6 mm-long zooids arising from large, cylindrical, and del- icately annulated basal sockets. The slender and predom- inantly rigid stalk bears a broad, cup-shaped calyx with the circle of 20-24 long tentacles facing straight up. The rigid part of the stalk, depending on the growth conditions, may be the same diameter over its entire length, or may widen slightly distally. Its smooth, yellowish to brownish cuticle is usually perforated by more or less numerous, minute, pore-like openings of subcuticular epithelial or- gans; the latter are presumably ion regulating cells ho- mologous to protonephridia (Emschermann, 1972. 1982). As is normal for Barentsiidae, the rigid portion of the stalk distally. just below the calyx, turns into a short mus- cular segment with a wrinkled flexible cuticle. This distal a ^ Figure 19. Barcnt\ia di\m-tti. a-c: three zooids from the Bransfield Strait (a) and the Weddell Sea (b. c): d: basal socket with a disc-shaped resting bud below it (part of a colony from the Bransfield Strait); e: calyx with the characteristic atnal retractor muscle indicated. ON ANTARCTIC ENTOPROCTA 181 stalk segment has the capacity for calyx regeneration. Un- der favorable growth conditions, after degeneration of a calyx, and before its regeneration, the muscular section may give rise to a second stalk segment, consisting in turn of a proximal stiff and a distal muscular portion. The primary muscular swelling in such a case persists as an intercalating muscular joint, separated from the next seg- ment by a cuticular hemiseptum. A stack of 8 to 10 star- shaped transverse muscle cells ("star cells" Emschermann, 1969) forms a sort of diaphragm between stalk and calyx. In older well-fed colonies, a cup-shaped secondary in- flation can develop below the bases of the zooid muscular sockets (Fig. 19d); the inflation is filled with storage cells and is separated by a diaphragm from the zooid base. These basal inflations function as resting buds, being re- sistant to mechanical damage, as well as temperatures up to 25C, and even against being embedded in ice or drying for at least a week. They give rise to new zooids after the primary ones have been damaged or have died. The structure of the calyx musculature, in particular the shape of the paired atrial retractor muscles, is a useful and reliable species character (Fig. 19e), as in most Bar- entsiidae (Emschermann. unpub.). In Barentsia discreta. three fine muscular strands on either side originate from the atrial floor just behind the mouth. Running down- wards, in line with the roof of the stomach, they unite to form a short muscular ribbon. This in turn bifurcates again into an anterior and a posterior branch, each splitting into 2 to 5 fine single fibers, which insert in the lateral calyx walls at either side of esophagal entrance into the stomach. These atrial retractors can best be visualized in contracted calyces with polarized light or Nomarski in- terference contrast. In the Antarctic samples, sexually mature zooids with both ovaries and, more rarely, testes were found. Measurements. Total length of zooids: 4-6 mm; length of muscular base: 0.8-1.1 mm; diameter of muscular base: 0.3-0.44 mm; length of the distal muscular portion of the stalk: 180-330 pm: length of calyx: 580-700 Mm: number of tentacles: 20-24. Additional remarks about the species. Barentsia dixcreta has been found worldwide, the size of the zooids varying considerably, not only from location to location, but also under different nutritive conditions at the same locality. The cylindrical (but never barrel-shaped), annulated muscular base, the stalk-rigid over nearly its entire length with only a short muscular portion below the cup-shaped calyx, and the typical structure of the atrial retractor mus- cles are reliable, if only morphological, species characters. In colonies of different origin (California, Florida, and the Mediterranean Sea) cultured in the laboratory under the same conditions, no significant morphological differences between the specimens of different origin were found (Emschermann, unpub.). Their range of variation falls within that of the Antarctic material. Interbreeding be- tween different populations can be observed in culture to the extent that the experimental populations are able to be active and become sexually mature under the same environmental conditions. In their physiological tolerance to environmental conditions, such as temperature, pop- ulations from different parts of the world can differ mark- edly. An Antarctic colony in my laboratory cultures did not remain active at temperatures above 4-5C, but in an inactive state, it tolerated temperatures up to 1 5C for several weeks. On the other hand, populations from tem- perate climates are able to tolerate low temperatures nearly to freezing, but they do not develop gonads under these conditions. To date, no long term attempts to gradually adapt colonies of different origin to lower or higher tem- peratures, have been carried out). Therefore the genetic exchange between the Antarctic populations and others may be considerably reduced, but not interrupted. Their morphological conformity can be seen as an indication that they are not genetically isolated, and the populations of Barentsia discreta reported world- wide may be thought of as belonging to the same species (cf. Franzen, 1973. p. 185). Habitat and distribution. In the Weddell Sea, especially in the eastern part, Barentsia discreta is found regularly, but never abundantly, at depths between 200 and 400 m. This species grows on every solid substrate, preferably on primary or secondary hard bottoms, basally on the stems of erect hydrozoan and bryozoan colonies, as well as on stones, shells, and even on brittle stars. But in the Brans- field Strait, the species occurred abundantly everywhere at depths from 80 to 500 m, presumably because of the more favorable nutrient conditions throughout the year in this region. In general, this species is distributed worldwide, missing only from the Atlantic-subarctic European coasts. Fur- thermore, it is reported circumantarctically, along the shelves of Antarctica itself and the subantarctic islands (Busk, 1886; Franzen. 1973; Johnston and Angel, 1940; Rogick, 1956; Vigeland, 1937/38; Waters, 1904). Along the South and North American coasts, its distribution extends, on the Atlantic side, from Tierra del Fuego, along the Argentinian and Brazilian coasts (Marcus, 1937, 1953), the Caribbean Sea (Osburn, 1914, 1940; Emscher- mann, unpub.), and Florida (Nielsen, pers. comm.), up to the Massachusetts Bay in the north (Hutchins, 1945: Mature. 1957; Osburn, 1912, 1932, 1944); on the Pacific side, it extends from southern Chile and along the coast of Central America (Osburn, 1953), to California (Rob- ertson, 1900; Emschermann, 1985). In the Atlantic region, and along the European coasts, the species is reported from the Bermuda Islands (Verrill, 1900; Mature and Schopf, 1968), from Madeira (Norman, 1907/10; Emschermann, unpub.) and the Azores (Em- schermann, unpub.), and from the Mediterranean Sea (Ehlers, 1890; Zirpolo, 1927; Emschermann, unpub.). 182 P. EMSCHERMANN In the Indo-Pacific region, Barentsia discreta seems to be common everywhere, from South Africa (O'Donoghue, 1920), the Indian Ocean (Annandale. 1915: Harmer, 1899;Kirkpatrick, 1888; Thornely, 1905) and South-Pa- cific (Marcus, 1922). to the Chinese- and Japanese Sea (Konno, 1971; Oka. 1895; Okada and Mawatari, 1938; Toriumi, 1949, 1951:Yamada, 1956). Finally the species was reported by Kluge (1946) from the Laptev Sea (Si- berian Polar Sea). Some General Concluding Considerations Besides representing merely a faunistical survey, four particular aspects of the above results are of special in- terest: ( 1 ) the detection of nematocyst-like organs in an entoproct; (2) the ability of a loxosomatid to regenerate its calyx; (3) some additional observations on the nature of host preference or host specificity of the Loxosomatidae; and (4) the bipolar occurance of several Loxosomella spe- cies. ( 1 ) The detection of extruding organs in an entoproct raises questions about their comparative morphological importance and their phylogenetic significance. Compa- rable, usually unicellular, extrusive glandular organs, which produce clearly structured secretions, have been described in quite a number of invertebrate phyla, in ad- dition to coelenterates: in Platyhelminthes (only in Tur- bellaria; Reisinger and Kelbertz. 1964; Smith et al.. 1982), Gastrotricha (Rieger cl al., 1974), Nemertini (Jennings and Gibson. 1969), Gnathostomulida (Rieger and Mein- itz, 1977), and the Archiannelida among the annelids (Martin, 1978). Except in the Cnidaria. Ctenophora, and Turbellaria, these extrusion organs do not represent typical characters of the above animal taxa, but occur in isolation in one or another species. Only in Cnidaria and Cteno- phora do the extrusive organs eject harpoonlike, poisonous or sticky threads. In all of the other above taxa the extru- sive gland cells produce rod-like mucous secretions of the rhabdiite type. The probably syncytial plurinuclear extru- sive capsules of Loxosomella brochobola seem, at present, to be unique in the animal kingdom and to differ re- markably, in development, structure, and extrusion mechanism, from comparable organs in other groups. Thus they must be considered as an isolated apomorphic character of this particular entoproctan species, rather than a character of phylogenetic significance. Probably they are derived, in a highly specialized form, from con- spicuous uni- or pluricellular mucous glands of unknown function, which occur in a number of loxosomatids around the margin of the tentacular crown. (2) The ability l/> regenerate calyces in Loxosomella antarctica is unusual for solitary entoprocts. Distinct from Loxokalypus socialis (Emschermann, 1972) another entoproct species with an enhanced regenerative capacity, and in which the budding zone has shifted from the calyx wall down to the stalk as a first evolutionary step towards the colonial growth pattern-normal asexual budding in Loxosomella antarctica proceeds as usual in two paired budding areas on the oral wall of the calyx. Therefore, the enhanced regeneration capacity of the distalmost tip of the stalk epithelum in L. antarctica (Figs. 11: 12) is an isolated secondary adaptation to the conditions of Ant- arctic life. This is important to the biology of entoprocts. but is without phylogenetic significance. (3) A marked host specificity is thought by several au- thors to be characteristic of, most of the epizoic Loxo- somatidae. For example, Nielsen (1966) describes Loxo- soma davenport i as normally settling inside the tubes of the maldanid polychaete Clyinenella zonalis. but as com- pletely absent from the tubes of the closely related Cly- inenella torqiiala. which is found much more frequently than Clyinenella zonalis on the same sandy bottoms. Consequently, many authors consider the host a sufficient species character for the identification of loxosomatids. But the host can only be employed as a reliable species character if its relationship with the loxosomatid is specific: i.e.. determined by a strict physiological dependence. A shared preference of the host and its epizoan for the same microenvironment, or some structural feature of the host that offers the epizoan an ideal complex of life conditions (C.K.. a combination of mechanical shelter and a water current supplying food and oxygen and removing detritus) are situations in which host specificity is not a reliable species character. A majority of the guest-host relations in the loxosomatids seem to be of this latter type. Only three of the loxosomatids discussed above are known to show a preference for specific hosts independent of the respective localities: Loxosomella varians for neph- tyid polychaetes, Loxosomella antarctica for the brittle star Opliiurolepis gelida. and Loxosomella compressa for errant polychaetes of the family Polynoidae. The latter two loxosomatids were very abundant at many locations. Loxosomella antarctica is found predominantly on silty bottoms, on the oral disk between the arms of Opliiuro- lepis gelida. never on other ophiurids abundant in the same place. If nutrients are abundant, it also builds up crowded aggregations on the aboral side of its host and laterally along its arms. At adequate sites, where Opliiuro- lepis is lacking or very rare, Loxosomella antarctica does not switch to an other ophiurid, but rather to an aphroditid polychaete, Laelmonice producta. On this second host, it occupies exclusively the tips of the dorsalmost notopodial setae in the first segments as well as the posterior dozen body segments. The large robust zooids of Loxosomella antarclica are quite resistant to mechanical lesions (cf. regeneration ca- pacity), as well as low oxygen supply. As can be seen from their stomach contents, consisting mainly of detritus par- ticles and some larger ciliates mixed with fine mineral material, they are sediment feeders. Their requirements ON ANTARCTIC ENTOPROCTA 183 are for a nutrient-rich fine sediment and a solid settling substrate offering a certain protection against predators and against being buried irreversibly under sediments. So this species thrives on hosts like Ophiurolepis and Lact- iii* mice which creep on. or dwell in. the upper sediment layer. Loxosomella compressa on arctic and subarctic shelves as well as in antarctic waters was detected exclusively on polychaetes of the family Polynoidae. attached basally to the notopodial setae of their hosts. A more detailed anal- ysis of the microhabitat of L. compressa reveals that the only polynoidan species infested by this epizoan guest are those with notopodial setae that are thick and short, not too densely arranged, and covered by the elytrae. This loxosomatid has only exceptionally been found on po- lynoids with bushy, thinner notopodial setae or with parapodia not covered by the elytrae. Usually only smaller species, up to 10 cm in length, or younger specimens of larger polynoids are chosen as hosts. In culture experi- ments, the newly detached buds also settled on diverse non-living substrates (cf. p. #) exposed to the current. Loxosomella compressa is smaller and less resistant to mechanical injury and low oxygen supply than L. ant- (irciica. As can be demonstrated by an examination of its stomach contents, its diet consists mainly of small algae; predominantly small pennate diatoms. Consequently, its delicate zooids can grow only in a microhabitat that offers shelter against predators as well as against mechanical injury, but which also exposes them to a continuous water current and provides enough space for optimal feeding positions. Such conditions are preferably offered by smaller polynoid polychaetes, not dwelling in the sedi- ment, but creeping on the exposed surface of sponges and on erect bryozoan and hydroid colonies. Other habitats, offering comparable physical conditions, may also be chosen as a substrate by L. antarctica and L. compressa But the small loxosomatids are not easily detected amidst the bulk of possible substrates in dredged material; pre- sumably they are usually overlooked during sorting. But when the hosts were kept for a while in well aerated aquaria, the loxosomatids were also found on various other non-living substrates. From these observations, one can speculate that the choice of settling substrate, at least for these loxosomatid species, is determined by the physical structure of the mi- crohabitat and the supply of an appropriate diet, rather than by specific physiological properties of the host itself. Thus, although most loxosomatids have preferred hosts, these can only be regarded as weak species characters. (4) The observed bipolar occurrence of Loxosomella antedonis, L. compressa, and L. varians in coastal waters suggests, at first glance, a discontinuous, exclusively bi- polar distribution of these species. Their northern distri- bution in the litoral and sublitoral of the continental coasts stretches from Greenland (L. antedonis) and the Eurasian polar shelf (L. compressa and L. varians), along the northern European coasts, south to about 54 N in the southeastern North Sea. In the South Atlantic and the Atlantic sector of the Antarctic Ocean, these three species are common from the Weddell Sea. and north to the Islas Malvinas and South Georgia (about 54S). To date, none of them has been found along the eastern or western mid- Atlantic coasts, although the entoproctan fauna of the Central European shelf, in particular, as well as of the Caribbean, Argentinean and Chilean coasts have been well investigated. At present, however, nothing is known about the depth range of these species and their possible distri- bution along the Atlantic deep sea ridges. Comparable examples of a suggested bipolar distribu- tion of a single species are extremely rare and still con- troversial, the best known being the bipolar occurrence ofPriapnlus caudatus (Ekman. 1935; van der Land, 1970). A discontinuous distribution of taxa above the species level can be explained by the break-up of an originally continuous area of distribution by geomorphic events, such as continental drift, and long term climatic changes. At the species level, on the other hand, it seems unlikely that populations separated over geological periods could remain uniform in their specific characters unless at least a limited amount of genetic exchange were maintained between them. But how can such an exchange take place in the present case? Under the conditions of an exclusively bipolar dis- tribution, such a genetic exchange between the North and South Atlantic populations of the above loxosomatids must be excluded, because the life span of individual lox- osomatid zooids does not exceed 4-6 weeks, and the mo- bile larval phase lasts scarcely more than 8 days. Neither passive drifting with currents, nor transport by fast swimming hypothetical hosts such as whales could proceed quickly enough to maintain a sufficient exchange between populations of the North and South Atlantic. Nor can the considerable increase of the shipping traffic in the past decades be responsible for this distribution. One might postulate that the Antarctic faunal region had been colonized only recently by these species. But at least for Loxosomalle compressa and L. varians, their distri- butions in both the Arctic and Antarctic regions were al- ready established in the 19th century, as documented by the evaluation of several samples from the turn of the century (Franzen 1973; this paper). Thus a recent continuous distribution by colonization along the Atlantic ridges, and possibly the deep sea basins, must be postulated as being responsible for the bipolar occurrence of these loxosomatids in shallow coastal waters. More deep sea samples should be obtained and evaluated so that this hypothesis can be tested. As far as can be judged to date, the three loxosomatid species mentioned above are distributed in the Atlantic sector of the Antarctic Sea only, and they seem to be ab- 184 P. EMSCHERMANN sent from the Pacific sector. The faunal connection be- tween the North and South Atlantic must, therefore, be much more intense than the circum-Antarctic faunal mi- grations. 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(Pans) 5: 305-356. \\aters, A. \\'. 1904. Bryozoa. Pp. 1-1 13 in Res I 'or. du S. Y Belgica en 1S97-ISW. J. E. Buschmann. Anvers. N amada, M. 1956. The Fauna of Akkeshi Bay: 24. Entoprocta. J. Fac. Sci. Hokkaido L'/uvers. 12: 237-243. /irpolo, G. 1927. Sulla presenza della Barenlsia iliscreta Busk nel Golfo di Napoli. Boll Soc Nat. Napoli 39: 413-419. Reference: Biol Hull 184: 186-202. (April, 1993) Control of Hatching in an Estuarine Terrestrial Crab. II. Exchange of a Cluster of Embryos Between Two Females MASAYUKI SAIGUSA Okayama University, College of Liberal Arts and Sciences. Tsushima 2-1-1. Okayama 700. Japan Abstract. The eggs of an estuarine terrestrial crab, Se- sarma haematocheir (akate-gani), are incubated by the female for about one month. In estuarine crabs larval hatching is synchronized with the nocturnal high tide. To investigate whether the female or the embryo controls the actual timing of the hatching, one cluster of embryos was detached from each of two ovigerous females and recip- rocally transplanted. Hatching of the transplanted em- bryos was divided into the following three patterns ac- cording to the number of nights until either (or both) of the females released their larvae. In Pattern I. the trans- planted clusters both hatched on the same night that the donor females released their larvae. In Pattern II. the hatching of one of the transplanted clusters was not con- trolled by the host female, whereas hatching of the other transplanted cluster was obviously induced. Finally, in Pattern III. not only the induction of hatching, but also the time of hatching, was controlled by the female. Hatching profiles of transplanted embryos transferred to aerated conditions indicated that hatching requires three nights, and that each embryo also has an endogenous rhythm for hatching. The female seems to play two roles in hatching: i.e.. initiation of the hatching process, and enhancement of hatching synchrony in each embryo. A plausible hypothesis explaining the mechanism of induc- tion and the synchronization of hatching is presented. Introduction Clearly demarcated rhythmicities are often observed in the reproductive behaviors of both marine and terrestrial animals. A persistent question in reproductive rhythm research is whether the female or the embryo controls the Received 28 May 1 99 1 ; accepted 25 January 1993. actual timing of these behaviors. Rhythms of spawning (i.e.. shedding of gametes or fertilized eggs) or oviposition following gametogenesis must be controlled by the female alone. Examples of this phenomenon are the circadian rhythm of oviposition in the pink bollworm Pectinophora gossypiella (Pittendrigh and Minis, 1971), egg laying in the teleost Oryzias latipes (Egami. 1954; Ueda and Oishi. 1982), and the daily, tidal and lunar rhythms of spawning in many kinds of marine invertebrates (Korringa, 1947; Pearse, 1990). Embryonic development proceeds within the eggs ovi- posited by the female, and hatching occurs after a certain period. A circadian rhythm of hatching appears in the bollworm P. gossypiella. Eggs of this species, maintained at 20C, hatched 10-13 days after they were oviposited; the eggs were transferred from constant light (LL) to con- stant darkness (DD) every 5.5 h during embryonic de- velopment, and hatching was monitored (Minis and Pit- tendrigh. 1968). This experiment suggested that a circa- dian pacemaker controlling hatching is differentiated at least around the midpoint of embryogenesis, i.e., 6-7 days after oviposition. Obvious rhythmic patterns are also observed in the hatching of marine crustaceans (Sastry, 1983; DeCoursey, 1983). But eggs of most marine and freshwater crustaceans are incubated by the female until hatching occurs. This phenomenon complicates the control of the larval hatch- ing rhythm. Indeed, the timing of larval hatching is syn- chronized with day-night, tidal, and lunar cycles, but whether it is the female or the embryo that controls the actual timing of hatching remains unclear. This question has only been investigated with respect to eggs already detached from the female (Saigusa, 1992c), the role of the female is still unknown. This paper focuses on the female control of larval hatching in an estuarine 186 LARVAL HATCHING IN AN ESTUARINE CRAB 187 terrestrial crab, Sesarma haematocheir, and reports on an embryonic exchange method that was used in the inves- tigation. Eggs of this species consist of eight clusters. Two of such clusters, one from each of two ovigerous females, were detached and exchanged by reciprocal transplanta- tion. The transplanted eggs survived on the host females, and most of them successfully hatched. Hatching in the transplanted eggs was clearly divided into three patterns depending upon the number of nights intervening between the exchange and the occurrence of hatching in both or either of the females. These results suggest that the female triggers the hatch- ing process in each embryo, but that each embryo has also an endogenous rhythm of hatching. In response to some (unknown) stimuli released from the female, each embryo must initiate its hatching process around the time of nocturnal high tides, and hatching occurs 48-49.5 h later. Since all of the female-attached embryos hatch within a very short time, the female should have some mechanism for enhancing hatching synchrony just before the larval release. This paper provides evidence that the control of hatch- ing involves cooperation between female and embryo. Based on the data reported here, I present a hypothesis that explains the mechanism controlling the daily timing of larval hatching in Sesarma haemaloeheir. Materials and Methods Maintenance of crabs and monitoring of larval release in experimental rooms Experimental animals were ovigerous females of the terrestrial red-handed crab (akate-gani) Sesarma hae- malocheir, randomly collected on 9, 19, 31 July, 16 Au- gust 1990, 19 July, and 8 August 1991 from the thicket along a small estuary at Kasaoka, Okayama Prefecture. The crabs were immediately brought into the experimental rooms in the laboratory, and were kept in plastic con- tainers (70 cm long, 40 cm wide, and 25 cm high) with shallow water ( 1 cm deep) at the bottom, and with hiding spaces above it. Light and temperature in the experimental rooms were controlled. A 1 5-h light: 9-h dark photoperiod, the same phase as that in the field (light-off at 20:00 and light-off at 5:00), was employed for all experiments. The intensity of illumination in the light phase was 700-1200 lux at the floor, and in the dark phase, less than 0.05 lux. Temperature was constant at 23 1.5C and the crabs were fed every few days. A female of S. haematocheir incubates 20,000-50,000 eggs on her abdomen. When embryonic development is complete, all of the larvae hatch simultaneously. Hatching is completed within a very short time, within 5-30 min in the laboratory (Saigusa, 1992c). As soon as hatching is finished, the female releases her larvae into the water. The time of day of larval release can easily be monitored by the photoelectric-switch method (Saigusa, 1992a). Under the above-mentioned light conditions, larval release ac- tivity shows a circa-tidal rhythm, the phase of which co- incides roughly with nocturnal high waters. Exchange of egg clusters between two females The females of 5. haematocheir incubate their eggs for about one month. During this time, the color of the em- bryos changes from dark brown to brownish green, ac- cording to the stage of development which can, therefore, be estimated by visual inspection. In these experiments, females with mature embryos (brownish green color) or near mature embryos (light brown color) were used. The reciprocal exchange of a cluster of embryos between paired females is carried out as follows. Two females with similar carapace sizes were taken from containers. The walking legs and body, except the portion where the em- bryos are incubated, were wrapped in a paper towel, and the claws were then secured with a rubber band (Fig. 1 A, upper panel). To prevent the crabs from removing the A 1cm r >t i V cl 1cm Figure 1. Embryo exchange between two females. (A) Upper photo: females with their chelae and walking legs restrained with a rubber band (rb). Lower photo: a cluster of embryos (cl) detached from each female. The base of the ovigerous seta is tied with a thread (/). (B) Females after the embryo exchange: a view from behind. //: paper towel. 