Ascidian News*

Charles and Gretchen Lambert
Dept. of Biol. Sci., Calif. State Univ., Fullerton, CA 92834-6850
tel. (714)278-3481 Fax (714)278-3426
e-mail: or

Number 41
May 1997

Here it is spring again and time for another Ascidian News. Please note that our telephone and fax numbers have changed. We are grateful to those of you who sent us reprints for our New Publications section (88 new papers listed in this issue) and abstracts of meeting presentations. Please send all contributions for AN by e-mail so they can be incorporated without retyping.
We will be in France from May 31 to August 22, continuing work on fertilization of Phallusia at the Station Biologique, BP 74, 29682 Roscoff Cedex, France. Tel.; fax Our email addresses will remain the same.

*Ascidian News is not part of the scientific literature and should not be cited as such.


1. More ascidian invaders: Our research these days is focussed on the problem of introduced species; we have just finished our spring surveys of southern California harbors. Gretchen has identified several that were mysteries to us, the most recent of which is the Japanese species Symplegma reptans (confirmed by Dr. Nishikawa) and which we observed for the first time in April in Mission Bay, San Diego, where it has undergone an incredible population explosion this spring.

1.Books for sale: We have an extra copy (unbound) of W.G. Van Name's 1945 monograph, The North and South American Ascidians. Bull. Amer. Mus. of Nat. Hist. vol. 84. We would like to sell it for the price we paid, $65 some years ago.

3. It is with great sadness that we report that Dr. John Berrill passed away last October 15, 1996 at the age of 93, peacefully at the home of his daughter Lynn in New Hampshire, with whom he had been living. We never met him but felt that we knew him through our reading of his books and papers and our correspondence; he told us he continued to enjoy reading AN long after he retired. John certainly had a major impact upon several generations of biology graduate students working on ascidian development and all aspects of regeneration. His book on the ascidians of great Britain is still in use in laboratories all over the world. His book on the origin of the vertebrates has influenced our understanding of the similarities and differences in the development of the chordate egg and has had a very large impact upon the questions we ask in developmental biology. His textbooks on animal development have influenced a whole generation of biologists. although they may not be aware of this. He was also instrumental in founding the Society For Developmental Biology, an organization with members from throughout the world and a major impact upon the direction of current research in this field. With his popular books he impacted how we as a society approach environmental concerns; he also influenced at least two biologists to stay close to the sea in all our research. John lived a long fruitful life and made many contributions to science; he will be missed by all of us.


Abbas, S.A., M.B. Hossain, D. vander Helm, F.J. Schmitz, M. Laney, R. Cabuslay, et al. 1996. Alkaloids from the tunicate Polycarpa aurata from Chuuk Atoll. J. Org. Chem. 61:9072.

Acuna, J.L., D. Deibel & C.C. Morris 1996. Particle capture mechanism of the pelagic tunicate Oikopleura vanhoeffeni. Limnol. Oceanogr. 41:1800-1814.

Aiello, A., E. Fattorusso & M. Menna 1996. Low molecular weight metabolites of three species of ascidians collected in the lagoon of Venice. Biochem. System. & Ecol. 24:521-530.

Aizenberg, J., G. Lambert, L. Addadi & S. Weiner 1996. Stabilization of amorphous calcium carbonate by specialized macromolecules in biological and synthetic precipitates. Advanced Materials 8:222-226.

Arnoult, C., M. Albrieux, A.F. Antoine, D. Grunwald & M. Villaz 1997. A ryanodine-sensitive calcium store in ascidian eggs monitored by whole-cell patch-clamp recordings. Cell Calcium 21:93-101.

Azumi, K. & H. Yokosawa 1996. Humoral factors and cellular reactions in the biological defense of the ascidian Halocynthia roretzi. pp. 43-54 in New Directions in Invertebrate Immunology, ed. vol., ed. by Soderhall, K., S. Iwanaga & G.R. Vasta.

Benslimane, A.F., Y.F. Pouchus, J. Le Boterff, J.F. Verbist, C. Roussakis & F. Monniot 1988. Substances cytotoxiques et antibacteriennes de l'ascidie Aplidium antillense. J. Nat. Prod. 51:582-583.

Bergmann, T., D. Schories & B. Steffan 1997. Alboinon, an oxadiazinone alkaloid from the ascidian Dendrodoa grossularia. Tetrahedron 53:2055-2060.

Biard, J.F., C. Grivois, J.F. Verbist, C. Debitus & J.B. Carre 1990. Origin of bistramide A identified in Lissoclinum bistratum (Urochordata): possible involvement of symbiotic Prochlorophyta. J. Mar. Biol. Ass. U. K. 70:741-746.

Biard, J.F., S. Guyot, C. Roussakis, J.F. Verbist, J. Vercauteren, J.F. Weber, et al. 1996. Lepadiformine, a new marine cytotoxic alkaloid from Clavelina lepadiformis Muller. Tetrahed. Lett. 35:2691-2694.

Biard, J.F., C. Malochet-Grivois, C. Roussakis, P. Cotelle, J.P. Henichart, C. Debitus, et al. 1994. Lissoclimides, cytotoxic diterpenes from Lissoclinum voeltzkowi Michaelsen. Nat. Prod. Lett. 4:43-50.

Biard, J.F., C. Roussakis, J.M. Kornprobst, D. Gouiffes-Barbin, J.F. Verbist, P. Cotelle, et al. 1994. Bistramides A, B, C, D, and K: a new class of bioactive cyclic polyethers from Lissoclinum bistratum. J. Nat. Prod. 57:1336-1345.

Bingham, B.L. 1997. Light cycles & gametogenesis in three temperate ascidian species. Invert. Biol. 116:61-70.

Bruno, J.F. & J.D. Witman 1996. Defense mechanisms of scleractinian cup corals against overgrowth by colonial invertebrates. J. Exp. Mar. Biol. Ecol. 207:229-241.

Cammarata, M., V. Arizza, M. Vazzana & N. Parrinello 1996. Prophenoloxidase activating system in tunicate hemolymph. Ital. J. Zool. 63:345-352.

Cima, F., L. Ballarin, G. Bressa, G.B. Martinucci & P. Burighel 1996a. Embryotoxic effects of organotin compounds on Styela plicata (Tunicata; Ascidiacea). Fresenius Envir. Bull. 5:718-722.

Cima, F., L. Ballarin, G. Bressa, G. Martinucci & P. Burighel 1996b. Toxicity of organotin compounds on embryos of a marine invertebrate (Styela plicata; Tunicata). Ecotoxicol. & Envir. Safety 35:174-182.

Cima, F., L. Ballarin & A. Sabbadin 1996. New data on phagocytes and phagocytosis in the compound ascidian Botryllus schlosseri (Tunicata, Ascidiacea). Ital. J. Zool. 63:357-364.

Cohen, C.S. 1996. The effects of contrasting modes of fertilization on levels of inbreeding in the marine invertebrate genus Corella. Evolution 50:1896-1907.

Cooper, E.L. 1996. The immunology of earthworms and tunicates. Lab Animal Nov. 1996:38-43.

Corbo, J.C., M. Levine & R.W. Zeller 1997. Characterization of a notochord-specific enhancer from the Brachyury promoter region of the ascidian, Ciona intestinalis. Development 124:589-602.

Dallai, R., P. Burighel, G.B. Martinucci, & N.J. Lane 1997. Scalariform junctions: a revised model. Cell Biol. Intl. 21:23-34.

Davis, A.R. 1996. Association among ascidians: facilitation of recruitment in Pyura spinifera. Mar. Biol. 126:35-41.

Davis, A.R., D.J. Ayre, M.R. Billingham, C.A. Styan & G.A. White 1996. The encrusting sponge Halisarca laxus: population genetics and association with the ascidian Pyura spinifera. Mar. Biol. 126:27-33.

Davis, A.R. & D.J. Campbell 1996. Two levels of spacing and limits to local population density for settled larvae of the ascidian Clavelina moluccensis:a nearest neighbour analysis. Oecologia 108:701-707.

Degnan, B.M., C.N. Souter, S.M. Degnan & S.C. Long 1997. Induction of metamorphosis with potassium ions requires development of competence and an anterior signalling centre in the ascidian Herdmania momus. Dev. Genes & Evol. 206:370-376.

DeLeo, G., N. Parrinello, D. Parrinello, G. Cassara, D. Russo & M.A. DiBella 1997. Encapsulation response of Ciona intestinalis (Ascidiacea) to intratunical erythrocyte injection. 2. The outermost inflamed area. J. Invert. Pathol. 69:14-23.

Esnal, G.B., F.L. Capitanio & L.C. Simone 1996. Concerning intraspecific taxa in Fritillaria borealis Lohmann (Tunicata, Appendicularia). Bull. Mar. Sci. 59:461-468.

Foster, M.P., C.L. Mayne, R. Dunkel, R.J. Pugmire, D.M. Grant, J.M. Kornprobst, et al. 1992. Revised structure of bistramide A (bistratene A): application of a new program for the automated analysis of 2D INADEQUATE spectra. J. Amer. Chem. Soc. 114:1110-1111.

Glardon, S., P. Callaerts, G. Halder & W.J. Gehring 1997. Conservation of Pax-6 in a lower chordate, the ascidian Phallusia mammillata. Development 124:817-826.

Greaves, A.A., A.K. Davis, J.E. Dallman & W.J. Moody 1996. Co-ordinated modulation of Ca2+ and K+ currents during ascidian muscle development. J. Physiol. 497:39-52.

Hirose, E., Y. Saito & H. Watanabe 1997. Subcuticular rejection: an advanced mode of the allogeneic rejection in the compound ascidians Botrylloides simodensis and B. fuscus. Biol. Bull. 192:53-61.

Holyoak, A.R. 1997. Patterns and consequences of whole colony growth in the compound ascidian Polyclinum planum. Biol. Bull. 192:87-97.

Hopmann, C. & D.J. Faulkner 1997. Lissoketal, a spiroketal from the palauan ascidian Lissoclinum voeltzkowi. Tetrahed. Lett. 38:169-170.

Ishida, K., T. Ueki & N. Satoh 1996. Spatio-temporal expression patterns of eight epidermis-specific genes in the ascidian embryo. Zool. Sci. 13:699-709.

