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ROCKET SCIENCE: 800 446 5967 olympusamerica.com/microscopes Cover Dinoflagellates are a diverse and ecologically im- portant group of unicellular protists. Some of them are free-living, photosynthetic or heterotrophic con- stituents of the plankton; others are symbiotic. Dinoflagellates in the genus SvnihioJiniiuu. com- monly called zooxanthellae, are intra- or intercellu- lar syrnbionts of diverse marine invertebrates, in- cluding foraminiferans, sponges, cnidarians. and molluscs. The image on the cover shows a throng of Symhio- i/iniiiiu ku\\'ci",iitii cells that were isolated from their symbiotic host, the Hawaiian stony coral Montipora cupitata ( = M. verrucosa) 1 ; the coral appears in the inset. In 1987, on the basis of cytological evidence, R. J. Blank 2 speculated that the vegetative cells of M. vermcosii are haploid; but this finding was never corroborated. The question of ploidy is important, Muraj>os, J. E. 1995. Revised checklist of extant shallow-water stony coral species from Hawaii (Cnidaria: Antho/oa: Scleructiniu). Bishop Museum Occasional Papers 42: 54-55. Blank, R. .1. 1987. Cell architecture of the dinotlagellate Symhio- diniwn sp. inhabiting the Hawaiian stony coral Montipora vcrnicosa. Mar. Biol. 94: 143-155. for it is central to our understanding of genome evolution and population genetics. Now. for the first time, the methods of molecular genetics have been applied to the problem of hap- loidy in dinoflagellates. In this issue of The Biolog- ical Bulletin (p. 10). Scott R. Santos and Mary Alice Coffroth report that vegetative cells of Symhio- iliniuin clade B symbiotic with gorgonians (sea fans and sea whips), and cultured cells from a range of hosts and locations, are haploid. Moreover, since Symbiodinium is monophyletic. S. kawagutii and other members of the genus must also be haploid. The Svnihiotlininiu cells on the cover are about 10 jam in diameter; they were photographed by Scott R. Santos (State University of New York at Buf- falo). The photograph of Montipora capitcita was taken by Frank Stanton (University of Hawaii) at a depth of about 1.5 meters; the coral is about 1 m in diameter. Materials and information for the cover and legend were provided by Fenny Cox (Univer- sity of Hawaii). The cover was designed by Beth Liles, Marine Biological Laboratory, Woods Hole, Massachusetts. THE BIOLOGICAL BULLETIN FEBRUARY 2003 Editor Associate Editors Section Editor Online Editors Editorial Board Editorial Office MICHAEL J. GREENBERG Louis E. BURNETT R. ANDREW CAMERON CHARLES D. DERBY MICHAEL LABARBERA SHINYA INOUE, Imaging and Microscopv JAMES A. BLAKE, Keys to Marine Invertebrates of the Woods Hole Region WILLIAM D. COHEN, Marine Models Electronic Record and Compendia PETER B. ARMSTRONG JOAN CERDA ERNEST S. CHANG THOMAS H. DIETZ RICHARD B. EMLET DAVID EPEL KENNETH M. HALANYCH GREGORY HINKLE NANCY KNOWLTON MAKOTO KOBAYASHI ESTHER M. LEISE DONAL T. MANAHAN MARGARET MCFALL-NGAI MARK W. MILLER TATSUO MOTOKAWA YOSHITAKA NAGAHAMA SHERRY D. PAINTER J. HERBERT WAITE RICHARD K. ZIMMER PAMELA CLAPP HINKLE VICTORIA R. GIBSON CAROL SCHACHINGER WENDY CHILD The Whitney Laboratory, University of Florida Grice Marine Laboratory. College of Charleston California Institute of Technology Georgia State University University of Chicago Marine Biological Laboratory ENSR Marine & Coastal Center, Woods Hole Hunter College. City University of New York University of California, Davis Center of Aquaculture-IRTA, Spain Bodega Marine Lab., University of California. Davis Louisiana State University Oregon Institute of Marine Biology, Univ. of Oregon Hopkins Marine Station. Stanford University Auburn University, Alabama Millennium Pharmaceuticals. Cambridge. Massachusetts Scripps Inst. Oceanography & Smithsonian Tropical Res. Inst. Hiroshima University of Economics. Japan University of North Carolina Greensboro University of Southern California Kewalo Marine Laboratory, University of Hawaii Institute of Neurobiology, University of Puerto Rico Tokyo Institute of Technology, Japan National Institute for Basic Biology, Japan Marine Biomed. Inst., Univ. of Texas Medical Branch University of California, Santa Barbara University of California, Los Angeles Managing Editor Staff Editor Editorial Associate Subscription & Advertising Administrator Published by MARINE BIOLOGICAL LABORATORY WOODS HOLE, MASSACHUSETTS http://www.biolbull.org CONTENTS VoLUMH 204, No. I: FEBRUARY 2003 SYMBIOSIS AND PARASITOLOGY Nixon, Julie E. J., Jessica Field, Andrew G. McArthur, Mitchell L. Sogin, Nigel Yarlett, Brendan J. Loftus. and John Samuelson Iron-dependent hydrogenases of Kntamix'bn lii^tnlytirn and (iitndin laniblia: activity of the recombinant ent- amoebic en/yme and evidence for lateral gene trans- fer . CELL BIOLOGY Santos, Scott R., and Mary Alice Coffroth Molecular genetic evidence that dinoflagellates be- longing to the genus Synibiodinium Freudenthal are haploid Coursey, Yvonne, Nina Ahmad, Barbara M. McGee, Nancy Steimel, and Mary Kimble Amebocyte production begins at stage 18 during em- bryogenesis in l.nnnlus /iiil\/>/ii'inii\. the American hoist-shoe nab . NEUROBIOLOGY AND BEHAVIOR 10 DEVELOPMENT AND REPRODUCTION Walker, Anna, Seichi Ando, and Richard F. Lee Synthesis of a high-density lipoprotein in the devel- oping blue crab (CaUinectes mpidus) 50 McBride, Richard S., and Paul E. Thurman Reproductive biology o( Hemiramphus brasiliensis and H. balao (Heniiramphidae): maturation, spawning frequency, and fecundity 57 Raskoff, Kevin A., Freya A. Sommer, William M. Ham- ner, and Katrina M. Cross ( lollection and culture techniques for gelatinous zoo- plankton 68 PHYSIOLOGY AND BIOMECHANICS Gainey, Louis F., James C. Walton, and Michael J. Greenberg Branchial musculature of a venerid clam: pharmacol- ogy, distribution, and innervation 81 ECOLOGY AND EVOLUTION Mann, Roger, and Juliana M. Harding Salinity tolerance of larval Rapnna venosa: implica- tions for dispersal and establishment of an invading predatory gastropod on the North American Atlantic coast. . 96 Buskey, Edward J., and Daniel K. Hartline High-speed video analysis of the escape responses of the copepod Acartni ttmvi to shadows 28 McGaw, I. J. Behavioral thermoregulation in Hem'tgi'tipsus mains. the amphibious purple shore crab 38 RESEARCH NOTE Aoyaina, Jun, Sam Wouthuyzen, Michael J. Miller, Ta- dashi Inagaki, and Katsumi Tsukainoto Short-distance spawning migration of tropical fresh- water eels 104 THE BIOLOGICAL BULLETIN THE BIOLOGICAL BULLETIN is published si\ limes a year by the Marine Biological Laboratory, 7 MBL Street, Woods Hole, Massachusetts 02543. Subscriptions and similar matter should be addressed to Subscription Administrator. THE BIOLOGICAL BULLETIN. Marine Biological Laboratory, 7 MBL Street, Woods Hole, Massachusetts (12543. Subscription includes both print and online journals. Subscription per year (six issues, two volumes): $280 for libraries; $105 for individuals. Subscription per volume (three issues): $140 for libraries; $52.50 for individuals. Back and single issues (subject to availability): $50 for libraries: $20 for individuals. Communications relative to manuscripts should be sent to Michael J. Greenberg. Editor-in-Chief, or Pamela Clapp Hinkle. Managing Editor, at the Marine Biological Laboratory. 7 MBL Street, Woods Hole, Massachusetts (12543. Telephone: (508) 289-714". FAX: 508-289-7422. E-mail: pclapp/ Entamoeba histolytica anil Trichomonas \aginalix Fe-hydrogencutes Sample Hydrogenase activity (nmol/min/mg of protein)'' Bacteria transformed with E. histolyticti Fe-hydrogenase Transfected E. hixiolytica with Fe- hydrogenase I ru humnmis Yu^nidlis T. viif>inalis + 0.47 mg E. histolyticti lysate" 36(2) 3.5 0.7 (3) 167 32 (41 114(2) a Averages +/ standard deviations, where possible. Number of deter- minations in parentheses. '"Calculated A", = 0.56 mg (amount of E. lii\in/yhm Usate in mg of protein to cause 50% reduction of 7". ru.i;im///.v hydrogenase activity). 1 2 3 SOObp the T. vaginalis Fe-hydrogenase was greater than that of the recombinant entamoebic GST-entamoebic Fe-hydrogenase and was inhibited by a lysate of non-transfected entamoebas (Table 1). This may explain why it was difficult to detect Fe-hydrogenase activity in lysates of nontransfected ent- amoebas, even though Fe-hydrogenase 1 mRNAs were identified from them by RT-PCR (next section). Cultured entamoehas and giurdiiifi exprexx mRNAs en- coding short Fe-hydrogenases. We isolated an fe-hydroge- nase gene of G. lanihlia. because we have frequently com- pared the fermentation enzymes of this diplomonad with those of E. hixtolytieu (Rosenthal et ul.. 1997; Field et ul., 2000; Nixon et ul., 2002). A search of the contigs predicted from the G. lamblia shogun sequences suggested that this gene, which predicts a short Fe-hydrogenase. is the only hydrogenase gene present within the giardial genome. Like the entamoebic Fe-hydrogenase 1. the predicted giardial Fe-hydrogenase lacked an N-terminal organelle-targeting sequence and had two ferredoxin-like iron-sulfur centers and a hydrogenase iron-sulfur center like those present in the short Fe-hydrogenases of T. vugimilis, Desulfovibrio sp., and Clostridiu sp. iCammark. 1992; Thompson et ai, 1994; Bui and Johnson. 1496; Homer el ul., 1996; Nicole! et ai. 1999). RT-PCR showed that cultured entamoebas and giar- dias contain mRNAs, which encode short Fe-hydrogenases (Fig. 1 A, B). Negative controls without RT showed that the RT-PCR was not amplifying DNA from the extracts of cultured entamoebas and giardias. Because the giardial con- tigs predicted only one Fe-hydrogenase, which is expressed, it is likely that the hydrogenase activity recently detected in cultures of giardias derives from this enzyme (Lloyd and Harris. 2002). In contrast, entamoebas appear to have a second long hydrogenase (see next section), so if entamoe- bic hydrogenase activity is present, it might derive from one or more enzymes. These results suggest the possibility that entamoebas and giardias use protons as electron acceptors 3 4 SOObp B Figure 1. Agarose gels of ethidium-stained RT-PCR products from entamoebic and giardial mRNAs. Images are reversed for clarity of reproduction. (A) RT-PCR of amoebic mRNAs encoding malic enzyme (lane 1). Fe-hydrogenase (lane 2). alcohol dehydrogenase E (lane 3). and ferredoxin (lane 4). Size markers are shown in lane 5. Lanes 6-9 are the negative controls for malic enzyme, hydrogenase. ADHE. and ferredoMii. respectively. (B) RT-PCR of giardial mRNAs encod- ing ADHE (lane 3). Fe-hydrogenase (lane 4). and malic enzyme (lane 5). A negative control (no RT) tor Fe-hydrogenase is shown in lane d. A positive control for Fe-hydrogenase. using Giardia lumhlia WB strain DNA. is shown in lane 2. Size markers are shown in lane I. ACTIVITY AND ORIGIN OF THE ENTAMOEBIC AND GIARDIAL Ft^-HYDROGENASES Eh2 MSTQLTPLRNKIISEWKCFKSGRFIEDIDKLPTILTDGDGWKPTSKFVHSREQEEGIYR Td IKREILVRIAKLQFEGKLQEGVHYIPREMVPRN.STPI.RCCIFHDR. .EIMR Bf VRHKLLAKLVNLWKENKLTNEIDRLPIELSPRR.SRPLGRCCIHKER. .AVYK Eh2 EKVLSVLGF.VDGEYDDITPLHVYAQKALERT.SLHEPVFGISQKGCNKCHFNGYFVTQA Td HRVIARLGCSLENYDEEKT . LAQFAKEALERE . KPTWPMLTVLDEACNACVKSKYMITNA Bf YKLFPLLGFDMTDETDELTSLSEYARQALERKNKQKENILCVIDEACSSCVQVNYEVTNL Eh2 CEGCTSRPCSVNCPKKCISFGEDGRAVINQNNCIKCGRCYKFCPYGAIISKSVPCVKACP Td CQACVARPCMMNCPKTAIAIS . GGRARIDEEKCINCGICLKNCPYHAVIKIPVPCEEACP Bf CRGCVARSCYMNCPKDAIRFRKNGQAKIDHDACISCGKCHQSCPYHAIVFIPVPCEEACP Eh2 CGAMLDSPEGVKTIDFEKCINCGGCMRACPFGAILPRSNLIDVLK. ILPTKKVVACPAPS Td VGAISKDENGKERIDYHKCIFCGNCMRECPFGAMMDKGQIVDVIKHLMSGKKVSALYAPA Bf VKAISKDENGIEHIDESKCIYCGKCLNACPFGAIFEISQAFDVLEGIRSGEKMIAIPAPS X x Eh2 IAAHFGKYDLALVSGGLIQVGFTSVEDVSYGADLCALNEAKEFEERIVKNKKDFMTTSCC Td VAAQF . KAVPGQLESALKKAGFNKVWEVAIGADITADREASEFEERMEHGHI . LMTTSCC Bf ILGQF. NTSIEAVYGALRQMGFADWEVAQGAMDTVSHEAAELKEKLEEGQP . FMTTSCC o Eh2 PAYINAINKHMPELKENVSHTPTPMHFATQAVKDRDQETVTVFIGPCNAKRWETLQDSTT Td PAYVRAVKKHVPALVPCISDTRSPMHYTAELAKKEDPDCVTVFIGPCLAKRREGLEDEFV Bf PSYIELVNKHIPGMKPYVSSTGSPMYYAARIAKERHPDAKIVFIGPCVAKRKEARRDECV o Eh2 DYCLTFDEIFGLFEGSGIDLSKVQPYTFVDKAHKEGKIFAVSGGVASAVASLLPKEVPDG Td DYVLSIEELGALLTAKEIDISKEEALPGKITPTSSGRGFAASGGVAEAVRVRL.KKPEN. Bf DYILTFEEMASIFEGLDIQLEQTQPFSVLYTSVREAHGFAQAGGVMGAIKAYLGEEAKK. Eh2 VIKPTIIDGFSQENFKRLKNFKKNI TGNLVEVMVCEGGCAYGPGCPGLNTP Td . LRPVLINGLNKEGMKQLASYGKIQSGELPHDSSTPNLVEVMSCEGGCIGGP BF . FSAIQVSDLNKKNIGLLRAAAKTG KAQGQFIEVMACEGGCISGP o o Eh2 ATSAKIKIAVDKMEAHPEGRWVGLPNSQIKPIKVEN 504 Figure 2. Alignment of the predicted Entumnebu liixralvticu long Fe-hydrogenase 2 (Eh2) with predicted long Fe-hydrogenases ot Treponema ilcniici>lu (Td) and Bacteroides fi'ii^His (Bf). Conserved Cys residues, which are shaded, include those that coordinate putative |4Fe-4S] iron-sulfur centers (marked with x's) and those that coordinate putative hydrogenase iron-sulfur centers (marked with o's). Other conserved Cys residues, which may be involved in coordinating iron-sulfur centers, are marked with asterisks. Amino acids at the beginning and end of the conserved Fe-hydrogenase domain are underlined. when the organisms are growing under strictly anaerobic conditions in the bowel lumen (Brown et ai. 1998; Huston and Petri. 2001; Lloyd and Harris, 2002). E. histolytica has a hydrogenase 2 gene encoding a long Fe-hydrogenase. The assemblies of the shotgun sequences of the E. histolytica genome predicted a long Fe-hydroge- nase 2 (Fig. 2) in addition to the short Fe-hydrogenase 1. The entamoebic Fe-hydrogenase 2 was 504 amino acids long and had an N-terminal sequence, which included pos- itively charged Lys and Arg that are often present at the N-termini of organellar proteins (Claros and Vincens, 1996). In addition, the N-terminus of Fe-hydrogenase 2 contained Ser and Leu residues, which are present at the N-termini of crypton and hydrogenosomal proteins (Bui et til.. 1996; Mai et /., 1999). However, in the absence of experimental evidence, we cannot be sure that the entamoe- bic long Fe-hydrogenase is targeted to the crypton. The entamoebic Fe-hydrogenase 2 was much more sim- ilar (>38% amino acid identities) to predicted long Fe- hydrogenases of Bucteroides frag His and Treponema den- ticola than to short Fe-hydrogenases of entamoebas. giar- dias. trichomonads. and other anaerobic bacteria (<28% amino acid identities; Fig. 2). The entamoebic Fe-hydroge- nase 2 and the predicted long Fe-hydrogenases of B. fragilis NIXON ET AL. 96/95 |C. reinhardtii 1 [C. reinhardtii 2\ 96/1 OO T. maritima 1 |P/romycessp.| | N. frontalis\ 99/100 */100 80/65 T. gallinae] L \T. vaginalis 2\ T. vaginalis 3 \T. vaginalis 4\ M. elsdenii T. maritima 2 100/100 |S. barkhanus] 99/691 IG. lamblia \ |E histolytica 1\ 95169 96/99 */99 T. tengcongensis 1 T. maritima 3 T. denticola B. fragilis R. albus C. perfringens 1 C. acetobutylicum 98/100 c~ C. perfringens 2 D. vulgaris 1 r V. tengcongensis 2 "jr C. thermocellum * E. acidaminophilum 98/100 64/* C. saccharobutylicum C. pasteurianum C. perfringens 3 I/V. ovalisl D fructosovorans D. desulfuricans 1 D. vulgaris 2 D. desulfuricans 2 0.1 ACTIVITY AND ORIGIN OF THE ENTAMOEBIC AND GIARDIAL FE-HYDROGENASES and T. dcnticola each contained Cys residues that likely coordinate two ferredoxin-like [4Fe-4S] iron-sulfur centers (marked with x's in Fig. 2) and hydrogenase iron-sulfur centers (marked with o's). which have previously been identified in structures of short and long Fe-hydrogenases (Peters et ai. 1998; Nicolet et ai, 1999). In addition, the predicted entamoebic Fe-hydrogenase 2 had eight other N-terminal Cys residues, which aligned with those of the bacteroides and treponema long Fe-hydrogenases (marked with asterisks). Although these Cys residues probably co- ordinate other iron-sulfur centers, they remain unidentified, because they do not align with the N-terminal iron-sulfur centers of the long Fe-hydrogenase of C. pastenriannni. which has been crystallized (Peters et a/.. 1998). The entamoebic and giardial short fe-hydrogenase I genes appear to share a common ancestn: while the ent- tiinoehic lout; fe-hydrogenase 2 gene appears to have been laterally transferred from a prokaryote. Phylogenetic trees of Fe-hydrogenases from eubacteria and eukaryotes are star-shaped and contain few basal nodes that are strongly supported (Fig. 3). This result suggests that the Fe-hydro- genases are widely divergent and that little phylogenetic signal remains. For example, Fe-hydrogenases of closely related eukaryotes either trichomonads, green algae (Chlamydomonas reinhanltii, Scenedesinus obliqiiiis. and Chlorella fused), or chytrid fungi (Piromyces sp. and Neo- callimastix frontalis) each grouped together, but Fe-hydrog- enases of unrelated eukaryotes did not group together. In particular, our analysis does not support recent conclusions that hydrogenases of trichomonads are monophyletic with those of chytrid fungi ( Voncken et at., 2002) or with those of E. histolytica and 5. barkhanus (Horner et cii, 2000). The short fe-hydrogenase genes of G. Iambi ia, S. barkha- nus, and E. histolvtica appear to share a most recent com- mon ancestry, although a particular bacterial donor was not identified. Remarkably, the short Fe-hydrogenase of G. Iain- Miii was more similar to that off. histol\ticu than to that of S. harkhaims. Because G. lumblia and S. barkhanus are diplomonads, which share a recent common ancestor in phylogenetic trees of rRNA and proteins (Sogin and Silber- man, 1998), a possible explanation of these results is that the E . histolytica fe-hydrogenase gene was laterally trans- ferred from a diplomonad (Rosenthal et ai. 1997; Doolittle, 1998, 1999; Miiller, 1998; de Koning ft til.. 2000; Field et al.. 2000; Nixon et ai. 2002). This lateral gene transfer would not have occurred recently, because the Fe-hydroge- nases of entamoebas and giardias showed only a 40% amino acid identity with each other, and each fe-hydrogenase gene has the codon usage of its host. Alternatively, the diplomonad-E. histolytica sub-clade could be incorrectly rooted by the long branch connecting it to the remainder of the tree. The common ancestry of genes encoding the E. histo- lytica long Fe-hydrogenase 2 and those of B. fragilis and T. denticola is strongly supported. This appears then to be an example of lateral gene transfer, as Entamoeba is not a close relative of either of these eubacteria (Rosenthal et ill., 1997; Doolittle. 1998, 1999; Muller. 1998; de Koning et ai. 2000; Field et ai, 2000; Nixon et ai, 2002). There was weak support for the pairing of Fe-hydrogenases of the ciliate N. avails and Desulfavibrio sp., as has been previously noted (Horner et ai. 2000; Voncken et ui, 2002). This suggests that the ciliate hydrogenase was derived by lateral gene transfer, but does not prove it. Conclusions This is the first time that an Fe-hydrogenase from a protist has been expressed as a GST-fusion protein in bacteria. This is also the first time that an fe-hydrogenase gene (encoding the long hydrogenase of entamoebas) has been inferred to have been laterally transferred from a bacterium, although numerous genes encoding fermentation enzymes (e.g.. al- cohol dehydrogenases, malic enzyme, and acetyl-CoA syn- Figure 3. Phylogenetic relationships of Fe-hydrogenases, inferred using a distance matrix generated by the Dayhoff+I + r model and the Fitch-Margoliash algorithm. Bootstrap values obtained using PUZZLEBOOT and PROTPARS, respectively, are shown at the relevant nodes. Bootstrap values below 50% are marked with an asterisk if the other bootstrap value is >50%. If both bootstrap values are below 50%, neither is marked. The scale bar indicates estimated sequence divergence per unit branch length. Sequences from eukaryotes. which are boxed, were from Chlamydomonas reinhunltii 1 and 2 [accession # 16945126 and 18026272]: Chlorella fusca [21732235]; Enuinioebti histolytic.i 1 and 2 [9963974 and AY172963]; Giardia Uimhlin [13506793]: Neocal- limastix fruntalis [19547863]: Nyctnlherus avulis [4034791]; Piromyces sp. [19548180]: Scenedesinus obliquus 1 and 2 [12581498 and 13311187]; Spinmitcleiix harkhamis [11127703]; Trichoimmas gallinae [19548182]; Trk-hiinimiiix vuximilix 1.2, 3, and 4 [19547859. 1 1 127701. I 171 1 17. and 1345094]. Eubacterial sequences were from Bacteroides fragilis [unfinished microbial database]; Closlridium acetobutylicum [15896476]; Clostridiitm fiiiMcnrUmiim [557064]; Chsiridiiini pcrfrin^ns I. 2. and 3 [18311557, 18309258. and 1831 1328]; Closlridium saccharobutylicum [488597]; Clo.ilridiiim thermncellnm [4927278]; Desulfavibrio fructosovorans [1914864]; Dfsiilfovibrio destilfiiricans 1 and 2 [4930044 and 13022069]; Desulfovihrio vulgaris 1 and 2 [97381 and 66319, respectively]; Eiihucieriiini acidominophilum [14250935); Megasphaera elsdenii [66509851; Ruminococcui albus [unfinished microbial database); Theriiuiaiwerubiicter teiif-coiitn'iM* 1 and 2 [20807184 and 20515894]; Thermotoga marilimu 1. 2. and 3 [15644177, 7433127. and 4981985]; and Treponema Jenticnlii [unfinished microbial database]. 8 NIXON ET AL. thases) appear to have been laterally transferred from pro- karyotes to amoebas and giardias (Rosenthal et oi. 1997; Field et til.. 2000; Nixon et til.. 2002). Although the evi- dence is weak, this may also be the first time that a gene (encoding the short hydrogenase of entamoebas) has been inferred to have been laterally transferred from another protist. Because the hypothesized lateral gene transfer would probably have occurred after the acquisition of the fe-hydrogenase gene by the diplomonad lineage, this par- ticular result does not disprove the hydrogen hypothesis (Martin and Miiller, 1998). However, the failure to demon- strate that the eukaryotic Fe-hydrogenases share a common ancestry, or to identify an a-proteobacterial donor for these eukaryotic fe-hydrogenase genes (Horner et ai. 2000), dampens our enthusiasm for the hydrogen hypothesis. These results suggest that the mitochondria! endosymbiont was selected for a property other than hydrogen production (e.g.. its ability to consume oxygen) (Andersson and Kur- land, 1999) and that the presence of Fe-hydrogenases and other fermentation enzymes of microaerophilic eukaryotes may reflect a secondary adaptation to their anaerobic envi- ronment (Rosenthal et til.. 1997; Doolittle, 1998, 1999; de Koning et til., 2000; Field et til.. 2000; Lloyd and Harris, 2002; Nixon et til.. 2002). Acknowledgments This work was supported by NIH grants ( AI33492 to J.S., AI43273 to M.L.S.. and AI46516 to B.J.L.). Literature Cited Altschul, S. F., T. L. Madden, A. A. Schaffer, J. Zhang, Z. Zhang, W. Miller, and D. ,). Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25: 3389-34(12. Andersson, S. G., and C. G. Kurland. 1999. Origins of mitochondria and hydrogenosomes. (.'HIT. Opin. Microhiol. 2: 535-541. Bapteste, E., H. Brinkmann. J. A. Lee, D. V. Moore, C. \V. Sensen, P. Gordon, I,. Duruflc, T. Gaasterland, P. Lopez, M. Miiller. and H. Philippe. 20(12. The analysis of 100 genes supports the grouping of three highly divergent amoebae: DictytKtcliiini. Eniinnnchii. and A/II.V- tif>innoehti. Piot. Nail. Acini. Sci. USA 99: 1414-141'). Bradley, P. J., C. J. Lahti, E. Plumper, and P. J. Johnson. 1997. Targeting and trunslocation of proteins into the hydrogenosome of the prolix! Tnehoiiionii\: similarities with mitochondria! protein import. EMBO J.\f>: 3484-3443. Brown, D. M., J. A. Upcroft, M. R. Edwards, and P. Upcroft. 1998. Anaerobic bacterial metabolism in the ancient eukaryote Gil\phei>nis embryos consist of relatively few cell types. In the ventral region and the growing appendages, the epider- mal cells are typically columnar in shape, while the under- lying mesodermal cells are flattened in appearance. Coelo- mic cavities have formed within some of the developing appendages, and within these we occasionally observe cells that have a fibroblast-like appearance. Also within the ven- tral plate are occasional cells having dark-staining cytoplas- mic inclusions. These cells are most likely muscle precur- sors. In later stage embryos and larvae, cells with similarly stained inclusions are often seen adjacent to developing muscles. As one moves dorsally away from the ventral plate, there is a gradual transition in the epidermal cells from columnar through cuboidal to a flattened appearance. Similarly, the mesodermal cell layers in the dorsal region are very thin flat sheets that are often difficult to discern. By stage 18, the extension of the mesoderm over the central yolk mass appears to be complete or nearly so. The central region of the embryo is filled with yolk, within which are distributed numerous yolk nuclei. No evidence of internal organs is seen at this stage, although hemocoel cavities have begun to form. The cavities are located between the meso- dermal cell layers, and within the cavities granular pro- amebocytes and elements of connective tissue are fre- quently observed. Also located in the dorsal regions of the embryo are cells that appear to be producing chitin-like material (based on the staining properties of the material). Finally, as mentioned before, the lateral organs, composed of distinct goblet-shaped cells, have begun to develop. Thus we are able to identify at most seven to eight distinct cell types in the stage 18 L polyphemus embryos. How this compares with T. triilenUitns embryos at the same stage of development remains to be determined. Do the yolk nuclei represent u pool of multipotent cell precursors? In contrast to many arthropods, horseshoe crabs retain significant numbers of yolk nuclei after cellular blastoderm formation (Kishinouye. 