188 M. SAIGUSA exchanged cluster, 2-3 mm of one of the (paired) tips of both claws was removed with scissors (see the female at the left side of Figure 1A). Bleeding was stopped with a small soldering iron. Females of S. liuciniiiin heir have four pairs of abdom- inal appendages, each of them consisting of plumose and non-plumose setae (Fig. 2). The eggs are attached, just like grapes (8 clusters in all), to ovigerous hairs that grow from the non-plumose setae, and are ventilated by the female during development. The number of attached em- bryos in a cluster is 2000-6000. The first non-plumose seta on the right side was cut with scissors (Fig. 2). because it is the most convenient place to bind an exchanged clus- ter from another female. The excision of the egg cluster caused a small amount of bleeding from the base of the non-plumose seta, but hemostasis was induced with a sharpened soldering iron. These procedures, including the removal of an egg cluster, were applied in rapid succession to both females. Each cluster of removed eggs was tied at the cut end of its seta to the center of a long thread (Fig. 1 A, lower panel). Each tied cluster was then put into the space where the reciprocal cluster had been detached, and the free ends of the thread were passed around to the dorsal side of the abdomen and knotted at the articulation between the ab- domen and the thorax (Fig. 1 B). This prevented the trans- planted cluster from being squeezed out of the egg mass being incubated by the host female. There was no ex- change of blood between the transplanted non-plumose seta and the female. Paralleling the exchange of a cluster of embryos, another small embryo cluster (200-500 eggs) was removed from each female and placed in the glass beaker with aeration (Saigusa, 1992b). Under such conditions, embryos that were detached less than 48-49.5 h before the larval release, all hatch on the same night as the eggs incubated by the female; moreover, they develop and are able to swim. In contrast, embryos separated earlier than 48-49.5 h before the release, do not hatch during the experimental period. After more than a week of aeration, these embryos grad- ually hatch as larvae with no ability to swim (i.e.. the prezoea) (Saigusa, 1992c). To determine whether the hatching of transplanted embryos is triggered by the host female, hatching of the embryos detached from the female was monitored (i.e., control experiment). The time required for the removal and exchange of a pair of egg clusters was about 15-20 min. In addition, preparation of the control experiment i.e., detachment of a small egg mass, binding it with thread, and then setting it onto the apparatus for aeration took only about 5 min. To avoid nocturnal light, procedures were carried out in experimental rooms with a light phase of 24-h LD cycle. a*- an 1cm Figure 2. Abdominal appendages of Si'xurma haematocheir female. The abdomen is opened and drawn from the ventral aspect. (/. thorax, a: abdomen, an: anus, px: plumose seta, npx. non-plumose seta, ga: genital aperture). Ovigerous hairs growing from the non-plumose seta are omitted from the drawing. The unlabeled black arrow shows the place where the cluster of embryos is cut off with scissors. Inspections of hatching in transplanted embryos When the exchange of a cluster of embryos had been completed, the females were put into individual plastic cages with small holes in their sides. (These cages were either 1 1 cm in diameter and 1 0.5 cm in height, or 7 cm in diameter and 14 cm in height.) Each cage was then placed in a beaker containing 10%o clean seawater. The time of larval release was monitored with a photoelectric device: details of this apparatus have already been de- scribed elsewhere (Saigusa. 1992a). One of the most important questions in the present study was whether the transplanted cluster of embryos (Fig. 3A) would successfully hatch, and if so. whether this would occur simultaneously with the 7 clusters of female-attached eggs. For this purpose, hatching was also monitored, not only with the photoelectric apparatus, but also by visual inspections, described in detail below (Fig. 4A. B). In intact females of S. haemal ocheir (i.e.. females with- out embryo exchange), hatching occurs synchronously, possibly within 5-30 min in the laboratory. Eggs were frequently found to be wet from the diluted seawater in the beaker due to the female's movements within the cage. When the hatching started, several zoea larvae were ob- served swimming in the beaker (Fig. 4A, middle). As soon as hatching was completed, the female released all the larvae into the water within 3-5 s (Fig. 4A, right). This quick release is associated with an abdominal tanning be- havior, which triggers the photoelectric switch. Thus, if the seawater in the beaker is frequently checked, an ob- vious sign of hatching (i.e.. several swimming zoeas) will be noticed about 30 min before the larval release for most specimens. Such visual inspections were also applied to females with a transplanted cluster of embryos. LARVAL HATCHING IN AN ESTUARINE CRAB 189 A Figure 3. Eggs o( S. haematocheir and their hatching. (A) a cluster of embryos ((7.) the cut base of which is tied with a fine thread (.v). os: ovigerous seta. (B) empty egg-cases dr) remaining after larval release by the female. (C) very thin membranes dm} protruding from the egg-case upon the liberation of hatched larva. This membrane invests the embryo before hatching [described as the "third membrane" by Saigusa (1992b). but probably the cephalic portion of the so-called embryonic cuticle]. (D) embryos (cm) dropped from ovigerous hairs without hatching. Diameter of each egg. about 330-350 f/m. Observations were made every 15-30 min throughout the night, using a hand-held light (or head lamp) covered with a few sheets of red cellophane. (These red lights were used for all of the observations and manipulations carried out in the experimental rooms during the dark phase.) When several zoeas were found swimming, the beaker was ex- amined more frequently, i.e.. at intervals of 5-10 min. As the upper diagram of Figure 4B indicates, when the photoelectric switch monitoring one of the paired females with a transplanted cluster operated (i.e.. the sign of larval release), the female was taken out of the cage. The thread was cut, and the transplanted cluster was carefully re- moved from the female's abdomen. Since empty egg cases remain attached to the ovigerous hairs, as they do after a normal larval release, it was easy to determine whether all of the transplanted embryos had hatched. (The judg- ment as to whether all the eggs hatched at the same time as the other embryos carried by the female is described in the Results section.) This observation was made under normal light, outside of the experimental room, because a female that had completed larval release was never used for further experiments. If hatching had not yet occurred, the cluster was quickly transferred into vigorously aerated seawater ( 10%o), and examined for the subsequent occur- rence of hatching. Some of the aerated clusters were mon- itored for hatching every hour in constant darkness (DD) or in 24-h light-dark ( LD) conditions; the hatching of other clusters was sought during the light phase of the 24-h LD cycle. Just after the observations and manipulations men- tioned above, the other female was also checked to ex- amine whether her transplanted cluster had also hatched. This was done by observing the water in the beaker under red light. As indicated in the middle diagram of Figure 4B, when hundreds of zoeas were seen swimming in the beaker, the transplanted cluster was removed and its 190 M. SAIGUSA A Larval release in intact females B. Larval release in the females ', ;h transplanted clusters One fej r naicning removal of Ihe examination """" transplanted cluster ""*" of hatching L* no hatch-* removal of the examination transplanted cluster """" of hatching ' removal of the ' transplanted cluster examination of hatching Figure 4. Experimental procedures used to examine hatching in intact females and females with transplanted clusters. The female crabs (not depicted) are in perforated cages suspended in beakers containing dilute seawater (10%). A photoelectric device monitors the seawater for the presence of zoeas (see text). (A) The sign of hatching and larval release by intact females. Before larval release, the photoelectric switch is "off." Upon larval release, the switch operates ("on"): threshold of response: 10,000-20.000 zoeas in the beaker. (B) Examination of the transplanted clusters removed from host females. [ 'pper diagram: Larval release occurs in one of the paired females, and the procedures indicated are followed to monitor hatching in the transplanted cluster (described in Materials and Methods section). Middle diagram: The cluster transplanted to the other female has hatched, indicated by hundreds of zoeas swimming in the beaker, (observed visually the switch is "off'). The transplanted cluster is removed and examined, and the female is reset in the recording apparatus. Lower diUKram: No zoeas are seen in the beaker. The female is removed from the cage alter the release of her larvae, and the trans- planted cluster is then examined. hatching was confirmed; a stereo-microscope was used as necessary. The beaker was then replaced with another, and the female was reset to the recording apparatus. On the other hand, as shown in the lower diagram (Fig. 4B). there were many cases where no zoeas were seen swim- ming. In these cases, when the female finally released lar- vae, the transplanted cluster was removed and its hatching was examined. Notice that the transplanted clusters were examined under normal light to determine whether hatching had occurred. This was no problem when all of the embryos had already hatched. But a question remained in the other cases as to whether this light might have affected the timing of hatching. To reduce the effect of light, some of the transplanted clusters were removed from the female in the experimental room under red light and transferred into a vigorously aerated medium. The latter experiments were carried out in 1991 as specified in the figures. The embryo exchange experiments involving 5 1 pairs of fe- males were all done in 1990, although the year is not identified in the figures. Animals were never used for more than one experiment. Results Ovigerous females with a transplanted cluster released their zoea larvae between the night of embryo exchange (e.g.. Fig. 5A-a) and 1 1 days after. The release behavior was the same as that of intact animals. The results are clearly divided into the following three patterns (Pattern I. Pattern II. and Pattern III), which are related to the number of nights until larval release occurred. Pattern I (Fig. 5 A) Both females released their larvae within two nights after the embryo exchange. The detached eggs of the control experiment and the transplanted embryos, all hatched on the same night that the donor females released their larvae. The results of this data can be further divided into three sub-patterns. Sub-pattern 1-1 ( 1 pair). As shown in Figure 5A-a, larval release of both females (F-l and F-2) occurred on the night following embryo exchange. As soon as the release of one of the females (F-l ) was recorded, the transplanted eggs (cl:F-2) were removed. Almost all eggs remained un- hatched, and were quickly transferred to aerated condi- tions and monitored. As shown in Figure 5B-a, all of the embryos had hatched by about 6:00 on 18 August. On the other hand, the larval release of the paired female (F- 2) occurred 50 min later than F-l (Fig. 5A-a). When the implanted cluster (cl:F-l) was removed from F-2, it had already hatched. The eggs detached from both females (i.e., ae:F-l and ae:F-2) also hatched during the same night. Sub-pattern 1-2 (6 pairs). Larval release of these females occurred on the first and second nights after embryo ex- change, respectively (Fig. 5A-b). The egg mass of the con- trol experiment (ae:F-3 and ae:F-4) hatched on the same night that the donor females released their larvae. The transplanted eggs (cl:F-4) were removed from the female (F-3) immediately after larval release. No eggs hatched, and this cluster was monitored under constant darkness. Hatching occurred on the following night (Fig. 5B-b). corresponding to the release of the donor female (F-4). On the other hand, in the beaker where the female F-4 was confined, swimming zoeas emerged around the time of larval release of female F-3. These larvae had clearly hatched from the implanted cluster (cl:F-3). When this cluster was removed from the host female F-4, almost every embryo had already hatched. The remaining em- bryos hatched within a few hours under aerated condi- tions. Sub-pattern 1-3 ( 1 pair). In only one instance did both females release larvae two nights after the embryo exchange (Fig. 5A-c). The embryos in the control exper- LARVAL HATCHING IN AN ESTUARINE CRAB DayO Day 1 Day 2 191 72 h i F- 1 - A r o Aug. 7 '! -(A)cl:F-2 - t aeration in darkness a z i F- ^ - A * p / Aug i ^ b ( O )cl' F .3 u Fr- t .( ) c 1 F- 6 F-fi - Aug.1 9 : r~ t c r o C ) cl F "-5 Figure 5A. Hatching of transplanted clusters: Pattern I (a) Sub-pattern I- 1 F-l and F-2 designate the paired females that were set into recording apparatuses. Excision and exchange of embryos occurred at 16: 55-17:15 on 17 August. Upward black arrows show the time of day of larval release of these females (at 23: 00 for F-l. and at 23:50 for F-2). cl:F-2 and cl:F-l are the transplanted clusters. ae:F-l and ae:F-2 are the control egg masses kept in vigorous aeration. O: Hatching of the egg-cluster or egg mass. A: Partial hatching of the egg cluster. (/)) Siih-puttern /-.? Detachment and exchange of eggs. 15:20-15:40 on 4 August. F-3: Larval release at 1:00 on 5 August: F-4: at 1:40 on 6 August. <: No eggs hatched from the transplanted cluster when it was removed from the host female, (c) Sub-pattern 1-3. Detachment and exchange of eggs: 12:55-13:10 on 19 August. F-5: Larval release at 23:50 on 20 August: F-fi: at 0:50 on 21 August. : These transplanted egg-clusters hatched at the same time as the female-attached eggs. Other symbols as in Fig. 5A- a and 5A-b. iment (ae:F-5 and ae:F-6) both hatched on the second night after the embryo exchange. A feature of this case was that hatching of the transplanted cluster (cl:F-6) seemed to be synchronized with that of the host female (F-5). On the other hand, no swimming zoeas were ob- served in the beaker of the female F-6 around the time of the release of F-5, although the beaker was checked often. Female F-6 released her larvae before the trans- planted cluster (cl:F-5) was removed. When this cluster was examined, all the egg cases were already empty (Fig. 3B). Thus, hatching of this transplanted cluster also seemed to be synchronized with that of the female-at- tached eggs. Pattern II (Figs. 6A and 7 A) In this pattern, only one of the paired females released her larvae within one or two nights after the embryo ex- change. The embryos of the control experiment all hatched on the same night as the release of the donor female. In contrast, the paired females released their larvae more than three nights after the embryo exchange. The control egg masses taken from these females did not hatch at all. A remarkable feature was that the hatching of the transplanted cluster was apparently induced by the donor female. The results were further divided into the following two sub-patterns. Suh-pattern II- 1 (II pairs). The first pattern occurred when one of the females released larvae on the first night after embryo exchange. Five instances are summarized in Figure 6A. For example, in Figure 6A-a, larval release of F-7 occurred on the night of embryo exchange. The trans- planted cluster of embryos (cl:F-8) was removed from the female 10 min after the release of F-7, but no eggs had yet hatched. This cluster was quickly transferred to aerated conditions, and was monitored every hour (Fig. 6B-a). The reciprocal cluster (cl:F-7), which had been trans- planted to the female F-8. hatched on the same night that the donor female (F-7) released her larvae. When the beaker of F-8 was observed with the red light shortly after the larval release of F-7, hundreds of zoea larvae were swimming. The transplanted cluster (cl:F-7) was quickly removed from F-8 under red light, and examined. The zoea larvae remaining attached to broken egg cases began to swim when the cluster was shaken by hand several times in the seawater, and hatching was completed within 192 M. SAIGUSA Dayl Day 2 A8h Figure 5B. Hatching of the transplanted egg-clusters. (<;) Hatching of the cluster (cl:F-2) removed from the host female (F-l). Downward black arrow: time of larval release of the host female (F-l ). (b) Hatching of the cluster (cl:F-4) removed from F-3. Open areas in histogram indicate the number of premature zoeas (prezoeas) that could not swim and thus sank to the bottom of the beaker. a few hours. The beaker of F-8 was replaced with another one containing 10%o seawater, and larval release of this female was monitored. The control egg mass (ae:F-8) never hatched even after five days (Fig. 6A-a). In contrast, the cluster of embryos (cl:F-8) successfully hatched two nights after the start of aeration (Fig. 6B-a), and all eggs hatched by noon on 10 August. Obviously, hatching of the transplanted embryos (cl:F-8) was induced by the host female (F-2). As four additional cases show (Fig. 6A-b-e), hatching of the transplanted clusters (cl:F-10. cl:F-12. cl:F-14, cl: F-l 6) was induced by the host females. Moreover, when these embryos were transferred into aerated conditions, they always hatched two nights after the removal from the host female (compare Fig. 6A and 6B). The pattern of hatching was similar, whether the clusters were mon- itored in constant darkness (Fig. 6B-a-b). or in LD cycles (Fig. 6B-c-e). Sub-pattern fI-2 (1 pairs). In these cases (Fig. 7A), the first larval rei occurred on the second night after the embryo exchange. The control egg mass detached from these females hatched on the same night. For example (Fig. 7A-a), female F-l 7 released her larvae first. The clus- ter transplanted to this female (i.e., cl:F-18) was quickly removed and examined with the stereo-microscope. No hatching had occurred, so this cluster was transferred into aerated conditions and monitored. After about 24 h. it had completely hatched (Fig. 7B-a). At about the time that F-l 7 was releasing her larvae, zoeas were observed swimming in the beaker of female F-l 8 (Fig. 7A-a). The cluster transplanted to this female (cl:F-17) was removed and examined. Most egg cases were already empty and the remaining embryos all hatched within a few hours in aerated dilute seawater. Female F- 18 was replaced in 10%o seawater, and monitored until the time of larval release (day 3; Fig. 7A-a). Other transplanted clusters put into aerated conditions (i.e., cl:F-20, cl:F-22, cl:F-24, cl:F-26) also hatched about 24 h after the larval release of their host females. In the first three experiments (Fig. 7A-a-c), the transplanted clusters (i.e., cl:F- 1 8, cl:F-20, and cl:F-22) hatched on the same night as the larval release of the donor females (i.e.. F-l 8, F-20, F-22). But in the other two instances (d and e), the donor females (F-24 and F-26) released their larvae two or three nights later than their complementary pairs (F-23 and F-25). These results further indicate that hatch- ing of the transplanted clusters is induced by the host female. Pattern III (Fig. 8) In these experiments (25 pairs), both females released their larvae more than three nights after the embryo ex- change. In these cases, none of the control egg masses ever hatched during the experiment. Five instances of hatching by the transplanted clusters are summarized in Figure 8. For example, one of the females (F-29) released her larvae five nights after the embryo exchange (Fig. 8- b). Within a few minutes after the photoelectric switch had operated, the transplanted cluster was removed from female F-29 and examined resulting in the discovery that all the embryos had already hatched (Fig. 3B). The beaker containing the paired female (F-30) was frequently checked on the night that female F-29 released, but no swimming larvae were seen, and larval release occurred on the following night. Female F-30 was removed from the cage 5 min after the release, and the transplanted clus- ter was examined. Again, it was observed that hatching was complete. Similar results were obtained from the other experiments shown in Figure 8-a and 8-c-e. The following evidence suggests that all of the trans- planted clusters in Pattern III hatched simultaneously with the attached clusters that had been incubated by the donor female. ( 1 ) In intact females, a small number of zoea larvae begins to swim in the beaker around 20-30 min before the larval release. Animals with transplanted embryos ex- hibited the same phenomenon. (2) When the transplanted cluster of eggs was examined just after the larval release of the host female, hatching had already been complete. (3) The date of larval release for the complementary Day _ 24 LARVAL HATCHING IN AN ESTUARINE CRAB 1234 193 48 T 72 96 120 144 h P 7 - , 4 cl:F-8 ae ration in DO a C" Q A i 'T < x > ug 7 r o - FQ hCO) ae:F-7. - O cl.F-7 cl:F-10 ae ration in DD -4 b y - Fin A ) ug i 'i ' I(J' p.i 1 . i ae:F-9 (O) -~ o cl:F-9 T A :l:F-l2 a, 'ration ir LD15.9 -~ + i "" * c p.i 9. A ug17 rp^' '4 ' r i L P n i ae:F-ll ae:F-!2 r ) Embryo exchange between F-9 and F-10. Detachment and exchange of clusters: 15:50-16:20 on 2 August. Larval release: F-9. at 1:50 on 3 August: F-10. at 23:45 on 5 August, (c) Embryo exchange between F-l I and F-12 at 17:50-18:10 on 17 August. Larval release: F- 1 1 , at 1:00 on 18 August; F-12, at 1:25 on 20 August, (d) Embryo exchange between F-l 3 and F-l 4 at 16:10-16:30 on 17 August. Larval release: F-l 3. at 21:45 on 17 August; F-l 4. at 23:40 on 20 August, (c) Embryo exchange between F-15 and F-16 at 11:40-12:00 on 17 August. Larval release: F-l 5, at 23:40 on 17 August: F-16. at 22:40 on 21 August. The transplanted clusters (cl:F-7. cl:F-9, cl:F-l 1, cl:F-13. cl:F-15) all hatched on the same night as the larval release of the donor females. Symbols are the same as in Fig. 5A. females was usually different. If these females carried their eggs for three nights or more after the embryo exchange, no swimming larvae appeared until the host female re- leased her larvae. (4) When the transplanted cluster was examined with the stereo-microscope, a very thin mem- brane that invests the embryo had emerged from the egg case (Fig. 3C). Emergence of this membrane always ac- companies hatching in this species (Saigusa, 1992b). so its appearance signifies that larvae had just emerged. Eventually this membrane is lost, and the inside of the empty egg cases are contaminated with fine detritus. To prove that the transplanted clusters hatched si- multaneously with the clusters attached to the host fe- male, it was necessary to make direct observations of the female releasing larvae. Such observations were only meaningful if the females could be sampled within 30 min after the larval release was monitored in the event recorder (44 females). Shortly after larval release, the females unfold their abdomens and begin eating the empty egg cases that remain attached to the ovigerous hairs. Thus, six females that were examined more than 30 min after release had already eaten most of the empty egg cases, or the cases had dropped to the bottom of the beaker, so the time of hatching of either the trans- planted or attached clusters could not be determined. In Pattern III, there were only three instances in which 194 M. SAIGUSA t OOO-i 500- Figure 6B. Hatching ol'the transplanted egg-clusters in vigorous aer- ation. In the upper two panels (a and h), the cluster was monitored in constant darkness. In the lower three panels (c. d, and e), hatching was recorded in 24-h light-dark cycles. Note that the embryos hatch around 48 h after the larval release of the host females (downward black arrows). The horizontal arrow in panel h indicates that the egg masses dropped from cluster cl:F-10 during aeration, so the hatching was not fully mon- itored. a portion of the transplanted cluster did not hatch, but remained attached to ovigerous hairs. These clusters were shaken by hand several times in 10%o seawater to remove adherent zoeas, and were quickly transferred to contin- uously dark (DD) conditions and monitored under aerated conditions. These eggs all hatched with peak hatching oc- curring about 24 h later (not illustrated). Loss of the <',, >n%e hy the incubating female Figure 9A incJK m example of egg loss, a curious phenomenon not usu seen in intact females, either in the laboratory or the nek'. In this experiment, one of the paired females (F-37) released her larvae on the night of embryo exchange. The egg cluster transplanted to this female (cl:F-38) was quickly removed and transferred into an aerated medium where it hatched about 48 h later. However, the reciprocally transplanted cluster (cl:F-37) was not removed quickly, therefore the egg sponge of the other female (F-38) dropped from her abdomen during the daytime of 2 August. At the time they were dropped, the embryos were still alive (Fig. 3D). Thus, if a transplanted cluster hatched and was not removed soon thereafter, the host female's own attached eggs often dropped within a few days; indeed, 7 out of 52 females dropped all of their eggs without hatching. This phenomenon appears to be due to a substance released outside of the egg membrane and associated with hatching (Saigusa, submitted). Nevertheless, as shown in Figure 9A, the control egg mass (i.e., ae:F-38) never hatched. So we can presume that female F-38 would have released larvae more than three nights after the embryo exchange. These results clearly belong to Pattern II- 1. Eggs were lost only by females of Pattern I and Pattern II, and never in females of Pattern III. This is indirect evidence that, in Pattern III, transplanted embryos hatch on the same night as do the female-attached eggs. Influence of the light used in monitoring on the hatching of the transplanted clusters Hatching of transplanted embryos can be induced, if they are incubated by a host female that releases her larvae within two nights (Figs. 6B, 7B). Indeed, all of the trans- planted clusters were removed from the host female under red light in the experimental chamber. But the determi- nation of hatching was carried out under the normal light outside of the experimental room, although the time re- quired for this observation was only 5 min. Hence, we must question the influence of this light on the hatching of these embryos. To address this issue, the hatching profile of egg clusters exposed to normal light was compared with the profile of those exposed to red light. In this experiment, the trans- planted cluster was removed from the host female just after larval release, and was kept under the light for 5 min. This cluster was then transferred into aerated conditions and was monitored every hour. In two other experiments, the transplanted clusters were also quickly removed from the host females just after larval release, but they were not exposed to the light outside of the experimental room. Instead they were tied to nylon thread under red light and monitored for hatching. The hatching profiles of the cluster treated with normal light showed that hatching occurred two nights after the transfer into aeration (data not shown). The other two clusters, which had been treated with red light, also hatched two nights after the aeration. Furthermore, a small peak of hatching was also observed on the third night for LARVAL HATCHING IN AN ESTUARINE CRAB 195 DayO 21* ~T~ 48 72 r~ 96 r~ 120 i 144 h I F-17 4 . )cl:F-!8 a ^ration ir 00 a p. iQ . A ug.7 ! 