Jefferies, R.P.S., N.A. Brown & P.E.J. Daley 1996. The early phylogeny of chordates and echinoderms and the origin of chordate left-right asymmetry and bilateral symmetry. Acta Zool. 77:101-122.

Kang, H.J. & W. Fenical 1997. Aplidiamine, a unique zwitterionic benzyl hydroxyadenine from the Western Australian marine ascidian Aplidiopsis sp. Tetrahed. Lett. 38:941-944.

Kawaminani, S. & H. Nishida 1997. Induction of trunk lateral cells, the blood cell precursors, during ascidian embryogenesis. Dev. Biol. 181:14-20.

Keough, M.J. & P.T. Raimondi 1996. Responses of settling invertebrate larvae to bioorganic films: effects of large-scale variation in films. J. Exp. Mar. Biol. Ecol. 207:59-78.

Kimura, S. & T. Itoh 1996. New cellulose synthesizing complexes (terminal complexes) involved in animal cellulose biosynthesis in the tunicate Metandrocarpa uedai. Protoplasma 194:151-163.

Koike, I. & t Suzuki 1996. Nutritional diversity of symbiotic ascidians in a Fijian seagrass meadow. Ecol. Res. 11:381-386.

Kozloff, E.N. 1993. Three new species of Stoecharthrum (phylum Orthonectida). Cah. Biol. Mar. 34:523-534.

Lambert, G. 1996. Chapt. 11 - Phylum Chordata: Subphylum Urochordata, Class Ascidiacea. pp. 261-293 in vol. 14 of the Taxonomic Atlas of the Benthic Fauna of the Santa Maria Basin and Western Santa Barbara Channel, Misc. Taxa. Ed. by J.A. Blake, P.H. Scott & A. Lissner. Publ. by Santa Barbara Mus. of Nat. Hist.

Lambert, G. & C.C. Lambert 1996a. Antibodies to echinoid larval spicule proteins cross react with the spicular complex in the ascidian Herdmania momus. Bull. Inst. Oceanogr. Monaco numero special 14: 253-261.

Lambert, G. & C. Lambert 1996b. Spicule formation in the New Zealand ascidianPyura pachydermatina (Chordata, Ascidiacea). Connective Tissue Res. 34:263-269.

Lee, I.H., C.Q. Zhao, Y. Cho, S.S.L. Harwig, E.L. Cooper & R.I. Lehrer 1997. Clavanins, alpha-helical antimicrobial peptides from tunicate hemocytes. FEBS 400:158-162.

Lindquist, N. 1996. Palatibility of invertebrate larvae to corals and sea anemones. Mar. Biol. 126:745-755.

Lindquist, N. & M.E. Hay 1995. Can small rare prey be chemically defended? The case for marine larvae. Ecology 76:1347-1358.

Lindquist, N. & M.E. Hay 1996. Palatability and chemical defense of marine invertebrate larvae. Ecol. Monog. 66:431-450.

Lubbering-Sommer, B., P. Compere & G. Goffinet 1996. Cytochemical investigations on tunic morphogenesis in the sea peach Halocynthia papillosa (Tunicata, Ascidiacea). 2. Demonstration of proteins. Tiss. & Cell 28:651-662.

Malochet-Grivois, C., P. Cotelle, J.F. Biard, J.P. Henichart, C. Debitus, C. Roussakis, et al. 1991. Dichlorolissoclimide, a new cytotoxic labdane derivative from Lissoclinum voeltzkowi Michaelsen (Urochordata). Tetrahed. Lett. 32:6701-6702.

McKinney, F.K. 1996. Encrusting organisms on co-occurring disarticulated valves of two marine bivalves: comparison of living assemblages and skeletal residues. Paleobiology 22:543-567.

Monniot, F. & C. Monniot 1996. New collections of ascidians from the western Pacific and southeastern Asia. Micronesica 29:133-279.

Naranjo, S.A., J.L. Carballo & J.C. Garcia-Gomez 1996. Effects of environmental stress on ascidian populations in Algeciras Bay (southern Spain). Possible marine bioindicators? Mar. Ecol. Prog. Ser. 144:119-132.

Niermann-Kerkenberg, E. & D.K. Hofmann 1989. Fertilization and normal development in Ascidiella aspersa (Tunicata) studied with Nomarski optics. Helgolander Meeresunters. 43:245-258.

Ogasawara, M., K.J. Tanaka, K.W. Makabe & N. Satoh 1996. Expression of endostyle-specific genes in the ascidian Halocynthia roretzi. Dev. Genes & Evol. 206:227-235.

Okamura, Y., F. Ono & R. Okagaki 1997. Regulation of voltage-gated ion channels during ascidian embryogenesis. Develop. Neurosci. 19:23-24.

Pancer, Z., E.L. Cooper & W.E.G. Muller 1996. A tunicate (Botryllus schlosseri) cDNA reveals similarity to vertebrate antigen receptors. Immunogenetics 45:69-72.

Pancer, Z., U. Scheffer, I. Muller & E.G. Muller 1996. Cloning of sponge (Geodia cydonium) and tunicate (Botryllus schlosseri) proteasome subunit epsilon (PRCE): implications about the vertebrate MHC-encoded homologue LMP7 (PRCC). Biochem. Biophys. Res. Commun. 228:406-410.

Parrinello, N., V. Arriza, M. Cammarata & D.M. Parrinello 1996. Expression and modulation of immunological activities by tunicate hemocytes. pp. 391-405 in Modulators of Immune Responses: The Evolutionary Trail, ed. vol., ed. by Stolen, J.S. et al.

Patil, A.D., A.J. Freyer, R. Reichwein, B. Carte, L.B. Killmer, L. Faucette, et al. 1997. Fasicularin, a novel tricyclic alkaloid from the ascidian Nephteis fasicularis with selective activity against a DNA repair-deficient organism. Tetrahed. Lett. 38:363-364.

Perissinotto, R. & E.A. Pakhomov 1997. Feeding association of the copepod Rhincalanus gigas with the tunicate salp Salpa thompsoni in the southern ocean. Mar. Biol. 127:479-484.

Powell, J.F.F., S.M. Reska-Skinner, M.O. Prakash, W.H. Fischer, M. Park, J.E. Rivier, et al. 1996. Two new forms of gonadotropin-releasing hormone in a protochordate and the evolutionary implications. Proc. Natl. Acad. Sci. 93:10461-10464.

Raftos, D. & A. Hutchinson 1997. Effects of common estuarine pollutants on the immune reactions of tunicates. Biol. Bull. 192:62-72.

Reddy, M.V.R., D.J. Faulkner, Y. Venkateswarlu & M.R. Rao 1997. New lamellarin alkaloids from an unidentified ascidian from the Arabian Sea. Tetrahedron 53:3457-3466.

Riisgard, H.U., C. Jurgensen & F.O. Andersen 1996. Case study: Kertinge Nor. Coastal & Estuarine Studies 52:205-220.

Riisgard, H.U., C. Jurgensen & T. Clausen 1996. Filter-feeding ascidians (Ciona intestinalis) in a shallow cove: implications of hydrodynamics for grazing impact. J. Sea Res. 35:293-300.

Robinson, W.E., K. Kustin, K. Matulef, D.L. Parry, X. He & E.K. Ryan 1996. 14C- and 3H-tyrosine incorporation into Ascidia ceratodes tunichrome in vivo. Comp. Biochem. Physiol. 115:475-481.

Sanamyan, K. 1996. Ascidians from the north-western Pacific region. 3. Pyuridae. Ophelia 45:199-210.

Sato, S., H. Masuya, T. Numakunai, N. Satoh, K. Ikeo, T. Gojobori, et al. 1997. Ascidian tyrosinase gene: its unique structure and expression in the developing brain. Developmental Dynamics 208:363-374.

Satou, Y. & N. Satoh 1996. Two cis-regulatory elements are essential for the muscle-specific expression of an actin gene in the ascidian embryo. Dev., Growth & Differ. 38:565-573.

Sawada, H., H. Kawahara, Y. Saitoh & H. Yokosawa 1996. Physiological functions of proteasomes in ascidian fertilization and embryonic cell cycle. pp. 229-232 in Intracellular Protein Catabolism, ed. vol., ed. by Suzuki, K. & J. Bond.

Sawada, H., E. Kodama, M.R. Pinto, R. De Santis & H. Yokosawa 1997. Structure and functions of sperm proteases involved in fertilization of ascidians, Halocynthia roretzi and Ciona intestinalis. J. Repro. & Dev. 43:suppl.

Scippa, S., G. Iazzetti & M. de Vincentiis 1996. Vacuole-containing cells in the body cavity of Phallusia mammillata larvae before and after hatching. Invert. Repro. & Develop. 29:231-234.

Sims, D.W. 1996. A rare record of the salp, Thetys vagina (Tunicata: Thaliacea) from western Scottish waters. J. Mar. Biol. Ass. U. K. 76:833.

Sings, H. & K.L. Rinehart 1996. Compounds produced by potential tunicate-blue-green-algal symbiosis. J. Industrial Microbiol. 17:385-396.

Stoner, D.S. & I.L. Weissman 1996. Somatic and germ cell parasitism in a colonial ascidian: possible role for a highly polymorphic allorecognition system. Proc. Natl. Acad. Sci. 93:15254-15259.

Swalla, B.J. & W.R. Jeffery 1996. Localization of ribosomal protein L5 mRNA in myoplasm during ascidian development. Dev. Genetics 19:258-267.

Tanaka, K.J., S. Chiba & T. Nishikata 1996. Two distinct cell types identified in the ascidian notochord. Zool. Sci. 13:725-730.

Tanaka, K.J. & T. Nishikata 1994. Specification in the primary lineage of ascidian muscle cells analyzed with a specific monoclonal antibody. Mem. Konan Univ. 41:89-97.

Tanaka, K.J., M. Ogasawara, K.W. Makabe & N. Satoh 1996. Expression of pharyngeal gill-specific genes in the ascidian Halocynthia roretzi. Dev. Genes & Evol. 206:218-226.