1893; Kingsley, 1892, 1893; Kimble et al., 2002). The yolk nuclei persist throughout embryonic development. During the mid- to late stages of embryogenesis. some yolk nuclei probably function as vitel- lophages. After hatching, the residual yolk is incorporated into the developing midgut and digestive diverticulum. a network of blind-end caeca that extends throughout the prosoma. We have previously shown that some of the re- sidual yolk nuclei cellularize to form the columnar epider- mal lining of the digestive caeca, while others form a layer of flattened cells that surround the individual caeca (Kimble et til.. 2002). In most arthropod species, the yolk nuclei or yolk cells function only as vitellophages, degenerating before the end of embryonic development (Anderson, 1973; Campos-Or- tega and Hartenstein, 1997). In the terrestrial chelicerates, spiders and scorpions, most of the cleavage nuclei partici- pate in blastoderm formation. Subsequently some cells re- populate the yolk mass, where they function as vitello- phages. Eventually the vitellophages migrate to the surface of the yolk mass and form the endoderm epithelium (Ander- son. 1973). Thus, a role for the yolk nuclei or vitellophages in formation of the gut endoderm appears to be common to most if not all chelicerates. However, participation in for- mation of the mesodermal components of the gut is appar- ently unique to the Xiphosura. If. as suggested here, some yolk nuclei cellulari/.e and differentiate as amebocytes dur- ing late embryogenesis, it would suggest that retention ot large numbers of yolk nuclei in horseshoe crab embryos provides the embryos with a pool of undetermined nuclei thai can be utili/ed in a variety of distinct tissues during development. Acknowledgments We thank undergraduate students Evelyn Wurth, Carrie Ottoson, Nicole Tremblay, Kimberly Demon. Patrick Mella, AMEBOCYTES IN L1MVLUS EMBRYOS 27 and Victoria Davis for assistance with sectioning and stain- ing of the embryos. These studies were supported in part by grants to MK from the USF Research Council, and from NOAA, Office of Sea Grant, Department of Commerce. Grant # NA76RG-0120. The U.S. government is authorized to produce and distribute reprints for governmental pur- poses not withstanding any copyright that may appear hereon. YC was supported by grants/scholarships from Sigma Xi. Sigma Delta Epsilon Graduate Women in Sci- ence. Aylesworth/Old Salt, Sea Space, The American As- sociation of University Women, and the Florida and Tampa Garden Clubs. NA was supported in part by the McNair Post-Baccalaureate Achievement Program. Literature Cited Agarwala, K. L., S. Kawahata, Y. Miura, Y. Kuroki. and S. Iwanaga. 1996. Limulus intracellular coagulation inhibitor type 3. Purification, characterization. cDNA cloning, and tissue localization. J. Biol. Chem. 271: 23,768-23.774. Anderson, D. T. 1973. Embryology und Phytogeny in Annelids and Arthropods. Pergamon Press. New York. 495 pp. Armstrong, P. B. 1985. Adhesion and motility of the blood cells of Limulus. Pp. 77-124 in Blood Cells of Marine Invertebrates: Experi- mental Systems in Cell Biology and Comparative Physiology, W. D. Cohen, ed. Alan R. Liss. New York. Armstrong, P. B., and F. R. Rickles. 1982. Endotoxin-induced degran- ulation of the Limulus amebocyte. Exp. Cell Res. 140: 1 5-24. Bang, F. B. 1956. A bacterial disease of Limulus po/vphemus. Bull. Johns Hopkins H,,sp. 98: 325-351. Bang, F. B. 1979. Ontogeny and phylogeny of response to gram-negative endotoxins among the marine invertebrates. Pp. 109-123 in Biomedi- cal Applications of the Horseshoe Crab (Limulidae), W. D. Cohen, ed. Alan R. Liss. New York. Campos-Ortega, J. A., and V. Hartenstein. 1997. The Embrvonu Development of Drosophila melanogaster. 2nd ed. Springer-Verlag. Berlin. 405 pp. Dumont, J. N., E. Anderson, and G. Winner. 1966. Some cytologic characteristics of the hemocytes of Limulus during clotting. J. Morphol. 119: 1X1-208. Hilly, J. B., and D. G. Gibson. 1989. Culture of amebocytes on opened gill lamellae of the horseshoe crab. Limulus po/yphemu.s. (abstract). Am. Zoo/. 29: 1 1 2A. Iwanaga, S. 20(12. The molecular basis of innate immunity in the horse- shoe crab. Curr. Opin. Immunol. 14: 87-95. Kimble, M., Y. Course), N. Ahmad, and G. W. Hinsch. 2002. Behav- ior of the yolk nuclei during embryogenesis, and development of the midgut diverticulum in the horseshoe crab. Limulus polyphcmiis. In- vcrtcbr. Biol. 121: 365-377. Kingsley, J. S. 1892. The embryology of Limulus. ./. Morphol. 1: 35-68. Kingsley. J. S. 1893. The embryology of Limn/us. Part II. ./. Morphol. 8: 195-268. Kishinouye, K. 1893. On the development of Limulus longispina. ./. Coll. Sci. Imp. Univ. Japan 5: 53-100. Levin, J. 1985a. The history of the development of the Limulus amebo- cyte lysate test. Pp. 3-28 in Bacterial Endotoxins: Structure. Biomed- ical Significance, and Detection with the Limulus Amebocyte Lysate Test. Prog. Clin. Biol. Res. 189. Levin, J. 1985b. The role of amebocytes in the blood coagulation mech- anism of the horseshoe crab Limulus polvphcnn/s. Pp. 145-163 in Blood Cells of Marine Invertebrates: Experimental Svstems in Cell Biology and Comparative Physiology, W. D. Cohen, ed. Alan R. Liss. New York. Liang, P., T.-K. Cheng, Y.-Q. Wu, and W.-H. Wu. 1990. Ultrastruc tural observations on hemocytopoiesis in embryos of the horseshoe crab. Tachvpleus tridenlatus. Proceedings of the XII International Congress of Electron Microscopy. August 12-18, 1990, Seattle, WA. 3: 506-507. Miura, Y., S.-I. Kawabata, Y. Wakamiya, T. Nakamura, and S. Iwanaga. 1995. A Limulus intracellular coagulation inhibitor type 2. Purification, characterization. cDNA cloning, and tissue localization. J. Biol. Chen,. 270: 558-565. Miyata, T., M. Hiranaga, M. Umezu, and S. Iwanaga. 1984. Amino acid sequence of the coagulogen from Limulus po/yphemnx hemocytes. J. Biol. Chem. 259: 8924-8933. Ornberg, R. L. 1985. Exocytosis in Limulus amebocytes. Pp. 127-142 in Blood Cells of Marine Invertebrates: Experimental Systems in Cell Biology and Comparative Physiology. W. D. Cohen, ed. Alan R. Liss, New York. Sawada, T., and S. Tomonaga. 1996. The immunocytes of protostomes and deuterostomes as revealed by LM. EM and other methods. Adv. Comp. Environ. Physiol. 23: 9-40. Sekiguchi, K. 1973. A normal plate of the development of the Japanese horseshoe crab, Tachvpleus tridentatits. Sci. Rep. Tokvo kvoikti Daigaku Sect. 15: 153-162. Sekiguchi. K. 1988. History of the study. Pp. 1-9 in Biology of Horse- shoe Crabs, K. Sekiguchi. ed. Science House. Tokyo. Sekiguchi, K., Y. Yamamichi, and J. D. Costlow. 1982. Horseshoe crab developmental studies. 1. Normal embryonic development of Limulus polyphemu.s compared with Tachvp/ens tndentatiis. Pp. 53-73 in Physiology and Biology of Horseshoe Crabs: Studies on Normal and Environmentally Stressed Animals, J. Bonaventura. C. Bonaventura. and S. Tesh, eds. Alan R. Liss, New York. Sekiguchi, K., Y. Yamamichi, H. Seshimo, and H. Sugita. 1988. Nor- mal development. Pp. 133-181 in Bio/ogv of Horseshoe Crabs, K. Sekiguchi, ed. Science House. Tokyo. Yeager, J. F., and O. E. Tauber. 1935. On the hemolymph cell counts of some marine invertebrates. Biol. Bull. 69: 66-70. Reference: Bio/. Bull. 204: 2X-37. (February 2003) 2003 Marine Biological Laboratory High-Speed Video Analysis of the Escape Responses of the Copepod Acartia tonsa to Shadows EDWARD J. BUSKEY 1 * AND DANIEL K. HARTLINE 2 1 Marine Science Institute. 750 Channel View Drive, Port Aransas, Texas 78373: and ' Bekesv Laboratory of Neurobiology, Pacific Biomcdical Research Center. Universitv of Hawaii at Manoa. 1W3 East-West Road. Honolulu. Hawaii Vf>S22 Abstract. The copepod Acartia tonsa exhibits a vigorous escape jump in response to rapid decreases in light intensity, such as those produced by the shadow of an object passing above it. In the laboratory, decreases in light intensity were produced using a fiber optic lamp and an electronic shutter to abruptly either nearly eliminate visible light or reduce light intensity to a constant proportion of its original inten- sity. The escape responses of A. tonsa to these rapid de- creases in visible light were recorded on high-speed video using infrared illumination. The speed, acceleration, and direction of movement of the escape response were quan- tified from videotape by using automated motion analysis techniques. A. tonsa typically responds to decreases in light intensity with an escape jump comprising an initial reori- entation followed by multiple power strokes of the swim- ming legs. These escape jumps can result in maximum speeds of over 800 mm s~' and maximum accelerations of over 200 m s~ 2 . In .4. tonsa. photically stimulated escape responses differ from hydrodynamically stimulated re- sponses mainly in the longer latencies of photically stimu- lated responses and in the increased number of power strokes, even when the stimulus is near threshold; these factors result in longer escape jumps covering greater dis- tances. The latency of responses of A. tonsa to this photic stimulus ranged from a minimum of about 30 ms to a maximum of more than 150 ms, compared to about 4 ms for hydrodynamically stimulated escape jumps. Average re- sponse latency decreased with increasing light intensity or increasing proportion of light eliminated. Little change was Received 7 June 2(102: accepted 26 November 2002. To whom correspondence should he addressed. H-m;iil: buskeyfs'utmsi. utexas.edu observed in the vigor of the escape response to rapid de- creases in visible light over a wide range of adaptation intensities. Introduction Planktonic copepods are an important link in marine food webs between microplankton and higher trophic levels. Copepods are well known for their vigorous escape re- sponses (e.g., Singarajah. 1969; Fields and Yen, 1997). which play an important role in predator avoidance (e.g.. Drenner et a/.. 1978; Viitasalo et al.. 1998). These escape responses can be elicited by both hydrodynamic (Hartline et al.. 1999; Kiorboe et al.. 1999; Lenz and Hartline, 1999) and photic stimuli (Buskey et al., 1986. 1987). Despite the scarcity of direct evidence that chemosensory stimuli, by themselves, can produce vigorous escape responses in cal- anoid copepods, there is evidence that such stimuli can cause copepods to exhibit changes in swimming activity (e.g.. Katona, 1973; Buskey, 1984). Predator-specific chem- icals have also been shown to alter vertical migration be- havior in freshwater zooplankton (e.g., Tjossem, 1990; Rin- gelberg, 1991) and marine crab larvae (Forward and Rittschof, 2000). but similar effects are yet to be demon- strated in marine copepods (e.g.. Bollens et al.. 1994). Light has long been known to have an important effect on the behavior of planktonic organisms, with much research emphasizing the effects of light on vertical migration (re- viewed in Forward, 1988). Photophobic responses of cope- pods are thought to play a role in planktonic predator-prey interactions both in terms of a predator-deterrent role of bioluminescence in dark-adapted copepods (Buskey and Swift. 1983, 1985) and in terms of a predator-avoidance role for copepods exposed to shadows in light-adapted copepods (Buskey et a/.. 1M86). Alterations in behavior of planktonic COPEPOD ESCAPE RESPONSES TO SHADOWS organisms in response to decreases in light intensity have been demonstrated in neritic calanoid copepods (Buskey et al.. 1987) and crab larvae (Forward. 1977). Recent studies have used strain gauges and high-speed video to provide the high temporal resolution necessary to describe, in detail, the kinetics of the escape responses of both tethered (Len/ and Hartline. 1999; Hartline H.M' parameters t<"' tululi A'mii/o ami malc\ <>/ Acartia lonsa cv/xwJ Jo plume Miniiili Parameter Females Males P Latency (ins) 68.2(1.8:29-159) 62.2(1.3:35-138) / Inverichrutes. W.H. Freeman. San Francisco. 1714 pp. (Ch. 19). Burkenroad, M. D. 1943. A possible function of bioluminescence. J. Mar. Res. 5: 161-164. Buskey, E. J. 1984. Swimming pattern as an indicator ul the roles of copepod sensory systems in the recognition ol fond. Mar. Biol. 79: 165-175. Buskey, K. .1. 1993. Annual pattern of micro- and mesozooplankton abundance and biomass in a subtropical estuary. J. Plankton Res. 15: 9(17-924. Buskey, E. J., and E. Swift. 1983. Behavioral responses of the coastal copepod Acurtiu Inidsonica to simulated dinoflugellale hiolumines- cence. / F..\p. Mar. Hi, !. '>/. 72: 43-5S. COPEPOD ESCAPE RESPONSES TO SHADOWS 37 Buskey, E. ,)., and E. Swift. 1985. Behavioral responses of oceanic zooplankum lo simulated bioluminescence. Biol. Bull, 168: 263-275. Buskey. E. J., L. Mills, and E. Swift. 1983. The effects of dinoflagellate bioluminescence on the swimming behavior of a marine copepod. Limnol. Oceanogr. 28: 575-579. Buskey, E. J., C. G. Mann, and E. Swift. 1986. The shadow response of (he estuarine copepod Acania tonsa. J. .v/>. Mar. Biol. Ecol. 103: 65-75. Buskey, E. J., C. G. Mann, and E. Swift. 1987. Photophobic responses of calanoid copepods: possible adaptive value. ./. Plankton Res. 9: 857-870. Buskey, E. J., P. H. Lenz, and D. K. Hartline. 2002. Escape behavior of planktonic copepods to hydrodynamic disturbances: high speed video analysis. Mar. Ecu/. Prog. Ser. 235: 135-146. Camhi. J. M.. and T. G. Nolen. 1981. Properties of the escape system of cockroaches during walking. J. Coinp. Phvsiol. 142: 339-346. Davis, A. D., T. M. Weatherby, D. K. Hartline, and P. H. Lenz. 1999. Myelin-like sheaths in copepod axons. Nature 398: 571. Drenner, R. W., J. R. Strickler, and W. J. O'Brien. 1978. Capture probability: the role of zooplankton escape in the selective feeding of planktivorous fish. J. Fish. Res. Board Can. 35: 1370-1373. Eaton, R. C., R. A. Bombardier!, and D. L. Meyer. 1977. The Mauthner-initiated startle response in teleost fish. J. Ev/>. Biol. 66: 65- M. Eaton, R. C.. R.K.K. Lee, and M. B. Foreman. 2001. The Mauthner cell and other identified neurons of the brainstem escape network of fish. Prog. Neumbinl. 63: 467-485. Elofsson, R. 1966. The nauplius eye and frontal organs of the non- Malacostraca (Crustacea). Sarsia 25: 1-128. Fields, D. M., and J. Yen. 1997. The escape behavior of marine cope- pods in response to a quantifiable fluid mechanical disturbance. / Plankton Res. 19: 1289-1304. Forward, R. B., Jr. 1977. Occurrence of a shadow response among brachyuran larvae. Mar. Biol. 39: 331-341. Forward, R. B., Jr. 1988. Diel vertical migration: zooplankton photo- biology and behavior. Oceanogr. Mar. Biol. Annu. Rev. 26: 361-393. Forward, R. B., Jr., and D. Rittschof. 2000. Alteration of photore- sponses involved in diel vertical migration of a crab larva by fish mucus and degradation products of mucopolysaccharides. J. E.\p. Mar. Biol. Ecol. 245: 277-292. French, A. S. 1992. Mechanotransduction. Annu. Rev. Phvsiol. 54: 135- 152. Goldsmith, T. H. 1991. Photoreception and vision. Pp. 171-245 in Neural and Integrative Animal Physiology. C.L. Prosser, ed. Wiley- Liss, New York. Hartline, D. K., E. J. Buskey. and P. H. Lenz. 1999. Rapid jumps and bioluminescence elicited by controlled hydrodynamic stimuli in a me- sopelagic copepod. Pleuromamma xiphias. Biol. Bull. 197: 132-143. Hess, K., and F. White. 1974. A numerical tidal model of Narragansett Bay. Sea Grant Marine Technical Rep. 20. University of Rhode Island. Kingston. RI. Katona, S. K. 1973. Evidence for sex pheromones in planktonic cope- pods. Limnol. Oceanogr. 18: 574-583. Kiarboe, T., and A. Visser. 1999. Predator and prey perception in copepods due to hydromechanical signals. Mar. Ecol. Prog. Ser. 179: 81-95. Kiorboe, T., E. Saiz, and A. Visser. 1999. Hydrodynamic signal per- ception in the copepod Acania tonsa. Mar. Ecol. Prog. Ser. 179: 97-111 Laverack, M. S. 1969. Mechanoreceptors. photoreeeptors. and rapid conduction pathways in the leech. IliruJo nicilii -inulis. J. E.V/J. Hiol. 50: 129-140. Lenz, P. H., and I). K. Hartline. 1999. Reaction times and force production during escape behavior of a calanoid copepod Uiulinula vulgaris. Mar. 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Enhancement of the phototactic reaction in Daph- iiin Inalina by a chemical mediated by juvenile perch ( Perca fluviati- lis}. ./. Plankton Res. 13: 17-25. Singarajah. K. V. 1969. Escape reactions of zooplankton: avoidance of a pursuing siphon tube. J. Exp. Mar. Biol. Ecol. 3: 171-178. Stearns, D. E., and R. B. Forward, Jr. 1984a. Photosensitivity of the calanoid copepod Acartia tonsa. Mar. Biol. 82: 85-89. Stearns, D. E., and R. B. Forward, Jr. 1984b. Copepod photobehavior in a simulated natural light environment and its relation to nocturnal vertical migration. Mar. Biol. 82: 91-100. Stubblefield, C. L., C. M. Lascara, and M. Vecchione. 1984. Vertical distribution of zooplankton in a shallow turbid estuary. Contr. Mar. Sci. 27: 93-104. Tett, P. B., and M. G. Kelly. 1973. Marine bioluminescence. Oceanogr. Mar. Biol. Annu. Rev. 11: 89-173. Thurm, II. 1965. An insect receptor potential. Cold Spring Harbor Symp. Quant. Biol. 30: 83-94. Tjossem, S. F. 1990. Effect of fish chemical cues on vertical migration behavior of Chaohorus. Limnol. Oceanogr. 35: 1456-1468. Tomita, T. 1970. Electrical activity of vertebrate photoreeeptors. Quart. Rev. Biopliy. 3: 179-222. Viitasalo, M, T. Kinrboe, J. Flinkman, L. Pedersen, and W. W. Visser. 1998. Predation vulnerability of planktonic copepods: consequences of predator foraging strategies and prey sensory abilities. Mar. Ecol. Prog. Ser. 175: 129-142. Weeks, F. L, and G. Duncan. 1974. Photoreception by a cephalopod retina: response dynamics. Exp. Eve Res. 19: 493-509. Wine, J. J., and F. B. Krasne. 1982. The cellular organization of crayfish escape behavior. Pp. 241-292 in The Bio/ogv of Crustacea Vol. 4. Neural Integration and Behavior. D.C. Sandeman and H.L. Atwood, eds. Academic Press. New York. Wyman, R. J., J. B. Thomas, L. Salkoff, and D. G. King. 1984. The Dmsopliilii giant fiber system. Pp. 133-161 in Neural Mechanisms oj Startle Behavior. R.C. Eaton, ed. Plenum. New York. Yen, J., P. H. Lenz, D. V. Gassie, and D. K. Hartline. 1992. Mech- anoreception in marine copepods: eleetrophysiological studies on the first antennae. J. Plankton Res. 14: 495-512. Reference: Biol. Bull. 204: 3S-44. (February 2003) O 2003 Marine Biolo-jic.il Lahoraton Behavioral Thermoregulation in Hemigrapsus nudus, the Amphibious Purple Shore Crab I. J. McGAW Department of Biological Sciences, University of Nevada Las Vegas, 4505 Maryland Parkway, Las Vegus, Nevada S9 1 54-4004; and Bainfie/d Marine Sciences Centre. 100 Pachena Road. Bamfield, British Cohtnihiu VOR 1BO. Canada Abstract. The thermoregulatory behavior of Hanigrap- sns niuliis. the amphibious purple shore crab, was examined in both aquatic and aerial environments. Crabs warmed and cooled more rapidly in water than in air. Acclimation in water of 16 C (summer temperatures) raised the critical thermal maximum temperature (CTMax): acclimation in water of 10 C (winter temperatures) lowered the critical thermal minimum temperature (CTMin). The changes oc- curred in both water and air. However, these survival re- gimes did not reflect the thermal preferences of the animals. In water, the thermal preference of crabs acclimated to 16 C was 14.6 C, and they avoided water warmer than 25.? C. These values were significantly lower than those of the crabs acclimated to 10 C; these animals demonstrated temperature preferences for water that was 17 C, and they avoided water that was warmer than 26.9 C. This temper- ature preference was also exhibited in air. where 10 C acclimated crabs exited from under rocks at a temperature that was 3.2 C higher than that at which the 16 C accli- mated animals responded. This behavioral pattern was pos- sibly due to a decreased thermal tolerance of 16 C accli- mated crabs, related with the molting process. H. niidiis was better able to survive prolonged exposure to cold tempera- tures than to warm temperatures, and there was a trend towards lower exit temperatures with the lower acclimation (10 C) temperature. Using a complex series of behaviors, the crabs were able to precisely control body temperature independent of the medium, by shuttling between air and water. The time spent in either air or water was influenced more strongly by the temperature than by the medium. In the field, this species may experience ranges in temperatures Received 10 May 2002: accepted 17 October 2002. F.-mail: IIIIL>:,I\\ '"ccmail nevada.edu of up to 20 C; however, it is able to utilize thermal microhabitats underneath rocks to maintain its body tem- perature within fairly narrow limits. Introduction Intertidal organisms experience abrupt, frequently large, changes in temperature as a result of alternating episodes of exposure to air and water (Vernberg and Vernberg, 1972). These changes in temperature may pose an additional bur- den to amphibious organisms that are already challenged by the switch between ventilatory media (Greenaway et ai, 1996). Hemigrapsus audits, the purple shore crab, is a common species in the mid- to high-intertidal zone of rocky shores along the northeastern Pacific (Schmitt, 1921; Dehnel, 1960; Low, 1970; Daly, 1981). These crabs are involun- tarily exposed as the tide recedes, but they are active in air (Burnett and McMahon, 1987). The species can tolerate temperatures up to 33.6 C for short periods of time (Todd and Dehnel, 1960); however, exposure to suboptimal tem- perature regimes is associated with compensatory physio- logical responses in decapod crustaceans. The aerobic metabolism of crustaceans, like that of most other aquatic organisms, is temperature dependent. Oxygen uptake increases in Carcinus inaenas, the green shore crab. as the temperature of the water is raised (Taylor and Wheatly, 1979); likewise, oxygen consumption in Homanis f>tiininunts. the European lobster, decreases (for a short time) as temperature is lowered (Whiteley et ai. 1995). Increases in temperature also influence oxygen delivery to the tissues by causing a reduction in the carrying capacity ot the hemolymph for oxygen and the binding of oxygen to the hcmocyanin (Taylor. 1981 ; Truchot. 1983). Heart rate is directly related to temperature in a number 38 THERMOREGULATION IN HEMIGRAPSUS NUDUS 39 of crustacean species (deFur and Mangum, 1979; Taylor and Wheatly. 1979: DeWachter and McMahon, 1996; Still- man and Somero. 1996; Pirro et a/.. 1999: Jury and Watson, 2000: Fredrich etui., 2000). Heating increases heart rate and cardiac output but decreases stroke volume in Cancer nui- gister, the Dungeness crab (DeWachter and McMahon, 1996). This is associated with an increase in hemolymph perfusion of the carapace, gonads. and musculature of the pereiopods (DeWachter and McMahon. 1996). Cooling causes a decrease in cardiac parameters: heart rate and cardiac output drop sharply in low temperature, and hemo- lymph flow is directed away from anterior structures to more ventral structures (Fredrich et al., 2000). Since exposure to high or low temperatures can be met- abolically costly, the ability of crabs to sense temperature and orient to a "thermal niche" should be advantageous in minimizing physiological stress. In addition, many pro- cesses such as molting, growth, reproduction, and matura- tion of eggs are temperature dependent (Sastry, 1983a, b); therefore, selection of optimal temperatures should also maximize growth and reproductive potential (Hutchison and Maness. 1979). A number of crustacean species are known to exhibit behavioral thermoregulation. Homarus america- iiiis, the American lobster, can thermoregulate precisely for up to 6 days, preferring temperatures in the 15-21 "C range (Reynolds and Casterlin, 1979a; Crossin et nL 1998). Lob- sters are able to detect water temperature differences of as little as 1 C and exhibit directional taxis (Jury and Watson, 2000). Carcinus maenas, the green shore crab, avoids ad- verse temperatures, showing emersion responses at 28 "C in the laboratory (Taylor and Wheatly, 1979). Procambarus clarkii. the red swamp crayfish, has a broad temperature tolerance (Payette and McGaw. 2001) and prefers water with a mean temperature of 23-24 C (Espina et ai, 1993; Ramirez et ai. 1994). An animal's thermal preference can be also be influenced by the acclimation temperature. Ac- climation to warm temperatures results in a higher temper- ature preference in Homarus americanus, the American lobster (Crossin et ai. 1998), and in Astacus astacits, a crayfish (Kivivuori, 1994). Temperature acclimation has an opposite effect on the crayfish Orconectes immunis, with animals acclimated to warm water selecting cooler temper- atures than those acclimated to cold water (Crawshaw, 1974). Most of the articles on behavioral thermoregulation in decapod crustaceans have concentrated on fully aquatic species (see Crossin et ai, 1998). Much less information exists on amphibious species that are emersed in the inter- tidal zone twice daily (Thurman, 1998). H. nuthis is exposed to a wide range of temperatures on both a tidal and diurnal basis (Todd and Dehnel. 1960; Greenaway et al., 1996). Therefore, the aim of this study was to investigate the thermal ecology of this amphibious species and to assess the role of behavioral reactions, in both water and air. in min- imizing the effects of thermal stress. Materials and Methods Adult male and female purple shore crabs, Hemigrapsm nudiis, of 25-40-mm carapace width, were collected inter- tidally in Barkley Sound, British Columbia, during the months of May to August in 2000 and 2001. They were transferred to 40-liter aquaria at the Bamfield Marine Sci- ences Centre and maintained in aerated seawater at a salin- ity of 33 ppt 0.5 ppt on a natural light-dark cycle. The crabs were held at water temperatures of either 16 C 0.5 C or 10 C 0.5 C for at least 2 weeks. These temper- atures approximated those measured in the field during summer and winter respectively (Gosselin and Chia, 1995). More extreme temperatures were not used because the an- imals tended to molt at higher temperatures and become lethargic at lower temperatures. The crabs were fed sea lettuce, Ulva lactuca, ad libitum. Approximately equal numbers of each sex were used, and individual crabs were not re-used in any experiment. Rate of change of hod\ temperature Changes in the body temperature of H. nudiis (n = 10) were studied in water and air. To measure blood tempera- ture, a catheter-mounted (PE90) thermocouple (Physitemp IT18) was inserted through a small hole drilled in the first abdominal segment and guided to lie against the sternal artery. The crabs were returned to the holding tank and allowed to settle for 15 min. Animals (n = 10) were then transferred to water or air of 5 C or 20 C. The amount of time required for the body temperature to equilibrate with the surrounding medium was recorded at 30-s intervals using a BAT 12 digital thermometer (Physitemp Instru- ments). Critical thermal maximum and minimum temperatures The critical thermal maximum (CTMax) and critical ther- mal minimum (CTMin) temperatures of H. niiclim were assessed in air and in water (n = 30). Crabs were accli- mated to 10 C or 16 C and were studied separately, with the starting temperature being 10 or 16 C, respectively. The temperature of the air was raised (or cooled) at 0.5 C/min in an incubator (Percival Instruments [Boone. Iowa]; model 135LL), and the temperature was monitored at 1-min intervals with a Physitemp BAT 12 digital thermom- eter. A volume of 5 liters of water was used, and the temperature was raised (or cooled) at 0.5 C/min by way of a recirculating water bath (VWR Scientific Instruments). The water was aerated, and the temperature was monitored with the Physitemp thermometer. At random intervals, the crabs were turned on their backs until the first animal 40 I. J. McGAW reached its CTMax or CTMin; that is. until the animal could no longer right itself within 1 min (Cuculescu ct al., 1998). Thereafter, all remaining crabs were inverted together every minute and the CTMax or CTMin was recorded for each individual. Temperature preference hehavior The temperature preference range of H. inulus was de- termined using an elongated (length, 300 cm) cylindrical (diameter. 