1 F-1Q - ae;F-17 / /-, )ct:F-17 J ^ :l:F-20-*ae ration in DD b r iy F-7D- Ju ' :,:, 1.22 ,r^ . F-71- ae ae 1 cl:F-19 F "'O . . A .r- 11 aeratic n ^^ c F-97- Jul.20 I,' F ' 22 LD15:9 ' F-7T- :F-21 1 A r F JL aerat ' c >n -j^ 4 d F-7A- J j!20 LD15:9 w F.9R- ;F-23 ae - F ?? ] A (x)cl:F - oc aeratio n e r zo F 9C J ( ji.2o ; r LD15:9 1 /D F-25 Figure 7A. Induction of hatching in the transplanted cluster of embryos: Suh-ptitlern 1 1-2 (a) Embryo exchange between F-17 and F-18 at 19:45-20:00 on 7 August. Larval release: F-17, at 4:00 on 9 August; F-18, at 2:40 on 10 August. (/>) Embryo exchange between F-19 and F-20 at 1 1:30-12:00 on 22 July. Larval release: F-19. at 2:30 on 24 July; F-20, at 1:30 on 25 July, (c 1 ) Embryo exchange between F-21 and F-22 at 18:30-19:00 on 20 July. Larval release: F-21. at 0:10 on 22 July; F-22. at 23:20 on 22 July, (d) Embryo exchange between F-23 and F-24 at 16:30-17:00 on 20 July. Larval release: F-23, at 0:20 on 22 July; F-24, at 1:10 on 24 July, (e) Embryo exchange between F-25 and F-26 at 17:10-17:40 on 20 July. Larval release; F-25, at 22:30 on 2 1 July; F-26, at 23:50 on 24 July. For hatching of egg-clusters (cl:F-18, cl:F-20) after they were removed, see Figure 7B-a and 7B-b. The other transplanted clusters (cl:F-22, cl:F-24, cl:F-26) hatched during one night later (hourly data not obtained). one of these clusters. Such a secondary peak of hatching was often observed in other experiments treated with nor- mal light (e.g.. Fig. 6B-e), so it cannot be attributed to the influence of red light (data not shown). Some experiments related to the induction of hatching by the female In two instances, the implanted egg-cluster did not hatch at all. Although egg loss occurred in both experi- ments, these results clearly belong to Pattern II- 1 (see Fig. 9B for the result of one of these experiments). A feature common to these two experiments is that the interval be- tween the embryo exchange and the larval release of the host female was very short. The transplanted clusters were incubated by the host females for 4 h and 4.5 h, respec- tively. In every case of induced hatching, the minimum period was 5.5 h (e.g.. Fig. 6A-d). These results suggest that at least 5-6 hours are required to induce hatching of the transplanted cluster. The possibility that some stimulus of hatching in the female-attached eggs induced the hatching of transplanted clusters was also examined. For this purpose, the trans- planted cluster was removed from the host female some hours before the larval release (Fig. 9C). In this experi- ment, the clusters were incubated by the host females for about 1 7 h, and were then transferred into aeration. The cluster (cl:F-4 1 ) hatched on the same night that the donor 196 M SAIGUSA Day 1 Day 2 0,2000- ns ulOOO- 'r5 -C 0. .D 1 1000 500- cl:F-18 Aug 8 cl:F-20 JuL23 I- inure 7B. Distribution of hatching in the transplanted embryos in vigorous aeration: (a) cl:F-18; (/>) cl:F-20. The release of larvae by the host females (F-17, F-19) is shown by a downward arrow. (Egg masses dropped from the cluster at the time indicated by the horizontal arrow, and hatching could not be perfectly monitored during this period.) The other transplanted clusters from Figure 7A (cl:F-22. cl:F-24, cl:F-26) hatched one night later (hourly data not obtained). female (F-41 ) released their larvae. The cluster (cl:F-42) also hatched two nights after the aeration. Since no hatch- ing was observed in the control experiment (ae:F-42), hatching of cl:F-42 can be regarded as having been induced by the host female (F-41). The possibility that egg-clusters kept under aerated conditions for more than three nights after detachment may lose their ability to hatch was then tested. In Figure 9D, a cluster (cl:F-43) was detached from a female and placed in aeration for five days. This cluster was then transplanted to another female (F-44). This cluster hatched in synchrony with the attached eggs of the host female F-44. Similar results were obtained in another ex- periment (not illustrated). Thus, eggs that were detached and transferred into aeration retained their ability to hatch. Hatching was obviously inhibited under aerated condi- tions. The interaction between the female and transplanted embryos was also examined. A few clusters were covered with a skirt made of thin cellophane. This skirt was open at the bottom, ensuring an interchange of water at the surface of the eggs. As seen in Figure 10, the transplanted clusters were induced to hatch (upper left in each panel). But the hatching profiles of the egg-clusters that were transferred into aeration (Fig. 10) showed a somewhat different pattern. In these three experiments, the cluster hatched in two peaks, about 24 h apart. Almost all of the eggs hatched in one experiment (Fig. 10-a), but many eggs failed to hatch in the other two (Fig. 10-b-c). These results are difficult to interpret; one possible explanation is that the stimuli recognized by the embryos are attenuated by the cellophane, which caused the occurrence of two peaks and the decrease of the number of larvae hatched. In any event, such a splitting of the hatching pattern has never been observed in intact females. Does the hatching of transplanted clusters affect the day or the time of hatching of eggs attached to the host female? For example, the hatching of the transplanted cluster (Figs. 5A, 6A) might release a stimulus that acts on the female to disturb the time of hatching or to advance the day of hatching of the female-attached eggs. To ex- amine the former possibility, the time of day of larval release by host females (except the females whose eggs had dropped) was monitored with the event recorder. As shown in Figure 11, the larval release of these females coincided roughly with the time of night high water in the field, showing a clear circa-tidal rhythm. This suggests that at least the daily timing of egg hatching was not dis- turbed, either by the exchange of embryos or by the hatching of the transplanted eggs. To examine whether the hatching of transplanted egg- clusters advances the date of hatching in the host female, the number of nights between the larval release of paired females was compared with respect to differences between Pattern II and Pattern III The range was 0-9 days in Pattern III (25 pairs), but only 0-4 days in Pattern II ( 19 pairs with no egg loss). Since the experimental crabs were chosen randomly, this difference might suggest that hatching of the transplanted cluster can advance the day of hatching in the host female. Discussion The embryos of most marine crustaceans are incubated by the female for a certain period before hatching. An endogenous factor has been suggested as operating in the hatching rhythms of many kinds of marine animals (Sai- gusa, 1992c). But does the endogenous component con- trolling rhythmicity occur within the embryo, its mother, or both? To answer this question, larval hatching must be examined, not only in the embryo, but also in the female. Although the embryos of crustaceans are attached to non-plumose setae by a funiculus that is possibly com- posed of chorion, there is no circulation of blood between embryo and mother (Yonge, 1937, 1946; Cheung, 1966; Goudeau and Lachaise, 1983). Thus, the embryo exchange experiments were aimed at revealing the site of the en- dogenous clock. An endogenous clock times hatching in each embryo The present experiments were primarily aimed at de- termining whether the implanted embryos would hatch LARVAL HATCHING IN AN ESTUARINE CRAB 197 Day 24 h 10 p. 97 ( ( cl:F-< cl:F- !8 17 a r L 1 p. OQ A ug.5 , r zo F-7Q- a a [ e F-27* . s () 1 cl:F-30 A K,:F. 25 b r /a F-^O- A jg.3,; | F-T1 - as. an. 1 : r 70. F-30 . ' ^ )cl:F-: 2 4 k )cl:F 31 c > c r j i F-"}?- k ii.22 :r p.-jo. a< a i- > F-32J ) 1 ) cl:F-3 3 d A jg.ljf p.oc. a< a< 1 , x , . 4 )cl:F-36 X e :l:F-35 p.oc. Jc " t . i /5 i.23,r r jb ae at i -n^ F-lfi*_ Figure 8. Induction of hatching in the transplanted cluster of embryos; Pallcrn III (<;) Embryo exchange between females F-27 and F-28. Detachment and exchange of clusters: 19:20-19:40 on 5 August. Larval release: F-27, at 0:30 on 8 August; F-28. at 0:40 on 8 August, (b) Embryo exchange between F-29 and F-30 at 14:45- 1 5: 10 on 3 August. Larval release: F-29. at 0:35 on 8 August; F-30, at 3:05 on 9 August, (c) Embryo exchange between F-31 and F-32 at 16:30-17:00 on 22 July. Larval release: F-31. at 2:10 on 26 July; F-32, at 3:20 on 28 July. (gg spong n ae:F-37 ae F-38 -iO)ct: -0 F-37 not removed c 1 (x)cl:F jin c le rat ion n LD15.9 h. Y jut. 20 'r 1 > of egg sponge i * ae:F-39 - (O)cl:F -O "39( no ' remov A I at ion J f aer ul.29 I~ 1990 ; cl:F-4; ) ; * on cl:F-4 | aerat B 24 h F-43 h --43 ) : -:'[ ) Jut. 27 cl:F (19? 1} aer atk )n i ae:F-43 1 til in :ubat on Figure 9. Hatching profile of transplanted clusters. (A ) Loss ol the egg sponge by the host female. Embryo exchange between females F-37 and F-38 at 16:10-16:25 on 1 August. Larval release: F-37. at 23:35 on I August. (B) Failure to induce hatching. Embryo exchange between females F-39 and F-40 at 17:50-18:20 on 20 July. Larval release: F-39, at 22:30 on 20 July. (O Removal of the transplanted cluster before the release of the host female. Embryo exchange between females F-41 and F-42 at 16:05-16:25 on 29 July. Larval release: F-41. at 3:45 on 31 July; F-42, at 1:35 on 1 August. (D) Hatching of the egg-cluster kept in aeration for five days. Detachment of cl:F-43, at 19:50 on 27 July: binding to F-44, at 16:20 on 1 August. Larval release: F-43, at 4:55 on 2 August; F-44, at 23:10 on 6 August. circadian rhythm of emergence in the fly Drosophilu (Pit- tendrigh and Bruce, 1959). But the validity of using such a population to demonstrate a circadian rhythm has been questioned (Saunders. 1976, chapter 3). So Pittendrigh and Skopik (1970) used populations that were develop- mentally synchronous at pupation to study the emergence rhythm in the fly Drosophila pseudoobuscura, and sug- gested that a circadian pacemaker in each developing fly dictates the circadian time of emergence, but not that of the intermediate developmental stages, such as head ever- sion and eye pigmentation. The eggs of St'tumia luicmatochcir are oviposited within a short time. So the developmental embryos in- cubated by a female clearly do not constitute a mixed- age population. Like the data presented by Pittendrigh and Skopik (1970), the hatching of the egg clusters re- moved from the host females was often split into two distinct peaks almost 24 h apart (Figs. 6b-e, lOa-c). To explain such a splitting of hatching pattern, we can assume an allowed zone: (see Pittendrigh and Skopik, 1970) related to hatching in the endogenous pacemaker of each embryo. In Figure 12, this zone is expressed by the acrophase (shown by a dot) in the embryo's pacemaker. The pre- ceding paper (Saigusa, 1 992c) indicated that each embryo undergoes a hatching process that continues for 48-49.5 h prior to egg-membrane breakage. If the embryos were detached from the female early enough that this time in- terval were exceeded, hatching would not occur. One speculation is that, in the larval hatching rhythm in Se- sanna. a gated phenomenon occurs at the start of the LARVAL HATCHING IN AN ESTUARINE CRAB 199 Day 1 Day 2 Day 3 Day 4 96 h 600 n Figure 10. Hatching of egg-clusters from which the cellophane skirt was removed before transfer into aeration, (a) Hatching of cl:F-46. Embryo exchange between F-45 and F-46 at 17:25-17:40 on 9 August. Larval release: F-45. at 23:45 on 10 August; F-46. at 23:50 on 12 August. (/) Hatching of cl:F-48. Embryo exchange between F-47 and F-48 at 14:25-14:35 on 9 August. Lar\al release: F-47. at 1:30 on 1 1 August: F-48. at 1:50 on 15 August, d) Hatching of cl:F-50. Embryo exchange between F-49 and F-50 at 19:10-19: 20 on 9 August. Larval release: F-49. at 0:20 on 10 August: F-50. at 2:20 on 15 August. Downward arrows indicate time of dav of release bv the host females. hatching process, and not hatching itself. If the hatching process is not initiated at a certain acrophase (Fig. 12). the embryos must wait until the next allowed zone to start the process. Induction of hatching in the transplanted cluster While each embryo has an endogenous rhythm of hatching, the present study indicates that the hatching of exchanged clusters is induced by the host female, provided that the incubation was longer than 5-6 h (compare Figs. 6A, 7A and 9B). Since the start of the hatching process may be a gated event, it is reasonable to speculate that this process begins 48-49.5 h before the hatching, re- sponding to some signal to each embryo. But we do not know what stimuli trigger the hatching process in each embryo. One possibility is mechanical stimuli generated by the female perhaps some special movements of the abdomen or ovigerous setae as the embryonic develop- ment is completed. Another possibility is a hatch-inducing substance, produced by the female and recognized by the embryos. We also do not know when hatch-inducing stimuli are released from the female. Females could gen- erate such stimuli at any time of day, or at a particular phase of her circatidal rhythm (see the question mark on the female pacemaker in Fig. 12). Synchronization of hatching between transplanted embryos and female-attached embryos For most intertidal and estuarine crustaceans, female- attached eggs hatch within a very short period, although the exact duration cannot be determined because of the mass. In S. haematocheir, hatching is completed in 5-30 min in each female (Saigusa, 1992b). Because the hatching synchrony of the embryos detached from the female is perturbed (Saigusa. 1992c), some mechanism must un- derlie the highly-synchronous hatching in female-attached eggs. This cannot be an endogenous clock in each embryo; 200 M. SAIGUSA ' 18 Time of day It, 12 1990 Jul.20- 25- e 30- Aug.l - 5- O 10- 15 20 D a t 55 t SR Figure 11. Time of day of larval release by females carrying an exchanged cluster and by females from which the cluster had been removed. Records were made in the laboratory under 24-h LD cycle, but under no tidal influence. Solid diagonal lines connect the times of high water (//III in the field. D: larval release on the first night of embryo exchange. A: larval release on the second night after embryo exchange. : larval release occurs more than three nights after the embryo exchange, .v.v and AT connect the times of sunset and sunnse, respectively. the female must produce some unknown stimulus that enhances synchronous hatching. Females of S. haematocheir release their larvae with vigorous abdominal movements. This same behavior is observed in other terrestrial crabs (Saigusa, 198 1 ). In species that release their larvae under the water, the release is effected by the pumping behavior of the ab- domen (DeCoursey, 1979; Forward ct ai. 1982: Saigusa, 1992a). Forward and Lohmann (1983) suggested that this behavior enhances hatching synchrony. In contrast. hatching in terrestrial species occurs prior to larval re- lease. Clearly, larval release behavior itself does not en- hance the hatching synchrony in female-attached em- bryos. One possible mechanism is that the female kneads the egg-clusters several times around the time of night high tide. In addition to such physical stimuli, the hatching of a few embryos might release a substance like the hatching enzyme suggested by De Vries and Forward (1991), and thus stimulate the hatching of the remaining embryos. LARVAL HATCHING IN AN ESTUARINE CRAB 201 Tide Female Embryo Female-attached eggs hatching process *>() Detached eggs detachment detachment -o - x Figure 12. Proposed mechanism of induction of hatching and syn- chronization of hatching with nocturnal high water. Endogenous pace- makers related to hatching are shown with by a sine curve (female) and a rectangle (embryo). Small circle above the female's pacemaker indicates time of nocturnal high water. Stippled area: stimuli that induce the hatching process in each embryo are released during this period. The heavy downward arrow represents stimuli by the female to enhance hatching synchrony among embryos. The success of hatching in detached embrvos is shown under the horizontal line. See the text for details. Timing mechanism of hatching: a hypothesis (Fig. 12) As in most other decapods, the oviposited eggs of S. haematocheir are incubated by the female until hatching occurs. Most of this period is probably related to embry- onic development. But hatching does not immediately follow the completion of development; i.e.. embryos wait for stimuli that initiate the hatching process. So when the eggs are detached from the female during this period, hatching should be inhibited. As demonstrated in this study, one or more hatch-inducing stimuli are produced by the female. But once these signals have been received, the start of the hatching process would be determined by an endogenous clock within each embryo. We can assume that a self-sustained oscillation under- lies most endogenous rhythms (Pittendrigh and Bruce, 1 959). As shown in Figure 1 2, we can express the embryo's pacemaker for hatching as a rectangular wave with 24.5- h period. Similarly, females would also have 24.5-h pace- maker for hatching and larval release. Both pacemakers would be synchronized with nocturnal high tide in the field. Since eggs that are detached from the female more than 48-49.5 h before hatching of the female-attached eggs do not hatch, the "allowed zone" related to the start of this process should be positioned at the phase corre- sponding to the time of nocturnal high tide. i.e.. the ac- rophase of the embryo's pacemaker in Figure 12. When some (unknown) stimuli (stippled area in Fig. 12) have been transmitted to the embryos for several hours (at least more than 5-6 h), the hatching process starts at this phase, and zoeas hatch 48-49.5 h later; i.e.. around the time of nocturnal high tide. If the stimuli from the female are insufficient to start the hatching process, embryos must wait for the next acrophase. This would have resulted in the split hatching peaks seen in vigorously aerated dilute seawater (Figs. 6B-e and 10 a-c). As shown in the lower diagram of Figure 12. if the embryos are detached from a female before the hatching process, they would not hatch at all. If they are separated from a female while the hatching process is in progress, then those embryos will hatch at about the same time as the embryos that remain attached to the female. But hatching synchrony is perturbed in this condition, so the process is extended by several hours (Saigusa, 1992c). Since female-attached eggs hatch synchronously, the fe- males must have some mechanism (a downward arrow in Fig. 12) for enhancing hatching synchrony while they are still on the hillside awaiting the time of hatching. Acknowledgments The zoea larvae found after hatching were counted by Mr. A. Shiomi and Miss H. Yunoki, students of Okayama University. They also helped with some of the daytime procedures, such as exchanging clusters, placing females in the recording apparatuses, and collecting ovigerous fe- males. Supported by a Grant-in-Aid for Scientific Re- search (C) (No. 02640582) from the Ministry of Educa- tion, Science and Culture. Literature Cited Cheung, T. S. 1966. 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(April, Asymmetry in Male Fiddler Crabs is Related to the Basic Pattern of Claw-waving Display SATOSHI TAKEDA 1 AND MINORU MURAI 2 ^Marine Biological Station. To/iokn University. Asamushi, Aomori 039-34. Japan and 2 Department of Biology. Faculty of Science, Kyiislni University, Hakoiaki. Fukitoka SI J, Japan Abstract. Morphological asymmetry was correlated with the pattern of claw-waving display in males from five spe- cies of fiddler crabs: three vertical wavers ( Uca unillei. U. dussumieri, U. vocans), a lateral waver ( U. annulipes), and an intermediate waver (U. tetragonon). On the first, second and third ambulatory legs of male lateral waver crabs, the distance between the inner edge of the basis and the outer edge of the merus was larger on the side bearing the major cheliped than it was on the side with the minor cheliped. A similar asymmetry was observed in male intermediate waver crabs, but only the first am- bulatory leg was involved. This morphological asymmetry is clearly related to the style of waving adopted by these crabs. When lateral wavers display, the weight of the major cheliped (which forms about one-third of the total body weight) is carried largely by the anterior ambulatory legs on the same side of the body, but the imbalance of weight during display is less in the intermediate waver. In the vertical waver crab horizontal motion of the major che- liped occurs relatively rarely; thus there is hardly any ad- ditional load on the ambulatory legs, which showed no asymmetry. However, the total length of the five sterna bearing tho- racic legs tended to be larger on vertical waver males than on the female crabs. Thus the sterna of male crabs bulge outwards more than those of female crabs, and the angle between the sternum bearing the cheliped and the ground surface is larger in male crabs than in females. This may be an adaption enabling the cheliped of the male to be raised higher during the waving display. Introduction A characteristic feature of the genus Uca (Ocypodidae; Brachyura) is hypertrophy of one of the male chelipeds. Received 5 November 1992; accepted 25 January 1993. resulting in a striking asymmetry (Crane, 1975). The hypertrophic, or major, cheliped plays a very important role in antagonistic and courtship behaviors (Crane, 1957. 1975). especially as a distinctive indicator of the male sex during the breeding season (Salmon and Stout. 1962). The mechanism determining which of the chelipeds becomes hypertrophic has been examined in some species (Morgan, 1923, 1924; Yamaguchi, 1977; Ahmed, 1978), and the development of the asymmetry has been analyzed by monitoring the growth rate of the major cheliped rel- ative to that of the carapace (Huxley and Callow, 1933; Tazelaar, 1933; Miller, 1973). In addition, the first and second ambulatory legs are longer on the side bearing the major cheliped than they are on the contralateral side in male U. pugilator (Yerkes, 1901; Duncker, 1903; Huxley and Callow, 1933; Miller, 1973), and in U. pugna.\ (Yerkes, 1901; Tazelaar, 1933) in North America. This asymmetry of the ambulatory legs was thought to help in raising the major cheliped higher, thus displaying it to more crabs (Miller, 1973). Crane ( 1957, 1975) divided Uca into two groups based on the male's claw-waving display; i.e., into vertical and lateral waving species. U. pugilator and U. pugnax, with asymmetry of the ambulatory legs as well as the chelipeds, form the lateral waving group (Crane, 1957, 1975). On the other hand, the ambulatory legs of male crabs of the vertical waving group have not yet been examined for possible asymmetries. In this study, the degree of asymmetry of certain morphological characters, including the length of the ambulatory legs, was determined and compared among five species of Ucu with different patterns of claw-waving display. Three of the five species were vertical wavers, one species was a lateral waver, and one exhibited an intermediate type of waving dis- play. 203 204 S. TAKEDA AND M. MURAI Materials and Methods Crabs of five species. ('. urvillei, U. dussumieri, U. vo- cans, U. tetragonon and U. annulipes, were collected on the seashores in Thailand (Table I). Larger individuals were selected for examination, since the degree of asym- metry in male fiddler crabs increases with body growth (Miller, 1973). Crabs lacking thoracic legs or those with degenerate, atypical thoracic legs were excluded. U. urvillei, U. dussumieri and U. vocans are vertical wavers, U. annulipes is a lateral waver, and U. tetragonon is an intermediate (Crane, 1957, 1975). The proportion of right-handed and left-handed male crabs was nearly equal in I', urvillei. U. dussumieri and U. annulipes. and almost all male U. vocans and L'. tetragonon are right- handed (Takeda and Yamaguchi, 1973; Frith and Frith. 1977). Two indices of body-size were measured: carapace width (Fig. 1 A); and the whole body wet-weight. The wet weight of the cheliped cut off between ischium and basis was also measured. Several morphological measurements were made (Fig. 1 ). These measurements on the right and left side of each individual include: three dimensions on the minor cheliped or ambulatory leg (Fig. 1 B); body depth (Fig. 1 A); carapace depth (Fig. 1 A); and the length of each sternum bearing thoracic legs (Fig. 1C). These measured values were normalized with respect to the wet-weight or carapace width. To determine the degree of asymmetry, the ratio of the values on the major cheliped side to the values on the opposite side in male crabs, and the ratio of the values on the right side to those on the left side in female crabs, were calculated for each individual. These data were examined for significance with Student's Mest. Results The carapace width and body wet-weight of individuals differed among the five species of Uca (Table II). The weight of the major cheliped relative to the whole body wet-weight increased in the following order: L'. te- tragonon ^ U. dussumieri ^ U. iinillei ^ U. annulipes ^ U. vocans (Table III). The relative weight did not differ widely among the species of vertical, intermediate, and lateral wavers. The relative weight of the minor cheliped did not differ significantly (P > 0.05) among individuals of the same sex in four species, but U. tetragonon had a larger minor cheliped than the other four species (P < 0.05). The relative dimensions of the minor chelipeds were larger in female crabs than in male crabs in each species (P < 0.05). The smaller values in male crabs resulted from their larger whole body wet-weight, which included the weight of the major cheliped. The chelipeds of male crabs had a remarkable asym- metry (P < 0.001 ). and the ratio of major cheliped weight to minor cheliped weight increased in the order of U. tetragonon < L'. annulipes 2= U. dussumieri ^ L'. wvillei S= U. vocans. The smaller ratio in male crabs of U. tetra- gonon (P < 0.05) was caused by their having heavier minor chelipeds than the other four species. However, the in- tensity of asymmetry did not differ among the species with the different display patterns. In female crabs of all five species, the asymmetry ratio was near 1.0, which in- dicates the chelipeds were the same size. Considering the total length of the ambulatory legs, which was calculated by adding the lengths of the I, II and Ill-sections of each ambulatory leg (Fig. IB), asym- metry was perceptible (P < 0.05) only in first (ratio = 1 .04 0.02 (mean the 95% confidence interval)) and second ambulatory legs (ratio = 1.02 0.01) of male U. annulipes (Fig. 2). and the ambulatory legs of the side bearing the major cheliped were longer than those of the opposite side. Moreover, for each section of the ambu- latory legs, asymmetry was present in the I-section of the first, second and third ambulatory legs of male U. Table I Materials, display form and handedness in Uca xpp. Species 1 750' N; 9824' E - 1328'N; IOO55'E 3 744' N; 9825' E Display Handedness Specimens Location Date L'. urvillei vertical random 6 males Ao Nam Bor 1 1990. Sep. 7 females L ' dussumieri vertical random 9 males Smare Kaow 2 1987. Oct. 9 females L vocans vertical right 8 males Ao Nam Bor 1 1990. Sep. 6 females U. leli'iixonon intermediate right 1 1 males Ao Tang Khen' 1990. Sep. 9 females U. annulipes lateral random 10 males Ao Nam Bor 1 1990. Sep. 6 females Ao Tang Khen 3 1991. Jan. ASYMMETRY IN MALE FIDDLER CRABS 205 Figure 1. Diagrams of frontal view of carapace (A), ventral surface of left ambulatory leg (B), ventral view of male crab (C). and dimensions measured in this study. (1) carapace width: the minimum distance between both tips of the anterolateral angles. (2) body depth: the minimum distance between the carapace and a straight line in contact with the plane of the sterna with the first and second ambulatory legs. (3) carapace depth: the minimum distance between the tip of one anterolateral angle and the edge of the buccal region on the side of the Milne-Edwards opening. (4) I-section: the minimum distance between the inner edge of the basis and the outer edge of the merus of the thoracic legs. (5) II-section: the minimum distance between the inner edge of the carpus and the outer tip of the propodus ot the thoracic legs. (6) Ill-section: the minimum distance between the inner edge and the tip of the dactylus of the ambulatory legs. (7), (8). (9). ( 10). and ( 1 1 ) the length of each sternum with cheliped, first, second, third and fourth ambulator, leg, respectively. anmdipes (P < 0.001), and in the I-section of the first ambulatory legs only of male V. tetrugonon (P < 0.01). The asymmetry ratios of U. annulipes male crabs were 1 .09 0.0 1 , 1 .06 0.02 and 1 .05 0.02, and the degree of asymmetry decreased posteriorly. The ratio for male U. tetragonon was 1 .02 0.0 1 . Total lengths of the minor cheliped and the first am- bulatory leg on minor cheliped side tended to be greater in males than in females, and the difference was significant (P < 0.05) in V. dussumieri, L'. letragonon and U. an- nulipes for the minor chelipeds. and in U. dussumieri and U. letragonon for the first ambulatory legs (Fig. 2). In addition, the same tendency (P > 0.05) was observed for the total length of the second ambulatory leg in the four species other than U. annulipes. The total length of the third ambulatory legs did not differ between male and female crabs. The total length of the fourth ambulatory leg was greater in female U. letragonon than in male crabs (P < 0.05). The body depths of male crabs were larger on the major cheliped side than on the other side (P < 0.001) (Table IV). The asymmetry ratios were between 1.05 and 1.07 for all species, indicating no difference between the species. The body depths of female crabs of all five species were symmetrical (P < 0.05). The relative body depths on the major cheliped side of male crabs were larger than those of female crabs (P < 0.05). The relative body depth on the side bearing the minor cheliped in male crabs showed a similar tendency, especially in U. dussumieri and U. tetragonon (P < 0.05). Table II Carapace width and wet weight in Uca spp. Carapace width (mm) Wet weight (g) Species Male Female Male Female C itn'illei 25.19 1.43 (23.40-26.75) 23.07 1.49 (20.85-25.35) 5.030 0.709 (4.115-6.169) 2.989 0.604 (2.309-4.136) 1 '"' """' 29.80 1.18 (27.65-32.85) 23.31 1.03 (21.70-24.95) 9.273 1.161 (7.371-12.553) 3.490 0.508 (2.680-4.646) (.' >WUn .v 23.05 0.63 (22.15-24.55) 17.17 0.66 (16.15-18.05) 5.503 + 0.298 (4.865-5.930) 1.487 0.294 (1.191-1.955) I' letragonon 2 1.00 0.87 (19.40-23.35) 21.03 0.93 (19.10-22.95) 3.754 0.563 (2.956-5.465) 3.090 0.339 (2.333-3.605) U annulipe\ 15.49 0.79 (14.35-17.50) 11.98 + 0.60 (10.95-12.50) 1.3 19 0.229 (0.978-1.910) 0.437 0.048 (0.355-0.495) Mean the 95% confidence interval. Numbers in parentheses indicate the range. Table III Weight of chelipeds relative to whole body wet-weight in Uca spp. Male crab Female crab Species Major side Minor side Right side Left side V. urvillei 0.366 0.047 (31.48 7.15)' U. dussumieri 0.358 0.015 (31.07 2.12)* U. means 0.432 0.034 (40.18 4.35)* T tetragonon 0.340 0.013 (17.58 1.13)* U. annulipes 0:380 0.032 (28.41 4.41)* 0.012 0.001 0.016 0.001 0.016 0.001 (1.02 0.04) 0.012 0.001 0.015 0.001 0.015 0.001 (1.01 0.07) 0.011 0.000 0.015 0.002 0.016 0.001 (0.98 0.04) 0.0 1 9 0.00 1 0.022 0.00 1 0.022 0.00 1 (1.01 0.02) 0.014 0.001 0.018 0.001 0.018 0.001 (1.00 0.00) * Student's (-test, P < 0.00 1 . Mean the 95% confidence interval. Numbers in parentheses (mean the 95%' confidence interval) indicate the asymmetry ratio based on weight (male crab = major cheliped side/minor cheliped side: female crab = right side/left side). 