Tsukamoto, S., H. Kato, H. Hirota & N. Fusetani 1997. Seven new polyacetylene derivatives, showing both potent metamorphosis-inducing activity in ascidian larvae and antifouling activity against barnacle larvae, from the marine sponge Callyspongia truncata. J. Nat. Prod. 60:126-130.

Vervoort, H.C., S.E. Richards-Gross, W. Fenical, A.Y. Lee & J. Clardy 1997. Didemnimides A-D: novel, predator-deterrent alkaloids from the Caribbean mangrove ascidian Didemnum conchyliatum. J. Org. Chem. 62:1486-1490.

Wada, H., P.W.H. Holland & N. Satoh 1996. Origin of patterning in neural tubes. Nature 384:123.

Wada, S., Y. Katsuyama, Y. Sato, C. Itoh & H. Saiga 1996. Hroth, an orthodenticle-related homeobox gene of the ascidian, Halocynthia roretzi: its expression and putative roles in the axis formation during embryogenesis. Mechanisms of Development 60:59-72.

Yamada, A. & H. Nishida 1996. Distribution of cytoplasmic determinants in unfertilized eggs of the ascidian Halocynthia roretzi. Dev. Genes & Evol. 206:297-304.

Yasuo, H., M. Kobayashi, Y. Shimauchi & N. Satoh 1996. The ascidian genome contains another T-domain gene that is expressed in differentiating muscle and the tip of the tail of the embryo. Dev. Biol. 180:773-779.

(Some abstracts have been edited slightly for AN to conserve space.)

1. Ecology of Estuaries and Soft Sediment Habitats conference, Warrnambool, Victoria, Australia, Feb. 1997.
G. Clapin, JWTJ Lemmens and L Nielsen. CSIRO Mar. Labs, PO Box 20, North Beach 6020, W. Australia.
It has been regularly suggested that phytoplankton forms the main component of the food for marine filter feeders. However, ascidians are non-discriminating suspension feeders, so their food source largely depends on the food composition available. Phytoplankton levels in Marmion Lagoon, Western Australia are relatively low but large fields of kelp (Ecklonia radiata) potentially generate a substantial amount of detrital material that may be available as an organic food source for suspension feeding communities. A preliminary study of stable isotope levels in dominant ascidian species from seagrass beds in the lagoon showed that (13C levels were close to that of kelp (Ecklonia radiata and Sargassum sp.) but dissimilar to that of seagrass (Posidonia sp.). Thus although the ascidians are located in seagrass beds, kelp is a likely food source whereas seagrass is not. Because it was difficult to obtain sufficient natural phytoplankton to measure stable isotope levels, we conducted a controlled feeding experiment to determine whether the ascidians assimilate phytoplankton, fresh kelp or kelp detrital material. We examined the food preferences of two dominant ascidians from the lagoon, Pyura australis and Polycarpa viridis, collected from seagrass beds and maintained in flow-through, seawater aquaria for approximately one month. The incoming water was filtered to 1um and the subjects were fed on Rhodomonas cultures, fresh kelp, kelp detritus or filtered water only. Measurements of body length and dry wt were taken to determine growth rate, and stable isotope levels were used to determine if the food source had been assimilated. The preliminary results indicate that kelp detritus may be an important food source for ascidians in seagrass beds of Marmion Lagoon.

2. Soc. for Integrative & Comp. Biol. annual meeting, Dec. 26-30, 1996. Published in Amer. Zool. 36 (5), 1996.

MB Saffo, Arizona State Univ. West, Phoenix, Arizona.
Nephromyces is a heterotrophic protist inhabiting universally, and exclusively, molgulid ascidians. Unusually among horizontally transmitted marine endosymbioses, its host range is limited to a single taxon. Has Nephromyces co-evolved with molgulids? To supplement rDNA data, I investigated morphological variation and cross-infectivity in Nephromyces from several molgulids. Nephromyces cells do vary morphologically, even when different host species co-occur. In infectivity, Nephromyces shows strict host specificity. Lab-raised, aposymbiotic M. manhattensis, bred from Pacific (San Francisco Bay) populations and inoculated with Nephromyces from 3 other host species (M. citrina, M. occidentalis, and M. provisionalis), remained uninfected. Controls (inoculated with symbionts from SF Bay M. manhattensis) yielded 100% infection rates. Pacific M. manhattensis inoculated with Nephromyces from Atlantic (Cape Cod) M. manhattensis showed early infective cells after 2 weeks, but only sparse, deteriorated cells after 4 weeks, suggesting that Atlantic Nephromyces is only partially compatible with Pacific hosts. These data suggest genetic differentiation in Nephromyces, not only among different host species, but also among geographically isolated populations of a single host species.

S. Cohen, Y. Saito and I. Weissman, Stanford Univ., Calif. and Univ. of Tsukuba, Japan.
Botryllid ascidians show an intriguing diversity in reproductive and developmental characters, including: the timing and locations of gamete maturation, location of fertilization and development, length of embryonic development and degree of maturation of larvae upon hatching, and number of eggs per zooid. Ancestral states of these traits are determined by mapping them onto a molecular phylogeny based on small ribosomal subunit nuclear DNA sequence data. The Botryllus clade is ancestral to the Botrylloides group. There is a trend in the family towards increasing egg size, fewer eggs per zooid, longer development time and larger larvae upon hatching. Comparison of these traits with world-wide invasion patterns shows the highly successful Botrylloides species run counter to predictions on reproductive traits conducive to species introductions.

BJ Swalla, Vanderbilt Univ., Nashville, TN.
There are 3 classes of urochordates or tunicates: thaliaceans, larvaceans and ascidians. Although there is no fossil record for these organisms, ascidians comprise by far the largest group with about 3000 described extant species, allowing comparison of a wide variety of evolved body plans and life histories. There have been several important vertebrate developmental genes described recently which appear to have homologous functions during ascidian larval development. The expression of these genes in ascidian embryos will be reviewed and compared to the novel maternal genes manx, a zinc finger nuclear protein and cymric, a nonreceptor tyrosine kinase. In addition, distalless, a gene that is known to be expressed during appendage formation in a wide variety of phyla, is shown to be expressed during ampullae and siphon formation in ascidians. The implication of urochordate appendages which may contain secondary axes will be discussed with respect to the evolution of paired appendages in tetrapods.

JE Dallman and WJ Moody, Univ. of Washington, Seattle, WA.
Muscle cells of the ascidian, Boltenia villosa, exhibit 2 distinct phases during differentiation. These phases are created by expression of voltage-gated ionic currents. During the first phase, just after neurulation, inward Ca and outward K currents (mediate action potentials) are first expressed while the inwardly rectifying K current (sets the resting potential) is transiently absent. An unstable resting potential leads to spontaneous activity. During the second phase, the return of the inwardly rectifying K current along with the addition of a Ca-dependent K current terminates the spontaneous activity. To test the role of spontaneous activity in muscle differentiation, we blocked activity during the first phase with the Ca channel blocker Cd. When we compared treated and control cells at maturity, the treated cells lacked the Ca-dependent K current, while all other currents were normal. In addition, treated cells were less contractile than control cells, even though their actin cytoskeleton was similar. These results suggest that regulation of ion currents causes spontaneous activity and Ca entry that is required for muscle differentiation.

3. Intl. Soc. of Differentiation 9th Intl. Conf., Pisa, Italy, Sept. 28-Oct. 2, 1996. #61. VITELLOGENESIS IN ASCIDIANS. L. Manni, M. Della Barbera, G. Zaniolo, P. Burighel. Dipt. di Biol., Univ. di Padova, Italy.
The ovarian oocyte of ascidians possesses one of the most complex set of envelopes known in the animal kingdom, being surrounded by test cells encased in its surface, a fibrous vitelline coat, and inner and outer follicular cells. The size of the oocytes and differentiation of the envelopes seem to depend on the reproductive mode of the species, which may be oviparous, ovoviviparous or viviparous. Oviparous species usually produce many small oligolecithal eggs (about 150 µm), the ovoviviparous comparatively few, large yolked eggs (up to 720 µm), and the viviparous ones few, very small alecithal eggs (25 µm). Vitellogenesis was investigated ultrastructurally in several species representative of the different modes of reproduction. Two mechanisms of yolk synthesis are involved, according to the different types of oocyte. In oligolecithal eggs, such as those of Ciona intestinalis, yolk is accumulated by means of autosynthesis. At the beginning of vitellogenesis, the oocyte shows signs of intense nucleus-cytoplasm exchange and nuage, and has numerous mitochondria, annulate lamellae, RER cisterns and Golgi fields. These organelles co-operate in the synthesis of yolk, which accumulates in previtellogenic bodies to form progressively larger and denser granules, mainly distributed in the central area of the oocyte. Endocytosis is scarce and the few ovular microvilli establish only occasional relations with the inner and outer follicular cells. In species with yolked eggs, like Botryllus schlosseri, early autosynthesis is followed by heterosynthesis, as shown by the progressively increasing features of endocytosis and exchange between the oocyte and its environment. Tracers for electron microscopy also show that molecules coming from the blood are endocytosed by oocytes. The microvilli are long and polymorphic, and joined by means of gap junctions to cytoplasmic protrusions of the outer follicular cells. The latter are more columnar and richer in RER and Golgi fields than in oviparous species, and seem to be involved in the production of proteins to be passed to the oocyte. In viviparous species, such as Botrylloides violaceus, dense yolk granules are absent, although the oocyte produces numerous apparently empty previtellogenic bodies. Variations in the reproductive strategies of ascidians therefore seem to involve variations in the mechanism of vitellogenesis and envelope activities.