12 cm) chamber that was orientated horizontally. Heating and cooling recirculating water baths at either end of the chamber maintained the temperature gradient be- tween 7 C and 30 C. The placement of the heating and cooling water baths was alternated between each trial, to eliminate any bias for either end of the chamber. Airstones minimized any vertical thermal stratification in the gradient and ensured that the water did not become hypoxic. Shelters (broken glass beakers) were placed along the length of the chamber to reduce stress, H. niultis is highly thigmotactic and will remain active, attempting to escape, unless there is a place to shelter (McGaw, 2001). This atypical behavior could obscure thermoregulatory responses. Crabs a max- imum of five at any one time (8 repetitions; total // = 40) were introduced into the gradient at random locations; using this number of H. inulits in experiments does not affect the thermotolerance of an individual (Todd and Dehnel. I960). After 3 h. a temperature reading was taken at the position of each individual crab. Those crabs accli- mated to either 10 C or 16 C were studied separately. In control experiments, the temperature was maintained at either a constant 10 'C or a constant 16 C; crabs were then introduced randomly into the apparatus, and their position was recorded after 3 h. Temperature avoidance The following two experiments were designed to test the responses of the crabs after they had sensed a change in temperature. Behavioral responses consisted of migration from underneath a shelter as the temperature changed. Ex- periments were performed in both aquatic and aerial envi- ronments. The first experiment (aquatic) was carried out in a mod- ified two-choice chamber (Fig. 1 ), which contained seawater (32 ppt), in one side, as well as pieces of broken glass beakers for shelter. The chamber was held in an incubator (Percival Instruments Model 135LL). which allowed inde- pendent control of air temperature. Five animals per trial (5 repetitions; /; :: 25) were placed in the seawater and allowed to settle for 30 min. Any animals that exited the water within this period were not used in the experiments. The starting temperature of experiments was either 10 'C or 16 C for each group of acclimated crabs. The temperature of the seawater was raised at 0.5 C/min usinu a recirculat- Ramp Aerial environment Mesh screen To water bath Glass shelters Figure 1. Modified two-choice chamber used to measure exit temper- atures from water into air. shuttling behavior between air and water, and behavioral control of body temperature. ing water bath (VWR Scientific Instruments). The temper- ature at which the crabs made a voluntary migration into air was recorded; this behavior was defined as "emigration" (Taylor and Wheatly. 1979). The experiment was repeated with air temperatures of 5 C, 20 C, and 35 C, each at 50%-70% relative humidity. Experiments were then carried out to assess the lower preference range. The water was cooled at 0.5 C/min, and emigration temperature from the seawater was recorded at the three air temperatures. The water side of the chamber was alternated between trials to avoid any preference associated with either side of the chamber. For the second experiment, temperature avoidance was tested in air using a chamber measuring 45 cm X 45 cm X S cm deep, with a gauze bottom to allow air to circulate. Flat tiles were placed in the chamber. Five animals were then introduced into the chamber and allowed to settle under the tiles. Any animals that migrated from under the tiles within 30 min were not used in the experiments. The chamber was held in an incubator (Percival Instruments), with the starting temperature for the two acclimated groups being either 10 C or 16 C. The air temperature was then raised by 0.5 C/min, and the temperature (measured under the tiles) at which the crabs exited from under the tile shelters was recorded (n = 25). The experiment was repeated by low- ering the temperature, by 0.5 C/min, and observing the temperature at which the crabs exited from under the shel- ters. All recordings were made in constant dim red light. Shuttling behavior A time-lapse video recorder and camera (Panasonic AG- RT600AS VCR and Panasonic WV-BP120 camera) was used to monitor the shuttling behavior of individual crabs THERMOREGULATION IN HEMIGRAPSUS NUDUS 41 25 -i air/water temperature \ air/water temperature 10 20 30 Time (min) 40 50 60 Figure 2. Changes in body temperature (mean SEM) of 10 //<;/ i,'ni/i.v/.\ inulm. after transfer from 10 "C water to 20 C air (A), from 10 C water to 20 C water (A), from 16 C water to 5 C air (O), and from 16 C water to 5 C water (). In some cases error bars are smaller than the symbols. between air and water at temperatures of 10 C. 20 C, and 30 C. The choice chamber was set up in an incubator with glass shelters in both air and water. Four crabs (acclimated to 16 C) were placed in the water (2 repetitions, total n = 8 for each treatment). The number of shuttles, duration of shuttles, and total time spent in air and water were recorded over a 24-h period in constant dim red light. Behavioral control of body temperature The body temperature of eight crabs (acclimated to 16 C) was recorded with a thermocouple (Physitemp IT 18) introduced through the first abdominal segment. The ther- mocouple was connected to a BAT 12 digital thermometer (Physitemp Instruments); data were recorded on an ADIn- struments Powerlab data acquisition package. The two- choice chamber was placed in an incubator (Percival, model 135LL), and a recirculating water bath allowed independent heating or cooling of the seawater. An animal was initially placed in the shallow water, and the change in its body temperature was followed for 12 h as it shuttled between air and water. A variety of water and air temperature combi- nations were offered, separated by differing increments. Regulation of bod\ temperature in the field Regulation of body temperature was assessed in freshly collected crabs in the field. Crabs were fitted with thermo- couples (Physitemp IT18) on a 2-m lead (n = 5). Each crab was released on a falling high tide and allowed to settle; body temperature was recorded at half-hour intervals until the following high tide, using a BAT 12 digital thermometer (Physitemp Instruments). At the same time, air temperatures were recorded 5 cm above the rock surface, and seawater temperature was recorded at the low tide, using a Physitemp IT 14 thermocouple calibrated against a mercury thermom- eter. Experiments were repeated on days when air temper- atures were higher or lower than the ambient seawater temperature. Results Rate of change of body temperature An increase or decrease in water temperature of about 10 C resulted in a rapid change in body temperature (Fig. 2). Body temperature equilibrated with the surrounding water, within 2-3 min. In air, body temperature changed more slowly, and heat loss from the body was more rapid than heat gain. The body temperature took 25 min to equilibrate to a 10 C drop in air temperature, but it failed to reach equilibrium with the surrounding air within the 60-min experimental period when the temperature was raised by 10 "C. Although body temperature reached 90% of the final temperature within 20 min. it increased slowly thereafter. 42 I. J. McGAW Table 1 Tliennal preference o/Hemigrapsus nudus with incrcusmx temperature Air temperature (C) Water temperature I C) at emigration* Crabs acclimated to 10 C Crabs acclimated to 16 "C 5 20 35 25.5 0.81 27.4 061 27.9 0.6d 25.3 0.68 24.7 0.39 25.7 0.53 * Mean ( standard error of the mean) upper temperature at which crabs (n = 25 ) emigrated from water into air with a temperature of 5 C, 20 C, or 35 C as the temperature of the water was raised. Critical thermal maximum anil minimum temperatures In water, the CTMax of 31.1 C a standard error of the mean (SEM) of 0. 16 C for crabs acclimated to 10 C was significantly lower than the CTMax of 33.6 0.11 C for crabs acclimated to 16 C (Student's ? test = -2.32, P = 0.02). The difference between the two acclimation groups was greater in air. Crabs acclimated to 10 C had a CTMax of 33.2 0.34 C, which was significantly lower than the CTMax of 35.3 0.5 C for 16 C acclimated animals (t test = -3.45, P = 0.001). In addition, the CTMax values in water were significantly lower than those in air ( ANOV A, F ---- 7.55, P = 0.007). Acclimation to either 10 C or 16 C also affected the critical thermal minimum temperature. The CTMin in water of 3.5 0.14 C for crabs acclimated to 10 C was signif- icantly lower than the 4.82 0.14 C for 16 C acclimated crabs (/ test = -6.71. P < 0.001). A similar trend was observed in air, with CTMin values of 3.44 0.15 C and 3.99 0.12 C, for 10 C and 16 C acclimated crabs, respectively (nest = -2.89, P = 0.005). As with CTMax, there was a significant effect associated with the medium: the CTMin values in air were significantly lower than those in water (ANOVA, F = 10.41. P = 0.002 1. Temperature preference When 40 crabs (again, acclimated to either 10 C or 16 C) were placed randomly in a thermal gradient of 7 C to 30 C, there was considerable movement within the first 30 min. Temperature selection appeared to be complete after 3 h. with very little movement in the gradient thereafter. Although a small percentage of the crabs selected the ex- treme temperatures of 7 C C or 30 C. most were distributed between 1 1 C and 24 C. The mean preference range of 17.01 C 0.65 C SEM for 10 C acclimated crabs was significantly higher than the 14.60 'C 0.78 "C selected by 16 C acclimated crabs (t test = 2.37. P = 0.02). Control experiments were carried out for the two acclimation tem- peratures, with no thermal gradient. Control crabs did not show a preference for any area of the gradient tank. A similar effect of acclimation on temperature preference was observed in the temperature-avoidance experiments. When the temperature of the water was gradually increased, crabs exited from under the shelters and started to become active between 19-21 C. but did not leave the water at this temperature. Although there were three different air tem- peratures that crabs could emigrate into, the air temperature had no significant effect on emigration temperatures from water (Table 1) (ANOVA, F = 2.47. P = 0.088). Since air temperature had no effect on behavior, data for the three air temperatures was pooled. There was a significant behav- ioral effect based on acclimation: crabs acclimated to 10 C had a mean emigration temperature of 26.94 0.24 C; this was significantly higher than the mean emigration temper- ature of 25.25 0.19 C for crabs acclimated to 16 C (ANOVA. F = 10.47. P = 0.002). All crabs had left the water when the temperature reached 34 C. Although all crabs left the water when the temperature was raised, this was not the case when the water temperature was lowered. Only 45<7r of the crabs acclimated to 10 C and 30% of the crabs acclimated to 16 C emigrated from the water. The rest of the crabs remained in the water even though the temperature was reduced below their CTMin. incapacitating them. Statistical results for the animals that exhibited emigration behavior are given in Table 2. Air temperature had no significant effect on the emigration temperature of the crabs (ANOVA, F = 0.14, P = 0.87). Although the mean emigration temperature of 4.95 0.31 C for 10 C acclimated animals was lower than the 5.79 0.42 C for 16 C acclimated crabs, this difference was statistically insignificant (ANOVA. F = 2.58.P = 0.1 13). Therefore, in water, 10 C acclimated crabs have a pref- erence range between 4.95 C and 26.94 C, with a mean preference of 17.1 C. The crabs acclimated to 16 C have a mean temperature preference of 14.60 C with a narrower preference range between 5.79 C and 25.25 C. Acclimation to either 10 C or 16 C had a similar effect on temperature avoidance in air. When the temperature of Table 2 Tlifrnial preference of Hemigrapsus nudus with decreasing temperature Air temperature (C) Water temperature (C) at emigration* Crabs acclimated to 10 C Crabs acclimated to 16 "C 5 20 35 4.72 0.53 5.23 0.57 4.91 0.66 5.87 0.51 5.24 0.61 6.26 0.89 * Mean ( standard error of the mean) temperature at which crabs (;i - 25) emigrated from water into air with a temperature of 5 C, 20 C. or 35 "C as the temperature of the water was lowered. THERMOREGULATION IN HEM1GRAPSUS NUDUS 43 Table 3 Shiittliiif; behavior of Hemigrapsus nudus from water into air u-hen both media were maintained at 10 C 20 C or 30 "C Parameter* Temperature (C) Number of shuttles Duration of each shuttle (mini Percent time spent in air/24 h II) 20 30 26.4 5.1 18.0 4.3 27.3 6.2 8.6 0.9 21.2 8.1 28.5 7.5 16.6 11.3 24.5 23.5 64.3 16.5 : Values are the mean ( standard error of the mean) response for 8 crabs. the air was gradually raised, the crabs exited from under tiles in an attempt to escape. The mean exit temperature for crabs acclimated to 10 C was 27.39 0.62 C; this was significantly higher than the exit temperature of 24.17 0.58 C recorded for 16 C acclimated crabs (t test = -3.8, P < 0.001 ). Consistent with lower emersion temperatures in water, not all crabs exited from under shelters as the air temperature was gradually reduced. Five of the 25 crabs acclimated to 10 C remained under the shelters, while 10 of the crabs acclimated to 16 C did not exit. Statistical anal- ysis includes only the animals exhibiting this exit behavior. Although the mean exit temperature of 4.36 0.47 'C recorded for 10 C acclimated crabs was lower than the 5.85 0.7 C recorded for 16 C acclimated animals, this difference was statistically insignificant (t test = -1.82, P = 0.077). Therefore, the temperature preference range in air for 10 C acclimated crabs was 4.36 C to 27.39 C, which was broader than that for 16 C acclimated animals (5.85 C to 24.17 C). Shuttling behavior The shuttling movement of 16 C acclimated crabs be- tween air and water was studied during a 24-h period to determine the number and duration of excursions into air (Table 3). There was no significant difference in the number of shuttles between air and water as a result of ambient temperature (ANOVA, F = 0.96, P = 0.4). There was a trend towards an increase in the average duration of each excursion into air as the temperature increased, but it was not statistically significant (ANOVA, F = 3.01. P : 0.05). When the percentage of time (per 24-h period) that each crab spent in air was considered, a significant pattern emerged (Table 3). As the temperature increased, the crabs spent a significantly greater total percentage of time in air (ANOVA, F ---- 14.72, P < 0.001). The percent of time that the crabs spent in air at 30 C was significantly higher than time spent in 10 C and 20 C conditions, but there was no significant difference between 10 C and 20 C (Tukey test, q = 1.18, P = 0.684) Behavioral control of hod\ temperature When offered a choice of 20 C water with 8 C air (trial 5), the crabs remained in the water most of the time: the mean body temperature of 20.3 C 0.2 C SD was not significantly different from the water temperature (Fig. 3). The body temperature in experimental trial 5 was signifi- cantly higher than in the other trials (Tukey test. P < 0.05). In experimental trial 4 ( 14 C water and 24 C air), the crabs also remained in the water; again, the mean body tempera- ture of 14.2 0.3 C was not significantly different from that of the water (Fig 3). In all other trials, the crabs maintained the body temperature at levels between the temperature of the air and the water. In the shallow water of the chamber, periodically, a crab either raised or submerged itself to control its body temperature between mean values of 7.7 0.9 C and 14.6 1.5 C. Examination of the body temperature of individual crabs shows the thermoregulatory responses in more detail (Fig. 4a-d). When the water was held at 4-5 C and the air at 33-34 C (Fig. 4a), the crab spent the first 2 h shuttling O 40 - 35 - 25 - 20 - 10 - 5 - Water temperature Air temperature \ Body temperature I 1 . :' i Experimental Trial Figure 3. Mean body temperature ( SD) of Wf'.i;ra/>.Mf.\ niuhis ( n = 8) when offered a choice between air and water, maintained at different temperatures relative to one another. Hashed bars represent water temper- ature, and solid bars represent air temperatures. 44 I. J. McGAW a a a> o m 30- 20- 10- AIR WATER 30- 20- I m 10- 6 Time (h) AIR K 12 WATER 6 Time (h) 12 _. 30- o 8. E TJ O m 20- 10- WATER AIR 6 Time(h) 12 _ 30 o 20 o CO 10 AIR WATER 6 Time(h) 12 Figure 4. Representative examples of body temperatures of individual Hi'mixnipsiix nudux in a two-choice chamber with the ability to shuttle between air and water of different temperatures, (a) Water of 4-5 "C and air of 33-34 "C. (b) Water of 33-34 C and air of 4-5 C. (c) Water of 6-7 C and air of 30-31 "C. (d) Water of 8-9 C and air of 20-21 C. between air and water, after which it raised or submerged itself in the water to maintain a body temperature of about 8-13 C. When the temperatures of the air and water were reversed (4-5 C air and 33-34 C water), the crab (Fig 4b) was still able to maintain a body temperature between 8 and 13 C for most of the 12-h experimental period. When the difference between air and water was decreased (8-9 C water and 29-30 C air), body temperature fluctuated some- what during the first 2 h when the crab was active; there- after, body temperature was maintained between 10 C and 17 C (Fig. 4c). When air and water temperatures (20-21 'C air and 8-9 C water) approached limits within the animal's preference range (Table 1.2). the crab tended to shuttle back and forth between air and water, spending extended periods of time in either medium, where body temperature equilibrated with the medium (Fig. 4d). Bil\ temperature in the field Changes in body temperature of H. nndiis were recorded in the field during an intertidal period (Fig. 5). Body tem- perature was monitored on a cold day (Fig 5a), when the air temperature dropped below that of the seawater. Although air temperatures fell from 18 C to 10.7 C, body temper- atures decreased only slightly. The mean body temperature of the crabs (n = 5) dropped from 17.65 0.28 C SEM when initially emersed, down to 14.92 0.44 C at the end of the intertidal period. This was only a 16% drop in body temperature, compared to a drop of 41% in the temperature of the surrounding air. Body temperature increased rapidly when the crabs were re-immersed, reaching 16.5 0.27 C as it equilibrated with the seawater. On a warm day (Fig. 5b) the air temperature quickly rose THERMOREGULATION IN HKMIGKAPSUS NVOVS 45 24 "- 20 - 3 ro 16 s. seawater temp. 8 J r 1700 1900 2100 2300 0100 Time 25 U _ 20 o 3 | a I 15 10 -" Time Figure 5. Changes in body temperature (mean SEM) of 5 specimens of Hemigrapsus midiis (solid line) in the intertidal zone. Crabs were released during a falling tide and monitored until the following high tide. Seawater temperatures (dotted line, solid symbols) and air temperatures (dashed line, open symbols! were also recorded during this time. Times of emersion and immersion of the crabs, as well as low tide (LT). are indicated on the graphs. Recordings were made on (al 8 July 200 1. when surrounding air temperatures were lower than ambient seawater tempera- tures and (b) 23 July 2001. when air temperature was higher than seawater temperature. from 11 C in the morning to 22-24 C by early afternoon. Despite this 12 C rise in air temperature, the body temper- atures of the crabs did not change as rapidly, and reached only 16.86 0.51 C by the end of exposure period in air. The change in body temperature was similar to the observed increase in seawater temperature during the day (Fig. 5b). When the crabs were re-immersed, their body temperatures quickly equilibrated with the seawater. Discussion The observed rates of change in body temperature (Fig. 2) were similar to those reported previously for Hemigrapsus nudus (Greenaway et al., 1996). In lobsters, heat loss in air is more rapid than heat gain ( Whiteley el al.. 1995): this was also observed here for H. muhts (Fig. 2), probably due to the evaporative heat loss in air. During these experiments, sev- eral of the crabs regurgitated frothed fluids from the stom- ach, smeared this over the ventral carapace with the chelae, and raised their body above the substrate. This foaming behavior has been reported for a number of crab species (Lindeberg, 1980; Maitland. 1990) and can used to reduce body temperature (Jansen, 1970). However, there was no evidence to suggest that the H. nudus specimens were using this method to slow their rate of heating. The relative humidities of 60%-70% used during the experiments, which mimicked conditions measured in the field, could have reduced the effectiveness of (but not eliminated) evap- orative cooling (Edney, 1961). Although foaming behavior was not observed in the field, it is possible that it could reduce heating rates on warm days with low relative hu- midity (Thurman. 1998). Fiddler crabs (Uca species) are able to maintain a body temperature below that of the surrounding air by changing posture, blanching, and evap- orating water from the body surface (Wilkens and Finger- man, 1965; Smith and Miller, 1973; Thurman. 1998). In the present study, there was no difference in heating or cooling rates when comparing live and dead animals (not shown), suggesting that there is no active mechanism that allows H. nmlus to control the rate of heat gain or loss from the body. When H. nudus was acclimated to different temperatures, an increase in the upper survival limits occurred as a result of the higher acclimation temperature; this has been re- ported previously for H. nudus (Todd and Dehnel, 1960). as well as for other species of crustaceans (Mundahl and Benton. 1990; Lagerspetz and Bowler. 1993; Korhonen and Lagerspetz, 1996; Cuculescu el al.. 1998; Stillman and Somero, 2000). However, much less is known about the critical thermal minima. In the present study, acclimation to a lower temperature extended the CTMin. Acclimation to a wider range of temperatures has also been shown to extend the CTMin range in other crustaceans (Layne et al., 1987; Stillman and Somero, 1996). Survival limits in air as a function of temperature have not been investigated previously for H. nudus. Interestingly, both the CTMax and CTMin were greater in air than in water. The heating and cooling rate in the incubator (0.5 C/min) was adequate to allow equalization of the body with the surrounding air (unpubl. data; Fig. 2). These results are somewhat surprising: an animal would already be phys- iologically challenged by the switch in ventilatory media (Greenaway el al., 1996). because an increase in air tem- perature decreases the oxygen-carrying capacity of the 46 I. J. McGAW hemocyanin and results in thermal acidosis (Morris et al., 1996b). The CTMax was determined close to the body of the crab rather than by using internal temperature probes, which tended to tangle around the legs when the crabs were turned over, affecting the ability of the animal to right itself. Even though relative humidities in the incubator were high (60%-80^:), a degree of evaporative cooling could have kept the body temperature a degree or so cooler than the surrounding air (unpubl. data; Fig. 2). This would suggest that the upper lethal limits in air were probably similar to those measured in water. However, if evaporative cooling reduced the body temperature, then the CTMin in air would also be expected to occur at a higher temperature than in water. This did not happen in the present study. The thermal preference behavior of crabs acclimated to 10 C and 16 C, ascertained in a thermal gradient and by temperature-aversion experiments (Table 1 ), did not reflect their temperature tolerances: in both cases. 