206 S. TAKEDA AND M. MURAI Uca dussumien Chetiped Uca tetragonon Uca annulipes 1st ambulatory leg majo mino right 5 major minor igh left 2nd ambulatory leg 5 major minor ? right left 3rd ambulatory leg S major minor ? right left 4th ambulatory leg * major O minor nghi left ILJ 05 10 15 05 10 1.5 :-M-;->; , :::: 1 1 :-'.-:-:-:.: j :-:-:-: :-:-:-:-: : : 3 5 l.< ) 05 10 15 !- 1 ab L ;-', ab ::.-:-:-:- -:-; :-x : a ::-:-:- . i 5 15 Length relative to carapace width Figure 2. Length of the three sections of the thoracic leg relative to carapace width in L'ca spp. Each bar shows the relative length of the I-, II- and Ill-section (Fig. IB) from the left. (a), (b): asymmetry in the length of I-section of the thoracic leg and in the total length of the thoracic leg, respectively. The carapace depths of crabs was symmetrical (P < 0.05) in both sexes of the four species other than in male U. vocans (Table V). In the male crabs of all species, the sternum bearing the major cheliped was larger than that bearing the minor cheliped (P < 0.001 ), and the asymmetry ratios were be- tween 1.16 and 1 . 1 9 ( Fig. 3 ). The asymmetry is probably due to the hypertrophy of the coxa of the major cheliped, as indicated by the remarkable development of the pro- podus of the major cheliped (see Fig. 1C). In the male crabs of all species, the total length of the five sterna with thoracic legs was greater on the major cheliped side than it was on the opposite side, showing significant asymmetry (P<0.05). Male crabs of U. itnil/ei. I', dussitmieri and U. vocans had larger major cheliped-bearing sterna than did female crabs of the same species (P < 0.05). But, there was no significant difference between male and female crabs of U. tetragonon and U. annulipes (P > 0.05). The total length of the five sterna on the minor cheliped Table IV Length ot body depth relative to carapace width in Uca spp. Male crab Female crab Species Major side Minor side Right side U.un'illei 0.53 0.01 (1.06 0.01)* U iliissiiiiiten 0.58 0.01 0.50 0.01 ('. I ( tfiragmum 0.56 0.01 (1.05 0.00)* U. annulipes 0.55 0.01 (1.05 0.01)* Left side 0.48 + 0.01 0.48 0.01 (1.00 0.01) 0.55 0.01 0.52 0.01 0.52 0.01 (1.07 0.01)* (1.00 0.01) 0.56 0.01 1 .07 0.01) 0.53 0.01 0.49 0.02 0.49 0.03 (1.00 0.01) 0.54 0.01 0.51 0.01 0.51 0.01 (1.00 0.00) 0.52 + 0.02 0.52 + 0.01 (1.00 0.02) * Student's /-test. P< 0.001. Mean the 95% confidence interval. Numbers in parentheses (mean the 95^ confidence interval) indicate the asymmetry ratio (male crab = major cheliped side/minor cheliped side; female crab = right side/left side). Table V Length of carapace depth relative to carapace width in Uca spp. Male crab Female crab Species Major side Minor side Right side Left side U. itrvillci 0.34 + 0.01 0.34 0.01 0.32 + 0.01 0.33 + 0.01 (1.02 < 0.02) (1.00 0.01) I' lllUUllfllCI'l 0.33 . 0.01 0.33 0.01 0.32 0.01 0. 32 0.01 (1.01 f 0.01) (1.00 + 0.01) U. vocans 0.31 0.01 0.32 0.01 0.31 0.01 0. 31 0.01 (0.98 0.0 1)* (0.99 0.01) I' iclraxtinon 0.3 1 . 0.00 0.32 0.00 0.30 0.00 0. 31 0.00 ( 1 .00 0.0 1 ) (1.00 0.00) L' ttnnulipes 0.33 - 0.00 0.33 0.00 0.32 0.01 0. 32 0.00 ( 1 .00 1 0.01) (1.00 + 0.01 * Student's /-test, P<0.05. Mean the 95% confidence interval. Numbers in parentheses (mean the 95% confidence interval) indicate the asymmetry ratio (male crab = major cheliped side/minor cheliped side; female crab = right side/left side). ASYMMETRY IN MALE FIDDLER CRABS 207 r i Uca urvillei a c d ^^;JA......-..V^:.V^ I ' -. , ,>, i ','[-------------; v-v. .....'..'] 1 minor - :::^ -^ r'-''r' - 1 right 4 r . " . : * left H1M1SS ; ( I '. Uca dussumieri a c d * major wmmmm ".'- - ,'\ .----.. v:-:-. ":": .::! i O minor . . x :-::::-:::; : : : : : )T ,, ; ,.;- j - 1 -- . v . .-... ;, ; r '9 hl -:-: \ ^ ' ^plx-ftffffi "i S major b Uca vocans a d mmmmw, 1 ' -. '-",-t K/.\V. -..-..v.-.-i minor g right .if :.-;-* >. ' ^my'i ' * left rr~v:;'""4 ,i i, ...,...:: (yea tetragonon a d * major .1 . :. .:- .1 i O minor r ~ j .. E ."'.... -.-v.i i r '9 nt left . . .'.....-.-i-.-.-.-.- T -rt-ij i ..'-.i ' >.*. = : '. .**. . ;'.."; *. .' ;'..*; '. .' ; Uca annulipes a c d J.:-.::-.-.-.::-.-.-.-.-J.'.'.'l ' ' T I. ... ... ... II 1 minor , /.^ -'...'-.-'** i^.^.1- 1 J r '9 h t :.::". ^- -f |--v^v^ | i i I 025 0-50 Length relative to carapace width 0.75 Figure 3. Length of each sternum bearing thoracic legs relative to carapace width in L'ca spp. Each bar shows the relative length of the sternum bearing (from left to right) the cheliped, first, second, third and fourth ambulatory leg (Fig. 1C), (a), (b), (c). and (d) indicate asymmetry in the length of the sternum bearing the cheliped. first and second am- bulatory leg, and in the total length of five sterna with thoracic legs, respectively. side of male U. dmsitmieri and U. vocans tended to be larger than those of female crabs, and a similar, though weaker, tendency was observed in U. un'illei. The differ- ences between the male and female crabs of U. dussumieri and U. vocans were caused by the male crabs having more extensive sterna bearing the cheliped and first ambulatory leg. The slight difference in the total length of the sterna of male and female U. un'illei was due to the smaller degree of enlargement of the sternum with the first am- bulatory leg in male U. un'illei, compared with that of male U. dussumieri and U. vocans. On the other hand, female U. tetragonon and U. annulipes tended to have slightly longer sterna than the male crabs. This was be- cause the sternum bearing the third ambulatory leg is re- markably larger in female crabs than in males, although the male crabs tended to have a larger sternum bearing the first ambulatory leg than did the female crabs. Discussion Male crabs of U. annulipes, which display with lateral waving, had longer first and second ambulatory legs on the major cheliped side than on the other side (Fig. 2), corresponding with the asymmetry reported for the lateral waver males of U. pitgilator ( Yerkes, 1 90 1 ; Duncker, 1 903; Huxley and Callow, 1933; Miller, 1973) and U. put>na.\ (Yerkes, 1 90 1 ; Tazelaar, 1933). Moreover, comparing the major and minor cheliped sides, the I-sections of the first, second and third ambulatory legs were longer on the major cheliped side, resulting in asymmetry of total lengths of, especially, the first and second ambulatory legs. Male U. tetragonon have a form of display intermediate between the lateral and vertical waving types, and the I-section of their first ambulatory leg showed only asymmetry similar to that of the male U. annulipes. Asymmetry of the am- bulatory legs was not seen in U. un'illei, U. dussumieri and U. vocans males, which display in the vertical form, or in females of any of the five species. The sequence of the display performed by U. annulipes male crabs is as follows: the major cheliped, normally flexed in front of the buccal region, is extended laterally and subsequently raised, then flexed and brought down to the starting position from above the eyes and buccal region (Crane. 1957, 1975). The major cheliped consti- tutes about one-third of the whole body weight (Table III), and during such a display the proportion of its weight supported by each pair of ambulatory legs will change according to the angular position of the major cheliped. That is, while the flexed major cheliped is moving toward the side, its weight is borne on the anterior ambulatory legs and then on the ambulatory legs on the same side as the major cheliped. When the major cheliped is fully ex- tended laterally, it achieves its maximum loading weight. Subsequently, during the raising of the unflexed major cheliped and its return to the starting position, the weight carried on the side of the body bearing the major cheliped will decrease gradually. The degree of asymmetry in the I-section of the ambulatory legs was greatest for the first ambulatory leg, and decreased gradually until no asym- metry was apparent for the fourth ambulatory legs (Fig. 2). Thus, the distances between the dactylus tips of each pair of ambulatory legs are greater anteriorly, especially on the side bearing the major cheliped, since waving males hold the I-section horizontally rather than vertically (Crane, 1975, Fig. 92). In addition, the distance between the dactylus tips of the first and fourth ambulatory legs is greater on the major cheliped side than on the other side, since the ambulatory legs of both sides were extended in the antero-posterior direction during display. The in- creased horizontal distance on the major cheliped side may be a morphological adaptation to bearing most of the weight of the major cheliped as it moves laterally from the anterior position during display. On the other hand, in the sequence of the display of male U. iirvi/lei, U. dussumieri and U. vocans, the major cheliped initially remains flexed in front of the buccal region, and is moved up and slightly forward, without 208 S. TAKEDA AND M. MURAI unflexing (Crane, 1957, 1975). In such a display, the hor- izontal component of the motion of the major cheliped will be very small in both the antero-posterior direction and laterally, compared with the display of the lateral wavers. Therefore, the crab does not have to bear an ad- ditional load on the side of the major cheliped during waving. Vertical wavers, therefore, do not have an elon- gated I-section of the ambulatory legs on the major che- liped side. The display performed by male U. tetragonon is inter- mediate between the lateral and vertical waving types (Crane, 1957, 1975). That is, the major cheliped, flexed in front of the buccal region, is incompletely extended forward at an acute angle. Subsequently, the semi-flexed cheliped is raised so that its tip barely reaches to the eye. In such a display, the crab does not have to bear a load on the ambulatory legs of the major cheliped side, as the lateral wavers do. The asymmetry in the total length of the ambulatory legs or in the length of each section of the ambulatory leg was not seen in U. vocans, in which right-handed males are predominant (Takeda and Yamaguchi, 1973), or in male U. unillei and U. dussumieri in which the ratio of left and right handedness is similar (Fig. 2). These facts suggest that the asymmetry of the ambulatory legs cor- responds with differences in the form of display, rather than with differences in the mechanism inducing hyper- trophy of one of the chelipeds, whether innate or random. The ratio of body depth to carapace width was greater on the side bearing the major cheliped than it was on the opposite side, in male crabs of all species (Table IV). However, the relative carapace depth, which is considered to be related directly to the body depth, was the same on the two sides of the body (Table V). On the other hand, the length of the sternum bearing cheliped was much greater on the major cheliped side than it was on the minor cheliped side, resulting in the asymmetry of the body depth. Because the body depth was measured as the min- imum distance between the carapace and a straight line in contact with the planes of the sterna bearing the first and second ambulatory legs (Fig. 1A), the excessive in- crease in the body depth on the major cheliped side, re- sulting from the excessive enlargement of the sternum bearing the major cheliped, means that the plane of the sternum with the major cheliped was inclined more ver- tically than was that of the opposing sternum. This more vertical sternum position probably contributes to the smoother motion of the major cheliped in a vertical di- rection. The total length of the five sterna on the side with the minor cheliped in male U. urvillei, U. dussumieri and U. vocans tended to be greater than that of the female crabs (Fig. 3). But, there was no such sexual dimorphism in U. tetragonon and U. anmdipes. These results indicate that the sterna on the minor cheliped side of males extend further laterally, like those on the major cheliped side, and that the plane of the sternum bearing the minor che- liped becomes more vertical than is the case in females of the vertically waving species. Acknowledgments We thank the Phuket Marine Biological Center and the National Research Council of Thailand for providing fa- cilities for this research in Thailand. We thank Dr. M. Matsumasa, Department of Biology, School of Liberal Arts and Sciences, Iwate Medical University and Mr. T. Koga and Mr. T. Kosuge, Faculty of Science, Kyushu University, for their field assistance. This study was par- tially supported by Grants-in-Aid for International Sci- entific Research for the Japanese Ministry of Education, Science and Culture (Nos. 62042019 and 01041069). Literature Cited Ahmed, M. 1978. Development of asymmetry in the fiddler crab Vca ciumdanla Crane. 1943 (Decapoda, Brachyura). Cntstaceana 34: 294- 300. Crane, J. 1957. Basic patterns of display in fiddler crabs (Ocypodidae. Genus Uca). Zoologica 42: 69-82. Crane, J. 1975. Fiddler Crabs of the World. Ocypodidae: Genus Uca. Princeton University Press, New Zealand. 736 pp. Duncker, V. G. 1903. Uber Asymmetric bei Gelasimus pugilator Latr. Biiimctnka 2: 307-320. Frith, D. W., and C. B. Frith. 1977. Observations on fiddler crabs (Ocypodidae: Genus Uca) on Surin Island, western peninsular Thai- land, with particular reference to Uca letragonon (Herbst). Phuket Mar. Bwl Center Res. Bull, 18: 1-14. Huxley, J. S., and F. S. Callow. 1933. A note on the asymmetry of male fiddler-crabs (Uca pugilator). W RouxArch. Entwicklungsmech. 129: 379-392. Miller, D. C. 1973. Growth in Uca. I. Ontogeny of asymmetry in Uca pugilator ( Bosc ) ( Decapoda, Ocypodidae ). Crusiaceana 24: 119-131. Morgan, T. H. 1923. The development of asymmetry in the fiddler crab. Am. Nat. 57: 269-273. Morgan, T. H. 1924. The artificial induction of symmetncal claws in male fiddler crabs. Am. Nat. 58: 289-295. Salmon, M., and J. F. Stout. 1962. Sexual discrimination and sound production in Uca pugilator Bosc. Zoological!: 15-21. Takeda, M., and T. Yamaguchi. 1973. Occurrences of abnormal males in a fiddler crab Uca marionis ( Desmarest), with notes on asymmetry of chelipeds. Pmc. Jpn. Soc. Syst. Zool. 9: 13-20. Tazelaar, M. A. 1933. A study of relative growth in Uca ptignax. W. RI>IL\ Arch. Entwicklungsmech. 129:393-401. Yamaguchi, T. 1977. Studies on the handedness of the fiddler crab, Uca lactea. Biol. Bull. 152: 424-436. Yerkes, R. M. 1901. A study of variation in the fiddler crab Gelasimus pugilator Latr. Pmc. Am. Acad. Arts Sci. 36: 4 1 7-442. Reference: Biol. Bull 184: 209-215. (April. 1993) Studies of Intracellular pH Regulation in Cardiac Myocytes From the Marine Bivalve Mollusk, Mercenaria campechiensis W. ROSS ELLINGTON Department of Biological Science, B-157, Florida State University. Tallahassee, Florida 32306 Abstract. Myocytes were isolated from the ventricle of the marine clam Mercenaria campechiensis by enzymatic dispersion procedures. Intracellular pH (pH,) was mea- sured via fluorescence imaging techniques using an in- verted microscope interfaced with a high sensitivity tele- vision camera. Myocyte pH, was similar to values observed in other molluscan muscles measured by weak acid dis- tribution and nuclear magnetic resonance (NMR) tech- niques. Myocytes displayed a good capacity for defending pH, against changes in extracellular pH (pH e ) as the pH, remained unchanged in the pH c range of 7.1 to 8.0, but gradually declined at lower pH e values. Myocytes had a relatively high non-bicarbonate intracellular buffering ca- pacity. Further, these cells showed recovery from imposed acid loads. This recovery was accelerated by increasing HCOj concentrations, was not dependent on external Na + and was blocked by a stilbene transport inhibitor, suggesting that a HCO 3 ~:Cr transporter plays a central role in regulation of pH,. Collectively, these data show that ventricular myocytes of M. campechiensis have a rel- atively high capacity for dealing with potential metabolic proton loads associated with environmental anaerobiosis. Introduction Environmental hypoxia or anoxia imposes important energetic and acid/base stresses on marine invertebrates. When anaerobic energy yielding processes prevail, there appears to be an uncoupling of proton production and consumption. The extent of excess production of protons is dependent on the specific pathways operating (Portner el ai, 1984a; Portner, 1987a, 1989). The major evolu- tionary trajectory in highly anoxia-tolerant marine in- Received 13 November 1992; accepted 25 January 1993. vertebrates (bivalve/gastropod mollusks and certain worm groups) is the development and use of anaerobic metabolic pathways with a lower H + /ATP ratio, the ratio of proton release to ATP produced (Gnaiger, 1980). Because proton production will continue throughout anoxia, it is readily apparent that specific mechanisms are present in these organisms to minimize reductions in in- tracellular pH (pH,). Rates of intracellular acidification during anoxia (or air exposure) are generally quite low in the muscles of bivalves (Barrow el ai, 1980; Ellington, 1983a; Walsh el ai. 1984) and gastropods (Ellington. 1983b; Graham and Ellington, 1985). This is also true of the sipunculid Sipunatlm midiis (Portner el ai, 1984b; Portner, 1987b). Muscles of many of these species have moderately high non-bicarbonate intracellular buffering capacities (/C?NB) (Eberlee and Storey, 1984; Morris and Baldwin, 1984; Portner el ai. 1984a; Wiseman and El- lington, 1989). Furthermore, there is good evidence for ion exchange of acid/base equivalents between the intra- and extracellular compartments. For instance, in S. nudus Portner and coworkers (Portner el ai, 1984b; Portner, 1987b) have shown that the extracellular compartment serves as sink during anoxia for metabolically produced protons. This is also true of bivalves where the calcareous shell serves as an external buffering agent (Crenshaw and Neff, 1969; Booth el ai, 1984). In terms of ion exchange processes in marine invertebrates, a sodium-dependent Cr-HCO} exchanger appears to be the predominant ef- fector of regulation of pH, in two well-studied systems squid giant axon (Boron and Russell, 1983) and the giant muscle fibers of barnacles (Boron el ai, 1979). These ex- changers are blocked by stilbene derivatives and have low Kms for HCO 3 ~ (around 2-3 mA/). Recently, it has been shown that bivalve anterior byssus retractor muscle (ABRM) has a stilbene-sensitive anion exchanger (Zange el ai. 1990). 209 210 VV R ELLINGTON In the present study, regulation of pH, has been inves- tigated in myocytes isolated from the ventricle of the ma- rine bivalve Merccnaria campechiensis. This study uses fluorescent ratio imaging technology, which permits the observation of the dynamics of change in pH, in individual myocytes. Experiments focus on the measurement of J NB and observation of defense of pH, after exposure of cells to acid/base stress. Materials and Methods Animals and materials Specimens of M. campechiensis were collected via a dredge by a commercial fisherman from St. Joseph's Bay, Gulf County, Florida, and were transported to the Florida State University Marine Laboratory within a few hours after collection. Animals were maintained in raw (unfil- tered, unsettled), continuously flowing seawater. Prior to experiments, animals were transported to the main uni- versity campus and maintained in recirculating aquaria 20 1.5Cundera 12:12 (L:D) photoperiod. Dispersion enzymes and buffers were purchased from Sigma Chemical Co. (St. Louis, Missouri). Nigericin (free acid). 2',7'-bi-(2-carboxyethyl)-5-(and -6)-carboxyfluores- cein-acetoxymethyl ester (BCECF/AM) and BCECF (free acid) were purchased from Molecular Probes (Eugene, Oregon). The anion transport inhibitor, 4-acetamido-4'- isothiocyanatostilbene-2, 2'-disulfonic acid (SITS), was obtained from Sigma Chemical Co. All other chemicals were of reagent grade quality. Isolation of myocytes Procedures for myocyte dispersion were adapted from suggestions made by C. Bruce (Department of Pharma- cology, University of British Columbia, Vancouver, BC). Ventricles were dissected from 3 to 5 specimens of A/. campechiensis. After removal of the intestine, tissue was cut into very small pieces (1 mm 3 ), suspended in 45 ml myocyte artificial seawater (MASW, 440 mA/ NaCl, 10 mA/KCl, 7.5 mA/CaC! 2 , 23 mA/MgCl 2 , 25 mA/MgSO., and 10 mA/ HEPES adjusted to pH 7.75 with NaOH), and gently washed in a rotary shaker for 1 h. The sus- pension was placed in a 50 ml conical centrifuge tube and the tissue pieces were allowed to settle by gravity for a few minutes. After aspirating off the MASW, tissue was resuspended in 20 ml 0.1% protease VIII (Sigma) in MASW and incubated with gentle agitation for 30 min. The tissue was again placed in a 50 ml centrifuge tube followed by 30 ml MASW. After settling of the tissue, the MASW was aspirated off and 50 ml MASW added. After settling and aspiration, the tissue pieces were resuspended in 20 ml 0.1% collagenase (type 2, Sigma) and incubated with agitation for 90 min. Periodically during incubation, tissue was gently sucked in and out of a flame-polished Pasteur pipette. After incubation, the cell suspension was centrifuged for 45 s at low speed (400 rpm) in a clinical centrifuge. The supernatant was carefully decanted with- out disturbing the loose pellet and centrifuged for 4 min as above. The supernatant was discarded and the pellet resuspended in MASW and centrifuged for 4 min. The final pellets were resuspended in a small volume of MASW. Cells were seeded on circular coverslips (Ni- cholson Precision Instruments, Gaithersburg, Maryland) which were immersed in 15 ml MASW in 10 cm plastic culture dishes. Coverslips had been previously washed in acid, rinsed exhaustively and then polished with ethanol using lens paper. Dishes were placed in a humidified cul- ture chamber. Cells were always prepared during the af- ternoon and then used for imaging the following morning. All isolation and incubation procedures were conducted at 18-21C. Fluorescent ratio imaging The overall rationale and approach for BCECF imaging has been previously described (Rink el a/.. 1982; Bright et ai. 1987). Cells were loaded with 5 pM BCECF/AM for 45 min. After washing, the coverslip was mounted in a Dorvak-Stottler chamber (Nicholson Precision Instru- ments) which was attached to a Peltier device (Physitemp. Clifton, New Jersey) mounted on the stage of a Zeiss IM- 35 inverted microscope. Cells were superfused (0. 1 ml/min) by gravity flow from a manifold device consisting of a Hamilton (Reno, Nevada) eight-way valve, microbore tubing and eight reservoir chambers. Temperature was controlled at 20C. A xenon lamp (Optiquip, Highland Mills, New York) provided illumination. Fluorescence excitation was con- trolled via a dual filter wheel/shutter assembly (Ludl, Hawthorne, New York). One wheel contained excitation filters (490, 450 nm) while the other had a range of neutral density filters. A dichroic (530 nm) was positioned on the fluorescence emission side. All filters were from Omega Optical (Brattleboro. Vermont). Phase and fluorescence images were obtained using an Achrostigmat LD 32X/.4 PHI objective with light passing onto an iCCD camera (QUANTEX, Sunnyvale, California). Video signals were digitized and processed by IMAGE 1/FL software from Universal Imaging Corp. (Westchester, Pennsylvania) us- ing a 486-based computer with output on a color monitor (Trinitron, SONY). Software controlled the operation of the rilterwheel/shutter system. Isolated myocytes did not display any auto-fluores- cence. Preliminary experiments showed that BCECF- loaded myocytes went into contracture when illuminated with intense monochromatic light (490 or 450 nm). Un- loaded cells did not respond to light in this way. Fur- thermore, the ability to regulate pH, was impaired in BIVALVE MVOCYTE INTRACELLULAR pH REGULATION 211 loaded cells at high light intensities. Thus, during all ex- periments we used high range neutral density niters to reduce light intensities compensating for the reduced flu- orescence by employing high camera gain and intensity settings. Furthermore, the period of irradiation of cells with monochromatic light was reduced to a minimum consistent with camera lag. Images were acquired using shade (shade "mask" obtained using 25 n\ of 25 fiM BCECF sandwiched between coverslip and slide) and background correction capabilities of the IMAGE 1/FL software. Image pairs were acquired at specific time in- tervals (usually every 20 s). Individual cells were selected and fluorescence ratios (I^o/Uso) for each cell versus time were stored in a spread-sheet data base. Numerical data were transferred as ASCII files to a Macintosh Ilci and processed and analyzed using Sigmaplot ( Jandel, San Ra- fael, California). In vivo calibration of ratios Fluorescent ratios with respect to pH were calibrated by the nigericin pH clamp approach of Thomas el al. ( 1979). The calibration solution was identical to MASW except that it contained 290 mA/NaCl, 160 mAl KC1 and nigericin (5 fig/ml). The concentration of K.C1 chosen brackets values for intracellular K 1 as determined in the muscles of marine mollusks (Potts, 1958; Robertson, 1965; Burton, 1983). Cells were allowed to equilibrate with each solution until ratios stabilized (generally <5 min). In routine experiments, pH, was estimated for in- dividual myocytes. Mean values for acid-base measure- ments in each physiological treatment represent data from 3.0 2.8 2.4 2.2 2.0 1.8 1.6 6.4 6.6 6.8 7.0 7.2 7.4 7.6 7.8 pH F'igure 2. Relationship between the fluorescence intensity ratio (R = IWUso) and pH obtained using the nigericin approach with cardiac myocytes from Mcrcciuiria campechiensis. Each value corresponds to a mean I SD (n ranges from 8 to 12). individual cells from up to two independent cell disper- sions. Results Figure 1. Cardiac myocyte from the ventricle of the clam Aiercenaria campechiensis. Photograph was taken with Kodak TMAX 100 film. Bar corresponds to 100 Mm. Dispersion procedures produced a very high yield of myocytes. Cells were generally long and spindly (50-400 fj. X 10 n) (Fig. 1). Immediately after dispersion, a large fraction of cells showed spontaneous contractile activity but were generally quiescent after 12 h. Dispersed cells excluded trypan blue and were responsive to addition of 10~ 5 M 5-hydroxytryptamine (5-HT). In fact, many cells remained viable and responded to 5-HT up to 7 days after isolation. The addition of antibiotics (penicillin G. baci- tricin) and 5 mAI D-glucose did not enhance survival in the short term nor influence results of pH, determinations. Thus, these components were not added to MASW. Ven- tricles of the congeneric clam Mercenaria mercenaha have extremely high glycogen levels, on the order of 240 ^moles/g wet wgt (Ellington, 1985). Thus, it is clear that there is a sufficient endogenous reserve of metabolic fuels in these myocytes for the period over which they were used (15- 18 h). Nigericin pH clamp Myocytes were subjected to a nigericin pH clamp pro- tocol encompassing the range of pH from 6.6 to 7.6 in 0.2-unit increments. Typically, most cells responded to the clamping medium by contracting to approximately 60% of their initial length and remained so throughout the protocol. Fluorescence ratios varied linearly with pH (Fig. 2). A regression equation was calculated (RATIO = 1.1 1 1 pH -5.724, R = 0.994) and used to transform all ratios from the ratio versus time spreadsheets to pH, . The compressed ratio range was due to differential 212 W. R. ELLINGTON 7.5 74 7.3 pHi : 7.1 7.0 6.9 6.8 6.0 6.4 6.8 pHe 7.2 7.6 8,0 Figure 3. The relationship between pH, and pH e in cardiac myocytes from Mercenaria campechiensis. The continuous diagonal line corre- sponds to the iso-pH line. Each value corresponds to a mean 1 SD (n = 11). gain/level adjustments of the analog-digital converter at the two excitation wavelengths as well as the fact that the neutral density filter at 490 nm was higher than the one used at 450 nm. Under routine superfusion conditions, pH, of myocytes was observed to be 7.22 0.08 (mean 1 SD, n = 20). Relationship between intracellular and extracellular pH Cells were superfused with MASW (containing 20 mM HEPES) adjusted to various pH values (pH e ). Media were equilibrated with air. Ratios were observed for 1 5-20 min at each pH. pH, was essentially constant in the pH e range from 7.1 to 8.0 (Fig. 3). At lower pH e values, the pH, declined linearly but still was considerably above the iso- pH line indicating good capacity for defense of pH, against pH e (Fig. 3). Non-bicarbonate buffering capacity (@NB) Non-bicarbonate buffering capacity was estimated by the NH 4 C1 prepulse method of Boron (1977). Myocytes were superfused with MASW and then subjected to a pulse of 15 mM NH 4 C1-MASW (pH 7.75). After peak alkalin- ization, myocytes were superfused with MASW resulting in a pronounced acidification. /} NB values were calculated as described by Boron (1977). Buffering capacity is ex- pressed as Slykes (dH 4 /dpH). Since the MASW was equilibrated in air, [HCO.r] was low (around 0.7 mAf) so the contribution of this species to total buffering ca- pacity is negligible. Figure 4 is an example of a typical pre-pulse experiment. Following alkalinization, there was a gradual decline in pH,. After NH 4 C1 wash-out, there was a characteristic alkalinization back towards the initial condition (Fig. 4). J NB values for individual myocytes were somewhat variable ranging from 22 to 65 Slykes with a mean of 39.98 8.86 (1 SD, n = 31). The inherent limitation of the NH 4 C1 prepulse approach is that some recovery of pH, could occur during the early phase of the wash-out therebye producing an overestimate of /3 NB (Bo- ron, 1977). In the present study, wash-out was extremely pHi f . \J 1 1 1 I 7.7 1 7.6 - 1 I 7.5 ***4 7.4 o 7.3 1 7.2 - *- 7.1 la^v**^^ 7.0 ^ 6.9 - fi ft 1 1 1 1 10 20 30 40 51 time (min) Figure 4. Time course of changes in pH, in a single Mercenaria campechiensis cardiac myocyte during a typical NH 4 C1 pre-pulse experiment. The first arrow indicates the onset of superfusion with 1 5 mM NH 4 CI- MASW. The second arrow indicates the onset of washing with MASW. BIVALVE MYOCYTE INTRACELLULAR pH REGULATION 213 rapid as the peak acidosis occurred within 3 min (Fig. 4). Thus, it is likely that pH, recovery processes would po- tentially produce only minor errors in /} NB determination in this system. Recovery from acid loading Isolated myocytes regulate pH, after acid-loading as ev- idenced by the slow alkalinization following NH 4 C1 wash- out using normal MASW ([HCO, ] approximately 0.7 mM) (Fig. 5A; Table 1). However, the rate of alkaliniza- tion was greatly accelerated during wash-out using 0.3% CO : /4 mA/ HCO 3 MASW (Fig. 5B; Table I). Recovery from acid loading did not appear to be dependent on ex- ternal Na + , as the recovery rate was essentially the same for MASW and Na + -free MASW (Table I). SITS com- pletely blocked recovery. In fact, there was a gradual re- duction in pH, once the plateau acidification after wash- out had been attained (Table I). Collectively, these results show that it is likely that a SITS-sensitive HCO 3 ":CI~ ex- changer plays a major role in recovery from acid loading in M. campechiensis myocytes. Discussion Molluscan myocytes have been used on a number of occasions as experimental systems for investigating phys- iological phenomena ranging from ion channels (Brezden eta/., 1 986) to changes in intracellularCa :+ concentrations during contraction (Ishii el a/.. 