#221. DIFFERENTIATION OF THE NEURAL COMPLEX DURING ASEXUAL REPRODUCTION IN TUNICATES. P. Burighel, G. Zaniolo, L. Manni, Dipt. di Biol., Univ. di Padova, Italy.
In both the tunicates and the related vertebrates the embryonal neural tube originates from an ectodermal neural plate. But in tunicates it becomes completely rearranged when the larva metamorphoses in sessile filter-feeding zooid. The latter finally possesses a neural complex formed of the cerebral ganglion and the associated neural gland, a blind sac opened into the pharynx through the ciliated duct and extended posteriorly with the dorsal strand. In ascidians, the most studied tunicates, the cerebral ganglion has the unusual capacity to regenerate completely in few weeks after ablation and the possible contribution of cells from blood or dorsal strand to the process is under investigation. Numerous ascidians form colonies which, as single clones, are constituted of few to thousand genetically identical zooids, each one derived by asexual reproduction and possessing its own neural complex. Origin and differentiation of the neural complex was followed in the buds of Botryllus schlosseri colonies cultured in the laboratory. The early bud derived from a specialized area of the atrial tissue which folds to form a vesicle covered by the epidermis. Primordia of atrial and branchial chamber and gut derive through invaginations and extension of vesicle wall. A tube (neural primordium) evaginates from dorsal area of the vesicle and extends anteriorly to meet and open into the branchial chamber rudiment. Posteriorly, the original aperture is then closed. A laminar roof of mesenchymal cells is interposed between epidermis and the tube. These cells early show microtubules and cytoplasmic extensions with scattered junctional areas and differentiate as neurons. They move ventrally and, together with elements proliferating fom the ventro-lateral wall of the tube, give rise to the anlage of the cerebral ganglion. In the latter, cell bodies accumulate in a cortical layer around a fribrous medulla, from which the peripheral nerves start. Early the neural tube differentiate forming the anterior ciliated duct and the posterior sac whose wall is of vacuolated polymorphic cells. Thus in B. schlosseri cerebral ganglion differentiates early by contribution of elements from blood and neural gland primordium.

3. Italian Embryology Group 42nd annual meeting, Bressanone, June 6-8, 1996. Publ. in Anim. Biol. 5 (2), 1996.

NEURAL COMPLEX DEVELOPMENT IN BOTRYLLUS SCHLOSSERI BUDS. P Burighel, L Manni, & G. Zaniolo, Dipt. di Biol., Univ. di Padova
The neural complex of the ascidian B. schlosseri (Stolidobranchia) consists of the cerebral ganglion and the dorsally associated neural gland opening in the prebranchial region through the ciliated duct. At first, in the oozooid, the nervous complex is formed at metamorphosis, starting from a primordium to the left of the cerebral vesicle of the motile tadpole larva; but in every blastozooid it is newly formed. By light and electron microscopy, we analysed the origin and development of the neural complex taking the B. schlosseri bud as a model. Colonies of this ascidian were cultured in the laboratory on glass, and pieces of them were cut off at opportune stages and processed for study. We refer to the stages of bud development as proposed by Sabbadin (1955, Boll. Zool. 22:243-263). Botryllus buds are palleal and originate from two symmetrical regions of the parental mantle. They soon form a vesicle covered by epidermis with blood circulating in the interspace. Organ primordia derive mainly from the inner vesicle, by infoldings of its wall. Early nervous primordium occurs as tubular evagination from the prospective cloacal region and a mesenchymal lamina, probably of blood cells, insinuates itself between it and the epidermis. Neural evagination extends forwards as a dorsal tube which contacts and opens in the prospective branchial chamber. At the same time its original posterior aperture closes. The dorsal tube represents the primordium of the neural gland. In the mesenchymal band, cells begin to differentiate as neurons, extending cytoplasmic protrusions rich in microtubules and forming junctional areas. These cells move ventrally to the neural gland to form the anlage of the cerebral ganglion. However, morphological evidence was obtained to show that cells proliferating from the ventral and lateral sides of the dorsal tube also make important contributions to cerebral ganglion formation. When the bud heart begins beating and the primordia of the branchial stigmata become recognizable, the neural complex appears in its definitive configuration. The neural gland is a blind sac with a well-ciliated duct and extends as a very short dorsal strand. The neurons are arranged peripherally to the fibrous medulla and form the cerebral ganglion, which sends its nerves at organ primordia in the various body regions. In conclusion, our observations show that the neural gland originates as a posterior evagination of the inner vesicle and that material from this anlage also forms the cerebral ganglion, with the possible, early contribution of cells from the blood.

Many studies have dealt with the origin and differentiation of oocytes and their envelopes in ascidians and, in particular, with the origin and roles of test cells and the vitelline coat. Test cells differentiate in close association with oocytes and, although several functions have been attributed to them during oogenesis, none has ever definitely been demonstrated. Strong evidence has recently been reported that test cells synthetize and secrete thin structures called "ornaments" which, during embryo development, are deposited on the larval tunic, and make it hydrophilic. (Cloney 1996, Acta Zool. 17:73-78). Test cells are externally covered by the vitelline coat, the features of which differ among species and which contains the sites for gamete recognition. Its origin from oocytes has long been debated. We have now studied the appearance and differentiation of test cells and the vitelline coat in Clavelina lepadiformis by light and electron microscopy. C. lepadiformis is an ovoviviparous ascidian, whose branching tubular ovary is continuous with the single oviduct which opens into the atrial chamber. The ovarian epithelium is composed of a single layer of cells, joined apico-laterally by tight junctions and covered by a thin basal lamina towards the stroma. Early oocytes probably derive from blood cells which segregate within the ovarian epithelium after crossing the basal lamina. Unlike the contiguous epithelial cells, they do not establish tight junctions with any type of cell. They first become surrounded by one layer of primary follicular cells derived from and continuous with the ovarian epithelium. Later, several dark and undifferentiated cells insinuate themselves between the primary follicle cells and the oolemma, and gradually form a discontinuous inner layer. In the following stage the acellular vitelline coat becomes recognizable as a homogeneus, fibrous material externally covering several scattered dark cells which become encased in indentations in the oolemma and represent the test cells. At this stage, the oocyte possesses many small vesicles of RER filled with filamentous electron-dense material, several of which closely adhere to the oolemma. At the beginning of vitellogenesis all the egg envelopes are well developed (outer and inner follicular cells, vitelline coat and test cells). In particular, the test cells have giant Golgi with more than 20 piled cisternae with budding vesicles at their borders. In a later phase, the test cells contain a great number of round secretory granules filled with homogeneous and/or fibrous content. These granules can fuse one another; several of them release their fibrous content which intermingles with and is added to the vitelline coat. At the same time, the vitelline coat assumes a three-layered aspect, with the middle (central) layer more compact and dense than the other two; in particular, the inner layer, facing the oolemma, has a net-like structure, resembling that of the secreted material of the test cells. Our results suggest that the vitelline coat is built up with the contribution of oocyte vesicle contents, to which, secretions from test cells are then added. Nevertheless, because of the large number of test cell granules at ovulation, it is also possible that, during embryogenesis, the test cells of C. lepadiformis, like those of other ascidians, secrete hydrophilic ornaments on the larval tunic.

Three blastogenetic generations are usually present in colonies of the ascidian Botryllus schlosseri, namely adult zooids, buds and budlets. At 19 °C adult zooids are weekly resorbed and replaced by mature buds during the regression phase of the colonial life cycle. This period is characterised by an intense phagocytosis and a significant increase in the frequency of circulating macrophage-like cells is always coupled with a significant decrease in the frequency of circulating amoebocytes. Hyaline amoebocytes and uni- or multi-vacuolated macrophage-like cells are the two hemocyte types involved in phagocytosis. Our previous studies suggest that they represent two functional stages of a single cell type, the active phagocyting stage being the hyaline amoebocyte which withdraws its cytoplasmic projections and changes to macrophage-like morphology upon ingestion of foreign particles. The hypothesis is supported by in vitro experiments, using short term hemocyte cultures, as test particles appear inside hyaline amoebocytes after 5 min of incubation whereas they are visible inside macrophage-like cells after 30 min of incubation. During the resorption of old adult zooids circulating hemocytes containing apoptotic cells inside their vacuoles are easily seen. Therefore phagocytes, in a certain stage of their differentiation pathway must express surface molecules involved in the recognition of senescent cells. In mammals recognition of apoptotic cells by circulating macrophages is often mediated by thrombospondin, secreted by macrophages, which forms bridges between the membrane of senescent cells and both the receptor for vitronectin and the 88 kD monomer CD36 on macrophage surface. In order to verify whether a similar mechanism of apoptotic cell recognition is present also in ascidians, we tested the monoclonal antibody OKM5, anti CD36, on monolayer of B. schlosseri hemocytes. The results point to the presence of molecules recognized by OKM5 on the surface of hyaline amoebocytes. This indicates that hyaline amoebocytes are directly involved in the recognition of cells undergoing apoptosis thus confirming our hypothesis that they represent the actively phagocyting cells and suggests that a common mechanism of apoptotic cell recognition, involving thrombospondin and its receptors, has been maintained throughout the evolution of chordates.

A MORPHOLOGICAL STUDY OF THE PERICARDIAL BODY IN THE ASCIDIAN CIONA INTESTINALIS. S Scippa, C Izzo and M de Vincentiis, Dipt. di Genetica, Biol. Gen. e Molec., Fac. di Sci. dell'Univ. di Napoli e Stazione Zool. di Napoli.
The pericardial body is a globular structure present in the pericardial cavity of some ascidian species. This structure shows granular and large cells (Roule 1884, Ann. mus. hist. nat. Marseille Zoologia Tome II), degenerating cardiac fibres embedded in an amorphous matrix also containing some hemocytes (Millar 1953. Memoirs on typical British Marine Plants & Animals 35: 1-123), and degenerating lymphocytes in the middle of the aggregation (Kalk 1970. Tissue & Cell 2: 99-118). The presence of Cardiosporidium Cionae has been also reported (Van Gaver and Stephan 1907, C.R.Soc.Biol.Paris 62:556-557). The present note is a morphological study of the pericardial body of Ciona intestinalis at the light and electron microscope ((TEM). Cytochemical investigation at the light microscope revealed the presence of acid and neutral mucopolysaccharides and collagen fibres. At the electron microscope, numerous blood cells, as stem cells, clear vesicular granulocytes, microgranulocytes, unilocular granulocytes and globular granulocytes, were found at the periphery of the small-sized pericardial bodies. The amorphous matrix prevailingly contained degenerating cells and cardiac fibres. Large cells could be distinguished; they were almost completely occupied by one or more vacuoles with a generally homogeneous and strongly electron-dense content. Moreover, at TEM, the cells that, at the light microscope, appeared as granular cells showed a peculiar ultrastructure. We observed a sporogonium of a microsporidium containing numerous sporoblasts, i.e. nucleated cells which will form the lasting spores. The nuclei of these cells contained electron-opaque zones, probably richer in chromatin. A double membrane, or pansporoblast, enveloped the plasmodium. The central portion of the plasmodium showed tubular structures resembling the polar filaments typical of microsporidium spores. To conclude, the pericardial body of C.intestinalis is a mass of amorphous material containing degenerating elements surrounded by numerous blood cells in small-sized pericardial bodies; it is characterised by the presence of a microspiridium, irrespective of the size. As far as the formation of the pericardial body is concerned, we suggest that Ciona might be infected by the sporozoon; the latter would stimulate a reaction in the blood cells involved in defence mechanisms, which would start producing the amorphous substance. Alternating contractions of the heart would make the detachment of fibres and endothelial cells easier due to rubbing of the heart wall with the pericardial wall.