10 C accli- mated crabs had a higher temperature preference than those acclimated to 16 C. In the temperature-aversion experi- ments, the oxygen tension was maintained at constant lev- els, so the emigration from water was a direct consequence of temperature. Indeed, aquatic hypoxia is not an impetus for emersion in this species (Moms et ai, 1996c). The air temperature that the crabs could exit into did not affect the exit temperature from the water (Table 1 ). This was unex- pected, since acute exposure to higher air temperature (>15 C) is costly and is associated with thermal acidosis and compensatory increases in cardiac output to maintain ade- quate oxygen uptake (Morris et ai, 1996a, b). Acclimation to 10 C or 16 C also influenced aversion behavior in air. When the air temperature was raised, crabs acclimated to 10 C exited from under stones at a higher temperature than did 16 C acclimated crabs. The adaptive significance of this behavior is unclear, since 16 C acclimated crabs are more tolerant of higher temperatures (CTMax values). Thus, the effect of acclimation on behavior is apparently the opposite of its effect on survival regimes. In other reports on ther- moregulatory behavior, lobsters that are acclimated to warm water choose wanner temperatures than do cold-acclimated individuals, possibly to maintain an optimal thermal regime for metabolic activities (Crossin ft nl.. 1998). When Astiiftis astacus, a crayfish, is acclimated to cold or warm water, this also directly affects thermal preference (Kivivuori, 1994). Acclimation to either 15 C or 25 C has no effect on the emersion response of the shore crab Cardans macnas. which exits into air when the water temperature reaches 28 C (Taylor and Wheatly, 1979). Likewise, acclimation to differing temperatures has no effect on the temperature preference of Procambants clarkli. the red swamp crayfish (Espina ft ai, 1993). In contrast to these responses, when the crayfish Orconectes immunis is acclimated to cold wa- ter, it tends to choose higher temperatures than animals acclimated to warm water, yet no explanation is given for this paradox (Crawshaw, 1974). Several factors can be eliminated as causes for the unex- pected behavior observed in the present study. ( 1 ) The crabs were not responding to temperature increases of a particular magnitude, as occurs in lobsters (Cooke-Schreiber et ai, 2001 ). (2) The warming rate of the water (0.5 C/min) was slow enough to allow the body temperature to equilibrate with the surrounding medium (Fig. 2). (3) Although the crabs were introduced into the apparatus in groups of five, this was unlikely to have a substantial effect on their be- havior: they were roughly equal in size and there was ample shelter both of these factors would reduce aggressive in- teractions between animals (Jacoby, 1981). (4) Although Carcinus maenas exhibits a behavioral hypothermia when exposed to hypoxic conditions (DeWachter et ai, 1997), this is probably not the case for H. mtdus because oxygen levels were maintained during experiments and this species does not modify its behavior in response to hypoxia (Morris et ai, 1996c). (5) Finally, the acclimation period of 2 weeks should have been long enough for an increased temperature tolerance (Layne et ai, 1987: Cuculescu et ai, 1998). Indeed, rapid acclimation to thermal zones is an advantage for intertidul organisms: H. mtdus, which acclimatizes within 48 h (Todd and Dehnel, 1960), is no exception. When considering factors that could have influenced this overt behavior, it is worth noting that H. mtdus could not be acclimated to temperatures greater than 16 C without in- ducing widespread molting. Because the entire molting pro- cess can take several weeks (O'Halloran and O'Dor, 1988). it is possible that the 16 C acclimated crabs were just starting to molt (D, or D 2 stage) without visible signs. Early stages of the molting process are associated with biochem- ical and physiological changes (see Chang, 1995) and make H. midus less tolerant of high temperatures (Todd and Dehnel, I960). The crabs acclimated to 10 C would not undergo molting and could be expected to be more tolerant of the higher temperature regimes than the 16 C acclimated crabs, which would avoid warmer temperatures. In addition, activity levels of cold-acclimated Astacus astacus decrease when these crayfish are warmed in water (Lehti-Koivunen and Kivivuori, 1994); if this were the case here for H. mtdus, then cold-acclimated crabs would be less active and would not exhibit an escape response until a higher temper- ature. Though all animals showed avoidance behavior when the temperature was increased, this was not the case when temperature was lowered. Only 30%-45% of the crabs emigrated from water and 20%-40% remained under shel- ters in the air. As expected, crabs acclimated to 10 C appeared to emigrate at a lower temperature than those acclimated to 16 C. However, since only animals that emigrated from the water or from under shelters were used in the analyses, this difference was not statistically signiri- THERMOREGUl.ATION IN HEM1GRAPSUS NUDUS 47 cant (Table 2). The reason that not all the animals exited when the temperature decreased becomes apparent when their long-term survival in extreme temperatures is consid- ered. The crabs also did not recover after a few minutes of exposure at CTMax; they did, however, recover from cool- ing, even after several hours of exposure below CTMin. The same result is reported foiAstacus axtacnx (Lehti-Koivunen and Kivivuori, 1994). Since H. nudus can survive exposure to low temperatures these crabs would not benefit from leaving the water or a protective shelter, where they would become vulnerable to predation. The results of the shuttling experiments between air and water (Table 3) correspond to the behavioral patterns ob- served in the avoidance experiments. In cold water ( 10 C), H. nudus individuals made fewer excursions into air; there- fore the total time spent in air was also less (Table 3). At higher temperatures, the crabs were more active, making a greater number of excursions and spending a greater amount of time in air. In air, H. nudus is able to take up sufficient oxygen via an increased cardiac output (Morris et ai, 1996a, b). However, this is not without cost, especially at higher temperatures, where hemocyanin affinity and pH are affected to a greater degree, suggesting that oxygen delivery to the tissues declines when H. nudus breathes air at warm temperatures (Morris et ai. 1996a, b). Given these factors, the opposite behavior with respect to temperature may have been expected. However, as temperature increases in both air and water, so does oxygen uptake. The possible advan- tages of emigration from warm water into warm air could be a reduction in oxygen demand, as a consequence of evap- orative cooling across the gills (Taylor and Wheatly. 1979). In addition, the CTMax values showed that the crabs toler- ate higher temperatures in air than in water, which may explain why they spend more time in air at higher temper- atures. In the present study, only two or three animals (tested at the 30 C regime) spent longer than 5 h emersed (Table 3), whereas Greenaway et ai (1996) found that H. nudus can remain emersed for up to 8 h. These workers were using colder water temperatures (10-13 C) than this animal is normally exposed to in summer (Gosselin and Chia, 1995). Thus, the crabs were probably moving into the warmer air (19-22 C) due to a thermal preference rather than to the selection of a particular medium. To test this hypothesis, the behavior of H. nudus was investigated as crabs shuttled between air and water of differing tempera- tures, to determine if they were able to maintain the body temperature within a preferred range. Although purple shore crabs are unlikely to encounter such extreme differences in air and water temperatures as shown in Figure 3, the results obtained suggest that they possess well-developed thermosensory mechanisms. The crabs tended to migrate to the air-water interface; although a slight microhabitat may have existed there, they exhibited a complex series of behaviors that suggested they were using the thermal properties of both media to control body temperature (Fig. 3). The crabs raised or submerged their bodies in the shallow water of the chamber, thus gaining the benefits of evaporative cooling from the gills (Taylor and Wheatly, 1979) without the imbalances in pH and hemocy- anin affinity caused by longer emersion in adverse temper- ature regimes (Morris et ai, 1996a. b. c). Maintenance of an optimal body temperature, rather than selection of a partic- ular medium, appeared to be most important factor. In support of this conclusion, when water temperatures of 4-5 "C and air temperatures of 33-34 C were offered (Fig. 3, trial 1 ). the crabs were able to maintain a body temperature of about 8-12 C, independent of the two media (Fig. 4a). When air and water temperatures were reversed (Fig. 3, trial 7), the body temperature was still maintained within similar limits (Fig. 4b). It was also important to investigate the thermoregulatory behavior of H. nudus in the field, since this species displays different behaviors in its natural environment (McGaw. 2001 ). Greenaway et ai ( 1996) suggest that H. nudus may routinely experience 10 C differences in body temperature in the field. Certainly, the porcelain crab Petrolisthes cinc- tipes. which occupies a similar niche, may be exposed to temperatures under rocks in excess of 20 C (Stillman and Somero, 1996). And even though H. nudus voluntarily exits into air in the laboratory (Greenaway et ai, 1996; Burnett and McMahon, 1987), this was not observed in any of the experimental animals in the field. They remained under rocks or deep in crevices during the intertidal period. This behavior has adaptive significance in that it keeps crabs in close contact with cover, thus avoiding the threat of preda- tion (Low, 1970; Duly, 1981: McGaw, 2001). Indeed, H. nudus prefers to shelter underneath larger boulders, which provide the added advantages of heating or cooling more slowly (Stillman and Somero, 1996). Thus, using subtle movements within this thermal microhabitat, the crabs were able to maintain their body temperature independent of the surrounding air (Fig. 5). Additionally, the prevailing weather conditions can have a profound effect on the mi- crohabitat and behavior of animals (Stillman and Somero, 1996). I have observed crabs active at low tide on humid or dull days; clearly other factors, in combination with tem- perature, play a role in emersion behavior and deserve further investigation. H. nudus is well adapted for an existence in the intertidal zone (Morris et ai, 1996a. b, c; Greenaway et ai, 1996). The present study demonstrates that this species is able to detect differences in its thermal environment and use the thermal properties of both water and air to control its body temperature within a fairly narrow range. This study extends the work on thermoregulatory behavior in aquatic crusta- ceans (Crawshaw, 1974; Reynolds and Casterlin, 1979a, b, c, d: Lewis and Roer. 1988; Mundahl and Benton, 1990; Espina et ai. 1993; Kivivuori, 1994; Lehti-Koivunen and 48 I. J. McGAW Kivivuori, 1994; Crossin ct ai, 1998) by examining the responses of an amphibious species during exposure to temperature change in both aquatic and aerial environments. 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Some effects of temperature on respiration in decapodan crustaceans. J. Therm. Biol. 6: 239-248. Taylor, E. W., and M. G. Wheatly. 1979. The behaviour and respira- tory physiology of the shore crab, Carcinus maemns (L.) at moderately high temperatures. J. Comp. Physiol. 130: 309-316. Thurman, C. L. 1998. Evaporative water loss, corporal temperature and the distribution of sympatric crabs (Uca) from South Texas. Comp. Biochem. Phyxiol. 119A: 279-286. Todd, M. E., and P. A. Dehnel. 1960. Effect of temperature and salinity on heat tolerance in two grapsoid crabs Hemigrupsits midux and Hem- igrapsus oregonensis. Biol. Bull. 118: 150-172. Truchot. J. P. 1983. Regulation of acid-base balance Pp. 431-457 in The Biology of Crustacea, Vol 5: Internal Aiuitomv and PhvMnlnftical Regulation. L. H. Mantel, ed. Academic Press. New York. Vernberg, W. B., and F. J. Vernberg. 1972. Environmental Phyxiologv of Marine Organisms. Springer. New York. Whiteley, N. M., A. H. Al-Wassia, and E. W. Taylor. 1995. The effects of sudden changes in temperature on aquatic and aerial respiration in the lobster Homam.i gainmarns (L.). Mar. Frexlm: Behav. Phvsiol. 27(1 1: 13-27. \\ilkens, J. L., and M. Fingerman. 1965. Heat tolerance and temper- ature relationships of the fiddler crab, Uca pugilator. with reference to body coloration. Biol. Bull. 128: 133-141. Reference: Biot. Bull. 204: 50-56. (February 2003) 2003 Marine Biological Laboratory Synthesis of a High-Density Lipoprotein in the Developing Blue Crab (Callinectes sapidus) ANNA WALKER 1 , SEICHI ANDO 2 . AND RICHARD F. LEE 1 * ' Department of Patholog\, Mercer University School of Medicine, Macon, Georgia 31207: Lipoprotein Research Laboratory, Department of Fisheries Science, Kagoshima University, 4-50-20 Shimoarata, Kagoshima 890-0056, Japan: and Skida\ra\ Institute of Oceanography, Savannah, Georgia 31411 Abstract. An important lipoprotein in the hemolymph of crustaceans is Lpl. It transports lipid to peripheral tissues and also has a role in crustacean immune recognition. We employed a monoclonal antibody specific for the Lpl pep- tide to demonstrate by ELISA, western blot and immuno- histochemistry the appearance of Lpl during development of Callinectes sapidus, the blue crab. Lpl was first found in stage 5 embryos and appeared to be synthesized by lateral basophilic cuboidal cells that demonstrated cytoplasmic im- munoreactivity for Lpl at their interface with the yolk mass. The embryonic cuboidal cells bore a strong cytologic re- semblance to the hepatopancreas cells of later stages (zoea, megalopae, adults), which were also immunoreactive for Lpl. Introduction The hemolymph of male and female decapod crustaceans contains a high-density lipoprotein (Lpl) with concentra- tions ranging from 1.1 to 2.0 mg/1 (Lee and Puppione, 1988; Spaziani, 1988; Lee. 1991; Spaziani and Wang, 1991; Stratakis et ai. 1992; Tom et ai. 1993; Yepiz-Plascencia ct ill., 1995; Ruiz-Verdugo et ul.. 1997). It plays an important role in transporting lipids from the hepatopancreas to pe- ripheral tissues such as muscle, and functions as a j8-l,3- glucan-binding protein in crustacean immune recognition (Khayat ct ai, 1994; Hall ct ai. 1995; Kang and Spaziani, 1995). Embryos of Callinectes sapidus. the blue crab, develop in ess sacs through a series of 10 staues (Table I) over a Received 25 July 2002; accepted 8 November 2002. * To whom correspondence should be addressed skio.peachnet.edu E-mail: dick(S' period of 16-23 days. At stage 10, they emerge from the egg sacs as swimming zoea larvae; these metamorphose into megalopae. then into juvenile crab forms, and ultimately become adult crabs. Until they emerge from egg sacs, embryos are nutritionally dependent on lipids and lipo- vitellin stored within the eggs. Lipovitellin (LpII) is a high-density lipoprotein that differs from Lpl in density, sediment coefficient, and peptide components (Lee and Puppione, 1988; Lee and Walker, 1995). In adult blue crabs, Lpl is composed of phospholipids (45%). cholesterol (2%). triacylglycerols (3%), and one peptide (49%, molecular mass 1 12 kD) (Lee and Puppione. 1988). Although Lpl was reported in juvenile and adult blue crabs, it has not been previously reported in crab oocytes or embryos. We employed a monoclonal antibody specific for the Lpl peptide to demonstrate by ELISA. western blot, and immunohistochemistry the appearance of Lpl during blue crab development. In addition, we offer immunohisto- chemical evidence that the developing hepatopancreas is the site of Lpl synthesis in embryonic and larval stage blue crabs, and remains so in the adult. Materials and Methods Collection of crabs, isolation of Lpl. and purification of Lpl peptide Blue crabs were collected by trawling in the estuaries near Skidaway Island, Georgia (USA). Hemolymph was collected with a 5-nil disposable syringe from the base of the swimming leg and centrifuged in a low-speed centrifuge (3 C) for 10 min at 2000 X g to remove clotted materials and cells. Hemolymph lipoproteins were separated from other hemolymph proteins by adjusting the density of the 50 LIPOPROTEIN IN DEVELOPING BLUE CRAB 51 Table 1 Description of embryo stages o/Callinectes sapidus Stage Description Elapsed Time (hours at 27 C) 1 Fertilization T Early cleavage; morula 12 (random mass of yolk cells) 3 Late cleavage; blastula 36 (mass of undifferentiated yolk cells) 4 Embryonic naupliar stage; transparent 85 embryo above the yolk 5 Early appendage formation; embryo 111) invading ventral portion of yolk 6 Embryonic eye; eye appears as scarlet 160 crescent; elongating appendages 7 Presence of beating heart; pigmented ISO appendages 8 Oval, pigmented eye; 50% of yolk utilized; 210 clear appendages 9 Compound eye with dark pigmentation; 230 only small amounts of yolk 10 Protozoeae stage ready for hatching into 2X0 free-swimming zoea hemolymph and then centrifuging it (Beckman L5-40 ultra- centrifuge. 40.3 rotor). Salt solutions used to adjust the solutions densities were prepared according to the methods outlined by Lindgren (1975). Solution densities were veri- fied by refractometry using an Abbe refractometer (Bausch and Lomb). Consistent with an earlier study (Lee and Pup- pione, 1988), lipoproteins with densities less than 1.063 g/ml were not detected in blue crab hemolymph. Thus, blue crab high-density lipoprotein (Lpl) was isolated by adjust- ing the density of hemolymph to 1.21 g/ml with solid potassium bromide, followed by 40 h of centrifugation at 1 17,000 X g. The floating layer of high-density lipoprotein was removed and dialyzed for 24 h at 4 C against 0.22 M NaCl containing 1 mM EDTA and 2 mM sodium azide. After dialysis, lipoproteins were run on vertical slab gels (7% polyacrylamide. 0.1% sodium dodecyl sulfate (SDS). 0.8% mercaptoethanol), following the procedures of Laemmli ( 1970). The protein band (apoLpI) was visualized with 0.3 M copper chloride. The apoLpI was cut from the gel and eluted by electrodialysis (Electro-Eluter model 422. Bio-Rad). The eluted apoLpI was dialyzed against 0.22 M NaCl. SDS- polyacryamide electrophoresis of the purified peptide was carried out to verify its purity. Purified apoLpI was used as the antigen for the preparation of monoclonal antibodies. Monoclonal antibody production Four female BLB/c mice were immunized with 50 fil of apoLpI (0.1 mg/ml) mixed with Freud's complete adjuvant. The injections were repeated twice (4 weeks and 6 weeks after the original injection) with 25 /u,g of apoLpI in Freud's incomplete adjuvant. Three days after the last injection, the mice were sacrificed and spleens removed. The mouse spleen cells were fused with a mouse myeloma strain Sp2/(), using polyethylene glycol as the fusing agent, as described by Galfre and Milstein (1981). After fusing, cells were plated in hypoxanthine/aminopterin/thymidine selection medium in microplates on a feeder layer consisting of mouse peritoneal macrophages. The wells were screened by indirect ELISA (enzyme-linked immunosorbent assay) for antibodies to apoLpI. The positive hybridomas were cloned by limiting dilution. The hybridomas were grown on RPMI 1640 medium (Gibco) supplemented with 10% fetal calf serum and 0.01% streptomycin and penicillin. Indirect ELISA assa\ fur apoLpI An indirect ELISA assay was used to test antibodies, using the procedures described by Lee and Walker ( 1995), and absorbance was measured at 410 nm with an ELISA microplate reader (model EL307C. Bio-Tek Instruments). Indirect competitive ELISA for apoLpl A criss-cross serial dilution analysis was carried out to determine the optimal concentrations of apoLpI and anti- body (Hornbeck el i>ln>m ct-cnipiii: support of yolk formation and preparation tor (light. Insect Hinclicin. 21: 653-663. Tom, M., O. 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Ph\\inl. 121B: 309-314. Reference: Biol. Bull. 204: 57-67. (February 2003) 2003 Marine Biological Laboratory Reproductive Biology of Hemiramphus brasiliemis and H. balao (Hemiramphidae): Maturation, Spawning Frequency, and Fecundity RICHARD S. McBRIDE* AND PAUL E. THURMAN Florida Marine Research Institute, Florida Fish and Wildlife Conservation Commission, 100 8th Avenue SE, St. Petersburg, Florida 33701-5095 Abstract. Analyses of life-history data show that both the size-specific batch fecundities and the age-specific spawn- ing frequencies differ for two halfbeak species, Hemiram- phus hrasiliensis. the ballyhoo, and H. halao, the balao. Halfbeak ages were determined from sectioned otoliths; histological data was used to describe oocyte development and estimate spawning frequency; and batch fecundity was measured from counts of whole oocytes in final maturation. Hemiramphus hrasiliensis lived longer (4 versus 2 years) and had a higher survival rate ( 14.9% versus 7.5% annually) than H. halao did. Of the two species the larger and longer- lived congener, H. hrasiliensis, reached sexual maturity at a larger size (fork length 198 versus 160 mm). The spawning period of age-0 females was strongly related to season, whereas spawning by older females occurred throughout the year. Reproduction by both species peaked during late spring or early summer, and all mature females were spawn- ing daily during April (H. hrasiliensis) or June (H. halno). This is the first demonstration of iteroparity for the family Hemiramphidae. H. hrasiliensis had a lower batch fecundity (about 1164 versus 3743 hydrated oocytes for a 100-g female) than H. halao did. Such low batch fecundities are typical of the order Beloniformes, but quite different from those of other fishes that live in association with coral reel habitats. H. halao' s higher batch fecundity is consistent with the life-history theory that predicts higher numbers of eggs for shorter-lived species; this is possible because H. halao produces smaller hydrated oocytes than H. hrasilien- sis (modal diameter about 1.6 versus 2.4 mm). The high spawning frequency of Hemiramphus species compensates Received 21 June 2002; accepted 6 November 20O2. * To whom correspondence should be addressed. E-mail: richard. mcbride@fwc. state. fl. us for their low batch fecundity. The annual fecundity of both species is similar to that of other reef fish species, after adjusting for body size and spawning frequency. The life- time fecundity of H. halao was very similar to that of H. hrasiliensis, after accounting for the differences in survival for each species. This suggests a fine tuning of different reproductive traits over the entire life cycle that results in roughly equivalent lifetime fecundity for both species. Introduction Two pelagic halfbeak species. Hemiramphus hrasiliensis and H. halao, are conspicuous and abundant elements of the Atlantic Ocean's coral reef fauna ((Toilette, 1965; Ny- bakken, 1997, p. 368; McBride el al.. 2003). These conge- ners are similar in size and shape (about 30 cm maximum length: McBride el al, 1996) but differ in both habitat use and diet. Both halfbeak species intermingle above coral reef habitats; otherwise, H. hrasiliensis is found only inshore of reef habitats and H. balao is found only offshore of reefs (McBride et al., 2003). H. hrasiliensis preys on zooplankton and grazes on seagrasses, whereas H. halao is a planktivore (Berkeley and Houde. 1978). Berkeley and Houde (1978) also characterized both species as oviparous summer- spawners with low batch fecundities (i.e., the number of eggs released per spawning event: Hunter et al., 1985). and they reported that H. hrasiliensis lived longer but had a lower batch fecundity than H. halao. These life-history patterns (i.e., age, reproduction, and mortality) are particu- larly intriguing because such patterns suggest a trade-oft between survival and reproductive output. Comparing life-history traits within species and between morphologically similar species in different habitats is a powerful method for understanding life-history evolution 58 R. S. McBRIDE AND P. E. THURMAN (Partridge and Harvey, 1988). If the life-history patterns of fishes evolve largely in response to their environment, it is striking that H. hrasiliensis and H. balao have much larger eggs but lower batch fecundities (i.e.. egg diameter > 1 mm and thousands of eggs per batch: Berkeley and Houde. 1978) than other coral reef fishes (e.g., see Thresher, 1984). These large eggs and low batch-fecundity values may sim- ply reflect the evolutionary history of hemiramphids. Aver- age batch fecundities for other oviparous hemiramphids range from one hundred (Silva and Davies, 1988; Coates and Van Zwieten, 1992) to a few thousand eggs per female (Talwar, 1962, 1967). In contrast, an average female coral reef fish with a body size similar to that of a Hemiramphus species produces about 100,000 eggs (Thresher, 1984). Such low fecundities for Hemiramphus species imply either high fertilization success, high survival rates, or the produc- tion of multiple batches of eggs. Multiple spawning is a common life-history trait among marine fishes and can greatly increase lifetime reproductive output. Although multiple spawning has been suspected to occur in several hemiramphids, it has never been demonstrated conclusively (e.g.. Ling. 1958; Talwar. 1967; Coates and Van Zwieten, 1992). In this study, multiple spawning is demonstrated for both H. hmsiliensis and H. balao. and new measurements are made of other life-history variables, namely age, mortality, size at maturity, egg size, batch fecundity, and spawning frequencies. Such detailed measurements demonstrate the interaction of phenotypic traits that determine fitness in two congeneric hemiramphids. We also compare these traits for Hemiramphus species with those of other hemiramphids and other coral reef fish species to evaluate the importance of evolutionary history in constraining allocation of repro- ductive effort. Materials and Methods Fishes were collected in the coastal waters of southeast- ern Florida (approx. 26.0 N, 80.0 W to 24.5 N, 82.2 W). Hemiramphus brasiliensis and H. balao were collected to- gether near the surface in association with coral reefs. H. brasiliensis alone was collected in other inshore habitats such as bank habitats in nearby Florida Bay, and so it is more numerous in our collections overall. From July 1997 to October 1998, 100 to 200 fish were subsampled. on each of 4 days per months from the catch of commercial fishing operations. Additional specimens were collected indepen- dent of the commercial fishery for a target number of 4 additional trips per month and a sample size of 12 fish per trip. Fish were kept on ice and brought to the laboratory for processing. Fish lengths and weights were measured in the laboratory. Fork length (FL) was measured to the nearest millimeter from the tip of the upper jaw to the fork of the tail. Whole body weight was recorded to the nearest O.I g. Ages of halfbeaks were determined by examining annual increments deposited on otoliths. For each trip in the months from July 1997 to June 1998, 12 fish were selected, at random, for aging; their sagittal otoliths were removed and stored dry. A low-speed saw was used to cut multiple 500-/xm-thick sections along the transverse plane through the otolith core. Otoliths were cut only from fish larger than 200 mm in FL, because otoliths of smaller fish are known to be age-0 (Berkeley and Houde, 1978). Sectioned otoliths were mounted to coded glass slides and examined, usually at 40 X, with reflected light under a dissecting microscope. The annuli were counted as a measure of fish age. in years, by two readers. If the two independent counts did not agree, then a third reading was conducted, with both readers work- ing together. Only 5% of the otoliths were so difficult to evaluate by both readers that they were rejected (/; = 61 ). The frequency of annulus formation was confirmed as an- nual by a marginal increment analysis. In such an analysis, the percentage of age-1 ballyhoo with an opaque margin, which was interpreted as a second annulus, was calculated for each month; monthly frequencies were checked for periodicity of annulus formation. Annual survival estimates (S) for each species were de- rived using the estimator from Robson and Chapman s= where x is the coded age class (0 = youngest age [in years] fully vulnerable to fishing), / v is the number of fish per age-class .v, and A is the oldest age class observed. The data for this analysis were only from the period October-May, because age-0 fish are not fully vulnerable to the sampling gear during the summer months (Berkeley and Houde, 1978). Gonads from 1 2 randomly selected fish in each collection were removed during the period of July 1997 to October 1998 and prepared for histology. Ovarian tissue was ini- tially fixed in 10% buffered formalin; a section of tissue was then transferred to ethanol, embedded in glycol methacry- late, sectioned along the transverse plane, stained with the periodic acid-Schiff (PAS) reaction, iron-hematoxylin, and counterstained with metunil yellow (Quintero-Hunter et ul., 1991). Gonads were assigned a stage based on the most advanced stage of oocyte development, namely perinucleo- lar. cortical alveolar, vitellogenic, nucleus migration, or nucleus breakdown. Cellular atresia such as postovulatory follicles (POP) and PAS-positive melano-macrophage cen- ters were also noted. Characterization of POFs follows the descriptions of Hunter and Macewicz ( 1985). Identification of PAS-positive bodies follows the descriptions in Grier and Taylor (1998; pp. 531. 