1989). In this regard, car- diac myocytes from M. campechiensis appear to be an ideal model system for studies of regulation of pH, in that these cells are easily isolated, retain viability for extended time periods and, of course, can maintain acid-base bal- ance in spite of extracellular and intracellular pH distur- bances. The average pH, of 7.22 in these cells as deter- mined by BCECF-imaging is comparable to values ob- served in various marine gastropod muscles (Ellington, 1983b; Graham and Ellington, 1985; Wiseman and El- pHi 7.0 6.9 6.8 20 30 time (mm) 10 20 time (mm) Table I Recovery from acid loading in myocytes from Mercenaria campechiensis Washing medium Acid/base dpH/dt transport (pH units/min) (Aimoles/min) n MASW 0.0040 0.00 18 0.157 0.055 10 MASW-0.3%CO 2 : 4 mM HCOr 0.0 152 0.00 12 0.576 0.115 9 Na + -free MASW 0.0054 0.0030 0.236 0.159 9 MASW-0.5 mM SITS No Recovery Not estimated 10 (-0.0073 0.0056) Figure 5. Typical records of change of pH, in single Mercenaria campechiensis myocytes during NH 4 C1 pre-pulse experiments when MASW (A) or 0.3% CO 2 :4 mM HOV-MASW (B) were used in washing. Myocytes were superfused with MASW followed by 15 mM NH 4 C1- MASW. After the plateau alkalinization was acheived, myocytes were washed with various media. The rate of recovery (dpH/dt) was calculated using a regression (Sigma Plot) of the initial, linear portion of the recovery curve after NH 4 CI wash-out. Buffering capacity values (dhT/dpH) were calculated according to Boron (1977). Rates of acid/base equivalent transport (^moles/min) were calculated for each myocyteby multiplying the measured individual f? NB value times the corresponding dpH/dt value (^moles/mm = /} NB -dpH/mm). Each value represents a mean 1 SD. Sample size (n) is indicated. MASW-0.3% CO,: 4 mM HCO, was prepared by gassing MASW with 0.3% CO 2 (balance air), addition of solid NaHCO 3 followed by adjustment of pH. The reservoir was continuously gassed with hydrated 0.3% CO 2 in air and the superfusion line was contained within a gas jacket. Concentrations were calculated using appropriate apparent dis- sociation (Mehrbach cl a/., 1973) and solubility (Riley and Skirrow, 1975) constants. Na + -free MASW was prepared by replacing NaCl with three times crystallized choline chloride (Sigma Chemical Co.). SITS solutions were shielded from light to prevent photodecomposition. lington, 1989) and is slightly lower (0.1-0.2 units) than what has been observed for squid giant axon (Boron and Russell, 1983) and various tissues of the mussel Mytilus edulis (Walsh el al, 1984; Zange el a/.. 1990). The above data on other species were obtained by a variety of tech- niques including NMR, weak acid distribution and micro- electrode methods. Changes in pH e have minimal effect on the pH, of M. campechiensis myocytes over what can be viewed as a physiologically realistic range of pH e s (7.1-8.0). A similar high capacity for defending pH, against changes in pH e has been observed in M. edulis ABRM preparations (Zange el al., 1990) as well as in hemocytes from the squid Sepiateuthis lessoniana (Hemming el al.. 1990). In the present study, we have further seen that clam myocytes display recovery from experimentally imposed acid-base disturbances. Walsh and Milligan (1989) have pointed out that there are three potential avenues of regulation of pH, available to cells (a) intracellular physico-chemical buffering, (b) ion exchange of acids/bases between intra- and extracellular compartments, and (c) metabolic pro- duction or consumption of acids and bases. The present results with M. campechiensis myocytes provide strong 214 W. R. ELLINGTON evidence for operation of the first two of these mecha- nisms. The presence of non-bicarbonate intracellular buffers constitutes the first line of defense against acid/base stress in cells (see reviews by Burton, 1978, and Roos and Boron, 1 98 1 ). In vertebrate-muscles, there appears to be a general correlation between the magnitude of the /3 NB and the potential for anaerobic function (Castellini and Somero, 1981). This general correlation has been suggested for molluscan muscles (Eberlee and Storey, 1 984; Morris and Baldwin. 1984), however, the validity of these conclusions is somewhat in doubt due to the artifacts imposed by ho- mogenate titration methods used for /3 NB determinations (Wiseman and Ellington, 1989; Portner, 1990). In the present study we used the NH 4 Cl-prepulse approach and obtained a value of 40 Slykes, which is in the range of values determined by NMR-prepulse for whelk radula muscle (33 Slykes; Wiseman and Ellington, 1989) and mussel ABRM (26.5 Slykes; Zange et ai, 1990), both of which have impressive capacities for anaerobic metabo- lism. In contrast to these observations, the average /3 NB for squid giant axons was 1 1.2 (Boron and Russell, 1983). Thus, it is clear that M. campechiensis myocytes have a relatively high /j NB , which is consistent with the natural history of this species where exposure to hypoxic stress may be a regular phenomenon. The pH L . in bivalves is alkaline relative to pH, under normal conditions (Booth et al., 1984). Given a slightly alkaline pH e , the pHj of 7.22, and the undoubtedly neg- ative sign of the membrane potential in Al. campechiensis myocytes, it is clear that protons are not at equilibrium. These cells must continuously export protons or bring in base equivalents to maintain pH, . This problem becomes greatly exacerbated when the pH e is reduced (therebye decreasing or even reversing the transmembrane proton gradient) or when acid or base loads are imposed on the cells. The relative constancy of pH, with pH c and recovery from experimentally imposed acidosis in clam myocytes clearly show that such ion exchange processes are oper- ating in these cells. Our results show that A/, campechiensis myocytes ap- pear to regulate pH, via a SITS-sensitive ion exchanger which does not have a requirement for external Na + . Most likely, this transporter is a HCO 3 :C1~ exchanger as has been seen in M. editlis ABRM (Zange et ai, 1 990). Under the routine, normocapnic conditions in the present study the concentration of HCO 3 was estimated to be around 0.7 mA/, which appears to be sufficient to promote re- covery of pHj after acidosis. However, recovery was greatly accelerated when HCO 3 concentration was increased to around 4 mA/. Booth et al. (1984) estimated that [HCO-f ] in M. edulis hemolymph was 1.8 mA/ in normoxia and rose to nearly 3 mA/ during hypoxic stress. It is likely that physiological [HCO 3 ] in A/, campechiensis spans a higher range that 0.7 mA/, implying greater overall transport rates in vivo. The above results are in contrast to the work of Boron and Russell (1983) who found that there was an absolute Na + requirement (Km = 77 mA/) for HCO 3 : Cr exchange in squid axons. However, Hemming et al. (1990) found that acid recovery in squid hemocytes was Na + -independent. Zange et al. (1990) found that addition of 5-hydroxytryptamine (5-HT) elicited activation of a Na + :H + exchanger in A/, edulis ABRM. It was not possible to investigate this possibility in A/, campechiensis myo- cytes, as addition of 5-HT caused rather violent contrac- tions of myocytes, which interfered with imaging exper- iments. The present results show that myocytes from the clam M. campechiensis have a good capacity for regulation of pH,. This capacity is based a relatively high /3 NB and the presence of a SITS-sensitive anion exchanger. Clam myo- cytes also appear to be excellent candidates for long-term primary culture. Thus, future studies will focus on poten- tial phenotypic plasticity of /S NB and ion exchange capacity in cells cultured under conditions which might induce such changes (altered pH c , hyper- or hypo-capnia or hyp- oxia). Acknowledgments I wish to heartily thank Ms. Carolyn Bruce (University of British Columbia) for advice and encouragement in the development of the myocyte isolation protocol. I am also very grateful to Leavins Seafood, Inc. (Apalachicola, Florida) for providing animals. This research was sup- ported by NSF grants DIR-9014510 (Instrument and In- strument Development Program) and IBN-9 104548 (Functional and Physiological Ecology Program). Literature Cited Barrow, K. D., D. D. Jamil-son, and R. S. Norton. 1980. "P nuclear magnetic resonance studies of energy metabolism in tissue from the marine mollusc Tapes watlingi. Eur. J. Biochem. 103: 289-297. Booth, C. E., D. G. McDonald, and P. J. Walsh. 1984. Acid-base bal- ance in the sea mussel Mylilus edulis. I. Effects of hypoxia and air- exposure on the hemolymph acid-base status. Mar. Biol. Lett. 5: 347-358. Boron, \V. F. 1977. Intracellular pH transients in giant barnacle muscle fibers. Am. J. Physiol. 233: C61-C73. Boron, \V. F., and J. M. Russell. 1983. Stoichiometry and ion depen- dencies of the intracellular-pH-regulating mechanism in squid giant axons. ./. Gen. Physiol. 81: 373-399. Boron, \V. F., W. C. McCormick, and A. Roos. 1979. pH regulation in barnacle muscle fibers: dependence on intracellular and extra- cellular pH. Am. J. Physiol. 237: C185-C193. Brezden, B. L., D. R. Gardner, and G. E. Morris. 1986. A potassium- selective channel in isolated Lymnaea stagnulis heart muscle cells. ./. Exp. Biol. 123: 175-189. Bright, G. R., G. W. Fisher, J. Rogowska, and D. L. Taylor. 1987. Fluorescence ratio imaging microscopy: temporal and spatial measurements of cytoplasmic pH. J. Cell Biol. 104: 1019-1033. BIVALVE MYOCYTE INTRACELLULAR pH REGULATION 215 Burton, R. F. 1978. Intracellular buttering. Resp Physiol. 33: 51-58. Burton, R. F. 1983. Ionic regulation and water balance. Pp. 291-352 in The Mollusca. Vol 5. A. S. M. Saleudin and K.. M. Wilbur, eds. Academic Press. New York. Castellini, M. A., and G. N. 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Heisler. 1984b. Anaerobiosis and acid-base status in marine invertebrates: effect of environmental hypoxia on extracellular and intracellular pH in Sipunculus nudiis L.J Comp. Physiol 155: 13-20. Potts, W. T. W. 1958. The inorganic and amino acid composition of some lamellibranch muscles. J. Exp Biol. 35: 749-764. Riley, J. P., and G. Skirrow. 1975. Chemical Oceanography. Academic Press. New York. Rink, T. J., R. Y. Tsien, and T. Pozzan. 1982. Cytoplasmic pH and free Mg 2+ in lymphocytes. J Cell Biol. 95: 189-196. Robertson, J. D. 1965. Studies on the chemical composition of muscle tissue. III. The mantle muscle of cephalopods. J. Exp. Biol 42: 153- 175. Roos, A., and W. F. Boron. 1981. Intracellular pH. Physiol. Rev 61: 296-434. Thomas, J. A., R. N. Buschbaum, A. /.imniak, and E. Raeker. 1979. Intracellular pH measurements in Ehrlich ascites tumor cells utilizing spectroscopic probes generated in situ. Biochemistry 18: 2210-2218. Walsh, P. J., D. G. McDonald, and C. E. Booth. 1984. Acid-base bal- ance in the sea mussel Mytilus eiiulis. II. Effects of hypoxia and air exposure on intracellular acid-base status. Mar. Biol. Lett. 5: 359- 369. Walsh, P. J., and C. L. Milligan. 1989. Coordination of metabolism and intracellular acid-base status: ionic regulation and metabolic consequences. Can. J. Zool. 67: 2994-3004. Wiseman, R. W ., and W. R. Ellington. 1989. Intracellular buffering in molluscan muscle: superfused muscle versus homogenates. Physiol. Zool. 62: 541-558. Zange, J., M. K. Grieshaber, and A. W. H.Jans. 1990. The regulation of intracellular pH estimated by -"P-NMR spectroscopy in the an- terior byssus retractor muscle of Mytilus editlis L. J. Exp. Biol. 150: 95-109. Reference: BID/. Bull 184: 216-222. (April. 1993) Two S-Iamide Peptides, AKSGFVRIamide and VSSFVRIamide, Isolated from an Annelid, Perinereis vancaurica O. MATSUSHIMA 1 , T. TAKAHASHI 1 , F. MORISHITA 1 , M. FUJIMOTO 1 , T. IKEDA 2 , I. KUBOTA 3 , T. NOSE 4 , AND W. M1KI 4 1 Zoological Institute, Faculty of Science, Hiroshima University, Higashi-hiroshima 724, Japan, 2 Physiological Laboratory. Faculty of Integrated Arts and Sciences, Hiroshima University. Hiroshima 730. Japan. 3 Suntory Bio-Pharma Tech Center. Gunma 370-05. Japan. and ^Marine Biotechnology Institute, Shimiiu 424, Japan Abstract. Two peptides, H-Ala-Lys-Ser-Gly-Phe-Val- Arg-Ile-NH : (AKSGFVRIamide), and H-Val-Ser-Ser- Phe-Val-Arg-Ile-NH 2 (VSSFVRIamide) were isolated from a polychaete annelid, Perinereis vancaurica. Both the peptides evoked rhythmic contractions in the esophagus of Perinereis with a threshold as low as 10~'-10~ 9 M. suggesting that the peptides may be in- volved in the regulation of gut motility of the animal. The sequences of these peptides are very similar to those of other S-Iamide family peptides which have been pre- viously isolated from an echiuroid worm and some mol- luscs. In particular, the sequence of VSSFVRIamide is identical to that of an echiuroid S-Iamide peptide. All of the molluscan and echiuroid S-Iamide peptides, as well as the annelid peptides. were found to produce con- tractions in the esophagus of Perinereis. On the other hand, the annelid S-Iamide peptides, as well as the mol- luscan and echiuroid peptides, were found to inhibit or potentiate contractions elicited by electrical stimulation in echiuroid and molluscan muscles. S-Iamide peptides may be a typical neuropeptide family distributed inter- phyletically in the Protostomia. Introduction In annelids, pharmacological studies have been exten- sively done on the actions of classical transmitters such as 5-hydroxytryptamine, epinephrine, norepinephrine and dopamine mainly on somatic muscles, and these sub- Received 6 October 1992, accepted 25 January 1993. stances have been suggested to be present in the central and peripheral nervous systems (for review, Tashiro and Kuriyama, 1978). In addition, bioactive peptides found in vertebrates and other phyla of invertebrates have been suggested to be present in annelids (Carraway et a/., 1 982; Engelhardt et a/.. 1982: Dhainaut-Courtois et a/.. 1985; Diaz-Miranda et a/., 1991, 1992). Many peptides are known in vertebrates, especially in mammals which control the motility of the gut (Holm- gren, 1989). However, few gut motility-controlling pep- tides have been reported for invertebrates. Immunohis- tochemical or immunochemical studies have suggested that some vertebrate neuropeptides, such as enkephalin, /3-endorphin (Alumets et al. 1979), substance P (Dhai- naut-Courtois et al.. 1985; Kaloustian and Edmands, 1986), cholecystokinin/gastrin, 0-MSH (Engelhardt et a/.. 1982; Dhainaut-Courtois et al.. 1985) and neurotensin (Carraway et al., 1982) may be present in annelids. Ka- loustian and Edmands (1986) reported that substance P stimulated the rate of spontaneous contraction of intes- tinal tissues of the earthworm Lumbricus terrestris. It has also been shown that a tetrapeptide (WMDFamide) re- lated to cholecystokinin/gastrin has excitatory effects on the anterior intestine of a polychaete, Chaetopterus var- iopedatus (Anctil et a/.. 1984). Apart from immunocy- tochemical and pharmacological studies, most investi- gations of bioactive peptides in annelids have centered on those involved in reproductive events (Thorndyke, 1989). Thus, reports on authentic bioactive peptides in- volved in the regulation of gut-motility of annelids are very few in number. 216 S-IAMIDE PEPTIDES OF AN ANNELID 217 Recently, Krajniak and Price ( 1990) showed the pres- ence of FMRFamide which was first identified in a mol- lusc as a cardioexcitatory neuropeptide in the polychaete Nereis virens (Price and Greenberg, 1977). Krajniak and Greenberg (1992) showed that immunoreactive FMRFamide was present in various tissues including the gut in Nereis, and that FMRFamide had a relaxing action on both the spontaneously active and electrically stimu- lated esophagus, suggesting the involvement of the tet- rapeptide in the control of gut-motility. Furthermore, FMRFamide and its related peptides have been shown to be present in other annelid species such as Nereis diver- sicolor (Baratte el a/.. 1991) and Hirudo medicinalis (Evans et at.. 1991). In the present study, we isolated and sequenced two bioactive peptides, AKSGFVRIamide and VSSFVRIam- ide, from the polychaete Perinereis vancaiiricii, which in- duced contraction of the isolated esophagus of the animal. These peptides were found to be members of the S-Iamide peptide family ( Ikeda t'/ al.. 1991; Muneokaand Kobaya- shi, 1992). The name S-Iamide peptide was given after the common structure. -SSFVRIamide. Kuroki el al. (1992) first isolated one of the S-Iamide peptides, LSSFVRIamide, from the prosobranch mollusc Fitsinus ferrugineus, and up to the present, S-Iamide peptides have been found not only in molluscs but also in an echiuroid worm (Ikeda et al., 1991). We also examined the effects of several S-Iamide peptides on some invertebrate muscle tissues including the esophagus of Perinereis. Materials and Methods Purification Perinereis vancaurica tetradentata are commercially available as fishing bait. Approximately 380 worms (500 g) were rinsed twice with artificial seawater ( ASW). blotted lightly with tissue paper and boiled for 10 min in 4 vol- umes of 4% acetic acid (21). The animals were homoge- nized in 4% acetic acid by using a Waring blender and a Polytron. The homogenate was centrifuged ( 15,000 X g, 40 min, 4C), and the resulting precipitate was again ho- mogenized and centrifuged. The two supernatants were pooled and concentrated to a volume of about 100 ml by using a rotary evaporator (40C). To the concentrated supernatant, 1/10 volume of 1 N HC1 was added, and the precipitated material was centrifuged off( 1 5.000 X g, 40 min, 4C). Next, the supernatant was forced through two disposable C-18 cartridges in series (Mega Bond-Elut. Varian). The retained material was eluted with 50% methanol. The eluate was concentrated, loaded on a C- 18 reversed phase HPLC column (CAPCELL-PAK, Shi- seido; 10 mm X 250 mm), and eluted with a linear gra- dient of 0-60% acetonitrile (ACN) in 0. 1% trifluoroacetic acid (TFA) for 120 min at a flow rate of 1 ml/min. The chromatography was monitored at 220 nm. Aliquots of 2 mi-fractions were evaporated to dry ness, and the residues were dissolved in ASW and bioassayed on an isolated esophagus of Perinereis as described below. Two con- tractile peaks were detected. The fractions of each active peak were concentrated and subjected to HPLC using an- other C-18 reversed phase column (ODS-80TM, Tosoh; 4.6 mm X 150 mm) with a linear gradient of 10-20% ACN for one activity and 1 5-25% ACN for the other in 0.1% TFA (0.5 ml/min). Active fractions obtained from each HPLC were then loaded onto a cation-exchange col- umn (SP-5PW, Tosoh; 7.5 mm X 75 mm) and eluted in a linear gradient of 0-0.7 Al NaCl in 10 mM phosphate buffer (pH 7. 1 ) for 70 min at a flow rate of 0.5 ml/min. Then, the active substances identified as single peaks on the cation-exchange HPLC were chromatographed on the ODS-80TM column with a linear gradient of 10-16%. ACN and 15-25% ACN, respectively, and finally purified on the same column with an isocratic elution of 17% and 19% ACN. The two purified substances were subjected to amino acid sequence analysis by automated Edman degradation with a gas-phase sequencer (Shimazu PSQ-1 ). The results of the chemical analyses suggested that the substances were members of the S-Iamide peptide family. Therefore, the two peptides having the suggested structures were syn- thesized by a manual method followed by an HF-anisol cleavage and purified by reversed-phase HPLC. Then, the synthetic peptides were compared with native ones in the behavior on HPLC and in the bioactivity on the Perinereis esophagus. Bioassay The contractile activities of the native and synthetic substances were examined on the isolated esophagus of Perinereis. The method for contraction recording was essentially the same as that reported by Krajniak and Greenberg (1992). Both ends of the isolated esophagus were ligated with two cotton threads, one being secured to a stationary rod at the bottom of a trough (2 ml) and the other connected to a force-displacement transducer (NEC San-ei Instruments). The saline in the trough was constantly aerated through a syringe needle connected with an air pump to ensure uniform distribution of applied substances (dissolved in 0.1 ml saline) in the trough. In the present study, we did not apply electrical stimulation but just monitored in- ductivity of spontaneous contractions of the esophagus by test substances. For examination of the bioactivity of the material retained by the C-18 cartridges, two more assay systems, the inner circular body-wall muscle of an echiuroid worm Urechis unicinctus (Ikeda et al., 1991), and the radula retractor muscle of a prosobranch mollusc 218 O. MATSUSHIMA ET AL B "i JL 4 RM 1 min t RM 10 " y) I i I 1 1 g s .0 5 i! RM 1 min Figure 1. Effects of the retained materials (RM) on the three muscle systems. (A) the esophagus of Pe rinereix. (B) twitch contractions of the inner circular body-wall muscle of L'rechis. The twitch contraction was produced by an electrical pulse (20 V, 3 ms). (C) twitch contractions of the radula retractor of Rapana. The twitch contractions were produced by a train of electrical pulses (15V. 1 ms, 0.2 Hz, 5 pulses). In each case. 1/1000 of total RM. which corresponded with extracts from 0.4 worm, was applied to the assay system. The upward arrows indicate application of RM to the tissue. The downward arrows indicate washing-out of the RM. Rapana thomasiana (Muneoka et al., 1991), were em- ployed. In these cases, electrical pulses of stimulation were applied to the preparations. Pharmacology The methods used in the pharmacological experiments were basically the same as those in the bioassay experi- o CM 4.0 a 0) ra ^ O CO .0 2.0 60 CJ" A B '^lA^ I 1 1 I 1 I i i i 40 80 Time (min) 120 160 o Figure 2. HPLC profile of the retained materials (RM) on a reversed phase column. The RM loaded onto the column was eluted with a linear gradient of ACN concentration (0-60%/120 min) in 0.1% TFA (pH 2.2) at a flow rate of 1 ml/min and collected in 60 fractions of 2 ml each. Aliquots (10 n\ = 1/200) of each fraction were evaporated to dryness, dissolved in ASW and applied to the Perinereis esophagus. The contractile peaks were indicated by the horizontal bars (A and B). nO.7 20 40 60 20 40 60 Time (mm) Figure 3. HPLC profiles of active fractions (A and B in Fig. 2) on a cation-exchange column. Elution was performed in a 70-min linear gra- dient of 0-0.7 A/ NaCl in 10 m.U phosphate buffer (pH 7.1) at a flow rate of 0.5 ml/min (collected in 1-ml fractions). The activities (A and B) were detected in respective peaks indicated by arrows. ments. In these experiments, we used three kinds of mus- cles, the esophagus of Perinereis, the anterior byssus re- tractor muscle (ABRM) of the bivalve mollusc Mytilus edulis and the radula retractor muscle of the prosobranch mollusc Fusinus ferrugineus. Salines The saline used for Perinereis and Urechis muscles was ASW of the following composition: 445 mAl NaCl, 55 mM MgCl : , 10 mA/ CaCl : , 10 mA/ KG, 10 mAl Tris- HC1; pH 7.6. For the Rapana and Fusinus muscles, low Mg-ASW (20 mA/ MgCl 2 ) was used. The low-Mg ASW was prepared by replacing a part of MgCl : in the normal ASW with osmotically equivalent NaCl. Results The retained material (RM) eluted with 50% methanol was examined for its biological action on three muscle systems, the esophagus of Perinereis, the inner circular body-wall muscle of Urechis and the radula retractor muscle of Rapana (Fig. 1 ). The RM elevated a basal tone with rhythmic small contractions in the esophagus of Per- inereis and exerted inhibitory effects on twitch contrac- tions evoked by electrical stimulations in the latter two muscles. After the test solution was replaced with normal ASW, the contractions of Rapana radula retractor became greater than the control contractions and then returned to the control level. We decided to purify at first the sub- stance which elicited contractions of the Perinereis esophagus. At the first step of HPLC, two contractile peaks (peak A and B) were found. They were eluted at approximately 15% and 20% ACN, respectively (Fig. 2). At the second step, active substances of peak A and B were eluted at 13% ACN and 19% ACN, respectively (data not shown). Then, the fractions containing active substance A and B were respectively subjected to the cation-exchange HPLC S-IAMIDE PEPTIDES OF AN ANNELID 219 o CM "0.5 ra 20 J 40 Time 0.1 20 4 (min) 19 0.5 9 2 min Figure -4. Final purification by HPLC using a reversed-phase column (A. B) and the action of each purified substance on the Perincrcis esoph- agus (C, D). Isocratic elution with 17% ACN (A) and 19% ACN (B) in 0.1% TFA at a flow rate of 0.3 ml/min. Aliquots ( 1/100) of the purified substances were dissolved in ASW and applied to the isolated esophagus at the time indicated by arrows (C. D). (Fig. 3). The active substances appeared to be eluted as single peaks around 0.35 M NaCl (A) and 0.25 M NaCl (B). The final purification was performed on the C-18 column with an isocratic elution of 17% ACN (A) and 19% ACN (B) (Fig. 4). The respective single peaks with OD at 220 nm of 0.303 (A) and 0.085 (B) were eluted at 23 min and 16 min after injection. The purified substances (1/100) elicited rhythmic contractions of the esophagus (Fig. 4C, D). Amino acid sequence analysis of the purified substances (A and B) revealed the structure to be the octapeptide Ala (216)-Lys (43.3)-Ser (8.7)-Gly (7.0)-Phe (5.9)- Val (1.7)- Arg (1.3)-Ile ( + ) and the heptapeptide Val (181.5)-Ser (66.0)-Ser (45.8)-Phe (118. 6)- Val (150.2)-Arg (28.2)-Ile (0.9), respectively (the figures are expressed in pmoles). The peptides of the respective sequences with C-terminus amidated were synthesized, and HPLC profiles of the syn- thetic peptides were compared with those of native ones. The synthetic and native peptides showed identical reten- tion times on the C-18 reversed-phase column and the cation-exchange column (data not shown). Furthermore, the mixture of the synthetic and native peptides was eluted as a single peak on each column (Fig. 5). The respective synthetic peptides evoked contraction of the Perinereis esophagus in the similar manner to the corresponding native peptides (Figs. 6, 7). The threshold concentrations for the synthetic peptides to evoke contraction were found to be between 10~' M and 10~ 9 M for both peptides. Thus, the structures of substance A and B were con- cluded to be AKSGFVRIamide and VSSFVRIamide, re- spectively. Both AKSGFVRIamide and VSSFVRIamide were members of S-Iamide peptides which had been pu- rified from one echiuroid and four mollusks (Table 1 ). 10' 9 M S*N iLJ * .05 20 40 20 40 Time (min) Figure 5. HPLC profiles of mixtures of native and synthetic peptides. AKSGFVRIamide (A) and VSSFVRIamide (B) on a reversed phase col- umn with an isocratic elution of 17% ACN and 19% ACN at a flow rate of 0.3 ml/min. AKSGFVRIamide (C) and VSSFVRIamide (D) on a cation-exchange column with an isocratic elution of 0.27 At NaCl and 0. 