During early oogenesis in solitary ascidians each ovarian oocyte becomes enclosed within extensions of the ovarian epithelium known as the outer follicular epithelium. Gradually, accessory cells of maternal origin (probably hemoblasts) are recruited into the compartment around each oocyte. A vitelline coat is secreted around each oocyte. Some accessory cells, outside the vitelline coat, differentiate as inner follicular cells. Others, beneath the vitelline coat, in close contact with the oocyte, become test cells. The function of the test cells has been an enigma since the middle of the 19th century. The popular assumption that they transfer substances into oocytes has not been substantiated. In many ascidians the test cells synthesize and store submicroscopic particles or filaments (called ornaments) in vacuoles. Seven types have been identified in 8 families of ascidians (Cloney 1994). Many species with "multigranular ornaments" contain silica (Monniot et al.1992a). In species with ornaments the test cells deposit them on the outer cuticular layer of the larval tunic (C1) before hatching. The presence of these ornaments has been invaluable in tracing the secretions of test cells by TEM. The test cells of some species (ascidiids and Ciona intestinalis) do not synthesize ornaments. Instead, they attach themselves firmly to C1. The test cells of nearly all species are metachromatic. Their vacuoles contain acidic (usually sulfated) glycosaminoglycans (Monniot et al.1992b) or, based upon recent analyses, sulfated polysaccharides (M. Pavao, pers. comm.). I infer that the secretions of test cells are negatively charged regardless of the presence or absence of ornaments. What happens if ascidian larvae are deprived of test cells or their secretions before hatching? Embryos that develop normally and hatch in the presence of test cells (controls) are always hydrophilic and never become trapped at the surface of SW. When demembranated neurula are cultured alone or in small numbers in 10 ml of SW (without test cells, follicle cells or the vitelline coat), C1 does not form fins; the larvae are sticky, hydrophobic and cannot swim. But when demembranated neurulae are cultured in large groups (30-80) in 1 ml of SW, many form fins with normal morphology. They are not sticky, they swim normally, but are also hydrophobic, as demonstrated with 7 solitary species (Cloney 1990; Cloney & Hansson 1996). These larvae easily become trapped at the SW surface and cannot reenter. The test cells normally deposit a negatively charged hydrophilic "finishing coat" on the larval tunic. The significance of the diverse types of ornaments and silica (when present) is unknown. In Molgula pacifica, there is no swimming larva and no outer cuticular layer (C1); development is direct and test cells are completely absent (Cloney, 1995). Apparently, the test cells were eliminated during the evolution of this species when they had no significant function. (Refs: Cloney, R 1990. Acta Zool. 71:151-159; Cloney, R 1994. pp. 77-95 in Reproduction & Development of Marine Invertebrates. Wilson, Stricker, & Shinn, eds., Johns Hopkins Univ. Press; Cloney, R 1995. Acta Zool.76: 89-104; Cloney, R & Hansson, LJ, 1996. Acta Zool. 77:73-78; Monniot, F et al.1992a. Mar. Biol. 112:283-292; Monniot, F et al.1992b. Biochem. Systematics & Ecol. 20:541-552.)

ON THE SENSORIAL FUNCTION OF ASCIDIA MALACA ADHESIVE PAPILLAE. M Gianguzza*, G Dolcemascolo*, U Fascio° & F De Bernardi°. *Inst. of Biol., Univ. of Palermo; °Dept. of Biol., Univ. of Milano, Italy.
At the anterior end of the cephalenteron the swimming larvae of Ascidia malaca have 3 papillae that secrete an adhesive substance which allows the larvae to attach. Observations at the light microscope level reveal that papillae are cone-shaped, are arranged at the vertices of a triangular field and their anterior part ends in a hyaline cap. Ultrastructural (TEM) observations evidenced also, in the apical part of the hyaline cap, a mass of fine granular moderately electron-dense material which probably corresponds to the adhesive substance secreted by the papillae. Cells forming the papilla body are ovoid and elongated and can be distinguished ultrastructurally as types "A" and "B". A cells, whose ultrastructure is characteristic of secretory cells, are considered to produce the adhesive substance. Their cytoplasm presents a well developed RER, a Golgi in active synthesis phase and numerous granules in the apical region which undergo ultrastructural modifications; the material resulting from this process is later poured into the matrix of the hyaline cap (Gianguzza & Dolcemascolo, Eur. Arch. Biol. 105:51-62, 1994). Type B cells are ovoid and elongated and their main feature is the presence of several microvilli and a single cilium contained in a sort of "pocket" of the cytoplasmic membrane. Microvilli originate at the basis of the cell membrane, stretch all along the hyaline cap and sometimes come out of the same cap. Another characteristic of B cells is the long bundles of microtubules running parallel to the major cell axis; their presence was visualized through the use of tannic acid during fixation (cf. Hayat, "Stains and Cytochemical Methods" Plenum , 1993). The ultrastructure of B cells and, above all, the presence of cilia and microvilli let us hypothesize that the papillae somehow possess also a sensorial function which allows them to perceive environmental stimuli. This hypothesis was confirmed by confocal microscopy using antitubulin fluorescent antibodies. It was possible to evidence, in the larval cephalenteron, a wide network of innervations connecting the cerebral vesicle to the adhesive papillae. The ultrastructure of nervous fibres connecting the cerebral vesicle to the papillae could be seen with TEM. Ultrathin sections evidenced long bundles of microtubules running parallel to the axis of the fibre itself. Papillae being innervated confirm the hypothesis that they could exercise also a sensorial function through activity of B cells, which may perceive external environmental stimuli through their long microvilli and the cilium. The perception of these stimuli would create a signal causing A cells to secrete the adhesive substance allowing the larvae to attach to a substratum.

THE ADHESIVE PAPILLAE OF THE SOLITARY ASCIDIANS. F. De Bernardi, U. Fascio, S. Gropelli & C. Sotgia. Dept. of Biology, Univ.of Milano.
The swimming larvae of the solitary ascidians belonging to the Ascididae family bear 3 anterior, simple, coniform adhesive papillae arranged at the vertices of a isosceles triangular field. They secrete adhesives that are used to effect a transitory settlement at the beginning of metamorphosis. They are formed by the anterior epidermal cells and are induced together with the nervous system by the A 4.1 cells and by their descendants. In the method of both induction and differentiation the adhesive papillae may be considered homologous to the cement gland of the Amphibia Anura. The adhesive papillae of newly hatched Phallusia mamillata larvae examined at the SEM appear covered by the tunic. About 1-2 hours after hatching the tunic becomes fenestrated over the central part of the papillae and numerous bulb-ended microvilli protrude throughout the holes. The ultrastructure of this type of papillae as described for Ascidia malaca (Gianguzza & Dolcemascolo 1994, Eur. Arch. Biol. 105: 51-62) revealed two types of elongated cells: peripheral secretory-type cells and central cells with micovilli and bundles of microtubules along the major axis of the cells, probably with sensorial function. We performed immunofluorescence experiments with an anti-(-tubulin monoclonal antibody (clone 2-28-33; Crowther & Whittaker 1990, J. Neurobiol. 23:280-292) specifically reacting with axonal microtubules. Only the central sensory cells of the papillae were stained and the axons appeared to arise from the proximal end of these cells. These axons form a long nerve reaching the brain vesicle. Branches of the same nerve appear to connect the basal end of the peripheral secretory cells. By the confocal laser microscope we were able to follow the position of the papillary nerve both in Phallusia mamillata and in Ascidia malaca. A dorsal nerve connects the two dorsal papillae to a dorsal position of the brain vesicle. Another nerve connects the ventral papilla to a more ventral position of the brain vesicle. Dorsal papillae are also reached by axons coming from a distal position of the nerve and vice-versa. The central cells of the adhesive papillae could be primary sensory neurons and they may have both chemosensory and mechano-sensory function. The presence of two papillary nerves may be related to a heterochronic development of the thirds papilla.