539-540) and McBride et al. (2002). MARINE HALFBF.AK REPRODUCTION 59 Table 1 Maturity categories for female Hemirumphus ,v/i/>. Maturity Category Most Advanced Oocyte Stage Atresia and POFs'' Immature Perinucleolar stage Maturing Cortical alveolar (CA) Little or no atresia Little or no atresia Mature Vitellogenic or FOM h stage may be POFs; PAS+ bodies 1 Regressed Perinucleolar or CA stage PAS + bodies'" Females were scored according to their most advanced oocyte stage, and past spawning was inferred based on the presence of postovulatory follicles (POFs) and PAS-positive melano-macrophage centers (see text for details). The dashed line separates immature from mature stages, the division used for calculating size at 50% maturity. '' Postovulatory follicles (POFs) were observed as newly collapsed struc- tures after dusk. They were readily observed for about 24 h, after which they became more compact and darker. h Final oocyte maturation (FOM) began with migration of the nucleus, continued with breakdown of the nucleus ( = hydration), and ended with ovulation of eggs. L PAS-positive (PAS + ) melano-macrophage centers appeared as com- pact, bright purple bodies when our staining technique was used and are similar to yellow or brown bodies when other stains were used. The presence of vitellogenic oocytes was the primary indication of maturity (Table 1 ). Vitellogen, a protein se- creted by the liver and endocytosed by oocytes, accumulates in yolk globules that appear in the cytoplasm of oocytes during the spawning season (Wallace and Selman. 1978). Mature females with regressed ovaries were distinguished from immature virgin or maturing virgin females by the presence of PAS-positive melano-macrophage centers. Such PAS-positive bodies are involved in focal tissue deg- radation, and their presence increases during and after go- nad regression (Grier and Taylor, 1998). Size at 50% ma- turity was calculated as the inflection point of a logistic equation modeling the percent frequency of mature females: maturity = 1/(1 + exp(-/UFL - #])), where A -- the instantaneous rate of increase at the origin and B = the inflection point or the point where 50% of the individuals are mature. Model parameters were estimated by the logistic procedure of SAS software (SAS, 1990). Spawning frequency was estimated by the "post-ovula- tory follicle method" of Hunter and Macewicz (1985). We assumed that POFs became indistinguishable from other atretric bodies after about 24 h, so their presence as col- lapsed structures with identifiable thecal and granulosa lay- ers indicated that an individual female had spawned during the previous day. Batch fecundities and oocyte diameters were determined from examination of whole oocytes. Batch fecundities were estimated for 41 specimens of H. brasiliensis and 3 of H. balao collected in March 1997, May 1997, February-April 1998, and March 1999. About 1 g of tissue was removed from the anterior and posterior sections of the left and right ovaries, blotted dry. and weighed to the nearest 0.001 g. After the tissue was washed, teased apart, and placed in a solution of 33% glycerin:67% water, the number of hy- drated oocytes was counted. Batch fecundity was estimated according to the total weight of the ovary, following the methods of Hunter ft ul. (1985). To increase sample size, these data are presented together with data from Berkeley and Houde ( 1978), who used a similar method for estimat- ing batch fecundity and obtained similar results. Whole oocyte diameters, for at least 300 oocytes per female, were then measured to the nearest micrometer with the aid of a video system and image-analysis software. Results Age, growth, and xun'h'iil Sectioned otoliths revealed an alternating pattern of opaque and translucent bands (Fig. 1 ). The darker, translu- cent areas represented the periods of faster growth during the summer; the whitish, opaque bands reflected the periods of slower growth during the winter. Marginal increment analysis of Hemiramphus brasiliensis otoliths showed that a single annulus was formed each year and that annulus formation was complete by June of each year (Fig. 2). Although too few specimens of H. balao were available for the marginal increments of this species to be similarly analyzed, we chose June as the biological hatchdate for both species. Our aging results indicated that H. brasiliensis lives longer than H. balao (4 versus 2 years; Fig. 3). At a given age, individuals of H. brasiliensis were also larger on av- erage than those of H. balao, and females of each species were larger than male conspecifics. Of the 1022 specimens of H. brasiliensis aged, the largest was 294 mm FL, whereas of the 132 H. balao specimens aged, the largest was 251 mm FL. Annual survival of H. brasiliensis during the study period averaged 14.9% (95% confidence limits: 12.2%- 17.6%) and was double that of H. balao (7.5% on average; 2.01%-13.0%, 95% c.l.). Reproduction Ovaries of both halfbeak species were composed of two cylindrical lobes, roughly equal in size. During initial mat- uration of the specimens we examined, and again during spring recrudescence, these lobes increased in girth, devel- oped a pinkish color, and extended anteriorly along the coelomic cavity. Each lobe was a hollow sac with oocytes arranged in lamellae that extended into a central lumen (Fig. 4A-C). Virgin females lacked any PAS-positive melano- macrophage centers and had a thin gonad wall (Fig. 4A, C); mature, regressed females had PAS-positive melano-mac- rophage centers and a thick gonad wall (Fig. 4B). Oocytes 60 R. S. McBRIDE AND P. E. THURMAN Figure 1. Photomicrographs of sectioned utolilhs representing various age classes for Hemiramphus //liii* hni.\ilicnxix (ballyhoo), with a whitish, opaque annulus on the margin of the sectioned otolith, by month. Otoliths were collected from July 1997 to June 1998 (n = 1019). and fresh postovulatory follicles could be observed (Figs. 4J. M; 5C). Vitellogenic oocytes not in the final stages of oocyte maturation were observed together with oocytes that had migrating nuclei and with hydrated oocytes (Fig. 4N), which suggested a fairly rapid turnover of oocytes. The patterns of oocyte development for the two species did not appear to differ, except that the modal diameter of hydrated oocytes was only about 1 .6 mm for H. balao compared to about 2.4 mm for H. brasiliensis (Fig. 5B, D). In addition, females of H. halao matured (size at 50% maturity = 160 mm FL) at a smaller size than females of H. brasiliensis (i.e.. size at 50% maturity == 198 mm FL; Fig. 6). Both species became mature as age-0 fish (i.e., young-of-the- year). There were distinct seasonal trends in maturation, and the seasonal patterns of POP occurrence indicated prolonged, albeit slightly staggered, spawning periods for both species (Figs. 7, 8). During autumn, the incidence of immature and maturing individuals increased because age-0 fish were more frequently caught in the sampling gear; and the gonads of older fish were regressing as winter approached. Spawn- ing frequency peaked in April for H. brasiliensis and in June for H. balao. but spawning by some females was evident year-round based on the continued presence of POFs in older fish. Spawning frequencies were clearly age-specific, even for such short-lived species. All age classes of both species were spawning on a daily, or near-daily, basis, during spring or summer (Fig. 8). In other seasons, age-1 females spawned more frequently than age-0 females did, but age-2 ballyhoo females did not necessarily spawn more frequently than age-1 ballyhoo females. In terms of envi- ronmental associations, spawning activity peaked when the photoperiod was longest (i.e., June), and most juveniles were growing when temperatures were highest (/ e August' Fig. 9). Batch fecundity was higher for H. balao than for H. brasiliensis across the range of mature sizes (Fig. 10). Batch fecundity also increased more rapidly with increasing fish size in H. balao than in H. hrasiliensis. The average batch size for a 100-g female of H. halao (3743 oocytes) was more than three times that of a 100-g female of H. hrasil- ifiisis ( 1 164 oocytes). At a common size of 200 g, the batch fecundity of H. halao (8346 oocytes) was more than live times that of H. hrasiliensis (1538 oocytes). Discussion We report here, for the first time, the evidence that Hemiramphus species are multiple-spawners with group- synchronous oocyte development. Many general life-history patterns are similar for the two species of halfbeak. Both are short-lived, fast-growing, gonochoristic, and oviparous. Still, there were interspecific life-history differences, some of which varied in a manner predicted by life-history theory. The results of this study show the interplay between differ- ent phenotype-based life-history traits that are balanced out once lifetime reproductive output is calculated. Maximum ages for both species in our study were I or 2 years older than the ages determined in a previous study, but our results do not alter the conclusions that these are fast- growing and short-lived species (Berkeley and Houde, 1978). Berkeley and Houde (1978) reported that the maxi- mum age of 1 100 specimens of H. brasiliensis was age-2 and the maximum age of 135 specimens of//, balao was age- 1 . They read annuli on fish scales, and some researchers have noted that this method tends to underestimate ages compared to the use of otoliths (e.g., Lowerre-Barbieri et id.. 1994). Our results may also differ by mere chance from 1000 - 100 - 10 1 300 275 H. brasiliensis D H. balao |> 250 0) ^ 225 ; o "- 200 175 H brasiliensis -females H brasiliensis -males O H balao -females D H. balao -males 01234 Age (years) Figure 3. Number of Hcmirwnphus /j/W/mvu (balhhool and H. h,i/,i,i ( halao), collected by age class (upper panel I and the si/es ol each age class, by species and sex (lower panel; mean 95% confidence limits). 62 R. S. McBRIDE AND P. E. THURMAN ;" . .-.; j Figure 4. Hislological features of ovarian and oocyte development for Hcmirumplms hrasiliensis (bally- hoo). (A) whole cross section from a virgin, immature female; (B) partial cross section from a regressed, mature female; (C) whole cross section from a virgin, maturing female; (D) enlargement of detail of a late-stage "yolk vesicle" oocyte with cortical alveoli; (E) an even later-stage "yolk vesicle" oocyte; (F) a very early-stage "yolked" or vitellogenic oocyte; (G) a slightly more advanced vilellogenic oocyte; (H) a vitellogenic oocyte with yolk globules (probably a cell undergoing nucleus migration); (I) a vitellogenic oocyte with a migrating nucleus. MARINE HALFBEAK REPRODUCTION 63 0) CL D H balao, n-65 I H brasiliensis, n=522 0.00 0.50 1.00 1.50 2.00 2.50 3.00 Oocyte Diameter (mm) 3.50 Figure 5. Oocyte diameter frequencies for Hemimmfilws brasiliensis (ballyhoo) (A-C) and H. balao (balao) (D). in the final stages of oocyte maturation. The largest mode in each figure represents a developing batch of oocytes in final oocyte maturation, whereas the smallest mode represents a reservoir of primary growth oocytes and vitellogenic oocytes prior to nucleus migration. A third, middle mode of oocytes represents a batch of oocytes just entering final oocyte maturation. Measurements were made from (Ala mature H. brasiliensis whose most advanced oocyte stage was nucleus migration, (B) a hydrated H. brasiliensis whose most advanced oocyte stage was nucleus breakdown. (C) a spawning H. brasiliensis with ovulated eggs, (D) a hydrated H. balao whose most advanced oocyte stage was nucleus breakdown. At least 300 oocytes were measured per female. those of Berkeley and Houde; for H. brasiliensis we found only three age-3 individuals and one age-4; for H. balao we found only a single age-2 individual. Sample sizes in both E 9 iuu f 80- O 8 60- 'c 1 40 " 1 20- n 1! 1 Fork length (mm) Figure 6. Percent frequency of mature (vitellogenic) female Hcmir- tiniplni.t hrusilienxis (ballyhoo) and H. balao (bulao). Values are calculated by fork length intervals of 10 mm from fish collected during the peak spawning season (March-August), n = number of fish. studies were larger for H. brasiliensis because this species is more commonly caught and is found in a wider range of habitats than H. balao (McBride et al., 2003). At least one other hemiramphid, Reporhamphus inclainn'/iir, lives longer and grows larger (7 years, 380-mm FL; Ling, 1958). So even among hemiramphids, H. brasiliensis and H. balao are short-lived and grow to only modest lengths. Sizes of age-1 fish (mean = 248-mm FL for H. brasil- iensis and 226-mm FL for H. balao) were significantly different for the two species and were generally larger than previously reported. Berkeley and Houde (1978) reported that size at age-1 can vary between years for H. brasiliensis (1974 == 216-mm FL, 1975 = 230-mm FL) and that at age-1, H. balao was smaller (209-mm FL) than H. brasil- iensis. The sizes of both species overlapped (McBride et at.. 1996), and the mean sizes may vary from year to year naturally. Efforts to estimate the growth rates by nonlinear models failed because the short life spans of both species made it impossible to reasonably fit a growth model to the data. H. brasiliensis. however, spawns about 2 months earlier than H. balao (i.e., April versus June modes), so their growth rates may be fairly similar on a daily or monthly basis. We also found, as did Berkeley and Houde (1978), that females were longer than males at a common age. Survival rates differed between the two species. In our study, H. brasiliensis had a higher annual survival rate than H. balao did: 14.9% and 7.5%. respectively. We found the indicating the beginning of final oocyte maturation; (J) a vitellogenic oocyte with a migrating nucleus next to a fresh postovulatory follicle (POP); (K) a late-stage vitellogenic oocyte with the nucleus positioned against the chorion; (L) a fully hydrated oocyte after nucleus breakdown; (M) three hydrated oocytes. one without a follicle and adjacent to a fresh POF; and (Nl juxtaposition of a vitellogenic oocyte prior to nucleus migration, a vitellogenic oocyte undergoing nucleus migration, and a hydrated oocyte following nucleus breakdown. The major stages of oocyte development are indicated by capitalized letters: Perinucleolar (P). cortical alveolar (C) ( = yolk vesicle), vitellogenic (V) ( = yolked), hydrated (H). Other features include the chorion (ch), cytoplasm (cy), fibrils (fi), follicle (fo), lamellae (la), lumen (lu). nucleus (nu), periodic acid-Schiff reaction-positive (pas + ) melano-macrophage centers, post-ovulatory follicle (pot"), tunic (tu) (=gonad wall), yolk formation (yo). and yolk vesicles (yv). All scale bars, 0.250 mm. 64 R. S. McBRlDE AND P. E. THURMAN 100 80 60 40 c g '^ 20 20 1 10 o S. 80 60 ' 40 nd M A M J A S O N D FJ Immature Maturing I Mature I Regressed Figure 7. Seasonal reproductive cycles of (Al female Hemiramphus /'/iiw/ic/i.u.i (ballyhoo) and (B) female H. htiltio, (balao) based on histo- logical criteria for fish collected from July 1997 to October 199K. Values were calculated from mature tish sizes only (fork length S: 198 mm for H. i -H.W.V and >160 mm for H. balao). Nd = no data. was 2.2 mm. The modal size of hydrated H. balao oocytes ( 1.6 mm) was also very close to the size of the H. balao egg (1.5 mm) illustrated by Rass (1972). These independent descriptions and illustrations of mature oocytes and eggs confirm that interspecific differences in egg sizes exist for these two congeners. Sizes at maturity for both species were smaller than sizes attained by the first winter, and we agree with Berkeley and Houde ( 1978) that both species mature in their first year. In our study, the size at 50% maturity was 63.3% of the maximum body size for H. brasiliensis (313 mm FL) and 58.6% for H. balao (273 mm FL; McBride et al., 1996). To our knowledge, no study of hemiramphids has determined size at maturity with the precision that we achieved here, but other studies have reported the size of the smallest females with hydrated oocytes. Size at maturity in these other spe- cies is 56.8% maximum body length for Hyporhamphas iiiclanochir (Ling, 1958), 58.2% for Hemiramphus liinhntiix (Silva and Davies. 1988). and 58.0% for Zenarchoincnts kampcni (Coates and Van Zwieten, 1992). Size at maturity is very close to 60% maximum body size for a number of hemiramphids, and this percentage value may be useful for predicting the size at maturity of hemiramphid that have not been studied. Both H. hrasiliensis and H. halao spawn frequently, even daily, for at least a few months of the year. Although a same pattern when we calculated survival rates using data from Berkeley and Houde ( 1978), the only previous study available: estimates of H. brasiliensis survival varied be- tween the collection years ( 1974 = 23.8%. 1975 = 17.3%); these values were higher than that for H. balao (1974 = 12.4%). Even if the use of scales by Berkeley and Houde resulted in truncated ages (not identifying fish older than age-2), the number of fish aged was so large that the estimates of survival should not have been biased (Murphy, 1997). The more likely cause of the interannual variation is that survival rates may have declined since the mid-1970s or that violations of steady-state assumptions (variable recruit- ment of age-0 fish) may have biased the estimates of annual survival as calculated here. The macroscopic appearance and development of Henii- nunpluts species gonads were similar to those of Hypo- I'luunplnis (Reporhamphus) melanochir (Ling, 1958). The group-synchronous nature of oocyte development and large egg sizes have been noted for other hemiramphids (Ling, 1958; Taiwan 1967). The pattern of final oocyte maturation follows the diel cycle, and both species spawn at dusk (McBride ct al.. 2003). At sunset, the modal diameter of ovulated H. hrtmilienM'.'i eggs was 2.8 mm. Berkeley and Houde ( 1978) illustrated an H. hni.\ilicnsi.\ embryo that was about 2.5 mm in diameter and a near-hydrated oocyte that o c cr 0) C ra.w//tvi.us (ballyhoo), and //. luiliin (halao). by age class. Spawning frequency is based on the percent frequency of females with fresh postovulatory folli- cles (POFs). A value of 100% means that all females in a particular age class were spawning every day of that month. ;; = number ol tish. MARINE HALFBEAK REPRODUCTION 65 40 T Salinity, n=250 Temperature, n=252 20 2 13:00 - x Miami Harbor - Key West 10:00 M O N D Figure 9. Monthly average salinity and temperature (upper panel) measured at the sea surface at the time of fish collection. Error bars are 95% confidence limits. Seasonal change in photoperiod for Miami Harbor entrance and Key West (lower panel: data source: National Oceanic and Atmospheric Administration Tide Tables). prolonged reproductive season has been noted for other hemiramphids (Ling, 1958; Coates and Van Zwieten. 1992). this study provides the first conclusive evidence of multiple spawning within a year for any hemiramphid. Moreover, our age-specific analysis demonstrates that older fish spawn more frequently and for longer periods than age-0 fish do. In 10,000- o o 9,000 ' >s 8,000 ' ... -^ o ..'' c 7,000 - 13 o # ,-'"" CD 6,000 ' " ^ <$> -g 5,000 ' to o CQ 4,000 ' 3,000 ' ..'' o 2,000 ' o / Heniiramphus as the average number of eggs produced in the next generality b\ each ffiihiU 1 in the present generation Species A (years) 1, d, f, w. m, l,m x H. brasiliensis 1.01)1)1)1) 151 1 1 17.3 87.3 168,719 168,719 1 0.14S70 240 1264.6 126.7 303.507 45.133 2 0.02211 240 1353.2 150.4 324,757 7.181 3 0.00329 240 1493.4 187.9 358.419 1 . 1 74 4 0.00049 240 1445.1) 1 74.9 346.809 170 i 1,111, = 222,381 H. baliin 1.00000 124 2124.41 64.8 274.114 274.114 1 0.07527 240 4226.33 1 10.5 1.014,319 76,347 2 0.00567 240 2787.75 79.2 669,060 3,791 X 1,111, = 354.251 Variables are calculated by age classes in years (,v). Survival by age class (I,) was determined by the results of the Robson-Chaprnan survival estimate (see text for details). The number of days spawning by age class (d v ) was generalized from Figure 8. The batch fecundity by the average size female in each age class (f t ) was estimated from Figure 10. and the average weight of females in each age class (w,) was calculated from length-weight relationships (McBride, unpuhl.). Annual fecundity by age class (m,) is the product of the spawning frequency (d,) and batch fecundity (t,). and the expected reproductive output (I,m,) is the expected contribution of eggs produced by each age class after accounting for survival. fact, histological examination shows that some spawning by H. brasiliensis occurred year-round. Altogether, our data demonstrate that H. balao is a smaller, shorter-lived fish than H. brasiliensis. but it ma- tures at a smaller size and produces more, albeit smaller, eggs per batch. Morphological constraints of body size probably lead to this inverse relationship between size and number of eggs produced per spawning event (Elgar. 1990). and the larger batch size of H. Inilao is consistent with the life-history theory that predicts larger batch size for shorter- lived species (Stearns. 1976). H. balao is also more com- mon offshore of the reef tract, where food may be patchier than it is inshore; and various models predict that this would select for more numerous but smaller eggs ( Wootton, 1994). Ultimately, the life-history traits that typically represent trade-offs in evolutionary terms (i.e., survival and growth rates, age-specific spawning frequency, size-specific batch fecundity) are balanced so that the lifetime egg production of both Hemiramphns species is the same order of magni- tude; Table 2). Certainly the environment is shaping some elements of the reproductive traits of these Hemiraniphus species, but the influence of their phylogenetic history is clearly evident. Although Hemiramphus species are not structure-oriented, they are associated with coral reef habitat and thus are exposed to environmental cues similar to those encountered by coral reef fish. (<'.,?., warm temperature, high salinity. open coastal hydrodynamics, and tropical weather distur- bances). However, compared to most marine teleosts, and particularly other coral reef fishes. H. hrasiliensis and H. balao both have very large eggs, high spawning frequen- cies, and low batch fecundities U'.t,'., Thresher, 1984; Gross, 1987). Their reproductive style is typical of the order Be- loniformes, with its large eggs, low fecundity, multiple spawning events, and embryos that attach to floating vege- tation (Berkeley and Houde, 1978; (Toilette et ai, 1984). In fact. Hemiramphidae is a particularly interesting family to study while exploring trade-offs in reproductive traits be- cause there is remarkable variation in the egg size, fecun- dity, and reproductive mode of its species. For example, the diameters of hemiramphid eggs range from 1.3 to 3.5 mm, and reproductive modes include producing demersal eggs, buoyant pelagic eggs, or precocious young (Wourms, 1981; (Toilette et at., 1984; Meisner and Burns, 1997). Although many of the life-history traits of H. brasiliensis and H. balao are not shared by other coral reef fishes that have similar habitats, the annual fecundities of these two species might well be in line with those of other coral reef fishes. To illustrate this point, annual individual fecundities of H. brasiliensis and H. balao range from 169,000 to 1,014,000 eggs per year (Table 2), and these estimates are within the range of annual fecundity for a relatively small lutjanid, Rhomboplites aunmihcnx (140,000-3,000,000 eggs; Cuellar et rthern Anchovy, Engraulis mordax. R. Lasker. ed. NOAA Tech. Rep. NMFS 36. U.S. Dept. Commerce. Washington. DC. Hunter, J. R., N. C. H. Lo, and R. J. H. Leong. 1985. Batch fecundity in multiple spawning fishes. Pp. 67-78 in An Egg Production Method fur Estimating Spawning Biomass of Pelagic Fish: Application in the Northern Anchovy, Engraulis mordax, R. Lasker, ed. NOAA Tech. Rep. NMFS 36. U.S. Dept. Commerce, Washington, DC. Ling, J. K. 1958. The sea garfish. Reporhamphus melanochir (Cuvier & Valenciennes) (Hemiramphidae I. in South Australia: breeding, age determination, and growth rate. Atixt. ./. Mar. Freshw. Res. 9: 60-1 10. Lowerre-Barbieri, S. K., M. E. Chillenden, Jr., and C. M. Jones. 1994. A comparison of a validated otolith method to age weakfish, Cvnoscion rcgulis. with the traditional scale method. Fish. Hull. I US) 92: 555-568. McBride, R. S., L. Polisher, and B. Mahmoudi. 1996. Florida's hall- beak, Hemiramphus spp.. bait fishery. Mar. Fish. Rev. 58: 29-38. McBride, R. S., F. Stengard, and B. Mahmoudi. 2002. Maturation and diel reproductive periodicity of round scad (Carangidae: Decapterus puiictiitiix). Mar. Biol. 140: 713-722. McBride, R. S., ,). Styer, and R. Hudson. 2003. Spawning cycles and habitats for ballyhoo and balao {Hemiramphidae: Hemiramphus) in South Florida. Fixh. Bull. fUSI 101(3) (in press). Meisner, A. D., and J. R. Burns. 1997. Viviparity in the halfbeak genera Dermogenys and Nomorhamphus (Teleostei: Hemiramphidae). J. Morphol 234: 295-317. Murphy, M. D. 1997. Bias in Chapman-Robson and least-squares esti- mators of mortality rates for steady-state populations. Fish. Bull. (US) 95: 863-868. Nybakken, J. VV. 1997. Marine Biology: an Ecological Approach. 4lh ed. Addison Wesley Longman. Menlo Park. CA. 481 pp. Partridge. L., and P. H. Harvey. 1998. The ecological context of life history evolution. Science 241: 1449-1455. Quintero-Hunter, I., H. Grier, and M. Muscato. 1991. Enhancement of histological detail using metanil yellow as counterstain in periodic acid Schiff s hemotoxylin staining of glycol methacrylate tissue sec- tions. Biotech. Histochem. 66: 169-172. Rass, T. S. 1972. On the occurrence of ichthyoplankton in Cuban waters: pelagic eggs. Tr. Inst. Okeanol. Akml. Nauk. SSSR 93: 5-41. (In Russian). Roberts, C. M. 1997. Connectivity and management of Caribbean coral reefs. Science 278: 1454-1457. Robson, D. S., and D. G. Chapman. 1961. Catch curves and mortality rates. Traits. Am. Fish. Soc. 90: 181-189. SAS Institute Inc. 1990. SAS/STAT User's Guide. Vols. I and II. SAS Institute. Cary. NC. 1686 pp. Silva, E. I. L., and R. W. Davies. 