14 M NaCl in 10 mM phosphate buffer (pH 7. Data flow rate of 0.5 ml/mm. 2 min Figure 6. Comparison of the bioactivities of the native (N) and syn- thetic (S) peptides (AKSGFVRIamide) on the isolated esophagus of Per- inereis. The peptide solutions were applied at the time indicated by arrows. The concentration of the native peptide was estimated by comparing its peak height on HPLC with that of the synthetic peptide. 220 O. MATSUSHIMA ET AL A Ll B f ~^-i^-J^ , > * 10- 8 M 10' 7 F N 2 min Figure 7. Comparison of the bioactivities of the native (N) and syn- thetic (S) peptides (VSSFVRIamide) on the isolated esophagus of Pcri- nereis. The peptide solutions were applied at the time indicated by arrows. The concentration of the native peptide was estimated by comparing its peak height on HPLC with that of the synthetic peptide. These S-Iamide peptides and some fragment peptides were examined on the Pcrinereis esophagus (Fig. 8). All of the S-Iamide family peptides and the fragment peptides more or less elicited rhythmic contractions of the esophagus at 10~ 7 M. The biological activities of the two S-Iamide peptides isolated from Pcrinereis were examined on three muscle systems, the inner circular body-wall muscle of Urechis (Fig. 9). the ABRM of Mytilnx (Fig. 10) and the radula retractor muscle of Fusinus (Fig. 1 1 ). In the Urechis mus- Table 1 S-Iamide peptides Phyla Species Structures Annelida Pcrinereis vancaurica AKSGFVRIamide VSSFVRIamide Echiura I'rec/iis WHcvmvin ASSFVRIamide PSSFVRIamide VSSFVRIamide Mollusca Fiifinin Icirugineus LSSFVRIamide Helix pomatia TSSFVRIamide Achatina fulica SPSSFVRIamide APSNFIRIamide .inmlimta cygnea SGFVRIamide t t 4 10 ? M AKSGFVRIa 10 7 M APSNFIRIa 10' 7 M SPSSFVRIa 10 7 M ASSFVRIa I I 10 'M PSSFVRIa 10 7 M VSSFVRIa 10 7 M LSSFVRIa 10 'M TSSFVRIa 10 7 M SGFVRIa 10' 7 M SSFVRIa 10 7 M SFVRIa I 10 M FVRIa Figure 8. The actions of 1CT 7 M of various S-Iamide peptides and some fragment peptides on the isolated Pcrinereis esophagus. Each pep- tide was applied at the time indicated by arrows. cle. the twitch contraction evoked by electrical stimulation was inhibited by the S-Iamide peptides; AKSGFVRIamide was less potent than VSSFVRIamide (Fig. 9). The phasic contraction of the Mytilus ABRM evoked by repetitive electrical stimulation was inhibited by the peptides (Fig. 10). The effects of these two peptides on Fusinus muscle were somewhat complicated. AKSGFVRIamide poten- tiated twitch contractions at the concentration of 10~ 7 M. but inhibited at 10~ 5 M. VSSFVRIamide. on the other hand, did not show any augmentation of the twitch con- traction, but inhibited at concentrations higher than 10~ 6 M(Fig. 11). 10' 7 M AKSGFVRIa 1 min 4 10' 7 M VSSFVRIa Figure 9. Effects of AKSGFVRIamide and VSSFVRIamide on twitch contraction of the inner circular muscle of the body wall of Vrechis. The upward arrows indicate application of the peptides. The downward arrows indicate washing-out of the peptides. The twitch contraction was evoked by an electrical pulse (20 V, 3 msec). S-IAMIDE PEPTIDES OF AN ANNELID 221 t 10" 7 M AKSGFVRIa 10' 6 M * I J 5g 2 mm B t JV - * J 10' 7 M VSSFVRIa t I 10- 5 M * J 5g Figure 10. Effects of AKSGFVRIamide and VSSFVRIamide on phasic contraction of the ABRM of .\fytilus. The upward arrows indicate application of the peptides. The downward arrows indicate washing-out of the peptides. The phasic contraction was evoked by repetitive electrical pulses (15V. 3 msec, 10 Hz, 50 pulses). Discussion The principal aim of this study was to find out authentic bioactive peptides in annelids. We isolated two S-Iamide family peptides, AKSGFVRIamide and VSSFVRIamide, from the polychaete annelid, Perinercis. Both the peptides showed a contractile effect on the esophagus of the ani- mal. AKSGFVRIamide is the novel peptide, and VSSFVRIamide has previously been found in the ventral nerve cord of the echiuroid worm, Urechis. S-Iamide pep- tides have been found so far in one species of Echiura and four species of Mollusca (Ikeda et a/.. 1991; Kuroki el al.. 1992; Muneoka and Kobayashi, 1992; in prep, for Anodonta S-Iamide peptide), as listed in Table I. Thus, S-Iamide peptides have been proven to be distributed among at least three invertebrate phyla and may range throughout the Protostomia. The C-termini of the two S-Iamide peptides identified in the current study were concluded to be amidated. though the purified substances were not subjected to fast atom bombardment mass spectrometry. Since the C-ter- mini of all the S-Iamide peptides so far isolated from the echiuroid and molluscs have been known to be amidated. we synthesized AKSGFVRIamide and VSSFVRIamide and compared their behavior on HPLC and contractile activity with those of the purified native peptides. As a result, the identical properties of the native and synthe- sized peptides were confirmed. The synthetic tetra- and pentapeptides, FVRIamide and SFVRIamide, showed only a slight activity for induction of the spontaneous contraction in the esophagus of Per- inereis. However, the synthetic hexapeptide, SSFVRIam- ide (a common structure for most of the S-Iamide pep- tides), was active, suggesting that at least six amino acid residues would be important for the expression of the ac- tivity of S-Iamide peptides. However, SGFVRIamide which has been isolated from Anodonta showed weak contractile activity in the esophagus. The substitution of the amino acid residue, Ser, with Gly seems to be dele- terious for contractile activity, and the N-terminal elon- gation of SGFVRIamide by Ala-Lys might cancel the del- eteriousness. The effect of AKSGFVRIamide on twitch contractions of the radula retractor of Fusinus was somewhat compli- cated. That is, the peptide potentiated the contractions at 10~ 7 M, but inhibited at 10 5 M. The well-known mol- luscan neuropeptide FMRFamide has been known to po- tentiate the contractions of the same muscles (Kuroki et al.. 1992). Since the C-terminal tetrapeptide sequence of the S-Iamide peptide. -FVRIamide, is closely related to that of FMRFamide. the potentiating effect of 10~ 7 M AKSGFVRIamide may be attributable to the FMRFam- ide-like action of the S-Iamide peptide. The inhibition of the twitch contractions by 10~ 5 M of AKSGFVRIamide is probably the original action of the S-Iamide peptide. In this connection, Kuroki et al. ( 1992) reported that an- other S-Iamide peptide, LSSFVRIamide, isolated from the ganglia of Fusinus showed the same dose-dependent actions on the contractions as did AKSGFVRIamide. The physiological role of AKSGFVRIamide and VSSFVRIamide in Peri nereis is not elucidated at present. The threshold concentrations of these two S-Iamide pep- tides to induce contraction of the esophagus were between AJI_ 4 10' 7 M AKSGFVRIa IL^JIUL, 10 5 M JilL 19 1 min I 10' 7 M VSSFVRIa 0.5 g 10' 6 M 10' 5 M Figure 11. Effects of AKSGFVRIamide and VSSFVRIamide on twitch contractions ot the radula retractor muscle of Fusinus. The upward arrows indicate application of the peptides. The downward arrows indicate washing-out of the peptides. The twitch contractions were evoked by a train of electrical pulses (15 V, 1 msec, 0.2 Hz, 5 pulses). 222 O. MATSUSHIMA ET AL. 1(T 10 A/and 10 9 M. It seems to be probable that these S-Iamide peptides are neuropeptides which regulate the gut-motility in the annelid. It has been demonstrated that FMRFamide is present in annelids such as Nereis virens (Krajniak and Price, 1990), Nereis diversicolor (Baratte el al. 1991) and Himdo mcdicinalis (Evans el al.. 1991) and that the tetrapeptide relaxed spontaneous and elec- trically-induced contractions of the esophagus (Krajniak and Greenberg, 1992). Thus, the action of FMRFamide on the esophagus of polychaete annelid is opposite to those of the S-Iamide peptides. This was also the case for most of the molluscan muscles examined (Muneoka and Ko- bayashi, 1992). Therefore, FMRFamide and the S-Iamide peptides may regulate the esophagus-motility in an an- tagonistic manner, and this regulatory relation may be also applied to the molluscan muscles. The classical neurotransmitters such as norepinephrine, epinephrine, acetylcholine, 5-hydroxytryptamine and 7- aminobutyric acid also seem to regulate gut-motility in the polychaete annelid, Chaetopterus variopedatus (Anctil et al., 1984), and the presence of catecholamines (dopa- mine, norepinephrine and epinephrine) has been reported in the nervous and intestinal tissues of the same species (Anctil et al., 1990). Thus, peptides such as FMRFamide and the S-Iamide peptides, and classical transmitters seem to regulate the gut-motility harmoniously in annelids. Further study is necessary to reveal the relationship be- tween the physiological role of classical transmitters and peptides. Acknowledgments The authors wish to express their thanks to Professor Yojiro Muneoka (Hiroshima University) for his kind ad- vice regarding the present study. Literature Cited Alumets, J., R. Hakanson, F. Sundler, and T. Thorell. 1979. Neuronal localisation of immunoreactive enkephalin and 0-endorphin in the earthworm. Nature 279: 805-806. Anctil, M., M. Laberge, and N. Martin. 1984. Neuromuscular phar- macology of the anterior intestine of Chaetopterus variopedatus, a filter-feeding polychaete. Coinp. Biochem. Physiol. 79: 343-35 1 . Anctil, M., J.-P. De Waele, M.-J. Miron, and A. K. Pani. 1990. Monoamines in the nervous system of the tube-worm Chae- topterus variopedatus (Polychaeta): Biochemical detection and se- rotonin immunoreactivity. Cell Tissue Res 259: 81-92. Baratte, B., H. Gras-Masse, G. Ricart, P. Bulet, and N. Dhainaut-Cour- tois. 1991. Isolation and characterization of authentic Phe-Met- Arg-Phe-NH 2 and the novel Phe-Thr-Arg-Phe-NH, peptide from Nereis diversicolor. Ew. J Biochem. 198: 627-633. Carraway, R., S. E. Ruane, and H. R. Kim. 1982. Distribution and immunochemical character of neurotensin-like material in represen- tative vertebrates and invertebrates: Apparent conservation of the COOH-terminal region during evolution. Peptides 3: 1 15-123. Dhainaut-Courtois, N., M. P. Dubois, G. Tramu, and M. Masson. 1 985. Occurrence and coexistence in Nereis diversicolor O. F. Muller (Annelida Polychaeta) of substances immunologically related to ver- tebrate neuropeptides. Cell Tissue Res 242: 97-108. Diaz-Miranda, L., G. Escalona de Motta, and J. E. Garcia-Arraras. 1991. Localization of neuropeptides in the nervous system of the marine annelid Sabellastarte magnifica. Cell Tissue Res 266: 209- 217. Diaz-Miranda, L., G. Escalona de Motta, and J. E. Garcia-Arraras. 1992. Monoamines and neuropeptides as transmitters in the sed- entary polychaete Sabellastarte magnifica: Actions on the longitudinal muscle of the body wall. J Exp. Zooi 263: 54-67. Engelhardt, R. P., N. Dhainaut-Coutois, and G. Tramu. 1982. Im munohistochemical demonstration of a CCK-like peptide in the ner- vous system of a marine annelid worm. Nereis diversicolor O. F. Muller. Cell Tissue Res 227: 401-41 1. Evans, B. D.. J. Pohl, N. A. Kartsonis, and R. L. Calabrese. 1991. Identification of RFamide neuropeptides in the medicinal leech. Peptides 12: 897-908. Holmgren, S. 1989. Gut motility. Pp. 231-255 in The Comparative Physiology oj Regulatory Peptides. S. Holmgren, ed.. Chapman and Hall, New York. Ikeda, T., I. kubota, V. kitajima, and V. Muneoka. 1991. Structures and actions of neuropeptides isolated from an echiuroid worm, L're- chis vnicinctus. Pp. 29-41 in Comparative Aspects of Neuropeptide Function. E. Florey and G. B. Stefano, eds.. Manchester University Press. Manchester. Ikeda, T., V. kuroki, I. kubota, H. Minakata, k. Nomoto, \V. Miki, T. kiss, L. Hiripi, and V. Muneoka. 1991. SSFVRIamide peptides A new family of neuropeptides distributed interphyletically. Pp. 65- 70 in Peptide Chemistry. A. Suzuki, ed.. Protein Research Foundation. Osaka, Japan. kaloustian, k. V., and J. A. Edmands. 1986. Immunochemical evidence for substance P-like peptide in tissues of the earthworm Lumhnciis lerreslris: Action on intestinal contraction. Comp. Biochem. Physio/ 83C: 329-333. krajniak, k.. and D. A. Price. 1990. Authentic FMRFamide is present in the polychaete Nereis virens. Peptides 11: 75-77. krajniak, k., and M. J. Greenberg. 1992. The localization of FMRFamide in the nervous and somatic tissues of Nereis virens and its effects upon the isolated esophagus. Comp. Biochem Physiol. 101C: 93-100. kuroki, Y., T. kanda, I. kubota, T. Ikeda, Y. Fujisana, H. Minakata, and Y. Muneoka. 1992. FMRFamide-related peptides isolated from the prosobranch mollusc Fusinus femtgineus. J. Biol Hung. 43: 491- 494. Muneoka, Y., and M. kobayashi. 1992. Comparative aspects of struc- ture and action of molluscan neuropeptides. Experienlia 48: 448- 456. Muneoka, Y., Y. kuroki, H. Minakata, T. Ikeda, Y. Fujisawa, k. Nomoto, and I. kubota. 1991 . Structure and pharmacological characterization of a molluscan neuropeptide related to the crustacean RPCH. Pp. 274-279 in Molluscan Neurobiohgy. K. S. Kits, H. H. Boer, and J. Joose. eds. North Holland, Amsterdam. Price, D. A., and M. J. Greenberg. 1977. Structure of a molluscan cardioexcitatory neuropeptide. Science 197: 670-671. Tashiro, N., and H. kuriyama. 1978. Neurosecretion and pharmacology of the nervous system. Pp. 207-242 in Physiology of Annelids. P. J. Mill, ed. Academic Press, London. Thorndyke, M. C. 1989. Peptides in invertebrates. Pp. 203-228 in The Comparative Physiology of Regulatory Peptides. S. Holmgren, ed. Chapman and Hall, New York. Reference: Bio/. Bull. 184: 223-229. (April, 1993) Photosynthesis and Retention of Zooxanthellae and Zoochlorellae Within the Aeolid Nudibranch Aeolidia papillosa F. K. McFARLAND AND G. MULLER-PARKER 1 Shannon Point Marine Center, Western Washington University. 1900 Shannon Point Road. Anaeortes, Washington 98221 Abstract. Both zooxanthellae and zoochlorellae are found in the cerata of Aeolidia papillosa after it has in- gested symbiotic Anthoplewa elegantissima containing these algae. High rates of photosynthesis were found in algae present in the cerata and in algae isolated from nu- dibranch feces. For algal cells present in the cerata of nu- dibranchs collected in June 1991, carbon fixation by zooxanthellae ( 1 . 18 0.36 pg C/cell/h) was significantly greater than carbon fixation by zoochlorellae (0.55 0.32 pg C/cell/h). Algal densities within the cerata of laboratory fed nudibranchs were significantly greater for zoochlo- rellae (175 82 cells/Mg protein, light treatment; 131 106 cells/^g protein, dark treatment) than for zoox- anthellae (38 18 cells/^g protein, light; 53 30 cells/ ^g protein, dark). Ceratal densities of zooxanthellae (16 8 cells/Mg protein) in the field during January 1992 were low in comparison to ceratal densities in the labo- ratory several of the nudibranchs in the field lacked any symbiotic algae, and zoochlorellae were always absent. Nudibranch algal densities were not stable and dropped rapidly if the nudibranchs were starved. Both zoochlorella and zooxanthella densities dropped to cells///g protein within 1 1 days of starvation. While these results show that the relationship between A. papillosa and the two algae is not a stable symbiosis, the photosynthetic activity of the algae in the cerata suggests that the nudibranch and/or the algae may benefit from the association while it lasts. Introduction Several aeolid nudibranchs, as well as other nudi- branchs with cerata, contain zooxanthellae of the genus Received 6 July 1992; accepted 25 January 1993. 1 Author to whom reprint requests should be addressed. Symbiodinium (Rudman, 1981a. b, 1982; Kempf, 1984, 1991). Each ceras contains a diverticulum of the digestive gland within which the algal symbionts are both extra- and intracellularly located (Rudman, 1982; Kempf, 1984. 1991). Many of these nudibranchs obtain their algae through ingestion of marine cnidarians which are sym- biotic with zooxanthellae. Zooxanthellae in the cnidarian host fix carbon through photosynthesis, and then trans- locate much of this carbon to the animal's tissue (e.g.. Trench, 1979). The carbon available for translocation may represent as much as 95% of the amount fixed (Muscatine et ai. 1 984), and is used by the host for respiration, growth, and reproduction (Kevin and Hudson, 1979; Davies, 1984; Rinkevich, 1989). The ability of zooxanthellae to fix and translocate carbon in nudibranchs, as well as the benefits of such an association to the nudibranchs, have been described for several relationships. Grassland and Kempf (1985) reported that zooxanthellae in the tropical nudibranch Alelibe pilosa fixed large amounts of carbon (5.85 mg C/mg chlorophyll a/h), and that fixed carbon was translocated to the nudibranch for growth and re- production. Kempf (1990) reported that the aeolid nudibranch Berghia verrucicornis produced 1.7 times more eggs when in a symbiotic relationship with zooxanthellae than when algae-free. At high densities, zooxanthellae in the temperate nudibranch Pteraeolidia ianthina can supply carbon well in excess of the nudi- branch's respiratory demand during the spring and summer (H0egh-Guldberg and Hinde, 1986; H0egh- Guldberg et al. 1986). With the exception of the nu- dibranch Pteraeolidia ianthina. only tropical species have been studied, and all the studies have focused on species with zooxanthellae symbionts. 223 224 F. K. McFARLAND AND G. MULLER-PARKER The temperate nudibranch Aeolidia papillosa is found within the intertidal zone of the northeastern Pacific (Kozloff, 1983) where one of its preferred prey species is the symbiotic anemone Anthopleura elegantissima (Wa- ters, 1973; Edmunds el ai. 1974; McDonald and Nybak- ken. 1978). A. elegant issima forms symbiotic relationships with both zooxanthellae and the unicellular green algae called zoochlorellae (Muscatine, 1971). Fixation and translocation of carbon by zooxanthellae in A. elegant is- sima is substantial (Trench 1 97 1 a, b), and the contribution of these anemones to intertidal gross primary production is equal to that of temperate intertidal seaweed populations on an areal basis (Fitt ct ai, 1982). Zoochlorellae found in A. elegantissima and A. xanthogrammica are also pho- tosynthetically active (Muscatine, 1971; O'Brien, 1980). While both zooxanthellae and zoochlorellae fix carbon in their host species, zooxanthellae translocate much more of their fixed carbon to their host than do zoochlorellae. Zooxanthellae translocate on the order of 50% of the car- bon fixed while zoochlorellae translocate less than 5% (Muscatine, 1971; O'Brien, 1980). High densities of zooxanthellae and zoochlorellae are found in the cerata of A. papillosa after it has been fed symbiotic anemones containing these algae (Kellett and Wiederspohn, pers. comm.). The following study consid- ers the nature of the symbiotic relationship formed be- tween A. papillosa and both zooxanthellae and zoochlo- rellae. particularly the photosynthetic activity of these al- gae and the stability of their populations within the nudibranchs' cerata. Materials and Methods Collection and maintenance oj nudibranchs and anemones Specimens of A. papillosa were collected from the San Juan Islands, WA in June 199 1 and from the Port Orchard side of the Sinclair Inlet, WA in January 1992. All ane- mones used to feed the nudibranchs were collected from Skyline beach in Burrows Bay at Anacortes, WA. No nu- dibranchs were found on this beach. Individual nudi- branchs were maintained in separate plastic mesh con- tainers submerged in flow-through seawater tables at Shannon Point Marine Center (Anacortes, WA). Seawater tables were cleaned twice weekly. The temperature of the water during June 1991 ranged from 10.7C to 13.0C with a mean of 1 1.6C. During January 1992 the tem- perature ranged from 7.5C to 9.2C with a mean of 8.4C, and during February 1992 the temperature ranged from 8.2C to 10.9C with a mean of 9. 1 C. The average salinity during the study was 29 %o. Specimens of A. papillosa collected in June 1991 were used to determine the productivity of symbiotic algae. Continuous light provided to nudibranchs by a bank of two fluorescent lamps averaged 28 yumol photons/nr/s at the water's surface (LiCor cosine quantum sensor, 400- 700 nm PAR). One group of nudibranchs was fed brown A. elegantissima containing zooxanthellae, another group was fed green A. elegantissima containing zoochlorellae, and the control group was fed white (algae-free) A. ele- gantissima. Personal experience has shown that brown anemones always contain at least 98% zooxanthellae (on a cell basis) and green anemones contain at least 98% zoochlorellae. This was confirmed during the experiment by periodic microscopic examinations of tentacle squashes from brown and green anemones. Specimens of A. papillosa collected in January were separated into two groups of twelve to examine retention of zooxanthellae and zoochlorellae in the cerata. The ini- tial algal complement of field nudibranchs was determined by sampling two cerata from each nudibranch within 24 h of collection. One group was maintained in continuous darkness, and the other group was maintained under 12 h light/ 12 h dark. During the light cycle, irradiance at the water's surface averaged 33 /imol photons/m : /s. Each group in the light and the dark treatments was further separated into three treatments of 4 nudibranchs each. One group was fed brown anemones for 28 days, then had its diet switched to green anemones for 1 3 days, and was then starved. Another group was fed green anemones for 28 days and was then switched to a diet of brown anemones. The third group was fed brown anemones for 28 days and was then starved. Each fed nudibranch was given 5 anemones per week, provided individually on separate days. Fed nudibranchs had no more than two consecutive days without feeding. Productivity of symbiotic algae Algae within the cerata. To determine whether zooxan- thellae and zoochlorellae remain photosynthetically active within the cerata of the nudibranchs, 3 cerata (one an- terior, one middle, and one posterior) were removed from each nudibranch and incubated whole with I4 C in 20 ml glass scintillation vials. 2.0 ml of filtered seawater (FSW) and 0. 1 fid I4 C bicarbonate were added to each vial. The cerata were incubated at room temperature (20-24C) at 249 jumol photons/nr/s for 50-120 min. Control vials for dark carbon fixation were also maintained for each I4 C experiment. Replicate vials of each treatment were wrapped with black electrical tape and incubated under the same conditions as light vials. The dark vials were used to correct for dark carbon fixation. To determine total activity (TA), 100 fil was subsampled from each vial and placed in a 7 ml plastic scintillation vial with 5 ml of Ecolume (ICN) scintillation fluid. Incubations were terminated by removing the cerata and washing them with several rinses of FSW. The cerata were then homogenized SYMBIOTIC ALGAE IN A. PAPILLOSA 225 in 1.5 ml of FSW using a 5-ml Wheaton tissue grinder. Two 500 jul subsamples were taken from each homogenate solution and placed in separate plastic scintillation vials. All unfixed I4 CO : was evolved from the homogenate sub- samples by adding 300 n\ of 6N HC1 and then placing the vials under a heat lamp in a fume hood for 1 h. The subsamples were neutralized with 300 n\ 6N NaOH prior to the addition of 5 ml scintillation fluid. The homogenate subsamples were counted along with the TA subsamples in a Packard TR 1900 scintillation counter using the au- tomatic DPM mode. The remaining homogenate suspen- sion was used for algal cell counts and then frozen for future protein analysis. Algae isolated from nudibranchfeces. Nudibranchs fed symbiotic A. elegantissima produced green or brown fecal pellets consisting mainly of intact symbiotic algae. Fresh fecal pellets were collected from nudibranchs and were suspended in FSW. Fecal algae were washed three times in FSW by centrifugation and resuspension. The final suspension was sequentially filtered through 73 jum and 20 ,um Nitex screening to remove debris. After initial cell counts algal densities were adjusted to 4-6 X 10 5 cells/ ml. The productivity of fecal algae was measured using a protocol similar to that described for the cerata with the following exceptions: 2.0 ml of either the green or the brown fecal algae suspension was placed in each 20 ml glass vial and the algal cells were allowed to incubate for 30 min at room temperature (20-24C). Incubations were at an average irradiance of 249 ^mol photons/irr/s. Algae freshly isolated from anemones. The productivity of algae isolated directly from A. elegantissima was also determined. The oral disk and tentacles of individual anemones were excised and homogenized using a tissue grinder. Algal cell suspensions were washed and filtered as described above. Final algal densities ranged from 2.5-6 X 1 5 cells/ml. Incubations were performed as above except that cells were allowed to incubate for up to 1 h at an average irradiance of 102 ^mol photons/ nr/s. Algal densities and replacement within the cerata Algal population density within the cerata was mea- sured twice each week by removing 2 cerata (one posterior and one anterior) from each experimental nudibranch during January and February 1992. The cerata were ho- mogenized in 1.5 ml of cold FSW using a 2-ml Wheaton tissue grinder. Algal cell counts of the homogenate solu- tions were determined using a hemocytometer, and the remaining homogenate solutions were frozen for future protein analysis. Protein analysis was performed using the Lowry method (Lowry el al.. 1951) and bovine serum albumin (BSA) standards with the modification that the homogenates and standards were pre-treated at 30C for 30 min in 0. 1 N NaOH to solubilize the proteins. Cell counts and protein content were used to determine cell densities within the cerata of nudibranchs fed green and brown A. elegantissima. Statistical analyses Comparison of photosynthetic rates. Photosynthesis data for algae in A. papillosa cerata, algae freshly isolated from A. papillosa feces, and freshly isolated from A. ele- gantissima were analyzed to determine if there was a sig- nificant difference in the rates of carbon fixation for zoo- xanthellae and zoochlorellae. Zooxanthellae rates of carbon fixation were compared to zoochlorellae carbon fixation rates using two-sample t-tests. Algae in cerata. algae from feces, and algae from anemones were all com- pared separately. Comparisons were also made of the photosynthetic rates of zooxanthellae and zoochlorellae between the different treatments. Comparison of algal densities. Algal densities in A. papillosa cerata after 28 days of feeding the nudibranch either brown or green anemones were analyzed using two- sample t-tests to determine if there was a significant dif- ference in the densities of zooxanthellae and zoochlorellae found in the nudibranchs both under light and dark con- ditions. When zooxanthellar densities were compared to zoochlorellar densities, the data were logarithmically transformed to correct for differences in variance between the algal types. The effect of light versus dark on zooxan- thellar and zoochlorellar densities was also analyzed using two-sample t-tests. Comparison of treatment effect on algal replacement. Repeated-measures analysis of variance (Potvin and Lechowicz, 1990) was used to analyze the effect of light versus dark on the replacement (after switching diets) and expulsion (during starvation) of algae in the cerata. Zoo- xanthellae data for the replacement of zoochlorellae with zooxanthellae and the expulsion of zooxanthellae were logarithmically transformed to correct for differences in variance between the light and dark treatments. Results Productivity of symbiotic algae Both zooxanthellae and zoochlorellae remain photo- synthetically active within the nudibranch cerata (Fig. 1 ). where the mean rate of carbon fixation by zooxanthellae is significantly greater (P = 0.0216) than that of zoochlo- rellae. Cerata used for determining the photosynthetic rate of zooxanthellae contained 99.9% zooxanthellae on a cell basis. Cerata used for determining photosynthetic rate of zoochlorellae contained 99.5% zoochlorellae. Figure 1 also shows that algal symbionts isolated from nudibranch feces also had high photosynthetic rates. There is no significant difference (P = 0.078 1 ) between the pho- 226 1 k. VlcFARLAND AND G. MULLER-PARKER 3.5-, 3.0- = 2.5-1 . 300- Light Treatment ## '(n c Q) 250- Q 200 * \. ) 150- / ( ) 100- # rLl/ /I I 50- 0- f -- - I/I 'i- "Vi rS 1 1 S 1 R-^l A: --A ,A l /O . Al , 10 15 20 25 30 35 40 45 50 55 Day Figure 3. Mean algal densities in the cerata of nudibranchs initially fed anemones containing zooxanthellae, then switched to anemones containing zoochlorellae on day 28 of the experiment, and then starved after day 41 of the experiment. Closed symbols represent dark treatments and open symbols represent light treatments. A = zooxanthellae; O = zoochlorellae. Vertical lines represent 95% confidence intervals. The size of each treatment was 4 nudibranchs. # = day diet switched; ## = day began starving. 