C. Lambert1,3, G. Lambert1,3, A. Mc Dougall4, L. Robert1, L. Lucio1, and C. Goode2 . Depts. Biol.1 and Chem/Biochem2 , Calif. State Univ., Fullerton, CA 92834-6850; Sta. Biol., Pl. Georges Teissier, Roscoff, France3 ; Sta. Zool., Villefranche-sur-Mer, France4.
At fertilization ascidian eggs release a membrane-linked N-acetylglucosaminidase which is a principal block to polyspermy (Lambert 1989, Development 105:415-420) and also undergo cortical contractions which redistribute specific territories in the zygote (Sardet et al., 1989, Development 105: 237-250). We have examined both events in , Ascidia ceratodes eggs in California and Phallusia mammillata eggs in France. We use the fluorogenic substrate 4-methylumbelliferyl N-acetylglucosaminide to assay enzyme activity released and light microscopy to determine the cortical contractions. Both processes are inhibited by BAPTA-AM and thus involve increase in internal calcium concentrations. Glycosidase release requires extracellular calcium but the contractions do not when stimulated with ionomycin. We have detected the increase in internal calcium that precedes the cortical contractions in calcium green-loaded Phallusia mammillata eggs in the confocal microscope. We have thus far been unable to detect any change in intracellular calcium when we activate the eggs with ryanodine, which results in glycosidase release without the contractions ( Lambert et al., 1994, Growth Develop. & Differ. 36, 133-139). The glycosidase is released seconds after insemination but the contractions occur several minutes later. The calcium ionophore ionomycin induces cortical contractions within a minute in contrast to the 4.5 minutes required after insemination. This suggests that a multi-step pathway involving calcium is involved in the contractions but that the glycosidase release pathway is more direct (McDougall et al., 1995, Zygote 3: 251-258). The tyrosine kinase activator dimethylbenzanthracene (DMBA, Archuleta et al. 1993, Proc. Natl. Acad. Sci. 90, 6105-6109) causes glycosidase release without the contractions at 20(M. This release can be inhibited by the tyrosine kinase inhibitors genistein at 10(M and tyrphostin A-23 at 200(M. The tyrphostin blocks fertilization-induced glycosidase release without affecting sperm motility but genistein does not.. This is to be expected as genistein and tyrphostins affect different parts of the tyrosine kinase pathway (Robert et al. 1995, Mol. Biol of the Cell 6: 431a). Incubation of intact ascidian eggs in sea water containing the fluorogenic PLC substrate 4-methylumbelliferyl-phosphocholine discloses that they have a cell surface phospholipase C (PLC) which may be involved in release of the glycosidase. PLC activity is increased by DMBA, and we have detected on western blots an electrophoretic band that is phosphorylated in activated eggs but not phosphorylated in tyrphostin-treated eggs. Possibly this cell surface phospholipase is activated by tyrosine phosphorylation which then releases the lipid linked glycosidase. The cortical contractions would then be activated by an internal phospholipase which could generate inositol-tris-phosphate to release calcium from the endoplasmic reticulum (Dale 1988, Exp. Cell. Res. 177: 205-211). Thus activation of the ascidian egg involves two calcium dependent processes which may be under independent controls. The glycosidase release may have tyrosine kinase involved in its activation but the cortical contractions are initiated by a different trigger.

4. Zool. Soc. of Japan 67th annual meeting, 1996. (Publ. in Zool. Sci. ;some abstracts were edited slightly to conserve space.)

H. Sawada1, M. Rosaria Pinto2, and R. De Santis2. 1Dept. Biochem., Fac. Pharm. Sci., Hokkaido Univ., Sapporo; 2Dept. Cell Dev. Biol., Stazione Zoologica 'Anton Dohrn', Naples, Italy.
It is currently proposed that sperm trypsin-like proteases (spermosin and acrosin) and proteasomes (20S and 930-kDa) are involved in fertilization of the stolidobranch ascidian, Halocynthia roretzi. However, whether or not spermosin and proteasomes are involved in fertilization of the phlebobranch ascidian, Ciona intestinalis, is not yet known. Here, we found that two proteasome inhibitors (MG115 [Z-Leu-Leu-Nva-H] and MG132 [Z-Leu-Leu-Leu-H]) and two spermosin inhibitors (Z-Val-Pro-Arg-H and Dns-Pro-Arg-H), but neither acrosin inhibitor (leupeptin) nor cysteine protease inhibitor (E-64-d), inhibited the fertilization of C. intestinalis in a concentration-dependent manner. These results indicate that spermosin and proteasome may play a key role also in fertilization of the eggs of C. intestinalis.

M. Yoshida1, R. Deguchi2, M. Morisawa2, K. Mikoshiba134. 1Mol. Neurobiol. Lab., Tsukuba Life Science Center, Inst. of Phys. and Chem. Res., Tsukuba, 2Misaki Mar. Biol. Stn., School of Sci., Univ. of Tokyo, Miura, 3Dept. of Mol. Neurobiol., Inst. of Med. Sci., Univ. of Tokyo, Tokyo, and 4Mikoshiba Calciosignal Net Project, ERATO, JRDC, Tokyo.
In ascidian eggs, fertilization initiates a sequence of events including exit from meiosis, rearrangement of cortical and cytoplasmic domains with egg shape modification (ooplasmic segregation), first and second polar body extrusion, etc., and the elevation of intracellular Ca2+ is thought to initiate these processes. In the ascidian Ciona savignyi, transient Ca2+ wave just after fertilization and following repetitive Ca2+ oscillation are observed. In somatic cells, there are two mechanisms of the elevation of intracellular Ca2+; inositol 1,4,5-trisphosphate (IP3) -induced Ca2+ release (IICR) and Ca2+ -induced Ca2+ release (CICR). To study the role of IICR and CICR upon egg activation, we first examined the presence of IP3 receptor (IP3R), which mediated IICR, in eggs of ascidians. Monoclonal antibodies (mAb) against the type I IP3R recognized a protein band in C. savigyi membrane preparations by Western blots. Then we investigated the role of IICR by determining the effects of microinjected mAb 18A10, which binds to IP3R and inhibits IICR. The antibody did not inhibit the Ca2+ wave and egg shape modification, but inhibited the Ca2+ oscillation and 1st cell division. It indicates that IICR is essential for ascidian egg activation.

K. Kyozuka1, G.L. Lusso2, E. Tosti2 and B. Dale2. 1Asamushi Mar. Biol. Stn., Tohoku Univ, Aomori and 2Div. of Cell Biol., Stazione Zoologica 'Anton Dohrn', Naples, Italy.
Changes in [Ca2+]i during fertilization and meiosis in Ciona intestinalis oocytes were measured using the fluorescent calcium indicator. An initial large transient increase of [Ca2+]i followed by periodic changes of [Ca2+]i took place during egg contraction and first meiotic division. After the first polar body extrusion, Ca2+ oscillations took place again during the second meiotic division. Histone H1 kinase activity was high at metaphase I and metaphase II of meiosis. When heparin-injected oocytes were fertilized, the initial increase of [Ca2+]i occurred, however, subsequent Ca2+ oscillations were suppressed. Microinjection of BAPTA into the oocyte at telophase I of meiosis inhibited both the Ca2+ oscillations and the increase of histone H1 kinase activity at metaphase II. Protrusion of the second polar body was also inhibited. These results indicate that activity of metaphase promoting factor, estimated from the activity of histone H1 kinase, is regulated by [Ca2+]i. Periodic changes of [Ca2+]i are necessary for the egg contraction and protrusion of polar bodies during meiosis.

T. Oka1, R. Amikura2, S. Kobayasi2, H.Nishida1. 1Dept.of Life Sci., Tokyo Inst. Tech., Yokohama; 2Inst. Biol.Sci., Univ. of Tsukuba, Tsukuba.
Mitochondorial large ribosomal RNA(mtlrRNA) had been identified as a cytoplasmic factor that is involved in poll cell formation in Drosophila embryos. mtlrRNA is a component of germ plasm, and transported out of mitochondria. We examined the distribution of mtlrRNA in ascidian embryos by in situ hybridization. Different distribution between mitochondria and mtlrRNA was observed in the posterior-vegetal blastomeres at the 8-cell stage.

G. Kumano and H. Nishida. Dept. of Life Sci., Tokyo Inst. Technol., Yokohama.
In the previous meeting, we reported that we had purified endoderm-specific alkaline phosphatase and determined its N-terminal amino acid sequence in larvae of the ascidian, Halocynthia roretzi. In this meeting, we present the results of molecular cloning of the enzyme and the analysis of its temporal and spacial expression during embryogenesis. We sequenced the 3024-bp long cDNA clone of alkaline phosphatase, containing the complete open reading frame and polyadenylic acid. The expression of mRNA is observed in endoderm cells at tailbud stage.

Y. Nakatani1, H. Koide2, Y. Kaziro2 and H. Nishida1. 1Dept. of Life Sci. and 2Dept. of Biol. Sci., Tokyo Inst. Technol., Yokohama.
bFGF can induce notochord differentiation in ascidian embryos. Generally, bFGF signals are transmitted via membrane-bound receptor tyrosine kinases (RTKs). Ras is a small GTP-binding protein and play an important role in RTK signaling. Microinjection of dominant negative Ras (RasN17) protein into fertilized eggs blocked notochord differentiation. When the notochord precursor blastomere was co-isolated with the inducer blastomere and then RasN17 protein was microinjected, notochord formation was suppressed. The notochord precursor blastomeres microinjected with RasN17 protein were treated with bFGF. Many of them failed to develop notochord features. These results suggested that the Ras signaling pathway is involved in notochord induction during ascidian embryogenesis.

T.Hirano and H.Nishida. Dept. of Life Sci., Tokyo Inst. Technol., Yokohama.
To study the origin of adult tissues of the ascidian Halocynthia roretzi, we traced the cell fates by intracellular injection of horseradish peroxidase (HRP) into identified blastomeres at 110-cell stage. Descendants of the injected cells were histochemically detected at juvenile stage. We have reported that blood cells of juveniles are derived from trunk lateral cells of the larvae. Further detailed analysis revealed that trunk lateral cells give rise to body-wall muscle cells (in an oral siphon and a longitudinal mantle) and cells around 1st and 2nd gill slits, as well as blood cells.

T.Iseto and H.Nishida. Dept. of Life Sci., Tokyo Inst. Technol., Yokohama.
CAB is a structure that exists in the cortex of the posterior blastomere which shows successive unequal cleavage in ascidian embryos. It has been suggested that CAB attracts the centrosome and the nucleus with microtubules, thus it causes unequal cleavage. We observed CAB with transmission EM. CAB is a homogeneous electron-dense region that directly lines the plasma membrane. Some microvilli are observed in the surface area of the blastomere where CAB is just beneath the membrane.

S. Wada, Y. Ueno and H. Saiga. Dept. Biol., Fac. Sci., Tokyo Metropolitan Univ.
Previously we reported that homeobox genes, Hrdll-1, Hroth, HrHox-1 and Hrcad-1 of the ascidian Halocynthia roretzi are expressed in the neural tube and epidermis of the embryo with a distinct expression domain. The expression domains of these genes align in the order as described above along the anteroposterior axis, showing the presence of the anteroposterior patterning in these tissues. To understand the mechanism of anteroposterior patterning, we examined the involvement of vegetal cells in this patterning mechanism by cell ablation experiments. Embryos in which various sets of vegetal blastomeres were ablated using a sharpened needle at early cleavage stages were cultured until the middle tailbud stage and then gene expression was examined by whole mount in situ hybridization. We have found that expression pattern of above genes in the ectodermal tissues is severely disrupted in the operated embryos. This suggests that in the ascidian embryo, influence of vegetal cells plays an important role in patterning of the neural tube and epidermis along the anteroposterior axis.