1988. Notes on the biology of Hemiramphus limhatus (Hemiramphidae: Pisces) in Sri Lanka. Trap. Freshw. Biol. 1: 42-49. Stearns, S. C. 1976. Life-history tactics: a review of the ideas. Q. Rev. Biol 51: 3-47. Talwar, P. K. 1962. A contribution to the biology of the halfbeak, Hyporhamphus georgii (Cuv. & Val.) (Hemirhamphidae). Indian J. Fish. 9: 168-196. Talwar, P. K. 1967. Studies on the biology of Hemirhainphiix mar- ginatus (Forskal) (Hemirhamphidae-Pisces). J. Mar. Biol. Axxoc. India 9: 61-69. Thresher, R. E. 1984. Patterns in the reproduction of reel fishes. Pp. 343-388 in Reproduction in Reef Fishes, T.F.H. Publications, Neptune, NJ. Wallace, R.. and K. Selman. 1978. Oogenesis in Funilulux heiemclitiis. Dcv. Biol. 62: 354-369. Wootlon, R. J. 1994. Life histories as sampling devices: optimum egg size in pelagic fishes. J. Fish Biol. 45: 1067-1077. Wourms, J. P. 1981. Viviparity: the maternal-fetal relationship in fishes. Am. Zoo]. 21: 473-515. Reference: Binl. Bull. 2(14: 6S-80. (February 2003) 2003 Marine Biological Laboratory Collection and Culture Techniques for Gelatinous Zooplankton KEVIN A. RASKOFF 1 '*. FREYA A. SOMMER 2 , WILLIAM M. HAMNER 3 , AND KATRINA M. CROSS 4 Monterey Bay Aquarium Research Institute, Moss Landing, California 95039-9644: 2 Hopkins Marine Station. Pacific Grove, California 93950-3094: 3 University of California. Los Angeles, California 90095-1606: and 4 Monterey Bay At/iiariuni, Monterey. California 93940-1085 Abstract. Gelatinous zooplankton are the least under- stood of all planktonic animal groups. This is partly due to their fragility, which typically precludes the capture of intact specimens with nets or trawls. Specialized tools and techniques have been developed that allow researchers and aquarists to collect intact gelatinous animals at sea and to maintain many of these alive in the laboratory. This paper summarizes the scientific literature on the capture, collec- tion, and culture of gelatinous zooplankton and incorporates many unpublished methods developed at the Monterey Bay Aquarium in the past 15 years. Introduction Gelatinous zooplankton is a generic term for transparent and delicate planktonic animals with mesoglea-like internal (issues that aid in regulating buoyancy. These animals in- clude some radiolarians and foraminifera. as well as medu- sae, siphonophores, ctenophores, chaetognaths. pteropods, heteropods. appendicularians, salps, doliolids. and pyro- somes (e.g.. Hamner et y+ K. SK. PK Cassiopei ti Mimucliuna Anemia. Lighting 24-27 1 y+ RF Mastigias papua Anemia, Lighting 27-29 3 mo + K. PK. HP Hydrozoa Aequorea victoria Anemia. Rotifers (hydroid s). Juvenile Aurelia, l-'uit'iunu 10-15 6 mo + K. PK Eutonina indicans Artemiii. Rotifers 10-15 3 mo + K. PK Polyorchi x i>eiiicilltitn.\ Artciiihi 10-15 3 1110 + PK . K ( 'raspeda cn\iti sowerbii Wild Ires hwater plankton. Fro/en Daphnia Freshwater 27 <3 mo K. RT 1 Itlltl foil nosa Artemiii. Rotifers 24-27 6 mo + PK. K Data summarized from Sommer (IW2. 1943) for the Monterey Bay Aquarium. * K = Kreisel: PK = Pseudokreisel: SK = Stretch kreisel; RF = Reverse How; HP = Hon/onlal pseudokreisel; RT = Rectangular tank COLLECTION AND CULTURE OF GELATINOUS ZOOPLANKTON 75 Table 2 Selected culture techniques of non-cnidarian gelatinous zooplankton Species Diet Temperature (C) Lit'espan Tanks Reference Ctenophores Pleiinihrachia hachci Artemia 10-15 2 mo + K, PK Sommer, 1992 Wild-caught zooplanklon 15 nd Large jars Hirota, 1972 Pleurobrachia pileti.\ Wild-caught copepods 15 S 1110 + Modified PK Greve, 1970 Bolinopsis infundibulutn Artemia 10-15 8 mo + K. PK Sommer. 1992 Wild-caught zooplankton 16 7 mo Modified PK Greve. 1970 Mnemiopsis mccrailyi Wild-caught zooplankton 21-31 <2 mo 30 1 tanks Baker and Reeve. 1974 Beroe spp. Ctenophores, gelatin 10-15 <3 mo K. PK Sommer. 1992 Beroe gracilis Pleurobrachia pilcux 15 6 mo Modified K Greve, 1970 Beroe cuciimi\ Bolinopsis infundibulum 15 1 mo+ Modified K Greve, 1970 Molluscs Clionc limacina None 10-15 1 1110 + PK. K This study Wild-caught pteropods 12-14 4 1110 + Dishes Conover and Lalli. 1972 Cliopsis kmhni None 10-15 6 mo + PK, K This study Chaetogranths Sagitta hispida Copepods 17-31 2 mo + 30-1 tanks Reeve. 1970; Reeve and Walter. 1972 nd nd nd Modified K Greve. 1968 Pelagic Tunicates Oikoplt'iira dioicti Cultivated phytoplankton 13 8-12 days RJ Paffenhofer. 1973 Fritillaria boreal i\ Cultivated phytoplankton 12 nd RJ Paffenhofer and Hams, 1979 Thalia democratica Phytoplankton nd 8-20 days Large jars Heron, 1972 nd = no data. * K = Kreisel; PK = Pseudokreisel; RF = Reverse flow; RJ = Rotating jars. (such as Chrysaora fuscescens), since they will tend to congregate in the center of the tank, away from the walls (Tables 1 and 2). Any rectangular tank can be modified into a "pseudo- kreisel." but care must be taken to ensure that the height and width of the tank are about equal, or the water in the tank will not be able to rotate in a perfect circle and will create areas within the tank of limited flow where the animals may accumulate and contact the sides. Rectangular tanks are modified by glueing a screen across the upper corner at an angle of about 30-40 from vertical in front of the over- flow (Fig. 4). Water enters the tank through a perforated tube positioned so that the flow sweeps across the screen down towards the bottom of the tank. It is important that the tube is positioned so that the flow is parallel to the screen and covers the entire screen so that specimens are swept away rather than drawn against it. Curved plastic or vinyl inserts are glued with silicone into the bottom corners to round them into a more circular shape. Friction-fitting stiff screens can also be used to round the corners, although this option makes the tank more difficult to clean and maintain than one with solid corners. Water Several water quality issues are important for the suc- cessful culture and rearing of gelatinous organisms. Tem- perature and salinity must be kept within a range appropri- Screen \ Downwelling Inlet Outflow Inlet - \ Screen - ^Outflow Inlet Figure 3. Stretch kreisel design showing the two rotating Langmuir cells set up by the placement of the side and downwelling inlets. 76 K. A. RASKOFF ET AL. Inlet Inlet \ -Outflow Screen Outflow Plastic' or Vinyl Figure 4. Pseudokreisel design made from a standard tank. Bottom corners are filled in with silicone and solid pieces of plastic or vinyl. Outflow is separated from the tank by the inlet and screen. ate for the species being reared. The water must be relatively clean and filtered, especially if the animals are to be used for any display purpose. Small particles in the water will quickly clog the outflow screens. Filtering the water with 20-jum pleated cartridge tillers is usually sufficient; however, some cultures that are very sensitive to biological fouling (such as many hydroid species) may need additional filtration to the 3 jam level. Although air bubbles can be helpful in the culture of many small gelatinous animals by increasing water circulation, they can be detrimental to larger adult sizes (>3 cm). The bubbles can be ingested and collect in the gut and radial canals of medusae and cteno- phores, causing the animals to become positively buoyant, disrupting their normal swimming and feeding behaviors. A more serious problem is that these bubbles will slowly work themselves through the mesoglea. which can lead to infec- tion. A degassing system for the water may be needed if the incoming water tends to be supersaturated. A degassing tower in which the water trickles down through small plastic- balls or other material serves to degas the water before it enters the tank. Deep-sea animals may be sensitive to the high oxygen concentrations of surface waters. Reducing the oxygen concentration in tank water by bubbling nitrogen gas has been used in the past with some success, although it does not appear to be critical for most deep-sea species. Maintenance Throughout the course of feeding and rearing, tanks ac- cumulate debris that should be removed regularly. The use of pipettes, small brushes, basters, and siphons for removing larger debris, including waste, uneaten Anemia, and other food items, will help keep the tanks clean and discourage fouling growth. Pipettes of any size and type can be used to gently lift and collect debris. Kitchen basters work well for removing larger items because of their large reservoir vol- ume and wide bore. Siphons are best constructed from small-bore acrylic tubes with flexible plastic tubing at- tached, so that the tubing may be pinched to stop flow if an animal gets too close to the suctioning tip. Additionally, siphoning the "waste water" into a temporary container allows for the retrieval of any specimen that might inadver- tently be removed. To protect the insides of the tanks from scratches, it is helpful to dip the end of the acrylic tube into liquid plastic, available from most hardware stores; alterna- tively, a small ring of Nalgene tubing may be placed on the end of the siphon tube. Floating layers of lipid-rich mate- rials can be removed by skimming with small jars or beakers or fine-meshed nets, or by absorbing the material onto paper towels floated on the surface of the water. The sides of the tanks can be cleaned by wiping with brushes (firm paint brushes work well) or non-abrasive pads. For larger tanks (>75 1), painting or scrub pads can be covered with nonabrasive nylon mesh fabric and attached to poles for cleaning hard-to-reach areas of the tanks. The wood or metal handles of these scrubbers can be covered in plastic tubing to reduce the adherence of tentacles. Flow to the tanks can also be temporarily shut off and the animals allowed to collect on the bottom of the tank during cleaning. Also, tanks can be cleaned just after the animals have been fed, when tentacles are typically retracted and less apt to become ensnared (C. Widmer, Monterey Bay Aquarium, per. comm.). Screens in the tanks collect debris quickly and need to be scrubbed and cleaned at regular intervals. When screens become clogged, organisms are more likely to stick to them, possibly with fatal results. Even with proper cleaning and filtration, biofouling in COLLECTION AND CULTURE OF GELATINOUS ZOOPLANKTON 77 culture and rearing tanks can become a serious problem. In some cases of diatom and algae fouling, reducing the light that shines on the tank can help reduce growth, but typi- cally, scrubbing the tanks eventually becomes necessary. When diatom, hydroid, or other fouling organisms cannot be satisfactorily removed by any of the means discussed previously, bleaching is necessary. This can be especially useful on the screens, pumps, and waterlines, which can be very difficult to clean by other means. The entire tank system may need to be bleached every 1-6 months, depend- ing on the size and fouling rate. During bleaching, the occupants of the tank must be removed and transferred to a holding facility. The longer the tanks and lines are allowed to bleach, the more complete the fouling kill will be. Over- night is preferred, but bleaching for even an hour kills most fouling organisms. As a rule of thumb. 1 1 of standard 3%-6% sodium hypochlorite (NaOCl) bleach will treat about 200 1 of water ( = 1 gallon bleach/800 gallons of water), but this amount can be increased or decreased de- pending on the severity of the fouling and the time available to let the tank bleach. The water level in the tank should be dropped so that there is no overflow when the bleach is added. If the tanks have self-contained pumps, these should be run at a high flow rate to mix the bleach and flush it into the pump housings. To complete the process, the bleach must be neutralized. This can be accomplished by adding about 60 g of sodium thiosulfate (Na^S^O,) per liter of bleach used (=1 cup/ gallon). The sodium thiosulfate crystals may be dissolved in a bucket of water prior to adding to the tank. When the color of the water in the aquarium changes from yellow-green to clear, sufficient thiosulfate has been added for neutraliza- tion. Allow the thiosulfate several minutes to run through the entire tank and pumps. The treated water is then drained from the tank and discarded. While draining, thoroughly rinse out the tank with freshwater. Stubborn growth can be removed at this time by scrubbing. After all debris and treated water is removed, begin to refill the tank with seawater. minimizing turbulence and bubbles during the refilling since bubbles will stick to the walls of the tank and will have to be removed before gelatinous animals are returned. Discussion The use of the techniques described herein for the cap- ture, culture, and rearing of gelatinous zooplankton has allowed researchers to address many important biological issues. Historically, these contributions were limited pri- marily to the disciplines of systematics, developmental bi- ology, and evolution. More recently, new advances in our understanding of behavior, physiology, ecology, and ocean- ographic processes from the sea surface to the abyssal depths have also been possible. Through the use of culture methodologies, laboratory- based experimentation on salps and larvaceans has begun to address important ecological questions about the role these animals play in the nutrient cycling of the oceans and their impact on the ecosystem. These organisms have some of the fastest generation times and largest nutrient turnovers in the world, and their fecal pellets and associated "marine snow" are important sources of carbon transport into the deep sea (e.g., Alldredge, 1972; Silver et ai. 1998). Recent laboratory studies have shown that some species of medusa have chemically-regulated feeding behaviors (Arai, 1991, 1997; Tamburri et ul., 2000), with several different chemical stimuli controlling the feeding and swim- ming of both hydrozoan and scyphozoan medusae. Tank- based studies on the vertical migration of medusae (Mackie c? nl.. 1981; Mills, 1983) and on their swimming and feeding behaviors (e.g., Costello and Colin, 1995; Suchman and Sullivan, 2000) have provided much information on the physiological and behavioral components of medusa loco- motion as it relates to prey selection and capture. The interactions between gelatinous zooplankton and hu- mans are increasing, whether from envenomation (Burnett, 2001 ); blooms that clog power plant intakes (Masilamoni et ul.. 2000); interactions, both positive and negative, with fisheries (Mutlu et ul.. 1994: Mutlu. 1999; Mills. 2001; Purcell and Arai, 2001): or the general increase in gelati- nous zooplankton populations in perturbed or eutrophic environments (Mills. 1995. 2001; Arai. 2001). The oppor- tunities for scientific studies of gelatinous zooplankton are vast and largely untouched. We hope researchers can use some of the techniques presented here to expand the re- search being done on these important but poorly understood marine organisms. The public's fascination with and appreciation of gelati- nous zooplankton is growing rapidly. What were once con- sidered nasty animals that might sting or otherwise disturb beachgoers are now a major attraction in public aquaria all over the globe. The time and money spent by the aquarium industry to provide compelling exhibits on gelatinous zoo- plankton is a testament to their appeal. Over 3.4 million people visited the Monterey Bay Aquarium during the tem- porary "Planet of the Jellies" exhibit in 1992 and 1993 (Powell, 2001; J. Tomulonis, Monterey Bay Aquarium, pers comm.). Jellyfish and ctenophores were given permanent starring roles in the Outer Bay Wing, and in a new tempo- rary exhibit, "Jellies: Living Art." Aquarists in the United States and elsewhere are responsible for many of the tech- niques discussed in this paper. Aquariums around the world provide the bulk of the layperson's information on gelati- nous zooplankton. and we hope that the rising public ap- preciation of these important and beautiful animals may lead to increased financial and societal support for their continued study. 78 K. A. RASKOFF ET AL Acknowledgments F. Boero, A. Case. M. Coates, J. Connor, J. Costello, R. Hamilton, C. Harrold, G. Matsumoto. S. McDaniel, C. Priewe, K. Reisenbichler. B. Robison, R. Sherlock, J. To- mulonis, B. Upton, B. Utter. G. VanDykhuizen, C. Widmer. and D. Wrobel provided information and support for this review. J. Connor. B. Robison. G. Matsumoto, M. LaBar- bera, and two anonymous reviewers provided valuable com- ments on this manuscript. This work was supported by the David and Lucile Packard Foundation through MBARI/ MBA Joint Projects Committee. Literature Cited Abe, Y., and M. 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(February 2003) 2003 Manne Biological Laboratory Branchial Musculature of a Venerid Clam: Pharmacology, Distribution, and Innervation LOUIS F. GAINEY. JR. 1 *. JAMES C. WALTON 1 , AND MICHAEL J. GREENBERG 2 1 Department of Biological Sciences, University of Southern Maine, Portland, Maine 04104: and ' The Whitne\ Laboratory' of the University of Florida, 9505 Ocean Shore Blvd.. St. Augustine, Florida 32086 Abstract. This study was meant to analyze the neural control of the branchial muscles of the clam Mercenaria mercenaria. Gills isolated from the animal contract in re- sponse to 5-hydroxytryptamine (5HT). dopamine (DA), and acetylcholine (ACh); but the ACh contraction occurred only it" the gills had been pretreated with the cholinesterase inhibitor eserine. The 5HT antagonists cyproheptadine and mianserin blocked the contractile effects of all of the ago- nists. However, gills exposed to the 5HT antagonists and eserine relaxed in response to ACh. The DA antagonist SCH-83566 inhibited the effects of DA. but had no effect on contractions induced by 5HT and ACh. The ACh antagonist hexamethonium inhibited both the excitatory and inhibitory effects of ACh. but had no effect on contractions induced by 5HT and DA. 5HT and DA in gill tissue were visualized by using immunohistochemistry. Within each gill filament are dorsoventral neurons running adjacent to the epithelium and containing immunoreactive 5HT and DA. A complex net- work of 5HT-positive fibers is associated with the septa, blood vessels, and muscles, whereas DA-positive fibers are restricted to the septa. We propose that 5HT is the excitatory transmitter to the gill muscles, and that DA and ACh exert their excitatory effects by stimulating 5HT motor nerves. ACh may also be an inhibitory transmitter of the muscles. Introduction In most clams, the water current that supports respiration and feeding is driven through the gills by the beat of the lateral cilia. But the diameter and shape of the passages Received 8 July 2002; accepted 12 December 2002. * To whom correspondence should be addressed. Department of Bio- logical Sciences. University of Southern Maine. PO Box 9300. Portland. ME 04104-9300. E-mail: gainey@usm.maine.edti through the gill and thus the flow of water are controlled by contractions of the branchial musculature. These two fundamental activities of the branchial pump ciliary and muscular are regulated and coordinated by transmitters and modulators that are released at synapses by neurons that constitute an extensive network in the gill. The neural control of bivalve gill cilia has been extensively studied: this paper focuses on the musculature. The gills of the venerid clam Mercenaria mercenaria are eulamellibranch and plicate (Kellogg. 1892). That is. the filaments are connected to adjacent filaments via tissue junctions, and the descending and ascending lamellae are connected to each other and thrown into a series of folds (the plicae) by interlamellar septa (Fig. 1 ). The dorsoventral spaces within the gill, defined by adjacent septa and the intervening plicae, are the water tubes. The plicae exist in two conformations (Fig. 1C): either their contours are smooth the "primary folds" of Kellogg, or the "major plicae" of Eble (2001) or smaller depressions appear at the apexes of the plicae, giving rise to "secondary folds" (Kellogg), or "minor plicae" (Eble). We have seen the plicae alternate between these two conformations. Dorso- ventral blood vessels lie at the apex of each plica, within each septum, and within each filament (Kellogg, 1892; Eble, 2001). The blood channels of the branchial filaments are connected with the dorsoventral and septal blood vessels by a meshwork of horizontal blood vessels that are actually interlamellar abfrontal extensions of the filaments (see fig. 34 of the venerid Tapes unreus in Ridewood, 1903; fig. 4.20 in Eble, 2001; fig. 1 in Medler and Silverman, 2001). The horiz.ontal meshwork of vessels (collectively called the "subfilamentar tissue"; see Ridewood, 1903) lines the water tubes (Fig. ID). That bivalve "ills contain muscle fibers and are capable si 82 L. F. GAINEY ET AL cerebral ganglion visceral ganglion siphon pedal ganglion descending lamella ascending lamella foot exhalent (hyperbranchial) chamber visceral mass outer demibranch inner demibranch shell mantle cavity -gill mantle I) 5HTn water flow water flow gf Figure 1. Diagramatic anatomy of Mercenaria mercenaria: adapted from various sources on the basis of our own observations. (A) A clam on the halt shell. (B) Cross section of a clam. (C) Cross section of a water tube: with the musculature relaxed (left), and contracted (right). The water tube muscles are within the walls of the horizontal blood vessels; the vessels are not shown here. (D) Details of a relaxed demibranch (as in C, left). This cross section is slightly out of the horizontal plane; thus, the filaments to the left of the dashed line are at the level of the intern lamentar tissue junctions that contain the longitudinal muscles; whereas the filaments to the right of the line are at the level of the ostia and horizontal blood vessels. The walls of the horizontal blood vessels contain both the water-tube muscles and a dense network of serotonergic nerves; neither of these is shown. Abbreviations: bv = blood vessel; gf = gill filament; hbv = horizontal blood vessel; 1m = longitudinal muscle; o = ostium; s = septum; wt = water tube; wtm = water tube muscle; 5HTn = serotonergic neuron. of muscular activity has been known for over a hundred years since Kellogg (1892) published his observations on branchial anatomy and movement in a variety of bivalves, including Merci'miriu. Longitudinal muscle fibers have been described in the intertilament tissue junctions and septa of Mercenaria (Kellogg, 1892; Ridewood, 1903; Eble, 2001) and many other bivalve gills. In addition to these longitudinal muscles, called "horizontal muscles" by Atkins (1943) and others, Medler and Silverman (1997) noted the presence of a diffuse network of muscle fibers in the water- tube epithelium of the non-plicate gills of Dreissena poly- inorpha. The plicate gills of Mercenaria lack a water-tube epithelium, but they contain a similar network of muscle fibers in the walls of the horizontal blood vessels (Fig. 1 ). Neural elements occur within the filaments of both fili- branch and eulamellibranch gills (e.g., Setna, 1930; Aiello. 1990), but they also occur in the gills of eulamellibranchs (like Mercenaria) in association with the septa, blood ves- sels, and interfi lamentar muscles; structures that, by defini- tion, do not occur in tilibranch gills. Indeed, neurons have been reported in association with the longitudinal muscles in a unionid mussel, Ligiuniu subrostrata (Dietz et at.. 1985), in Merccmiria (Gainey et ai, 1999a), and in an oyster, Crassostrea virginica (Nelson, 1960). Nerves have GILL MUSCLE PHARMACOLOGY AND ANATOMY 83 also been observed in the interlamellar septa in Solen mar- ginatus and Ensis siliqua (Atkins. 1937), Mercenaria (Gainey et at., 1999a). and Crassostrea (Nelson. 1960, Galtsoff, 1964); and in the water-tube muscles and ostia of Mercenaria (Candelario-Martinez et ai, 1993). An extensive literature indicates that the flow of water through bivalve gills varies continuously within wide limits in response to both physical and biological factors (summa- rized by Dame. 1996; Jorgensen. 1996; Bayne, 1998). But the lateral cilia in both Mercenaria and Mytilus edulis beat only within a relatively narrow range of frequencies (about 10-25 beats/s) (Aiello, 1960; Catapane, 1983; Gainey etui.. 1999a), so the stimulatory and inhibitory motor nerves to the cilia seem to be activating a simple on-off switch. Medler and Silverman (2001 ) found, in Mercemiria. that the geometry of the water tubes changed, and their diameters decreased, in response to 5-hydroxytryptamine (5HT; sero- tonin). Such changes would tend to modify flow (Grunbaum et a!.. 1998). so changes in the tone of the branchial mus- culature might well be participating in the continuously variable regulation of water flow through the gill. Although the branchial muscles have the potential to modulate water flow through the gills, and neural elements are clearly present, the pharmacology and neural control of these muscles has received relatively little attention. In brief, acetylcholine (ACh) contracts the gill muscles in both Dreissemi polymorpha and Corbicula fluminea (Snow et ai. 1995; Medler and Silverman. 1997), whereas 5HT relaxes the gill muscles of Lignmia subrostrata (Gardiner et ai. 1991) and contracts those of Mercenaria (Gainey et ai, 1998; Medler and Silverman. 2001 ). In addition, the peptide FMRFamide contracts the gill muscles of Dreissemi (Medler and Silverman. 1997). The relationships between the effects of possible neurotransmitters on gill muscles, the distribution of these agents in identifiable neural networks, and the interactions among the elements of the networks are at present unexplored. We have been using the gill of the quahog Mercenaria mercenaria to study the neural control of branchial water flow. In a previous study, we found that 5HT and dopamine (DA), respectively, switch the activity of the lateral cilia on and off, and that YFAFPRQamide, an SCP-like peptide endogenous to Mercenaria. modulates the effects of DA (Gainey et al., 1999a). Now we report on the pharmacology of the branchial muscles, focusing especially on the actions of 5HT. DA, ACh. and their antagonists. We have also investigated the distribution of the branchial muscles and their innervation by immunoreactive serotonergic and do- paminergic nerves, expecting the findings to be consistent with our pharmacological observations. Preliminary results of this study have been presented to the Society for Inte- grative and Comparative Biology (Gainey et ai. 1998. 