228 F. K . McFARLAND 150-1 # # 1 125 ' "o - 100 1 75 ~ ^ 50- 1 / \ CO 1 25- n- f V 10 15 20 25 Day 30 35 40 Figure 4. Mean zooxanthellac densities in the cerata of nudibranchs initially fed anemones containing zooxanthellae. and then starved after day 28 of the experiment. Closed symbols represent dark treatments and open symbols represent light treatments. Vertical lines represent 95% confidence intervals. The size of each treatment was 4 nudibranchs. ## = day began starving. if the nudibranch is starved. The rapid expulsion of algae under both light and dark conditions suggests that A. pap- ///osa has little control over the retention or expulsion of algae from its cerata, even when starved. The large numbers of healthy algal cells present in fecal pellets and the photosynthetic rates of the fecal algae (Fig. 1 ) indicate that at least a portion of the algae consumed by the nudibranch pass unharmed through the digestive tract. Kempf (1984) found evidence of algal breakdown within the tissues of three tropical nudibranchs, but no evidence of active digestion in two additional species (Kempf 1984, 1991). Whether A. papillosa digests some of the ingested symbiotic algae is unknown, but at least a large number of the algae remain unaffected by passage through the nudibranch. Thus, the fecal material of A. papillosa may be important in the dispersal of algae and reinfection of temperate anemones as has been suggested for Berghia major, a tropical nudibranch that also feeds on symbiotic anemones (Muller Parker, 1984). Another possibility is that the algae are heterotrophic in the nudibranch and thus represent a liability. Zooxan- thellae isolated from the sea anemone Aiptasia pulchella are capable of heterotrophic growth under low light levels (Steen, 1987). The possibility of zoochlorellae being par- asitic in A. elcgantissima has been suggested by Muscatine (1971). Because of the generally low light levels in the Northeastern Pacific region, especially during winter months, algae within the cerata of the nudibranch may not be able to meet their carbon requirements photosyn- thetically. As such, it is possible that algae in A. papillosa are a benefit during the summer and a liability during the winter. Kempf (1990. 1991) suggested that the nudibranch Berghia verntcicornis has a primitive mutualistic sym- biosis with zooxanthellae based on the following obser- vations. Relatively high concentrations of zooxanthellae are found in all B. verntcicornis from the field. The zoo- xanthellae ( 1 ) reside in peri-algal vacuoles within the nu- dibranch's digestive cells, (2) do not appear to be digested along with their primary host Aiptasia pallida, (3) remain photosynthetically active within the nudibranch, and (4) appear to benefit the nudibranch in its reproductive effort. Kempf terms the relationship primitive because the sym- biosis is not permanent. The zooxanthellae are eventually exocytosed back into the gut and defecated in a healthy state when nudibranchs are starved in the laboratory. The relationship between zooxanthellae, zoochlorellae, and .-f . papillosa may also be a primitive form of symbiosis, possibly corresponding to a Type IV association as de- scribed by Kempf (1991). Both algae are photosyntheti- cally active within the nudibranch's cerata (Fig. 1 ). Rapid reduction in the density of each alga when it is no longer available in the nudibranch's food shows that frequent ingestion of symbiotic anemones is required to maintain the association. But A. papillosa does not appear to be obligately dependent on either alga at any period of the year. Several of the A. papillosa collected in the Sinclair Inlet lacked symbiotic algae in their cerata, and all of the A. papillosa collected in June 1991 on beaches where symbiotic Anthopleura sp. were not available, lacked symbiotic algae in their cerata. Ultrastructural investi- gations are needed to determine whether the algae are intracellular, and whether they reproduce while in the cerata. Translocation experiments to determine whether fixed carbon is utilized by the nudibranch for growth or reproduction will help to explain the nature of this rela- tionship. Our work to date suggests that A. papillosa will be a good model system for comparing and contrasting the symbiotic relationships between zooxanthellae and zoochlorellae and their animal host. Acknowledgments A portion of this study was supported by an NSF Re- search Experience for Undergraduates Site grant (OCE- 9000676) to Shannon Point Marine Center and an NSF Instrumentation and Laboratory Improvement Program award (USE-905 1 180). The technical assistance of Katie McFarland was critical to the successful completion of the project. The initial work on the symbiotic relationships of.-), papillosa by Michael Kellett and David Wiederspohn ( WWU undergraduates) provided the inspiration for this project. M. Kellett's assistance with collection of nudi- branchs is greatly appreciated. Brian Bingham provided invaluable assistance with the statistical analysis of the data. The suggestions of anonymous reviewers are also appreciated. SYMBIOTIC ALGAE IN ,1 PAPll.LOSA 229 Literature Cited Crossland, C. J., and S. C. Kempf. 1985. Carbon fixation and com- partmentation in the zooxanthellae containing nudibranchs. .\fclihc piloxa and Melihe -./> Proceedings of the Fifth International Coral Reel' Congress. Tahiti. Vol. 6: 125-130. Davies, P. S. 1984. The role of zooxanthellae in the nutritional energy requirements of Pocillopora eydmtxi. Coral Rech 2: 181-186. Edmunds, M.. G. \V. Potts, R. C. Swinfen, and V. L. Waters. 1974. The feeding preference of Aeolidia papillosa (L.) (Mollusca, Nudibran- chia). ./ Mar BID! .l.v.v. U.K. 54: 939-947. Fitt, VV. K., R. L. Pardy, and M. M. Littler. 1982. Photosynthesis, respiration, and contribution to community productivity of the symbiotic sea anemone Anthoplcura cIcKanlisfunni (Brandt. 1835). J. Y/>. Mar. Biol. Ecol. 61: 213-232. Hoegh-Guldberg, O., and R. Hinde. 1986. Studies on a nudibranch that contains zooxanthellae. I. Photosynthesis, respiration and the translocation of newly fixed carbon by zooxanthellae in Ptcracolidia iamhina Proc R Sue Loud B 228: 493-509. Hoegh-Guldberg, O., R. Hinde, and L. Muscatine. 1986. Studies on a nudibranch that contains zooxanthellae. II. Contribution of zoo- xanthellae to animal respiration (CZAR) in Pieraeolulia iamhina with high and low densities of zooxanthellae. Pmc. R Soc. Lond B 228: 511-521. Kempf, S. C. 1984. Symbiosis between the zooxanthella Synihiodinmm (=Gymnodinium) microadriaticum (Freudenthal) and four species of nudibranchs. Biol. Bull. 166: 110-126. Kempf, S. C. 1990. Is the association between the acolid nudibranch Berglua vemiacornis and a zooxanthella a true symbiosis'? Am. Zoo/. 30: 99 A. Kempf, S. C. 1991. A 'primitive' symbiosis between the aeolid nudi- branch Berglua verrucicornis (A. Costa, 1987) and zooxanthellae. J. Mull. Si ud. 57: 75-85. Kevin, K. M., and R. C. L. Hudson. 1979. The role of zooxanthellae in the hermatypic coral Plesiastrea iirvillea (Milne-Edwards & Haime) from cold waters. J. Exp. Mar. Biol. Ecol. 36: 157-170. Kozloff, E. N. 1983. Pp. 186 and 246 in Seashore Lite ol the \oriliern Pacific Coast, University of Washington Press, Seattle. WA. Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951 . Protein measurement with the Folin phenol reagent. ./. Biol. Chem. 193: 265-275. McDonald, G. R., and J. W. N> bakken. 1978. Additional notes on the food of some California nudibranchs with a summary of known food habits of California species. \'eliger 21: 110-119. Muller-Parker, G. 1984. Dispersal of zooxanthellae on coral reefs by- predators on enidarians. Biol Hull 167: 159-167. Muscatine. L. 1971. Experiments on green algae coexistent with zoo- xanthellae in sea anemones, Puc Sci. 25: 13-21. Muscatine, L., P. G. Kalkcmski, J. W. Porter, and Z. Dubinsky. 1984. Fate of photosynthetic carbon in light- and shade-adapted colonies of the symbiotic coral Slylophora pixtillata. Pine R Soc. Lond. 5222: 181-202. O'Brien, T. L. 1980. The symbiotic association between intracellular zoochlorellae (Chlorophyceae) and the coelenterate Aniliopleiira xanthogrammica J E\p. /ool 211: 343-355. Potvin, C, and M. J. Lechowicz. 1990. The statistical analysis of eco- physiological response curves obtained from experiments involving repeated measures. Ecology 1\(4): 1389-1400. Rinkevich, B. 1989. The contribution of photosynthetic products to coral reproduction. Mar. Biol. 101: 259-263. Rudman, W. B. I98la. Further studies on the anatomy and ecology of opisthobranch molluscs feeding on the scleractinian coral Forties. /.ool. .1 Linn Sue 71: 373-412. Rudman, W. B. I981b. The anatomy and biology of alcyonarian-feeding aeolid opisthobranch molluscs and their development of symbiosis with zooxanthellae. Zoo/. J Linn Soc 72: 219-262. Rudman, W. B. 1982. The taxonomy and biology of further aeolidacean and arminacean nudibranch molluscs with symbiotic zooxanthellae. /.ool. J. Linn. Soc. 74: 147-196. Steen, R. G. 1987. Evidence for facultative heterotrophy in cultured zooxanthellae. Mar. Biol. 95: 15-23. Trench. R. K. 197la. The physiology and biochemistry of zooxanthellae symbiotic with marine coelenterates. I. The assimilation of photo- synthetic products of zooxanthellae by two marine coelenterates. Proc R Soc Lond. B 177: 225-235. Trench, R. K. 1971 b. The physiology and biochemistry- of zooxanthellae symbiotic with marine coelenterates. II. Liberation of fixed I4C by zooxanthellae in \-nro Proc R Soc. Lond. B 111: 237-250. Trench, R. K. 1979. The cell biology of plant-animal symbiosis. Ami. Rev. PI. Phyxiol. 30: 4X5-531. Waters, V. L. 1973. Food-preference of the nudibranch Aeolidia pup- illoxa. and the effect of the defenses of the prey on predation. I 'eliger. 15: 174-192. Reference: Biol Bull 184: 2.10-242. (April, 1993) Biochemical Correlates of Estivation Tolerance in the Mountainsnail Oreohelix (Pulmonata: Oreohelicidae) BERNARD B. REES 1 AND STEVEN C. HAND Department of Environmental, Population ami Organismic Biology, University of Colorado. Boulder. Colorado 80309-0334 Abstract. Biochemical changes occurring over 7 months of estivation were studied in two species of land snail, Oreohelix strigosa (Gould) and O. subrudis (Reeve), to determine whether differential mortality during estivation is related to different energetic strategies. Laboratory- maintained snails, which were fed ad libitum prior to es- tivation, were compared with snails collected from the field and induced to estivate without augmenting their energy reserves. In all groups, polysaccharide was catab- olized early in estivation, and protein was the primary metabolic substrate after polysaccharide reserves were de- pleted. Lipid was catabolized at a low rate throughout estivation. Rates of catabolism were largely statistically equivalent between species. Urea and purine bases ac- cumulated during estivation as a result of protein catab- olism, with the former being quantitatively more impor- tant. In both laboratory-maintained and field-collected snails, the rate of urea accumulation was greater in O. subrudis. resulting in higher tissue urea contents in this species at the end of the 7-month experiment. The tissue concentrations of urea at 7 months ranged from about 150 to 300 mA/ and were positively correlated (r = 0.99, P = 0.006) with mortality in these snails. Methylamine compounds, a class of compounds that can offset disrup- tive effects of elevated urea, were measured in one group of O. strigosa at 7 months of estivation and found to be low relative to urea levels. We suggest, therefore, that in the absence of elevated levels of counteracting com- pounds, urea may reach toxic levels and may be one factor limiting the duration of estivation that is survived by these land snails. Received 2 June 1992; accepted 9 December 1992. 1 Present address: Hopkins Marine Station, Department of Biological Sciences, Stanford University. Pacific Grove. CA 93950. Introduction The success of gastropod mollusks in terrestrial habitats has been due to various structural, physiological, and be- havioral specializations (Riddle, 1983). One specialization that is well developed among the pulmonate land snails is the capacity to enter the dormant state of estivation during periods of hot and dry environmental conditions. By entering estivation, snails are able to endure potentially desiccating climatic conditions until the return of more favorable conditions. Some species are capable of esti- vating for remarkable periods of time, ranging up to sev- eral years in duration (Stearns. 1877; Machin, 1967). There are limits to the duration of estivation that can be tolerated, though, and mortality eventually increases as estivation is prolonged. Because there is no intake of foodstuffs during estivation, the period of estivation that can be survived may be limited by the exhaustion of en- dogenous energy reserves (Pomeroy, 1969; Schmidt-Niel- sen el til.. 1971 ). Metabolic rate reduction, which would serve to prolong the energy stores of the animal, occurs during estivation, and desert-dwelling species display lower rates than species from more mesic environments (Schmidt-Nielsen el a/.. 1971; Herreid, 1977; Rees and Hand. 1990). These observations have been taken as sup- porting the idea that energy reserves are limiting. But since the rates of metabolism and evaporative water loss are highly correlated in land snails (Barnhart, 1986), the re- duction of metabolic rate may reflect an adaptation to conserve water rather than energy. A comparison of sur- vivorship in snails with differing levels of energy reserves prior to estivation would more clearly address the question of energy limitation. The duration of estivation may also be limited by the accumulation of noxious end-products of protein catab- olism. Depending upon the species and activity pattern. 230 BIOCHEMICAL CHANGES IN ESTIVATION 231 land snails can dispose of nitrogen derived from protein catabolism in the form of uric acid and other purines, urea or gaseous ammonia (Bishop el Rifle Falls > Rifle Gorge. Animals and species identification Snails were collected in June and August of 1987 and in November of 1989. They were either sacrificed im- mediately for determination of the biochemical compo- sition of animals in the field, or brought into the laboratory and used for estivation studies (see below). The average shell-free tissue mass of snails prior to estivation in the laboratory was 0.453 0.014 g (SEM, n = 63) for O. strigosa and 0.394 0.013 g for O. siibntdis (n = 41). Both species are hermaphroditic and bear live young. Only individuals without developing young in their oviducts were used in this study. After the snails had been sacrificed for biochemical analyses (see below), the species was determined by starch gel electrophoresis of proteins (Rees, 1988). During the present study, additional, faster-migrating alleles were re- solved in O. strigosa at the phosphoglucomutase and phosphoglucose isomerase loci. This finding does not compromise the utility of this technique in species deter- mination, however, as the occurrence of the slow alleles at these loci remains diagnostic ofO. siibntdis. Individuals that were not electrophoretically genotyped (snails col- lected in June 1987 and those which died during the es- tivation series) were separated into species by their shell morphology (Rees, 1988). B. B. REES AND S. C. HAND Table I Climalu- iniul/lu>n/ IWtl in llic Mitchell Creek and Kust Ri/lc Creek drainages Site Month Daily low temp(C) Daily high temp (C) Daily low RH (%) Daily high RH (%) Rainfall (mm) Normal rainfall (mm) Mitchell Creek June 8 2 25 4 30 8 70 6 -n 31 July 11 2 23 2 37 8 75 6 48 30 August 10 2 23 3 36 9 72 8 15 36 June-Aug 10 2 23 3 35 9 72 7 85 97 East Rifle Creek June 11 3 28 5 26 6 52 15 18 21 July 13 2 28 2 31 6 65 15 31 19 August 122 27 4 30 7 61 17 7 32 June-Aug 12 2 28 + 4 29 7 60 17 56 72 Temperature and humidity readings were made continuously with hygrothermographs located at the Mitchell Creek and Rifle Falls Fish Hatcheries. Hygrothermographs were enclosed in Stevenson-style temperature cabinets approximately 10 cm above the ground and were calibrated against a hand-held temperature-humidity sensor that had been certified by the National Bureau of Standards. The data reported for June were recorded between June 5 and June 30: data for July and August are from all days in these months. Temperature and humidity are reported as the means and one standard deviation of the daily values. All monthly temperature and relative humidity averages are significantly different between field sites, except for June daily low relative humidity (t-test, P < 0.05). Monthly rainfall data for 1990 and normal rainfall (averages for the years 1951-1980) were recorded in the nearby communities of Glenwood Springs and Ritle (ca.. 5 and 20 km from fish hatcheries, respectively) and are taken from Climatological Data. Colorado (U.S. Department of Commerce). Estivation series Two experiments were carried out to assess the effects of estivation on the biochemical composition of these snails. One was performed with snails collected in No- vember of 1989 and fed ad libitum for 2 months prior to estivation. These snails were kept in damp terraria and fed lettuce and carrots. Chalkboard chalk was provided as a source of calcium. This feeding regime was designed to saturate the energy reserves of the snails prior to esti- vation and to minimize the variation in nutritional status due to differing conditions at the collection sites. After 2 months, these snails were transferred to dry terraria with- out food, which induced estivation. These snails are re- ferred to as the laboratory-maintained group. In the other experiment, snails collected in August 1987 were brought into the lab and induced to estivate immediately by place- ment in dry terraria. In this experiment, we wanted to determine the effect of estivation on snails that did not have their energy reserves augmented by laboratory feed- ing. These snails are referred to as the field-collected snails. In both series, snails were maintained at room temperature (23-28C) and humidity (ca. 20-60%) for the duration of the experiment. Under these conditions, snails were inactive within 2 days after being transferred to dry con- ditions, and there was no indication that any of the ani- mals became active again once they had entered quies- cence. Photoperiod was not controlled. Preparation ot snails for chemical analyses Snail extracts were prepared and maintained at 0-4C unless otherwise stated. Chemicals and biochemicals were of reagent grade, and water was purified with a Milli-Q Reagent Water System (Continental Water Systems, Inc.). At the start of the experiments and at 1, 2, 4, and 7 months following entry into estivation, snails were sam- pled randomly from the terraria. An additional sampling interval at 10 days was included in the experiment with the field-collected snails. The shell diameter of each in- dividual was measured, and the snails were then dissected from their shells, briefly blotted, and frozen in liquid ni- trogen. Tissues were kept at -70C until biochemical analyses could be performed, at which time a small portion (5-15 mg) of the digestive gland was removed for electro- phoresis, and the remainder of the tissue was lyophilized to a constant dry mass. The difference between fresh tissue mass and dry tissue mass was recorded as tissue water. Dry tissues were then pulverized with a mortar and pestle and divided into two subsamples: one fraction (approxi- mately 40 mg) was used for determination of protein, DNA, polysaccharide, urea, and for the lab-maintained snails, purines; and the other fraction (10-25 mg) was kept for lipid analysis. At the later time points in the es- tivation series, individuals were commonly less than 50 mg dry mass. This small amount of dry tissue could not be divided, so lipid was not measured in these individuals. Extracts for the determination of protein, DNA, poly- saccharide, urea and purines were prepared as follows. Dry tissues were homogenized in 1.0 ml of ice cold 1 N perchloric acid with a glass homogenizer. Two 50 ^1 ali- quots of the perchloric acid homogenate were removed: one was combined with 0.95 ml 0.5 N NaOH and saved at 70C for protein assays; and the other was combined with 0.95 ml 0.5% (w/v) lithium carbonate and saved at BIOCHEMICAL CHANGES IN ESTIVATION 233 70C for purine analysis. The remainder of the per- chloric acid extract was centrifuged at 10,000 X g for 15 min. The pellets were washed once with 0.7-0.8 ml of 1 N perchloric acid and centrifuged as above. The perchloric acid insoluble material was saved for DNA measurement. Perchloric acid supernatants for each individual were pooled, neutralized with 5 Af K 2 CO 3 , and centrifuged at 10,000 x g for 10 min to remove perchlorate salts. Two hundred to 400 n\ of the neutralized extract was combined with two volumes of 95% ethanol and stored at -70C for polysaccharide assays, and the remainder was saved at 70C for urea measurements. Biochemical analyses Protein was measured by the method of Lowry el al. ( 1951 ), as modified by Peterson (1977), with bovine serum albumin as the standard. For calculations of nitrogen bal- ance, it was necessary to determine the mass of nitrogen in snail protein. The protein in a perchloric acid homog- enate was recovered by centrifugation after the nucleic acids had been digested by heating (see below). Lipid was removed by washing the PCA-insoluble material with methanol. The amount of nitrogen in the PCA-insoluble fraction was determined by a micro-Kjeldahl procedure that includes direct nesslerization of ammonia following digestion of the proteins (Koch and McMeekin, 1924). The Nessler reagent was obtained from Sigma Chemical Company. The amount of nitrogen in protein determined in this manner was not different in the two species and was found to account for 16.8 0.9% (S.D., n = 4) of the protein mass measured by the Lowry assay. DNA was determined by the diphenylamine assay of Burton ( 1956) with modifications suggested by Giles and Myers (1965). Briefly, perchloric acid insoluble material was suspended in 1 .0 ml 1 .5 A' perchloric acid and heated at 70C for 20 min. Following centrifugation at 10,000 X gfor 20 min, an aliquot (50-100 ^1) of the supernatant was brought to 2.5 ml with 1.5 N perchloric acid and combined with 1.5 ml 4% (w/v) diphenylamine made in glacial acetic acid and 0.1 ml 0.16 mg ml ' acetaldehyde made in water. The color was allowed to develop for 20 h in the dark at room temperature. To correct for non- specific color development, an absorbance difference ( A 600 - A 700 ) was determined for each sample. Calf thymus DNA was the standard. Polysaccharide (glycogen plus galatogen), which pre- cipitated in the ethanolic extract, was collected by cen- trifugation at 10,000 X g for 20 min, washed once with 1.0 ml 95% ethanol and centrifuged again. The pellets were air-dried and redissolved in 1.0 ml water by heating at 70C. Polysaccharide was measured by the anthrone method described by Jermyn (1975), except that the ad- ditions of hydrochloric and formic acid to the samples were omitted. Polysaccharide content was expressed as 0.9 x glucose mass. For urea analysis, samples were thawed and clarified by centrifugation at 10,000 X g for 10 min. Urea was measured colorimetrically as ammonia after treatment of the samples with urease (Sigma Diagnostic Kit No. 640). Blanks without urease were subtracted from each sample. Purine bases were analyzed with high performance liq- uid chromatography essentially as described by Simmonds and Harkness ( 1981 ). A LDC/Milton Roy HPLC system was employed in conjunction with a Waters /uBondapak C-18 column (30 cm X 3.9 mm i.d.). The lithium car- bonate solutions were thawed, diluted, neutralized, and filtered through Gelman SuporO.45 ^m membrane filters. Twenty n\ were injected onto the column, and purines were eluted isocratically with a buffer of 4 mA/ potassium phosphate (pH 3.6) containing 1% (v/v) methanol. Ab- sorbance was monitored at 265 nm, and uric acid, guanine and xanthine were quantified by integration of peak area. Total lipid was determined after extraction of the tissues in chloroform:methanol (Folch el al.. 1957; Ways and Hanahan, 1964). For each snail, lyophilized tissues were homogenized in 4 ml chloroform:methanol (2:1) with a Virtis micro-ultrashear apparatus for 1 min and filtered through a fritted disc funnel. The residue was rehomog- enized in 4 ml chlorofornrmethanol and filtered. The res- idue was finally washed with another 2 ml of chloroform: methanol and the filtrates combined. The filtered chlo- roform:methanol homogenate was mixed with 0.25 vol- ume 0.88%- (w/v) KC1 in water, and after separation, the aqueous phase was aspirated. The remaining organic phase was mixed with 0.25 volume methanol:water (1:1), and the aqueous phase was aspirated after separation. The or- ganic phase was then decanted into a pre-weighed alu- minum planchet and evaporated to dryness under a stream of nitrogen. The dried lipid was held over Drierite a further 24 h and weighed to the nearest 0. 1 mg. In one group of estivating snails, methylamine com- pounds were measured by reineckate precipitation pro- tocol modified from Kermack et al. (1955). Lyophilized tissues from a whole snail were homogenized in 30 vol- umes of 40%> ethanol and centrifuged at 20,000 X g for 15 min. The pellet was washed with another 30 volumes of 40% ethanol, and the combined supernatants were boiled for 10 min to precipitate proteins. The ethanolic extract was centrifuged at 10.000 X g for 20 min, lyoph- ilized, and redissolved in 1.0 ml 0.1 TV HC1. Saturated ammonium reineckate, prepared in water and titrated to pH 1 with 5.0 N HC1, was added to the each sample in the ratio 3:1 (reineckate:sample). Reineckate salts were allowed to precipitate at 4C overnight and were collected by filtration on polycarbonate membrane filters (Nucleo- pore, 0.2 ^m). After washing the precipitate three times with 3 ml diethyl ether, the precipitate and membrane 234 B B. REES AND S. C. HAND Table II i^ilinn at lul'tiivlnry nuiinuiincil Orcohelix Compound O. strigiixa O subrudix mgg ' dry mass % dry mass mg g ' dry mass % dry mass Protein 512.6 19.2 51.3 509.4 13.7 50.9 Polysaccharide 216.2 11.3 21.6 230.1 8.8 23.0 Lipid 70.3 1.4* 7.0 78.2 1.9 7.8 DNA 14.9 0.3 1.5 16.7 0.5 1.7 jjmol g ' dry mass % dry mass ^mol g" 1 dry mass % dry mass Urea 0.98 0.29* <0.1 2.20 0.76 <0.1 Uric acid 55.1 3.9 0.9 45.1 4.0 0.8 Guanine 17.1 1.9 0.3 10.3 1.2 0.2 Xanthine 7.1 0.6 0.1 8.2 0.9 O.I Total dry mass accounted for 82.7 84.5 Values are given as the mean and standard error of the mean. The sample sizes were 27 O xtrigosa and 2 1 O xubritdix, except for the lipid analyses, where sample sizes were 1 8 and 1 2 for O slrigosa and O. subrudis. respectively. Asterisks indicate that species means for these biochemical constituents are significantly different. were dissolved in 70% acetone, and the absorbance was read at 520 nm. Betaine was the standard. Following the above protocols, the recoveries of known quantities of protein, DNA, urea, uric acid, guanine. xan- thine. and lipid were >88%, and we did not correct the results for differences in recovery. In the case of polysac- charide, this protocol led to a 77 2.5% (S.D., n = 4) recovery of glycogen standards, and the polysaccharide content of snails was corrected accordingly. Data analysis Examination of the total tissue contents of various bio- chemical compounds revealed a large degree of variation due to size differences among individuals. For snails prior to estivation (both laboratory-maintained and field-col- lected), biochemical constituents were expressed in terms of dry mass in order to standardize for size differences. Equality of sample variances was tested with Bartlett's Box-F (Zar, 1984), and differences among group means were evaluated with parametric or nonparametric analyses of variance accordingly (Zar, 1984). A posteriori testing was done with Scheffe's or Dunn's multiple comparison tests (Zar, 1984). During estivation, considerable dry mass was lost, so some variable other than dry mass was required as an index of snail size for standardization of biochemical composition. Data from non-estivating, laboratory- maintained snails showed that the relationship between shell diameter and snail size was quite good: coefficients of determination (r) for regressions of whole tissue and dry tissue mass versus shell diameter were 0.763 and 0.784, respectively. Furthermore, when all snails were consid- ered, there was no effect of duration of estivation on shell diameter (analysis of variance, P = 0.969), suggesting that shell diameter neither increases nor decreases during es- tivation. Therefore, tissue mass, water, and biochemical contents of estivating snails were adjusted to a snail of average shell diameter ( 15.63 mm) based upon the slopes of regression equations describing the relationship between each component and shell diameter. For each species, the rates of change in these adjusted values during various intervals of estivation were then determined by regression analysis. Differences between species-specific rates of change were evaluated with the test for homogeneity of slopes in an analysis of covariance package (Zar, 1984). Correlations between various biochemical measure- ments and mortality at 7 months of estivation were an- alyzed with Pearson's product-moment correlation. All statistical analyses were performed with SPSS-X, version 4 (SPSS, Inc.). and a probability < 0.05 was considered as statistically significant. Unless otherwise stated, data are presented as means and one standard error of the mean (SEM). Results Biochemical composition of laboratory-maintained Oreohelix Laboratory-maintained Oreohelix strigosa and O. sub- nidiswere composed of approximately 51% protein. 