K. Takamura, S. Ooyabu, Y. Yamauchi, Y. Inoue, M. Hashimoto, N. Ise and Y. Yamaguchi. Div. Biotech., Fac. of Engineering, Fukuyama Univ.
We isolated a cDNA clone for an antigen recognized by UA165 monoclonal antibody which was specific to test cells of C. intestinalis. This cDNA clone was about 600bp in length and showed high homology to sequence coding C`-half region of calreticulin of other animals. However, its 3`-region was very different from that of other animals. We also isolated another type of calreticulin homologue, which showed higher homology, by 3`-RACE method. These results suggest that C. intestinalis has at least two types of calreticulin homologue and we think that they seem to be different in their localization and function. Now, we try to isolate full-length cDNAs and analyze by in situ hybridization and Nothern blot analysis.

Y. Satou and N. Satoh. Dept. Zool., Grad. Sch. of Sci., Kyoto Univ.
Muscle cells in the anterior and middle part of the ascidian larva tail differentiate in a highly autonomous manner. Because this autonomy is due to so-called muscle determinants in the myoplasm of fertilized eggs, there is a genetic cascade for muscle cell differentiation in ascidian embryos, which begins with muscle determinants and ends with the expression of muscle-specific structural genes. We approached the molecular nature of the muscle determinants going upstream of the cascade by analyzing transcriptional control mechanisms of muscle-specific structural genes. The HrMA4a actin gene, a muscle specific structural gene, has been analyzed for this purpose. It has been demonstrated that a short upstream sequence up to -103 base pairs from the transcription start site is sufficient for the muscle specific expression of HrMA4a. We performed a more detailed analysis using the beta-galactosidase genes as a reporter. We demonstrated here that, within the proximal region (-103 ~ -66) of HrMA4a gene, there are two short sequences essential to the muscle specificity of HrMA4a promotor, one is 9-bp long (5`-TCGCACTTC-3`) and the other is 13-bp long (5`-GTGATAACAACTG-3`).

M. Ogasawara1, K. J. Tanaka2, K. W. Makabe1, N. Satoh1. 1Dept. Zool., Grad. Sch. of Sci., Kyoto Univ., and 2Dept. of Biol., Fac. of Sci., Konan Univ., Kobe.
Notochord, nerve cord, pharyngeal gill and endostyle are structures key to understanding of the molecular mechanisms underlying the origin and evolution of chordates. We made an ascidian endostyle cDNA library and isolated two endostyle-specific genes, HrEnds1 and HrEnds2 by differential screening of the library. Predicted amino acid sequences of these genes suggested that both genes encode novel secreted proteins. Transcripts of these genes were detected in the endostyle of 1-month young adult under our in situ hybridization condition. These genes may serve as probes for further analyses of molecular mechanisms involved in formation of the endostyle.

K. J. Tanaka1, M. Ogasawara2, K. W. Makabe2, N. Satoh2. 1.Dept. of Biol., Fac. of Sci., Konan Univ., Kobe and 2.Dept. of Zool., Grad. Sch. of Sci., Kyoto Univ.
Pharyngeal gill-slit, endostyle and notochord are key structures to understanding mechanisms underlying the origin and evolution of chordate. We made an ascidian pharyngeal gill cDNA library and isolated cDNA clones for two pharyngeal gill-specific genes (HrPhG1 and HrPhG2) by differential screening of the library. Transcripts of these genes were detected in pharyngeal gill wall a few days after metamorphosis. This expression pattern was retained to adult stage. These genes may serve as useful probes for further analysis of molecular mechanisms involved in the formation of the pharyngeal gill in chordates.

M. R. Wada, N. Tanimoto, Y. Ohtani and T. Nishikata. Dept. of Biol., Fac. of Sci., Konan Univ., Kobe.
Maternal mRNAs localized in the egg cytoplasm are very important to early embryogenesis. The cytoplasmic determinant which plays a key role in the differentiation mechanism in the ascidian egg are suggested to be an mRNA. Recently, the Y-box proteins in the Xenopus oocyte were proved to be RNA-binding proteins which prevent the RNA from translation. Therefore, we isolated the cDNA of the Y-box protein homolog from Ciona intestinalis gonads. This Y-box protein in the ascidian egg might be a good clue to identify the molecular nature of the cytoplasmic determinant.

H.Kato, K. Matsumura, M. Nagano, S. Tsukamoto, H. Hirota, and N. Fusetani. Biofouling Project, ERATO, JRDC, Yokohama.
Enzymes involved in hatching of ascidians have not been well-studied. Previously we reported that narain isolated from a marine sponge Japsis sp. inhibited hatching in the ascidian H. roretzi at a concentration of 10 uM. Narain did not inhibit the enzme reaction of commercially available trypsin and crude hatching enzyme, but inhibited dissolution of isolated fertilization membrane by them. Therefore, narain inhibited neither a trypsin-like proteinase activity in hatching enzyme nor its secretion.

M. Okuyama and Y. Saito. Shimoda Marine Res. Center, Univ. of Tsukuba, Shimoda.
There are two major genera, Botryllus and Botrylloides, in the family Botryllidae. An unknown botryllid collected from Shikine Island was thought to be a Botryllus, as it shows the feature of Botryllus in the arrangement of gonads, that is, the ovary is situated anterior to the testis. However, we found that a zooid of this botryllid forms a brood pouch as a brooding organ for its embryo. The brood pouch formation is one of the important features of Botrylloides. Histological studies on brood pouch formation in this botryllid show that the pouch is formed by extension of a part of the branchial sac. Thus, brood pouch formation in this botryllid is quite different from that of Botrylloides, in which the pouch is always formed by invagination of the atrial epithelium. On the other hand, it is known that a kind of brooding organ (not a brood pouch) is formed from the branchial sac in Botryllus sexiens. This undefined botryllid might have a close relationship with B. sexiens rather than the species of Botrylloides.

Y. Sato, M. Morisawa. Misaki Marine Biol. Sta., Univ. of Tokyo, Kanagawa.
In metamorphosis of ascidian tadpole larva, sequential degradation of the adhesive papillae (APBD) on its tip, tail resorption (TR) and differentiation of trunk occur. In Halocynthia in which the larval body is vertically cut off at various places before APBD, TR of posterior fragment of larva never occurs, but TR occurs when the amputation is carried out after APBD, suggesting that APBD is a signal for TR (Numakunai, 1967). In Ciona, APBD in the anterior part of fragments of amputated larvae occurred at any timing (before or after APBD) and in any other part of larval body. Furthermore, TR occurred in the posterior fragments of amputated larvae in any timing and any amputation, suggesting that contrary to Halocynthia, APBD is not essential for TR in Ciona. When the trunks of larvae were cut off anterior to brain vesicle (BV), both the posterior part of the amputated fragments including BV and tail and the anterior part of the trunk without BV were not fully differentiated. When the larvae were cut off posterior to the BV or mid part of the tail at any stage, the anterior fragments with BV or with BV and tail completely differentiated and became juveniles. These suggest that induction of the trunk differentiation does not need the part posterior to the BV.

S. Tsukamoto, H.Kato, H. Hirota, and N. Fusetani. Biofouling Project, ERATO, JRDC, Yokohama.
H. roretzi larvae do not metamorphose in fresh seawater (SW) when they are maintained at low densities in laboratory. Metamorphosis was induced, however, by addition of adult- or larvae-conditioned SW. The presence of metamorphosis-inducing compound(s) in the conditioned SW was confirmed by partial purification followed by physicochemical characterization. We have succeeded in isolation of 1mg of the metamorphisis-inducing compound from 2L of larvae. The same compound was detected in unfertilized eggs, larvae, and adult tunic, but not in other adult tissues.

S. Ohmori, J. Matsumoto, S. Fujiwara, K. Kawamura and T. Yubisui. Dept. of Biol., Fac. of Sci., Kochi Univ.
The activity of calcium-dependent (C-type) galactose- binding lectin is required for the aggregation of multipotent mesenchymal stem cells, called hemoblasts, around the morphogenetic region in the developing bud of the ascidian Polyandrocarpa misakiensis. We isolated two closely related cDNA clones encoding C- type lectins, named TC14-1 and TC14-2, from P. misakiensis. TC14-1 encodes a galactose-binding lectin TC-14, which had been previously purified from this species. TC14-1 and TC14-2 mRNAs were detected in hemoblasts. The amount of the mRNAs was increased during bud development. Two other C-type lectins, TCA18 and TCA15, were purified from this species, which had the activity to make cultured ascidian cells form large aggregations. Amino acid sequences of them were distinct but similar to those of TC14-1 and TC14-2. These results suggest that several types of C-type lectins synergistically act as a stem cell-aggregation factor in the developing bud of this species.

N. Harafuji, S. Fujiwara, K. Kawamura and T. Yubisui. Dept. of Biol., Fac. of Sci., Kochi Univ.
Retinoic acid is an endogenous determinant of anteroposterior axis in the developing bud of Polyandrocarpa misakiensis. Since the activity of aldehyde dehydrogenase (ALDH) is gradually enhanced in the morphogenetic region of the bud, local activation of this enzyme is thought to be one of the earliest events of bud development. Previously, we isolated 2 clones of the fragments of Polyandrocarpa ALDH genes that were expressed both in the adults and buds. But changes in the amount of mRNAs were not obvious during bud development. These results suggested that there was another ALDH gene which had not yet been cloned and was specifically expressed in the developing bud. We isolated several clones of ALDH cDNA fragments by RT-PCR, from the total RNA purified from developing buds. Amino acid sequences deduced from some of the cDNA clones were similar to those of mammalian ALDHs.