2001). Materials and Methods Animals Quahogs (Mercenaria mercenaria L.) that had been dug from various locales along the northeast Atlantic coast were purchased from Harbor Fish, Portland, Maine. The animals were held at 10 C in natural seawater (30 ppt) on a 12-h light/dark cycle. Individuals were held a minimum of 3 days before use. G/7/ preparation and apparatus Gills were dissected away from the body wall and sepa- rated into demibranchs, and the branchial nerves removed (Fig. 1 ). Muscular contractions were recorded as changes in the length of the anterior-posterior axis of the isolated demibranchs. Contractions of the branchial muscles were recorded in either of two ways: ( 1 ) Isolated demibranchs were sus- pended in organ baths and attached with thread to isometric force transducers (Grass FT03 and UFI 1030) equipped with springs; the resulting contractions were therefore semi-iso- tonic. The transducers were interfaced to Biopac DA 100 amplifiers and a Biopac MP100 analog-to-digital converter. (2) Ultratrasonic crystal transceivers (Sonometrics) were tied to the ends of demibranchs with thread. One end of the demibranch was pinned to a piece of rubber band that was glued with rubber cement to the bottom of a plastic petri dish (4.7-cm diameter); the petri dishes were placed on a cooling plate to maintain temperature. Under these condi- tions, the muscles were unrestrained and contracted against virtually no external load. The isotonic contractions were measured with a digital ultrasonic measurement system (Sonometrics TRX series 8). In both cases, the magnitude of the contractions was measured with AcqKnowledge version 3.5 (Biopac Systems). All experiments were earned out at 10 C in aerated artificial seawater (ASW; recipe in Welsh et ai. 1968). To retard the oxidation of dopamine (DA), the water was buff- ered with an ascorbic acid buffer as described by Malanga ( 1975); this buffered seawater was used in all of the exper- iments. Production and analyses of dose-response cnn'es Our initial experiments were performed with force trans- ducers; but prolonged contraction against the load of the springs used with these devices caused the gill muscles to fatigue. Consequently, we exposed each demibranch only once to a single concentration of agonist, and the dose- response curve was constructed from these individual re- sponses. In later experiments with the Sonometrics digital ultrasound measurement system, no external force was ap- plied to the muscles. No evidence of fatigue was observed. 84 L. F. GA1NEY ET AL. so a single demibranch could be used to construct an entire dose-response curve. Because the response to serotonin (5HT) and DA has a seasonal component (Gainey, pers. obs.). the dose-response data reported here were collected between November and July. All contractions and relaxations, measured in millimeters, were expressed as a percentage of the initial length of each demibranch. Regression lines were fitted with a logistic function of the form: response = n/1 + exp(/3 () + j3,* log( agonist)), where a is the asymptotic value of the max- imal contraction, and j3 and /3, are intercept and slope parameters. Initially, all three parameters were estimated using nonlinear regression (Systat, v 9); later, a was fixed in the model, reducing the error estimates of the remaining parameters. The concentrations of agonist giving half-max- imal responses (EC MI ) were estimated according to the following formula: EC ?0 = 1(> A ( - /3,//3, ). Effects of antagonists Each of the tour demibranchs from the same clam were suspended in an organ bath and attached to a force trans- ducer. After 15 min of relaxation, each of the demibranchs was exposed to an agonist at a standard concentration: 5HT = 2 X 1(T 5 M; DA and acetylcholine (ACh) = 5 X 1CF 5 M. After the resulting contractions had stabilized, the baths were flushed, and an antagonist at 10~ 4 M was added to three of the four demibranchs. After 60 min. the standard dose of agonist was reapplied to all four demibranchs. with the antagonist still present on the three demibranchs. The total number of demibranchs treated with a specific antag- onist is given in the data tables. The effect of the antagonist was expressed as the ratio between the second and first agonist-induced contraction (contraction ratio). Analysis of the contraction ratios of untreated controls with a Kolmogorov-Smirnov one-sample test revealed that these data were not normally distributed (P < 0.001, two-tailed, n = 139). The contraction ratios were therefore normalized by a logarithmic transformation, and the normality of this transformation was checked as above (Kolmogorov-Smirnov: P 0.614). The In trans- formed ratios of the controls were tested against a mean of (since In 1 =0) with a one-sample / test. This is mathe- matically equivalent to a paired / test because the contrac- tions used to construct the ratios were from the same demi- branch. Since the contraction ratios of the controls for 5HT, DA, and ACh were all significantly greater than 1, the normal- ized contraction ratios of the antagonists were compared to the normali/cd contraction ratios of the appropriate agonist control using post hoc paired Tukey HSD tests after an initial one-way ANOVA. But some of the antagonist con- traction ratios were 0. thus these ratios become undefined by a logarithmic transformation. To overcome this limitation. 0. 1 was added to all contraction ratios prior to the logarith- mic transformation. Although the statistical tests were per- formed on the In-transformed data, tabular data are pre- sented in the Results section untransformed for clarity. The P values reported for these tests are one-tailed probabilities: P values less than 0.05 were considered significant. In some of the experiments e.g., ACh after exposure to cyprohep- tadine or mianserin the gills relaxed rather than con- tracted; these data are coded in the tables as negative values. The concentration of antagonist that produced 50% inhi- bition (IC 5() ) was calculated using the experimental design described above, except that the demibranchs were exposed to lower concentrations of antagonists. Contraction ratios i.e.. the ratios of the second to the first contractions were regressed against the log of the concentration of antagonist. Because the contraction ratios were significantly greater than 1 for all of the controls, the IC ?(I was calculated by solving the regression equation for a contraction ratio that was 50% of the mean contraction ratio of the control. Branchial anatomy For relaxed specimens, isolated demibranchs were kept overnight, at 5 C, in isotonic MgCI 2 in ASW (7.6% MgCl : in distilled water added to an equal volume of ASW). For contracted specimens, the isolated demibranchs were placed in [Q M 5HT immediately after dissection. To observe the inner face of the water tubes, we cut dorsoventrally along several septa with fine scissors, separating a section of the demibranch into two layers. One of these was removed, and the remainder of the demibranch was then pinned to the bottom of a small petri dish, which had been coated with Sylgard. Fixation always carried out at 5 C varied with the object to be observed (e.g., muscle. 5HT. DA) and is described below. Because mammalian antibodies were used for the immunohistochemistry, subsequent rinses and solu- tions were made with mammalian phosphate-buffered saline (PBS). Crysostat sections were prepared as follows. After fixa- tion and a 15-min rinse in PBS (0.1 M sodium phosphate, 140 mM NaCl; pH 7.3). the demibranchs were placed in a solution of 30% sucrose/PBS overnight at 5 C. Pieces of demibranch were then placed in Tissue Tek OCT com- pound, frozen, and sectioned at 12 /urn. Sections were placed on gel-coated slides and stored at 20 C until used. Thick sections were prepared as follows. After fixation and three 15-min rinses in PBS. pieces of demibranch were placed in a plastic mold and covered with 12% Type A pigskin gelatin in 0. 1 M PBS that had been heated to 50 C. After the gelatin had cooled, the tissue was sectioned at 100 /j.m with a vibratome. The sections were heated briefly at 50 C on gel-coated slides to melt the excess gelatin. GILL MUSCLE PHARMACOLOGY AND ANATOMY 85 We usually processed whole mounts and sections simul- taneously and therefore followed a schedule designed for whole mounts. All of the steps in this protocol were carried out at 5 C. After fixation, rinse four times (1 h for each rinse) in PBS (O.I M. pH 7.3); or for DA, in 0.05 M PBS with 1% sodium metabisulfite. Incubate overnight in blocking solution (0.25% goat serum/ 1' BS A/PBS): for DA. in- clude \'7c sodium metabisulfite. Incubate overnight in primary antibody diluted appro- priately with PBS. Rinse four times in PBS (two 30-min rinses, one over- night rinse, one 30-min rinse). Incubate overnight in secondary antibody, phalloidin. or both, the reagents diluted appropriately in PBS. Rinse three times in PBS (1 h. overnight. 1 h). Mount the specimens under coverslips in 60% glyc- erol-1% n-propyl gallate/PBS. Muscle. The branchial muscles were visualized with phalloidin conjugated to the fluorescent probe Alexa Fluor 488 (Molecular Probes. Eugene. Oregon), the conjugate used in a concentration of 1 unit/100 jul in 0.1 M PBS. For single-stained preparations, whole mounts were fixed for 1 h in 4<7r formaldehyde with 0.01 M PBS (pH 7.3; 530 mM NaCl). rinsed twice, and then stained overnight. To double- label immunochemically stained preparations, the phalloi- din was added to the tissues at the same time as the sec- ondary antibody. 5HT and YFAFPRQamide. Pieces and sections of demi- branch were fixed overnight in 4% paraformaldehyde in 0.01 M PBS (pH 7.3; 530 mM NaCl); the fixative was prepared as described in Gainey et al. ( 1999a). For 5HT, the primary polyclonal antiserum was raised in rabbit to 5HT conjugated to BSA with paraformaldehdye (Diasorin. Still- water, Minnesota). For YFAFPRQamide. the primary poly- clonal antiserum was raised in rabbit to the peptide conju- gated to thyroglobulin (custom synthesis, etc.. by SynPep. Dublin. California). In both cases, the secondary antibody was raised in goat to rabbit IgG conjugated to Alexa Fluor 594 (Molecular Probes). Dopamine. Pieces and sections of demibranch were fixed for 2 h in 57c glutaraldehyde/19r sodium metabisulfite/PBS (0.01 M: pH 7.3; 530 mM NaCl). The primary polyclonal antiserum was raised in rabbit to DA conjugated to BSA with glutaraldehyde (Diasorin). The secondary antibody was raised in goat to rabbit IgG and conjugated to Alexa Fluor 594 (Molecular Probes). For negative controls, the primary antibodies were omitted from a slide in each series of preparations. Confocal images of 5HT distribution were made with a Leica LSCM SP2 microscope at the Whitney Laboratory. 5 min Figure 2. Traces of contractile activity recorded from three untreated demibranchs taken from a single clam. (A) Quiescent. (B) Occasional, spontaneous contraction. (Cl Arrhythmic, spontaneous contractions. Con- tractions were recorded with force transducers. St. Augustine, Florida. Fluorescent images were made with a Nikon Eclipse TE200 microscope equipped with a Spot RT digital color camera (Diagnostic Instruments). Images were prepared for publication with Adobe Photoshop. Drugs All chemicals were purchased from Sigma-Aldrich, St. Louis, Missouri, or ICN Pharmaceuticals. Costa Mesa, Cal- ifornia. The specificities of the antagonists listed in the tables were obtained from the Cell Signaling & Neuro- science catalog (2000/2001 ed.) of Sigma/RBI. Results Activity of isolated gills Most of the isolated demibranchs were quiescent in the organ baths (Fig. 2a), but occasionally gills would contract spontaneously and relax (Fig. 2b). and on rare occasions they would beat arrhythmically (Fig. 2c). All three demi- branchs in Figure 2 were from the same clam; the fourth, not pictured, was also quiescent. Of the hundreds of prepara- tions we have observed, only a handful showed the sponta- neous, arrhythmic contractions seen in Figure 2c. Pharmacology of branchial muscles Agonists. 5HT, DA, and ACh contracted the gill muscles in a dose-dependent manner, but the response to ACh was observed only in gills pretreated for 15 min with 10~ 4 M eserine (Fig. 3). The responses to all three agonists were 86 L. F. GAINEY ET AL LQG5HT(M1 RESPONSE -7 -6 -S -4 -3 -2 5 min Figure 3. Traces of contractions in response to increasing concentra- tions of 5HT; successive doses were added at the arrows. Each response is from a separate demibranch; data were recorded with force transducers. indistinguishable: the gills contracted tonically and, after 30 s to several minutes, reached their maxima. Dose-response curves for the three agonists were pre- pared (Fig. 4), and their characteristics are listed in Table 1. The rankings of the EC 5() values are ACh < 5HT < DA, and since the 95% confidence intervals do not overlap, the values are statistically different. The maximal contractions in response to 5HT and DA are equal, and both are signif- icantly larger than the maximal contractions induced by ACh. The comparative data noted above were independent of the method used to record the contractions. However, if we consider each agonist separately, then its EC 50 is signif- icantly less, and its induced contractions were larger (except for DA) when the contractions were recorded with the digital ultrasound system rather than with force transducers (Table I). The following neurotransmitters, all applied to the tissue at 1()~ 4 M. neither contracted nor relaxed the gills: ATP, GABA, histamine. and octopamine. Furthermore, the fol- lowing three peptides all found in Mercrmiriti and all applied at 10~ 6 M neither contracted nor relaxed the gills: FMRFamide, AMSFYFPRMamide, and YFAFPRQamide. Previously, we found that YFAFPRQamide modulates the effects of DA on the lateral cilia and those of 5HT on the frontal cilia (Gainey et til.. 1999a). Therefore, to determine whether the peptide would modulate the effects of 5HT or : Ultrasound recorder : Foree transducers : I : i * log |5HT(M) | -(. -5 -4 log|ACh(M)| Figure 4. Dose-dependent muscle contractions (as percentages of the resting length) in response to 5HT, DA, and ACh. Solid circles and lines: data recorded with force transducers; each datum is the response of a separate demihranch. Open circles on dashed lines: data recorded with an ultrasound system from either two (5HT, ACh) or five (DA) demibranchs; each preparation was exposed to increasing concentrations of agonist. Demibranchs exposed to ACh were pretreated with 10 4 M eserine. GILL MUSCLE PHARMACOLOGY AND ANATOMY 87 Table I Summary of dose-response effects for 5HT. DA. and ACh Agonist Type* EC 5() (M)t (95% CDI Cmax (%) (95% CI) 4 : 5HT II 1.1 x 1(T 4 (0.5-1.8 x 1(T 4 ) 20 (17-23) us 2.1 x 10~ 5 (1.9-2.5 x 10~ 5 ) 33 (28-38) DA ft 5.9 x 10~ 4 (2.3-9.5 x 1(T 4 ) 21 (14-29) us 1.4 x 1CT 4 (1.2-1.7 x 10' 4 ) 32 (17-48) ACh ft 1.5 x 10~ 5 (0.34-3.3 x 10" 5 ) 2 (1-3) us 8.6 X 10~ 6 (0.2-1.3 x 10~ 5 ) 9 (7-11) * f t = force transducers used to measure contractions; us = ultrasound used to measure contractions t Concentration of agonist giving a half maximal response. t 95% confidence intervals associated with the estimates. S Maximal predicted contraction. DA on the muscle, we applied YFAFPRQamide to the demibranchs before exposing them to 2 X 1CT 5 M 5HT or DA. At concentrations ranging from 1CT 9 to 1CT 6 M (5HT) or 10~ s to 10~ h M (DA), and exposures ranging from 15 min to 1 h (5HT) or 1 h (DA), the peptide had no effect upon contractions induced by either 5HT or DA. Antagonists. Because the three effective agonists contract the gill, and since the mechanical responses to 5HT, DA, and ACh are indistinguishable, we asked whether the mus- cles have receptors for each of the agonists, or whether one or more of the agonists are acting indirectly by stimulating the release of another agonist from motor nerves. To test these possibilities, antagonists were sought for each agonist, and these agents were cross-tested against the other ago- nists. Controls. In control experiments, each gill received two consecutive, equal doses of the same agonist. For each of these agonists, the second contraction in re- sponse to the same concentration was usually larger than the first, and the contraction ratios were signifi- cantly greater than 1 (Table 2). Moreover, when Table 2 The effect of antagonists on the actions of 5HT. DA, and ACh Antagonist Type* Agonistt Mean contraction ratio SD (n)t />$ None (control) 5HT 2.20 1.86 (72) < 0.001 ''' None (control) DA 2.18 1.74 (46) <0.001* p None (control) ACh 3.21 3.65 (18) 0.00 l* p Cyproheptadine 5HT, 5HT 0.324 0.495 (11) -o.oo ] DA 0.076 0.162 (10) 0.001 ' ACh -1.19 2.10 (9) *r Mianserin 5HT, 5HT 0.475 0.193(8) 0.002*' DA 0.494 0.490(8) <0.001*' ACh -0.449 0.701 (9) *r SKF-83566 DA, DA 0.483 0.300 (9) 0.003*' 5HT 0.869 0.233 (9) 0.32 ACh 0.881 0.530(3) 0.22 Hexamethomum ACh n ACh 0.215 0.262(5) 0.001 '" DA 1.05 0.593 (6) 0.49 5HT 2.14 0.850(8) 0.50 * The primary type of mammalian receptor blocked by the antagonist. t Agonist concentrations: 5HT = 2 x 10~ 5 M; DA & ACh = 5 x 10~ 5 M. Antagonist concentrations were 10' 4 M. t Contraction ratio: height of contraction after exposure to the antagonist/height of contraction before exposure to the antagonist. S P values are one-tailed probabilities: *i = significant inhibition; *p = significant potentiation; *r = relaxation, the second contraction was coded as a negative value. 88 L. F. GAINEY ET AL Table 3 .\iitiixniii.\t.\ thai Inn/ an we//Hni>itli\ Ostia F Gardiner el til.. IWI Ligumia subrostrata Ostia F (I Gardiner el ul .. Wl HETERODONTA Dreissemi polymurpha V/TR F Snow el til.. 1995 V/Ostia + Medler & Silverman . 1997 Corbicula ftuminea IFD/Ostia F Medler & Silverman . 2(101 Mei'centiriti mercenaria TR M This study * DVO. direct visual observation of isolated gills. IFD. intertilument distance, recorded on videotape. Ostia. change in diameter of ostia measured. TR, direct measurement with a transducer of movement or force development. V. measurement of length or area changes recorded on video tape. t M. marine; F. fresh water. Habitat is defined in terms of salinity. Note that the species listed here as "marine" are all at least moderately euryhaline (5-15%c to 30-40%r). The criterion for designation of habitat as "fresh water" (F) is the ability of animals to live and reproduce (or survive prolonged immersion) in fresh water. t +. excitation [increased tone (or ostia increased in diameter): or increased rate, regularity, or amplitude of contractions]. , inhibition [relaxation (or decreased diameter of ostia), or reduced rate, amplitude, or regularity of contractions]. 0, no response observed. The predominant responses of the tissues to each transmitter are listed. bearing the gill cilia, and in nerves running out to that region (Gainey et at.. 1999a). We have certainly not identified all of the transmitters in the innervation of the filaments, but the morphological restriction of YFAFPRQamide to the fila- ments, and its physiological restriction to effects on cilia, suggests that innervation of the branchial filaments may be exclusively in the service of the cilia, and that the remaining two neural divisions regulate the muscles. These consider- ations also support our hypothesis that the proposed syn- apses of dopaminergic and cholinergic neurons onto sero- tonergic neurons will be found in the septa. Coordination between the ciliary pump and the branchial muscles. Two video endoscopic observations suggest that the lateral cilia and the gill muscles act in a coordinated fashion. First, when the gills of a unionid, Pygunodon cataracta. stop pumping, the water tubes constrict, but re-open when pumping resumes (Tankersley, 1996). Sec- ond, when the valves of Mercenaria are closed, the lateral cilia are immobile, and the gills are tonically contracted, both longitudinally and dorsoventrally (Baker, pers. comm.). When the clam is actively pumping, we expect that se- rotonergic stimulation of the muscles is reduced and the muscles are relaxed. Under these circumstances, the ostia, water tubes, and blood vessels would be open, so the flow of water and hemolymph would be maximized. When the clam closes, the dopaminergic innervation would become active, switchini; the lateral cilia off and stimulating the serotoner- gic plexus. The longitudinal and dorsoventral muscles and the water-tube muscles would then constrict, closing the ostia and constricting the water tubes and blood vessels. Comparative aspects of branchial muscle pharmacology Although there is an extensive literature on the pharma- cology of bivalve muscles, it is largely focused on the anterior byssus retractor muscle of Mytilns and isolated ventricles of a variety of bivalves including that of Merce- naria. In contrast, the pharmacology of branchial muscles has been studied in relatively few species of bivalves, in part because the branchial musculature is not an advantageous model for the study of muscle cells per se. Branchial mus- cles are small and are embedded in a complex organ; thus they cannot be directly attached to a recording apparatus. Furthermore, their neural supply is complex, and the inner- vation of specific muscles is not readily accessible. How- ever, the pharmacology of these muscles has been studied by those interested in the physiology of bivalve gills; the available data are summarized in Table 4. The effect of 5HT on the muscle varies with species. The gills of Mytilns and Mercenaria are contracted by 5HT. whereas those of Dreissena polymorphic Anodonta gruiulis, and Ligumia subrostrata are relaxed. There is no taxonomic order in these data; but 5HT contracted the gills of the two marine species and relaxed those of the three freshwater species. Because the sample size is miniscule, however, the 94 L. F. GAINEY ET AL apparent relationship between 5HT action and habitat may be coincidental. ACh had a net excitatory effect on five of the six species on which it was tested; one species (Ligitmia subrostrata) showed no effect, but the gills were not pretreated with eserine. The effect of ACh on the gills of Mercenaria was revealed only after pretreating them with eserine; in addi- tion, the inhibitory effect of ACh became evident only when the gills were exposed to a 5HT antagonist and eserine. The relaxing effect of ACh has not been seen in any of the other gills tested, but then the pharmacological analysis reported here was not used in the other studies. Painter and Greenberg (1982) examined the effects of 5HT and FMRFamide on the ventricles of 50 species of bivalves and remarked that "the responses were strikingly diverse, varying qualitatively with dose as well as species." In their analysis, however, clear taxonomic relationships were discernable. In comparison to ventricles, gills are much more complex and interact directly with the environ- ment. For example, sodium transport in the gills of fresh- water mussels appears to be regulated by a serotonergic neural mechanism (data summarized in Dietz el ai, 1985). groundwork for studies of the integrated control of gill function. Acknowledgments We thank David Chicoine, Robert Pirone, and Kelly J. Vining (University of Southern Maine) for their assistance in performing the pharmacology experiments. In addition, David Chicoine helped assemble Figures 6 and 7. The following individuals at the Whitney Lab provided assis- tance: Dr. Dimitri Budko provided the basic protocol used for whole mounts; Dr. Paul J. Linser provided technical assistance with immunohistochemistry and confocal mi- croscopy and took the picture used for Figure 6E; Leslie Van Ekeris made the cryostat sections and provided tech- nical assistance with the immunohistochemistry; M. Lynn Milstead drew and prepared Figure 1 for publication. Fi- nally, Seth Tyler (University of Maine) advised on the phalloidin staining of muscle. Support was provided to LFG by grants from the Maine Science and Technology Foun- dation and the Bioscience Research Institute of Southern Maine (at the University of Southern Maine). The odd response of the control .y/Y/.v /;; experiments with antagonisls When two successive equal doses of any agonist (i.e., 5HT. DA, or ACh) were applied to the control gills, the second contraction was typically larger than the first, and this result was initially inexplicable. Later, however, we discovered that the gills produce nitric oxide (NO) in re- sponse to 5HT, and that NO potentiates gill muscle con- tractions (Gainey el ai, 1999b). This mechanism may also explain another experimental observation: that ultrasonic transducers record higher maximal contractions than force transducers, and they produce dose-response curves with lower EC 50 s. Thus, when force transducers were used, demibranchs could be exposed only to a single dose of agonist, so the individual contractions constituting the dose- response curves were not potentiated by NO. In contrast, when ultrasonic transducers were used, the demibranchs could be exposed to a set of increasing doses of agonist, so NO was produced, the contractions were potentiated, and the resulting dose-response curves were steeper. Summary The gills of Mercenuria are equipped with an array of muscles and four distinct sets of cilia, and the activity of these effectors coordinated by a complex neural net- work transports water and particles in support of respira- tion and feeding. This paper and a previous one on the modulation of ciliary activity (Gainey et /.. 1999a) lay the Literature Cited Aiello, E. L. 1%0. Factors affecting ciliary activity on the gill of the mussel A/vri'/ii.s etlulix. Pliysiol. Zoo/. 23: 120-135. Aiello, E. I,. 1970. 