22- 23% polysaccharide, 7-8% lipid. and about 1 .5% DNA (Table II). The levels of urea and purine bases were low prior to estivation. Urea averaged 1-2 ^molg~' dry mass, comparable to the level reported in Bulimulus dealbatus prior to estivation (Home, 1971). The levels of purine BIOCHEMICAL CHANGES IN ESTIVATION 235 bases totaled to 64-79 ^mol g~ ' dry tissue, similar to the tissue contents of other non-estivating snails (Jezewska el al.. 1963: Home, 1971). On a molar basis, uric acid ac- counted for about 70% of the total purine, with guanine and xanthine accounting for approximately 20 and 10% of the total purine, respectively, in both O. strigosa and O. subrudis. Hypoxanthine was not found in the tissues of these snails. Taken together, these compounds account for more than 80% of the dry mass of these snails. The unaccounted fraction is presumed to be other low mo- lecular weight organic compounds (e.g.. amino acids) and inorganic ash. Biochemical composition of field-collected Oreohelix Compared with the values obtained for laboratory- maintained snails, both O. strigosa and O. subrudis dis- played lower polysaccharide levels in the field-collected groups (Fig. 1A). Protein constituted a correspondingly larger portion of the dry mass in both species (Fig. IB), and lipid was somewhat higher in O. strigosa collected in the late summer (Fig. 1C). These differences in biochem- ical composition reflect the effects of ad libitum feeding in the laboratory-maintained group and suggest that snails feed less regularly or on food of differing qualities in the field. Of the snails collected in the late summer, O. strigosa displayed significantly higher levels of polysaccharide than O. subrudis. Differences in polysaccharide content may influence the capacity of these snails for long-term esti- vation (see Discussion). Snails of either species collected late in the summer demonstrated much more variable urea contents than snails in the laboratory-maintained or early summer groups (Fig. ID). Among the laboratory-maintained snails, only 17% had urea contents greater than 1 ^mol g ' dry mass, and among the snails collected early in the summer, this percentage was 22%. In these groups, the highest urea content measured was 11.7 /urno! g ' dry mass. Among the snails collected later in the summer, urea was higher than 1 /^mol g ' dry mass in 33% of the snails, and the highest value was 93.0 /xmol g" 1 dry mass. Since urea accumulates during estivation (see below), the occurrence of elevated urea in snails collected late in the summer suggests that many of these animals had been estivating in the field. Mortality during estivation Both species of Oreohelix experienced mortality during the later months of estivation. In the group of snails that had been maintained in the laboratory prior to estivation. 1 of the remaining 13 O. strigosa had died at 7 months, whereas 9 of 30 O. subrudis had died. For snails that were brought in from the field, the mortality at 7 months in both species was higher: 10 of 24 O. strigosa had died, whereas 28 of 34 O. subrudis had died. Among the field- collected snails, the proportion of dead O. subrudis at 7 months was significantly greater than the proportion in O. strigosa (G-test, P < 0.05). These results demonstrate that O. strigosa tolerates extended periods of estivation in the laboratory better than O. subrudis. ti O g E 200 ' A- (211 (27) |2 ' 120 E 80 o 51 -a 60 en 40 JL 500 0.250 o* 25 S 20 E ? 15 Figure 1. Biochemical composition of laboratory-maintained and field-collected O. strigosa (open bars) and O .subrudis (solid bars). A. Polysacchande content. B. Protein content. C. Lipid content. D. Urea content. Error bars indicate one standard error of the mean. Asterisks indicate that the content of this constituent is significantly different from that measured in laboratory-maintained snails of the same species, and the crosses indicate that species means are significantly different for that sampling interval. 236 Analysis oj changes during estivation We were interested in whether the two species have different rates of substrate depletion or end-product ac- cumulation during c>; vation. Because variation in the size of individuals among the sampling intervals and be- tween species would tend to obscure these rates, we have normalized the tissue mass, water content, and the content of biochemical constituents to an average snail size based upon shell diameter (see Materials and Methods). Note that, since dry mass, water content, and biochemical composition can be determined only once for any indi- vidual, the rates of change described below reflect average rates of loss or accumulation among groups of individuals rather than rates of change within individual snails. Fur- thermore, shell diameters were not measured on the field- collected snails sacrificed prior to estivation (day 0). and consequently the data for this group begin at 10 days of estivation. Loss of tissue mass and water during estivation Fresh tissue mass, dry tissue mass, and water decreased significantly in both species oWreo/ie/ix during estivation. When tissue mass and water content data were corrected for size differences among individuals, rates of loss in the two species were not significantly different. The loss of tissue was characterized by parallel decreases in both dry tissue mass and tissue water. These losses were biphasic, occurring more quickly at the onset of estivation as the snails entered estivation, and then reaching a steady slower rate after the initial drop. By 7 months of estivation, the tissue mass and water content of snails were reduced by approximately 35% in all groups. The loss of tissue water from estivating Oreolielix was not reflected in a decrease in the percent tissue water be- cause the dry mass decreased proportionately. The per- centage of tissue water remained between 78 and 81% tor both species in both experimental series. In fact, among the laboratory-maintained snails, there was a slight but statistically significant increase in the percent tissue water over the 7 months of estivation despite the overall loss of water. Thus a constant percentage tissue water cannot be interpreted as indicating no loss of water, as has been assumed previously for other species of estivating snails (Schmidt-Nielsen et at.. 1971). Catabolism of energy reserves during estivation Polysaccharide, protein, and lipid were all catabolized during estivation, but the substrates that were utilized changed as estivation proceeded (Figs. 2-4, Table III). Polysaccharide was the primary metabolic fuel for the initial months of estivation (Fig. 2). Snails that had been maintained in the laboratory began the estivation period B. B. REES AND S. C. HAND with large polysaccharide stores, and in these snails, ca- tabolism of this substrate continued for the first 4 months of estivation (Fig. 2A). During the first month of estiva- tion, the rate of polysacchande depletion was significantly faster in O. suhntdis (Table III). Between 1 and 4 months, carbohydrate catabolism continued at moderate rates that were similar in the two species. After 4 months, the poly- sacchande content of the snails was much reduced and its rate of utilization was correspondingly low. In the field- collected snails, the polysaccharide stores were smaller, and consequently they were depleted earlier (Fig. 2B). Al- though the initial rates of utilization were similar in the two species, carbohydrate lasted longer in O. strigosa. which had begun estivation with larger stores. As in the estivation series begun with laboratory-maintained snails, rates of polysaccharide utilization were much reduced during the later phases of estivation and statistically equivalent between species. Upon depletion of the polysaccharide stores, net protein catabolism occurred (Fig. 3). In the laboratory -maintained snails, the onset of net protein depletion occurred at about 2 months of estivation (Fig. 3 A). Before this time, no net Zb (26,21) i A. id 9n Q ^t-v < 1 4\ I -15 fc \\ co cMO \T ^Jl i^\V 2) 5 (12.12) ~?5^ ^\1 (10.14) 1,1 _ _. B. i i i o p\ cE-- < l I !=15 o (11.11) LO CP 1 J(14.9) ^ " R D_ 5 "\ \ U - 1 \ (12.9) (,2.6) *=-=^-==^^ : 01 234567 DURATION OF ESTIVATION (mo) Figure 2. Polysaccharide content during estivation in O. strigosa (O) and O .suhnulis (). All values have been adjusted to a snail of average size based upon shell diameter. A. Laboratory-maintained snails. B. Field- collected snails. Sample sizes are given in parentheses with the value for O. strigosa appearing first. Bars indicate one standard error of the mean. BIOCHEMICAL CHANGES IN ESTIVATION 237 Table III <>/ polysaccharide, protein, and lipid catabolism ami urea ami purine accumulation in Oreohehx v/>/> during estivation Compound Experiment Interval O suhnulis Polysaccharide A 0-1 month -7.81 2.16 -13.45 1.71 0.05 A 1-4 months -2.66 0.59 -2.23 0.41 0.54 A 4-7 months -0.23 0.22 NS -0.27 0.12 0.84 B 10 days-2 months -3.84 1.14 -2.25 0.45 0.23 B 2-7 months -0.23 0.08 -0.18 0.04 0.64 Protein A 0-2 months -0.72 1.56 NS 0.95 0.97 NS 0.37 A 2-7 months -1.95 0.63 -2.73 0.35 0.26 B 10 days-7 months -2.14 0.58 -3.03 0.43 0.26 Lipid A 0-7 months -0.33 0.06 -0.36 0.07 0.78 B 10 days-7 months -0.11 O.I2 NS -0.67 0.21 0.06 Urea A 0-2 months 0.98 + 0.21 1.25 0.32 0.47 A 2-7 months 6.20 0.87 8.66 0.67 0.03 B 10 days-7 months 5.52 0.59 8.75 0.53 <0.01 Uric acid A 0-7 months 0.43 0.12 0.56 0.08 0.36 Guanine A 0-7 months 0.1 1 0.04 0.15 0.02 0.40 Xanthine A 0-7 months 0.03 0.02 NS 0.05 0.02 0.51 Experiment A was done with snails after laboratory maintenance and experiment B with field-collected snails without prior laboratory maintenance. Values for rates of catabolism (negative values) and accumulation (positive values) are slopes and their standard errors from regression equations of the adjusted tissue content of each compound versus length of estivation over the intervals indicated (see also Figs. 2-6). Units are mg snail ' mo" 1 for polysaccharide. protein and lipid and /umol snail ' mo ' for urea, uric acid, guanine and xanthine. All slopes were significantly different from zero, except where indicated (NS). /"values are from tests of equality of species-specific slopes. protein catabolism occurred in either species, as indicated by the slopes of regression lines not significantly different from zero (Table III). After the onset of net protein ca- tabolism, the rates of utilization were fairly linear throughout the remainder of the estivation period. Among the field-collected snails, significant protein catabolism occurred from the beginning of estivation (Fig. 3B). While species-specific rates were not significantly different, there was a trend toward lower rates of protein catabolism in O. strigosa in both experimental series (Table III) a trend that likely influenced the rates of end-product accumu- lation (see below). Lipid was catabolized at a low rate throughout the du- ration of estivation in both experimental series (Fig. 4). The rates of lipid utilization in the two species were not significantly different during the 7-month estivation ex- periments (Table III). Accumulation oj nitrogenous end-products and nitrogen balance With the onset of protein catabolism. the nitrogenous end-products, urea and purine bases, accumulated in the tissues of estivating snails (Figs. 5-6. Table III). The tissue levels of urea increased dramatically in both species of Oreoheli.\ (Fig. 5). In the laboratory-maintained snails, protein was not catabolized early in estivation, and hence urea began to accumulate only after 2 months of estivation (Fig. 5 A). Between 2 and 7 months of estivation, the rate of accumulation was higher in O. subrudis than in O. strigosa (Table III). By 7 months of estivation, urea was 32.9 4.5 ^mol snail ' in O strigosa (n = 10) and 43.7 3.1 /umol snair 1 in O subrudis (n = 14). In the field- collected snails, urea began to increase almost immediately upon the commencement of estivation, reflecting the early dependence upon protein catabolism (Fig. 5B). Between 10 days and 7 months, the rate of urea accumulation in O. subrudis was again greater than in O. strigosa (Table III). The tissue urea contents of these snails after 7 months of laboratory estivation were 36.4 4.3 /xmol snair 1 in O. slrigosa (n = 12) and 58.2 6.1 yumol snail ' in O. subrudis (n = 6). The accumulation of purine bases was only measured in snails that had been maintained in the laboratory, and their patterns of change are shown in Figure 6. Over 7 months of estivation, uric acid increased by 3 to 4 j/mol snail' 1 (Fig. 6A), guanine increased by approximately 1 ^mol snair 1 (Fig. 6B), and xanthine increased by less than 0.5 ^mol snail ' (Fig. 6C). Hence the sum of the purines increased by only 5 to 6 j/mol snail~'. Over the 7-month estivation period, the rates for uric acid, guanine and xanthine accumulation were not statistically different between the two species (Table III). Ammonia production was measured as described by Speeg and Campbell ( 1968), except that estivating snails were kept in a closed chamber for a period of two days. 238 B B. REES AND S. C. HAND 60 50 ct: Q. o. 20 10 60 50 O rr 10 (26,21 ) (1 2.1 2) .0..4) (11.11) 1(14.9) (13.10) B. (4.9) (12.6) o 01234567 DURATION OF ESTIVATION (mo) Figure 3. Protein content during estivation in O sirigosa (O) and O xnhnidix (). All values have been adjusted to a snail of average size based upon shell diameter. A. Laboratory-maintained snails. B. Field- collected snails. Sample sizes are given in parentheses with the value for O. strigosa appearing first. Bars indicate one standard error of the mean. Over this period, the amount of ammonia produced by 8 snails of either species was below the limit of detection (0.02 Levels of urea-counteracting solutes Methylamine compounds were measured in one group of field-collected O. .strigosa after 7 months of estivation and found to be 2.68 0.27 ^mol snair ' (n = 5). HPLC analyses of selected extracts of both species have shown that betaine is the predominant methylamine compound, and that polyhydric alcohols, another class of protective compounds, do not significantly accumulate in snail tis- sues during estivation (data not shown). Discussion In the present study, we undertook an analysis of the biochemical changes that occur in Oreohelix strigosa and O. .siibniclis during a period of laboratory estivation. The temporal nature of substrate utilization and nitrogenous end-product accumulation were described for the first time in congeneric species of land snails that are dissimilar in their capacity for long-term estivation. Differences in the patterns of biochemical changes may account, in part, for the observed difference in mortality. Below, we evaluate the relationships between mortality and both the exhaus- tion of energy stores and the accumulation of nitrogenous end-products of protein catabolism. We also discuss the distributions of these Oreohelix species in the field in light of their different survivorship during desiccation stress. Mortality anil exhaustion of energy stores If the duration of estivation is limited by the depletion of energy storage compounds during estivation, then snails with larger stores prior to estivation would be predicted to survive estivation proportionately longer. We were able to elevate the level of polysaccharide, the primary meta- bolic substrate during early estivation, by feeding snails ad libitum in the laboratory prior to estivation. Subse- quently, when these snails were allowed to estivate, poly- saccharide stores lasted longer, and mortality in both spe- cies was lower than when snails collected from the field o 6 CO en Q CL 6 en en 9 3 Q. (17.12) 0123456 DURATION OF ESTIVATION (mo) Figure 4. Lipid content during estivation in O. slrigosa (O) and O xiihritdix (). All values have been adjusted to a snail of average size based upon shell diameter. A. Laboratory-maintained snails. B. Field- collected snails. Sample sizes are given in parentheses with the value for O. strigosa appearing first. Bars indicate one standard error of the mean. BIOCHEMICAL CHANGES IN ESTIVATION 239 estivated without prior laboratory feeding. In addition, among the field-collected snails. O. strigosa began with higher polysaccharide levels than O. suhrudis, and the former displayed only half the mortality by 7 months of estivation. With data from four groups of snails (2 species X 2 experimental series), we tested the correlation between pre-estivation polysaccharide stores and percent mortality at 7 months of estivation. Since snails with higher poly- saccharide stores were predicted to survive estivation bet- ter (i.e., show lower mortality), the test was one-tailed. The negative correlation between pre-estivation polysac- charide stores and mortality was statistically significant (r = -0.91, P = 0.045). The observation that polysac- charide stores were exhausted several months prior to the onset of mortality, however, suggests that mortality is not due to the depletion of this substrate in sensu stricto. Rather, the correlation between polysaccharide stores and mortality likely reflects other biochemical changes that are initiated upon the depletion of the polysaccharide re- serves (see below). Mortality and the accumulation of nitrogenous end-products Upon the exhaustion of polysaccharide, protein was catabolized, and both O. strigosa and O. suhrudis were found to accumulate urea as the major product of protein metabolism. Based upon rates of protein catabolism and end-product accumulation during the estivation interval of net protein depletion (2-7 months for laboratory- maintained snails and 10 days-7 months for field-collected snails), urea accumulation in the tissues accounted for approximately 50% of the nitrogen derived from protein catabolism, whereas the accumulation of purines only ac- counted for about 10% of the protein nitrogen. Ammonia production was below measurable levels, corresponding to less than 1% of the calculated nitrogen liberated from protein catabolism. A portion of the unaccounted fraction of nitrogen was probably lost during sample preparation (blotting of hemolymph can account for the loss of up to 25% of the urea nitrogen), and nitrogen may have accu- mulated in compounds not measured in this study (e.g., amino acids; c.f., Wieser and Schuster, 1975). Further studies of nitrogenous compounds in hemolymph of es- tivating snails may elucidate the nature of the missing nitrogen fraction. In both experimental series, the rate of tissue urea ac- cumulation was found to be faster in O. suhrudis than in O. strigosa. resulting in higher urea contents in the former species. Because urea can easily cross most cell membranes (Forster and Goldstein, 1976), the urea measured in ex- tracts of whole snails is likely to be uniformly distributed throughout the tissues of the snails. This assumption was supported by measuring urea in hemolymph, foot muscle 70 "650 ^40 o I 30 < 20 Ld 9510 A. (10.13) /u (12,6) fso B. i '550 X c ^x ^40 (12.9) X T o i 30 ^ ^ ^ ^20 (12.10) X }^^ LJ / * ^^^ ^ 9J10 (14.9) .X^ (11,11) fj$ n . %^^__ .... 01 234567 DURATION OF ESTIVATION (mo) Figure 5. Urea content during estivation in O xtrigosa (O) and O. subriuli.t (). All values have been adjusted to a snail of average size based upon shell diameter. A. Laboratory-maintained snails. B. Field- collected snails. Sample sizes are given in parentheses with the value for O strigosa appearing hrst. Bars indicate one standard error of the mean. and digestive gland of two laboratory-maintained O. stri- gosa after 7 months of estivation. In one snail, the urea concentrations were 131, 126, and 130 mM in hemo- lymph, foot muscle, and digestive gland, respectively, and the other snail had urea concentrations of 21 1, 155, and 198 mA/ in these tissues. When urea concentrations were calculated for all snails based upon a uniform distribution in the total tissue water, urea was found to rise from less than 1 mM prior to estivation to levels exceeding 150 mM by 7 months. The average urea concentrations in snails that had been estivating for 7 months were: 152 24 mM (n = 10) and 204 14 mM (n = 14) in labo- ratory-maintained O. strigosa and O. subrudis, respec- tively, and 203 + 15 mM(n = 12) and 288 21 mM (n = 6) in the two species when field-collected snails were used. When tested with correlation analysis, a significant positive correlation was found between tissue urea con- centration and mortality at 7 months in the four groups of snails (r = 0.99, P = 0.006). In a recent study of mam- malian cells in culture, Yancey and Burg ( 1990) showed a dramatic decrease in viability as the urea concentration 240 B. B. REES AND S. C. HAND o 8 c en |6 S' 4 o < 2 o rr => o c en o ~O E l_ '5 ^ 0.8 o E 0.4 0.0 01 234567 DURATION OF ESTIVATION (mo) Figure 6. Purine content during estivation in O slrignxu (O) and O. xuhriulix (). Measurements were only made with snails that had been maintained in the laboratory- prior to estivation. All values have been adjusted to a snail of average size based upon shell diameter. A. Urate content. B. Guanine content. C. Xanthine content. Sample sizes are given in 6A in parentheses with the value for O strigosa appearing first. Bars indicate one standard error of the mean. in the medium increased from 1 50 to 300 mM, the same range of concentrations across which survivorship de- creased sharply in Oreohelix. In other organisms that accumulate high levels of urea, there are also high levels of compounds that are capable of counteracting the potentially deleterious effects of urea (Yancey cl ai, 1982; Yancey, 1985). For example, in elas- mohranch fish, which display a tissue concentration of urea in this range, methylamine compounds occur in a 1:2 proportion with urea. At this ratio, methylamines are able to counteract the disruptive effects of high urea in vitro and are presumed to act this way in vivo. The me- thylamine content of estivating O. strigosa was low relative to urea. If methylamines were distributed uniformly in the tissue water, then their concentration would corre- spond to 12.2 1.9mA/. If methylamine compounds are concentrated intracellularly, as suggested by work with mammalian cells (Yancey and Burg, 1990), the intracel- lular concentration can approach 25 mM. Relative to the urea measured at this point in estivation, however, even 25 mAI methylamines is far below the ratio of 1 :2 at which methylamine effects counteract the perturbation of mac- romolecules by urea. Thus, we offer the hypothesis that urea toxicity is a factor that limits the duration of estivation that can be tolerated by these two species of land snail. While higher levels of urea have been reported in other species of land snail (DeJorge and Peterson, 1970; Home, 1971; Trammel and Campbell, 1972). in the absence of data on mortality and methylamine concentrations, we cannot evaluate the applicability of this hypothesis to these species. This hy- pothesis does not exclude the involvement of other factors (t'.,f., blood gases, pH or osmolarity) in setting the upper limit to estivation in these or other snails. Biological rationale for urea accumulation If urea does reach toxic levels, then it raises the question: why do estivating snails synthesize urea? One explanation is that the high tissue concentration of urea aids in water retention in arid environments (Home, 1971). This ex- planation is unlikely, though, for two reasons. First, urea concentrations of 300 mM only contribute a trivial amount to the gradient for water movement between the tissues and dry air (Machin, 1975). Secondly, urea ac- cumulates faster in humid environments than in dry ones (Home, 1973a). The osmotic effect of elevated urea could be beneficial in the uptake of water when conditions of high humidity return (Riddle. 1983). Alternatively, the synthesis of urea may simply serve as a means of ammonia detoxification. The LD 50 for am- monia in the land snail Bulimulus dealbatus is approxi- mately 16 Minol g ' wet weight (Home, 1973b). Based upon rates of protein catabolism measured for Oreohelix species during estivation, this amount of ammonia is gen- erated within 8 days. Clearly, if these snails are similarly sensitive to ammonia toxicity, then during prolonged pe- riods of high protein catabolism, ammonia must be re- moved. By producing the moderately less toxic urea, snails may be able to carry out protein catabolism for a longer period. But O. strigosa and O. siibrudis, as well as other BIOCHEMICAL CHANGES IN ESTIVATION 241 species that accumulate urea during estivation, appear to have the capacity to synthesize purines as nitrogenous wastes, a class of compounds considered completely in- nocuous. It is a paradox that urea synthesis, rather than purine synthesis, is the primary pathway for ammonia detoxification in these snails during estivation. Perhaps urea synthesis is a compromise between the toxicity of the terminal end-product and the loss of organic carbon and energy equivalents, both of which are greater in purine synthesis. Ecological implications of differential mortality during laboratory estivation Previous studies have demonstrated that species of land snail that are more tolerant of desiccation under laboratory conditions are typically found in more arid habitats in nature (Machin, 1967; Cameron, 1970: Arad el a/.. 1989). While the ecologies of O. strigosa and O. subrudis have not been studied in depth, our data describe the climatic conditions and the species distributions at three sites in western Colorado. Based upon summer temperatures, relative humidities, and precipitation, moisture avail- ability at these three sites decreases in the order Mitchell Creek > Rifle Falls > Rifle Gorge (see Materials and Methods; Table I). At the driest site, O. strigosa constitutes more than 60% of the snails collected (Fig. 7), suggesting that tolerance to prolonged estivation may influence the distribution of these species of land snail in nature. While O strigosa is the predominant species at the Rifle Gorge site, O. subrudis does constitute nearly 40% of the snails collected at this dry site. The survival of O subrudis, de- spite its lower tolerance to estivation under laboratory 1.00 0.00 MITCHELL CREEK RIFLE GORGE RIFLE FALLS Figure 7. Distribution of O. strigosa (open bars) and O subrudis (solid bars) at three collection sites in western Colorado. Data from the present study have been pooled with previous work (Rees. 1988; and unpub. obs.). Sample sizes were 157, 103, and 1 15 snails from the Mitchell Creek, Rifle Gorge, and Rifle Falls collection sites. At each site, the species proportions are significantly different from a uniform distribution (G-tests, P < 0.05). conditions, may be related to selection of moister micro- habitats, as described for land snail species of the Middle Eastern deserts (Arad ct ai. 1989). The other two sites were more mesic, and they were dominated by either O. strigosa (Mitchell Creek) or O. subrudis (Rifle Falls) (Fig. 7). Because the potential for desiccation stress is probably lowest at the Mitchell Creek site, the low abundance of O. subrudis at this site cannot be attributed to a lower tolerance of desiccation. Rather, other factors, either physical (e.g.. calcium availability) or biological (e.g., differential predation, different food preferences, or random effects associated with founding the colony), may explain their low numbers at the Mitchell Creek site. Similarly, factors other than desiccation tol- erance must be responsible for the low abundance of O. strigosa at the Rifle Falls site. Acknowledgments We would like to thank Mark Losleben (University of Colorado Mountain Research Station) and Drs. Jeffry Mitton and Cynthia Carey (Department of Environmen- tal, Population and Organismic Biology) for the loan of various pieces of equipment used in this study. We are grateful to the staff" of the Mitchell Creek and Rifle Falls Fish Hatcheries for allowing us to record climatic con- ditions at their facilities. Dr. Paul Yancey (Whitman Col- lege) is acknowledged for kindly performing the HPLC analyses of methylamine and polyol compounds. We also thank Dr. Michael Grant (EPO Biology) for statistical ad- vice and Dr. Shi-Kuei Wu (University of Colorado Mu- seum) for his help in locating populations of Oreohelix strigosa and O. subrudis. Financial support for this re- search was provided by the Kathy-Lichty Award for Graduate Student Research and a National Science Foundation Graduate Fellowship to BBR and NSF grants DCB-8702615 and DCB-90 18579 to SCH. Literature Cited Arad, 'L., S. Goldenberg, and .). 1 Idler. 1989. 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The utilization of foodstuffs and urea production by a land snail during estivation. Bio/. Bull. 144: 321-330. Home, F. R. I9