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Hull. 2(14: Wi-l().v il-'ebruar> 2 2003 Marine Biological Laboratory Salinity Tolerance of Larval Rapana venosa: Implications for Dispersal and Establishment of an Invading Predatory Gastropod on the North American Atlantic Coast ROGER MANN* AND JULIANA M. HARDING Department of Fisheries Science, Virginia Institute of Marine Science, College of William and Mary. Gloucester Point. Virginia 23062 Abstract. The lack of quantitative data on the environ- mental tolerances of the early life-history stages of invading species hinders estimation of their dispersal rates and estab- lishment ranges in receptor environments. We present data on salinity tolerance for all stages of the ontogenetic larval development of the invading predatory gastropod Rapana venosa, and we propose that salinity tolerance is the dom- inant response controlling the potential dispersal (= inva- sion) range of the species into the estuaries of the Atlantic coast of the United States from the current invading epicen- ter in the southern Chesapeake Bay. All larval stages exhibit 48-h tolerance to salinities as low as 15 ppt with minimal mortality. Below this salinity, survival grades to lower values. Percentage survival of R. venusu veligers was sig- nificantly less at 7 ppt than at any other salinity. There were no differences in percentage survival at salinities greater than 16 ppt. We predict that the counterclockwise, gyre-like circulation within the Chesapeake Bay will initially distrib- ute larvae northward along the western side of the Del- MarVa peninsula, and eventually to the lower sections of all major subestuaries of the western shore of the Bay. Given the observed salinity tolerances and the potential for dis- persal of planktonic larvae by coastal currents, establish- ment of this animal over a period of decades from Cape Cod to Cape Hatteras is a high probability. Introduction The Norway/United Nations Conference on Alien Spe- cies considers alien invasive species as the second most Receded 2 July 21)02; accepted 4 November 2002. ' ! To whom correspondence should be addressed. E-mail: rmann@vims.edu important threat, after habitat destruction, to indigenous biodiversity (Sandlund ct / various ages 10-27 days post-hatch) exposed to 20 veligers per treatment Veliger age (days post-hatch) Salinity (ppt) 2 4 6 9 11 13 7 76.9(3.8) 65.5 (5.6) 51,0(5.4) 20.5 (6.6) 62.3(6.8) 74(12.4) 29.4(8.0) 10 74.8(5.3) 70.9(5.5) 76.0(3.4) 67.1 (4.3) 72.1 (5.0) 92.4(1.8) 42.6(2.9) 13 77.9(2.4) 79.3 (2.S) 59.5(6.5) 75.9(9.7) 75.7 (6.5) 90.9(3.0) 69.2(2.6) 16 80.7(2.4) 80.7(3.1 1 90.7(3.1) 82.2(3.0) 83.8 (3.9) 89.2(1.7) 83.5 (2.2) 19 78.9(3.4) 82.6(3.4) 97.1 (0.8) 90.9(2.6) 77.3(3.7) 86.9(5.2) 83.1 (3.1) 22 85.7(3.0) 80.9(5.4) 97.8(1.4) 90.4(2.2) 69.5(3.2) 88.7(2.4) 87.8(1.8) 25 83.5(6.7) Sl.412.4) 94.7(2.8) 94.9 (0.9) 74.9(7.3) 93.5(1.5) 87.6(1.9) 32 59.5 (3.8) 90.5(4.1) 89.3 (1.4) 97.3 (1.4) 79.6(0.9) 85.8(6.5) 84.3(4.1) 15 17 19 21 -j ^ 25 27 7 2.3(2.3) 23.3 (14.4) 58.4(6.7) 95.8(2 1) 75.9(1.7) 97.2(2.8) 92.5(3.8) ID 42.5(12.2) 69.4(3.9) 82.2(4.2) 93.3(3.5) 66.4(4.5) 97.0(3.0) 88.7(8.0) 13 59.9(8.0) 54.6(14.4) 83.0(0.7) 88.9(5.9) 75.0(0.9) 89.9(1.4) 79.5 (8.8) 16 74.3(3.5) 59.7(5.9) 87.0(1| 93.9(6.1) 67.7(6.4) 97.2(2.8) 82.6(11.5) 19 68.8(12.5) 61.4(4.2) 84. 1 (2.1) 92.0(5.4) 74.8(4.1) 97.4(2.5) 95.2 (4.8) 22 78.8(4.3) 68.8 (4.9) 78.7(5.4) 86.1 (4.3) 81.0(1.9) 87.1 (6.5) 100(0.0) 25 71.5(2.6) 73.0(4.1) 72.7(3.8) 96.7(1.7) 80.9(1.0) 98.0(2.0) 98.7(1.3) 32 56.3(10.3) 67.6(3.3) 73.7(4.2) 94.5 (2.S) 69.9(2.4) 93.7(3.4) 96.4 ( 1.8) unclear. The limited observations from the Kerch Strait region suggest that an upstream limit of 12-13 ppt in the Chesapeake Bay is possible, and that low winter tempera- tures will not exclude Rapana from regions that infre- quently experience winter ice. Wu ( 1988) reports that in its native range, R. venosa can exploit estuarine regions that have warm summer temperatures and avoid possible surface freezing in winter by migrating into deeper water in these regions. Larvae of R. venoaa exhibit considerable plasticity in the duration of their planktonic development under experimen- tal conditions of temperature and salinity that mimic the summer conditions in the Chesapeake Bay, and they do not require specific metamorphic cues to complete the transition to the crawling, benthic post-larval phase (Harding and Mann, unpubl. data). Laboratory-cultured individuals can exploit a variety of native bivalves as prey, including the Table 2 Suiiiniars of two-factor AN() \'.\ n'elr^cr a^e salinity) used Jo \alpin.\ has no pelagic larval stage. Juveniles of Urosalpinx hatch and crawl away from the substrate-at- tached egg masses. Urosalpinx has been recolonizing its former Bay habitat over the past three decades by crawling up the Bay bottom over "islands" of suitable substrate. In the absence of an invader, the temporary displacement of Urosalpinx is but a minor perturbation in evolutionary time; however, the introduction of Rapana adds a new and op- portunistic component to this reestablishment process. There arguably now exists a race to reoccupy this tempo- rarily vacated niche; a race that may favor the invader because of the sequence of events that temporarily displaced the native species. Vermeij (1996) theorized that physiological tolerances are evolutionarily conservative parameters contributing to the determination of the range of survival. In this context we predict that, as a result of the counterclockwise, gyre-like circulation within the Chesapeake Bay, pelagic larvae of Rapana venosa originating from parents in the Hampton Roads region will initially be distributed northward along the western shore of the DelMarVa peninsula, and will eventually reach the lower sections of all the major subestu- aries of the western shore of the Bay. This entire region is within the salinity tolerance of the larval forms (compare Table 1 with Fig. 2). The potential for long-distance dis- persal within a single generation remains to be determined, although recent collections of small (<75 mm in length) adults on the Virginia Bay shore of the peninsula suggest that a distance of tens of kilometers per generation is pos- sible. Dispersal onto and along the coastal shelf outside of the Bay mouth may be influenced by both northward- and southward-flowing residual current. The effects on dispersal depend on depth, wind conditions, and time within the known egg laying period of the invader in the southern Chesapeake Bay. Establishment over a period of decades by natural dispersal in estuaries and coastal regions from Cape Cod to Cape Hatteras was considered a high probability by Mann and Harding (2000). This prediction still stands and is supported by the essentially continuous distribution of mol- lusc species suitable as prey in shallow waters throughout this range (for examples, see Theroux and Wigley, 1983). The time frame may, however, be considerably reduced by dispersal of larval forms in ballast water during intra-coastal maritime trade, a suggestion reinforced by the tolerance of the larval form (this study) and the location of both the Norfolk, Virginia. U.S. Naval base and an international 102 R. MANN AND J. M. HARDING container terminal within the extant adult range of invasion in Hampton Roads. If, as Vermeij (1996) suggests, factors such as "the presence of competitors, predators, or disease organisms, or the absence of a critical host, food, or sym- biotic species" prevent a species from extending its range, it is unlikely that Ru/wmi will be further restricted within the projected range. Large individuals of R. venoxu appear admirably equipped to compete with large native gastropods and have few obvious predators in the Middle Atlantic coastal region when they are full grown. We can find no reports of diseases of R. venosa in any of its native or introduced ranges. Finally, the only notable parasite of R. venosa in both its Black Sea and Chesapeake Bay popula- tions are shell-boring polychaetes of the genus Polydora (Gutu and Marinescu, 1979; Mann and Harding. 2000). The actions of Palydoni appear to have little, if any, detrimental effect on infected individuals in either location; may be limited to some individuals of R. venosa that forage epifau- nally; and may be terminated by burial of the host whelks as they grow and shift to an infaunal habit. Indeed, observa- tions on rapa whelk biology and physiological tolerances in the Chesapeake Bay strongly suggest that this animal is capable of successful colonization and establishment of viable populations within estuarine habitats up and down the East Coast of the United States. Acknowledgments Support for this project was provided by Virginia Sea Grant (R/MG-98-3), the Department of Fisheries Science, Virginia Institute of Marine Science, and partial support to RM by the National Science Foundation (OCE-98 10624). Special thanks are extended to local watermen and seafood processors who donated adult Ru/wmi to our research col- lection. We thank D. Bryn Jones, Dario Savini, Melissa Southworth, Rhonda Howlett, Peter Kingsley-Smith, Erica Westcott, Stephanie Haywood, and Catherine Ware for as- sistance in maintenance of adult brood stock and larval cultures. This manuscript is dedicated to the late Professor Ruth Dixon Turner, whose enthusiasm for the larval ecol- ogy of marine molluscs remains as an inspiration to us all. This is Contribution Number 2506 from the Virginia Insti- tute of Marine Science. Literature Cited Bombace, G., G. Kabi, L. Fiorentini. and S. Speranza. 1994. Analysis nl I he efficacy of artificial reefs located in five different areas of the Adriatic Sea. Fifth International Conference on Aquatic Habitat En- hancement. Bull. Mm. Sci. 55(2-3): 559-580. Carlton, ,|. 1996. Pattern, process, and prediction in marine invasion ecology. Binl. Con.scrv. 78: 97-106. Carlton, J. 1999. Molluscan invasions in marine and estuarine commu- nities. Malacologia 41: 439-454. Chukchin, V. 1984. Ecology of the (lastropo,! 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The Black Sea ecosystem changes related to the introduction of new mollusc species. Mar. Ecol. 17: 227-236. Reference: Biol. Bull. 204: 104-108. (February 2(1113) 2003 Marine Biological Laboratory Short-Distance Spawning Migration of Tropical Freshwater Eels JUN AOYAMA 1 -*. SAM WOUTHUYZEN 2 . MICHAEL J. MILLER 1 , TADASHI INAGAKI 1 . AND KATSUMI TSUKAMOTO 1 ' Ocean Research Institute, The University of Tokyo, 1-15-1 Minamidai. Nakano. Tokyo 164-8639. Japan: and : Research Center for Oceanography, Indonesian Institute of Sciences, Anchor. Jakarta, Indonesia The freshwater eels have fascinated biologists because of their spectacular long-distance migrations between their freshwater habitats and their spawning areas far out in the ocean (1. 2, 3). Recent progress on the molecular phytogeny of freshwater eels suggests that the\ originated in the trop- ics (4), and information on the reproductive ecologv and recruitment of tropical species will provide new insight into the evolution of the spawning migration of the freshwater eels (5, 6). However, the larvae (leptocephali) of the intinv sympatric tropical species are morphologically similar (7). so they are impossible to identify, and their spawning areas are thus virtually unknown. Recently, however, we have collected small leptocephali from around Sulawesi, Indone- sia, and have used species-specific genetic markers to iden- tify them as laiTae of Anguilla celebesensis and A. borne- ensis, which provides the first definitive information about the general spawning areas of these tropical eels. More- over, the discovery of a spawning area of A. celebesensis in Toinini Bay and the presence of small specimens of two species in the Celebes Sea indicate that, in contrast to the long migrations made b\ temperate eels, tropical eels make much shorter migrations to spawn in areas near their fresh- water habitats. This difference in migratory behavior may reflect an evolutionary dine among freshwater eels that extends from tropical to temperate regions. Early in the lust century, the Danish oceanographer Jo- hannes Schmidt succeeded in collecting small anguillid leptocephali in the Sargasso Sea thousands of kilometers away from their growth habitats in Europe and North Amer- ica, which indicated that the two species of Atlantic fresh- Received 20 June 2002: accepted 4 December 2002. * To whom correspondence should he addiessed. iaoyama@ori.u-tokyo.ac.jp [.-mail: water eels make remarkably long spawning migrations ( 1 ). After this finding, he and his colleagues shifted their efforts to search for the spawning areas of freshwater eels in the Indo-Pacin'c region where most of the species in the genus are found. They successfully collected more than 1400 leptocephali in the Indo-Pacific region (7) during the Carls- berg Foundation's oceanographic expedition around the world from 1928 to 1930. However, most of these lepto- cephali were relatively large, and the overlap in their mor- phological characters made it difficult to identify them exactly (7, 8). Since then, the spawning areas of the Indo- Pacitic anguillid species have remained a mystery, except for the Japanese eel, Anguilla japonica, which was found to spawn to the west of the Mariana Islands in the western North Pacific (3). Molecular phylogenetic research on the genus Anguilla has recently stimulated interest in the spawning migrations of tropical freshwater eel species by suggesting that the tropical eel. Angnilla borneensis. which is endemic to Borneo, is the most basal species, and that the genus radi- ated out from the tropics to colonize temperate regions (4). Because of these studies, species-specific genetic markers can be used to identify all species of anguillid leptocephali (9). Molecular techniques have, for the first time, allowed studies on the distribution of tropical anguillid leptocephali that can reveal the location of eel spawning areas and the nature of their migrations. In this study, we have collected the smallest tropical eel leptocephali ever reported and used molecular genetic methods to identify species of anguillid leptocephali from the Celebes Sea and Tomini Bay on the east side of Sulawesi. We provide the first definitive infor- mation on the spawning areas of tropical freshwater eels. A cruise of the R/V Hakuho Maru (Ocean Research 104 SHORT MIGRATIONS FOR TROPICAL EELS 105 Institute, University of Tokyo) in the western Pacific. Celebes, and Sulu Seas was conducted from 14 January to 10 March 2000 (Fig. I). A subsequent cruise of the R/V Banina Java VII (Research Center for Oceanography. In- donesian Institute of Sciences), made in the waters around Sulawesi from 8 to 30 May 2001, partly overlapped the sampling area of the Hakuho Muni cruise (Fig. 1). Lepto- cephali were collected during both cruises using identical Isaacs-Kidd midwater trawls with 8.7-irr mouth openings and 0.5-mm mesh. The collections usually consisted of 60-min oblique or step tows within the upper 300 m. Aboard ship, the leptocephali were tentatively identified on the basis of morphological characteristics (7, 8), but these character- istics could not always indicate one species. Total length and other measurements were recorded, and the specimens were preserved in 95% ethanol. In the laboratory, the spec- imens were identified by comparing their mitochondria! 16S rRNA gene sequences with those of morphologically well- identified adult specimens, as has been previously reported (9). Briefly, total genomic DNA was extracted from each leptocephalus according to a standard protocol (9). A por- tion of the mitochondria! 16S ribosomal RNA gene (about 500 base pairs) was amplified by the polymerase chain reaction (PCR) using two oligonucleotide primers, H2510 and H3058 (9). Amplification parameters were 30 cycles of denaturation at 94 C for 30 s, annealing at 55 C for 30 s, and extension at 72 C for 60 s. The PCR products were sequenced according to the manufacturer's protocol (Ap- plied Biosystems Inc.) on a 373A DNA sequencer (Applied Biosystems Inc.). Sequences were determined from the light strand only. The sequence data obtained from the leptoceph- ali were directly compared, without any alignments, to homologous data for anguillids in the Pacific region (A. japonica, A. aiistralis. A. borneensis, A. celebesensis, A. dieffenbachii, A. hicolor. A. megastoma, A. inarmontta, A. reinhanltii. A. obscura, and A. interioris). deposited in DDBJ/ EMBL/GenBank under accession numbers AB021748, AB021751-AB021754. AB021757. AB021758, AB021760- AB021762, AB021764. Within species the sequences were the same or had only one or two site differences, but among species the differences were more than threefold (6-74 sites). All of the sequences determined in the present study will appear in the DDBJ/EMBL/GenBank nucleotide se- quence databases with the accession numbers: AB097700- AB097767, in sequence. During the two research cruises, we collected 67 lepto- cephali and one glass eel (the transparent early juvenile stage of eels) of the genus Anguilla (15 leptocephali from the Hakuho Mam cruise and 53 from the Barium Java VII cruise, which included the glass eel). Genetic species iden- tification of the leptocephali clearly distinguished 12 A. marmoruta (34.0-50.7 mm in total length and one glass eel of 47.8 mm), 41 A. celebesensis (13.0-47.8 mm), 3 A. borneensis (8.5. 13.0, 35.4 mm). 4 A. hicolor (42.6-49.2 mm), and I A. interioris (48.9 mm), all from the waters around Sulawesi (Fig. 1). Also identified were 3 A. bicolor (31.3-46.0 mm) from the waters to the north of New Guinea, and 2 A. marmorata (28.0, 36.5 mm), 1 A. obscura (36.7 mm), and 1 A. aiistralis (47.0 mm) from the western South Pacific (their distributions are not shown). This is the first description of the distribution of anguillid leptocephali identified using genetic markers from Indone- sian waters, and these data suggest that as many as five species of the genus Anguilla may use the Indonesian waters as an area for spawning and larval development. Jespersen (7) reported on collections of relatively large anguillid lep- tocephali from many of these same areas, but could not make precise species identifications. Another more recent study used the same molecular genetic techniques used in this study to identify anguillid leptocephali as small as 16.3 mm in the western North and South Pacific, but did not make collections in the Indonesian Seas (9). Of particular interest in our study were the small leptocephali of A. borneensis that were 8.5 and 13.0 mm (indicating an age of about 16 and 26 days after hatching [10, I 1 1), which were collected in the Celebes Sea to the east of Borneo, and the specimen (35.4 mm) that was collected to the south in Makassar Strait, where water from the Celebes Sea is trans- ported (12). The freshwater growth habitat of A. borneensis is limited to the east-central part of Borneo (5. 6). which strongly suggests that this species spawns in the Celebes Sea and then migrates back to its growth habitat adjacent to the spawning area (Table 1 ). Another tropical anguillid species, Anguilla celebesensis, has a wider distribution that extends from Luzon of the Philippines to across Sulawesi (5, 6). Interestingly, the small leptocephali of this species collected about 25 days after hatching (10, 11) were found in two different seasons and in two different areas separated by the northern arm of Sulawesi: a I2.3-mm specimen was found at Station 50 in the Celebes Sea in February, and a 13-mm specimen was found at Station 21 in Tomini Bay in May (Fig. 1 ). Further, the collection of nine A. celehesensis leptocephali at Station 21 in Tomini Bay ranged in total length from 13 to 48.9 mm (fully grown larval stage [10, 1 1 1). These facts indicate that individuals of A. celebesensis inhabiting the watershed of Tomini Bay spawn over a relatively long period and that their leptocephali are retained in Tomini Bay because it is semi-enclosed and its waters apparently do not mix much with those of other areas (13). Therefore, these eels are probably geographically isolated from those in the Celebes Sea. The findings reported here indicate that freshwater eels living in tropical areas may have life-history characteristics that differ markedly from those of their temperate relatives, which have a single spawning site for each species, long spawning migrations in both the North Atlantic (1,2) and North Pacific Ocean (3), and distinct spawning seasons (Table I ). This distinction is supported by recent analyses of 120 E 125 E 120 E 125 E Pacific Ocean 10N 5N 5S A. celebesensis 10N 5N 5S A. borneensis A. marmorata A. bicolor A. interioris Figure 1. I In- study area, showing the sampling stations and the locations where the various species of anguillid leptocephali were collected. Upper left: Stations during cruises of the R/V Hakiiho Mam (circles) in February 2000 and the R/V Biininu Jnyu \'ll (squares) in May 2001. Only a few station numbers are shown to indicate the order of sampling or to identify those mentioned in the text. Upper right: Solid symbols indicate sampling stations where A. < r/< />( uv/w.v leptocephali were collected and open symbols indicate negative stations. Lower left: Collection locations of .A. hitrnecnsis. Lower right: Collection locations of ,4. marmorata. A. bicolor. and A. interioris. Symbols same as lor upper right panel. SHORT MIGRATIONS FOR TROPICAL EELS Table 1 Comparison of presumed spawning areas, ranges, and distant:? of migration of tempi-rate and tropical eels, (genus Anguilla) 107 Species Presumed spawning area Approximate distance to spawning area (6) Approximate latitudinal range (5) Nearest Endmost European eel .4. anguilla Sargasso Sea (2) 25N, 60W 28N-68N 4000 km Azores. Cape Verde Is. 8000 km Norway. Mediterranean American eel A. rostrata Sargasso Sea (2) 25N. 60W 10 : N-62N 900 km Greater Antilles 5500 km Iceland Japanese eel .4. japtmica West of Mariana Is. (3) 15N. 142E 18N^t3N 2000 km Taiwan 3500 km Northern Japan A. borneensis Celebes Sea 3N, 122E Equutor-7N 480 km Tawau, Borneo 650 km Mahakam Riv.. Borneo A. ce/ebesensis* Tomini Bay r j S. 121 : E ].4S-0.5N 80km Coastal areas around the bay 300 km Numbers in parentheses are reference citations; see Literature Cited. * For a part of a species or population found in the present study. otolith microstructure which showed that tropical anguillids in the Indonesian region may spawn (14) and recruit to freshwater (15) throughout much of the year. Catadromous freshwater eels have been suggested to have originated in the tropical waters near present-day Indonesia sometime around the late-Cretaceous to Eocene, and the endemic tropical species A. borneensis has been found to be the most likely basal catadromous eel species (4). Various character- istics of the migrations of present-day anguillid species clearly show at least a partial geographic cline (Table 1 ). For example, the most likely basal species, A. borneensis, is distributed narrowly over about 7 degrees of latitude (Equa- tor to 7 N) and spawns nearby in the Celebes Sea at a distance of only 480-650 km; in contrast, the growth hab- itat of the European eel extends widely over 40 degrees of latitude, and the distance to its spawning area in the Sar- gasso Sea ranges from about 4000 to 8000 km (Table 1 ). This and the short spawning migration of A. celebesensis in Tomini Bay suggest that freshwater eels of the genus An- guilla originally had migrated only short distances to local spawning areas in the warm waters surrounding their fresh- water growth habitats in the tropics. But following the passive, long-range dispersion of their leptocephalus stages by currents, freshwater eels may have had to evolve long- distance migrations to return from their temperate growth habitats to their tropical spawning grounds. The Atlantic species of freshwater eels are often used, even in basic biological textbooks, as a classic example of a species with a spectacular long-distance migration. How- ever, our findings provide the first evidence that this long- distance migration is an adaptation by eels that colonized temperate regions. Therefore, a new era of research on the ecology and behavior of tropical eels has begun, and it promises to unveil the mystery of the origin and evolution of the catadromous migrations of the genus Anguillci. Acknowledgments We thank all of the scientists who participated in the eel cruises and who worked together discussing the sampling design, operating the nets, and sorting samples. We wrote this paper on behalf of all the scientists aboard, and we also thank all the crew of the Hakuho Muni and the Baruna Jaya VII for their help during the cruise. This work was supported in part by Grants-in-Aid Numbers 07306022. 07556046. 08041139, 08456094, 10460081. and 11691177 from the Ministry of Education, Science, Sports and Culture. Japan, and by grant Numbers JSPS-RFTF 97L00901 from the "Research for the Future Program" of the Japan Society for the Promotion of Science. KT was supported by the Re- search Foundation "Touwa Shokuhin Shinkoukai" and the Eel Research Foundation "Noborika." Literature Cited 1 . Schmidt, J. 1922. The breeding places of the eel. ./. Philox. Trans. R. Sm: R 211: 174-208. 2. McCleave, J. D., R. C. Kleckner. and M. C'astonguay. 1987. Re- productive sympatry of American and European eels and implications for migration and taxonomy. Am. Fish. Soc. Symp. 1: 286-297. 3. Tsukamoto. K. 1992. Discovery of spawning area for the Japanese eel. Nature 356: 789-791. 4. Aoyama, J., M. Nishida, and K. Tsukamoto. 2001. Molecular phylogeny and evolution of the freshwater eels, genus Anguilla. Mol. Phylogenet. Evol. 20: 450-459. 5. Ege, V. 1939. A revision of the genus Anguilla Shaw. Dana-Rep. 16: 1-256. 108 J. AOYAMA ET AL 6. Tesch, F. W. 1977. The Eel: Biology ana 1 Management of Anguillid Eelx. Chapman and Hall. London. 7. Jespersen, P. 1942. Indo-Pacific leptocephalids of the genus An- guilla. Dana-Rep- 22: 1-128. 8. Castle, P. H. J. 1963. Anguillid leptocephali in the southwest Pa- cific. Zoo/. Pnbl. Victoria Univ. 33: 1-14. 9. Aoyama, J.. N. Mochioka, T. Otake, S. Ishikawa, Y. Kawakami, P. H. J. Castle, M. Nishida, and K. Tsukamoto. 1999. Distribution and dispersal of anguillid leptocephali in the western Pacific revealed by molecular analysis. Mar. Ecol. Prog. Ser. 188: 193-200. 10 Aral, T., J. Aoyama, S. Ishikawa, M. J. Miller, T. Otake, T. Inagaki. and K. Tsukamoto. 2001. Early life history of tropical Anguilla leptocephali in the western Pacific Ocean. Mar. Biol. 138: 887-895. 1 1. Ishikawa, S., K. Suzuki, T. Inagaki. S. Watanabe, Y. Kimura, A. Okamura, T. Otake, N. Mochioka, Y. Suzuki, H. Hasumoto, M. Oya, M. J. Miller, T. W. Lee, H. Fricke, and K. Tsukamoto. 2(101. Spawning time and place of the Japanese eel. Anguilla japonica, in the North Equatorial Current of the western North Pacific Ocean. Fish. Sci. 67: 1097-1103. 12. Miyama, T., T. Awaji, K. Akitomo, and N. Imasato. 1995. Study of seasonal transport variations in the Indonesian Seas. J. Geophys. Res. 100: 20.517-20,541. 13. Hatayama, T., T. Awaji, and K. Akitomo. 1996. Tidal currents in the Indonesian Seas and their effect on transport and mixing. J. Genphy.f. Re*. 101: 12.353-12.373. 14. Arai, T., D. Limbong, T. Otake, and K. Tsukamoto. 2001. Re- cruitment mechanisms of tropical eels Anguilla spp. and implications for the evolution of oceanic migration in the genus Anguilla. Mar. Ecol. Prog. Ser. 216: 253-264. 15. Sugeha, H. Y., T. Arai, M. J. Miller, D. Limbong, and K. Tsuka- moto. 2001. Inshore migration of the tropical eels, Anguilla spp.. recruiting to the Poigar River estuary on Sulawesi Island. Mar. Ecol. Prog. Ser. 221, 233-243. THE BIOLOGICAL BULLETIN 2003 Subscription Rates Volumes 204-205 *Paid Subscriptions include both print and electronic subscriptions at: www.biolbull.org Institutional* Individual* One year subscription (6 issues - 2 volumes) $280.00 $105.00 Single volume (3 issues) $140.00 $52.50 Single Issues $ 50.00 $20.00 *Surface delivery included in above prices. For prompt delivery, we encourage subscribers outside the U.S. to request airmail service. 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