T. Ishii. Biol. Lab., College of Education, Akita Univ.
Self or nonself recognition is well known in some species of ascidians. A colonial ascidian Aplidium yamazii, which lacks tunic vessels, shows self or nonself recognition. In this species, it is strongly suggested that phagocytic tunic cells play an important role in allorecognition and rejection. On the contrary, the solitary ascidian Halocynthia roretzi shows self or nonself recognition in hemocytes. In mixed-hemocyte-incubation (MHI-assay), alloreactivity appears in most nonself combinations. I wondered if allorecognition occurred as quickly also in the tunic of H. roretzi. Tunic fragments were fused in alloreactivity positive combination. After a day or two, in each of them, there was invisible response to the naked eye unlike MHI-assay. Histological observation also showed no particular response in the tunic in allogeneic combinations. In the tunic, alloresponse may be occurring slowly or there may be a different manner existing in the tunic.

M. Shirae and Y. Saito. Shimoda Marine Res. Center, Univ. of Tsukuba, Shimoda.
Colony specificity has been studied mainly in botryllid ascidians. We previously reported that the allo-rejection reaction of Botryllus scalaris is different from allo-rejection reactions of other botryllids. In order to understand the phylogenetic relationship among those rejection reactions, we examined colony specificity of Symplegma reptans that was thought to have a common ancestor with botryllids. In S. reptans, two types of rejection reactions were observed among the combinations of incompatible colonies. In one type, allo-rejection begins with the aggregation of blood cells after vascular fusion between two colonies, followed by the disintegration of vascular vessels in the fusion area. In the other type, rejection begins with the infiltration of blood cells from the tips of ampullae (vascular termini at the colony periphery) into the tunic without vascular fusion, followed by their disintegration there. In comparison with allo-rejection of botryllids, the former type resembles the allo-rejection reaction of B. scalaris and the latter resembles that of Botryllus schlosseri.

N. Tomita and M. Hoshi. Dept. of Life Sci., Tokyo Inst. Technol., Yokohama.
Hemocytes of the ascidian Halocynthia roretzi are known to exhibit allogeneic reaction called 'contact reaction'. The ascidians have several types of hemocytes, yet it remains unclear what type participates in each step leading eventually to the contact reaction. Besides morphological features, specific cell markers are required for the classification and identification of hemocytes. We raised a monoclonal antibody (mAb) that recognized only one type of hemocytes, namely the giant cells. The antigenicity was localized in the cell surface of the intact giant cell. Western blot analyses indicated that the mAb recognized a protein of more than 500kDa. The giant cell-specific mAb had no effect on the contact reaction.

M. Suzuki1, R. Kondoh 1, H. Ohba1, K. Tabaka2, and J. Chiba1. 1Dept. of Biol. Sci. and Technol., Sci. Univ. of Tokyo, Noda, and 2Dept. of Biol., Nihon Univ. Sch. of Med., Tokyo.
Hemocytes from H. roretzi undergo rapid lysis in vitro when those derived from different individuals come in contact with each other. We have prepared monoclonal antibodies (mAbs) against membrane fractions of hemocytes from H. roretzi and selected mAbs that inhibit this allogeneic cytotoxic reaction (ACR). Two groups of mAbs that significantly inhibit ACR, named CRB1 and CRB2, were found to react with different antigens in membrane fractions of hemocytes by Western blot analysis; CRB1 was reactive with many glycoproteins of high molecular weight (62, 129 kDa and more) and CRB2 was with similar those of 47, 58, 80, 100, 115 kDa and more. The ability of the protease to destroy the antigenic activity seem to support the possibility that both mAbs recognize a peptide epitope. The epitope recognized by CRB1 and CRB2 wre detected on the same 30, 38, 44, 49, 68, 86, and 98 kDa peptides after treatment of the membrane fractions with N-glycanase. In addition, a competitive binding ELISA using combinations of biotinylated and native CRB1 and CRB2 revealed that their binding to the deglycosylated membrane fractions was mutually competitive. These results indicate that the epitopes are located very close to each other on the peptide, and carbohydrate chain(s) attached through N-glycoside linkage exist(s) near the antigenic determinant and influence(s) the reaction with both mAbs. Enhancement of ACR by the addition of carbohydrates such as chondroitin sulfate in the reaction media and inhibition of ACR by pretreatment of hemocytes with heparinase suggest that carbohydrates on glycoproteins apparently involve in the cellular recognition process in ACR by H. roretzi hemocytes as known in natural cytotoxicity in higher vertebrates, The mAbs probably inhibit ACR through blocking of this process.

S. Ohtake1, T. Sawada2, M. Dan-Sohkawa3, and K. Tanaka. 1Dept. of Biol., Nihon Univ. Sch. of Med., Tokyo, 2Dept. of Anat., Yamaguchi Univ. Sch. of Med., Ube, & 3Dept. of Biol., Osaka City Univ.
There has been an argument on the hemocytic nature of the viriform cells of H. roretzi. We examined their distribution, behavior and surface features in vitro by light microscopy (LM) and by transmission and scanning electron microscopy (TEM, SEM). When cone-shaped pieces of the papilla of the animal were placed in a culture dish containing seawater, viriform cells formed a donut-shaped cluster exclusive of other cell types on the substratum facing the cut surface of each cone. When observed under SEM, their surface was rough. They attached to the substratum with their heads and tails. Under LM and TEM, viriform cells concentrated in the tunic matrix adjacent to the epidermis of the papilla. They constituted the major population in this area among the several cell types found in the tunic matrix. Interestingly, viriform cells showed no contact reaction to allogeneic viriform cells nor to allogeneic hemocytes. These features suggest that they are not hemocytes but a kind of tunic cells, or they may even be protozoan symbionts which live in the tunic of the ascidian.

T. Abe, F. Shishikura, S. Ohtake and K. Tanaka. Dept. of Biol., Nihon Univ. Sch. of Med., Tokyo.
A 58 kDa proteinase inhibitor from the hemolymph plasma of an ascidian, Halocynthia roretzi, inhibits activities of serine proteinases and is considered as a member of the serpin superfamily. SDS-PAGE and immunoblotting analysis showed that the inhibitor forms an equimolar SDS-stable complex with trypsin as well as other inhibitory serpins. Further investigation on the interaction of the inhibitor with bovine pancreas trypsin was performed. As the result of the relation between the [I] / [E] ratio and the inhibitory activity, another inhibitory path on trypsin was considered: The 58 kDa inhibitor was cleaved by trypsin. The reaction may proceed along two divergent paths: one is the formation of the stable complex and the other is the breakdown of the inhibitor. When a reaction was started by the addition of trypsin to the mixture of the inhibitor and MCA-substrate, the relatively rapid initial velocity decreased to slower steady-state rate after 10 min. It was therefore suggested that the ascidian 58 kDa inhibitor was a slow-binding inhibitor.

Y. Nose, M. Hayashi, T. Uyama, Y. Moriyama and H. Michibata. Mukaishima Marine Biol. Lab., Fac. of Sci., Hiroshima Univ.
Ascidians are known to accumulate high levels of vanadium in their blood cells. Among about 10 types of blood cells, signet ring cells are vanadium accumulating cells called vanadocytes. They contain vanadium in highest levels of about 350 mM, corresponding to 10,000,000 times higher than that in seawater. Recently, we found that, in the vanadium-rich ascidian Ascidia sydneiensis samea, the number of vanadocytes in the body fluid increased dramatically when the ascidians were immersed in seawater containing 10 mM or 20 mM NH4Cl for 20 hrs (Hayashi et al., 1996). NH4Cl is known to neutralize the acidic compartments in biological membrane systems. Therefore, this phenomenon might be caused by the neutralization of the acidic compartments in ascidian. To examine this possibility, we incubated A. sydneiensis samea with several salts, ionophores and inhibitors for ion-pumping ATPases. The ionophores which dissipate delta pH and/or delta psi in membrane systems, and the inhibitors for V-ATPase and F-ATPase caused an increase in number of vanadocytes, about 3-5x higher than the control. These reagents neutralized several acidic compartments in the ascidian body, which we think is the key to the increase in number of vanadocytes.

T. Adachi, T. Kanda, T. Uyama, Y. Moriyama, and H. Mitibata. Mukaisima Mar. Biol. Lab., Fac. of Sci., Hiroshima Univ.
We extracted a vanadium-associated protein (VAP) from the blood cells of the ascidian, Ascidian sydneinensis samea. VAP was estimated to associate with vanadium at an approximate ratio of 1 mol : 1 mol. SDS-PAGE and polyclonal antibody against VAP (anti-VAP) , revealed that VAP is composed of at least three types of peptides, 12.5kDa, 15kDa and 16kDa, whose proteins are hydrophilic and localized in the cytoplasm of the vanadocytes. In the present experiment, based on the partial sequence of amino acids of VAP, PCR primers were designed and the cDNA sequence encoding the 15kDa peptide of VAP was partially determined.

Y. Suhama 1, K. Takamura 2, T. Uyama 1, Y. Moriyama 1 and H. Michibata 1. 1 Mukaisima Mar. Biol. Lab., Fac. Sci., Hiroshima Univ. 2 Dev. of Biotechnol., Fac. of Engin., Fukuyama Univ., Hiroshima.
Ascidians have about ten types of blood cells. Among them, vanadocyte is cell characterized by a single, fluid-filled vacuole and has the unique and unusual functions of containing both extremely high levels of vanadium ions in the +3 oxidation state and sulfate ions under pH 2 in the vacuole. V-ATPase has already been revealed to be present in the vanadocyte and a monoclonal antibody S4D5 recognizing the vanadocyte specifically was produced. In the present experiment, the other monoclonal antibody reacted with a 100 kDa hydrophilic protein was produced and the antigen confirmed immunohistologically to be present in the vanadocyte.

E. Hirose1, M. Aoki2, and K. Chiba3. 1Biol. Lab., Col. Bioresource Sci., Nihon Univ., Fujisawa; 2Shimoda Mar. Res. Ctr., Univ. of Tsukuba, Shimoda, and 3Dept. Life Sci., Tokyo Inst. Technol., Yokohama. In the colonial ascidian Clavelina miniata, physical stimulations induce strong luminescence in the tunic. We describe here the tunic cell morphology and distribution in the tunic which is a luminous tissue of this species. Three types of tunic cells are morphologically discriminated as morula-like tunic cells, tunic phagocytes, and tunic granulocytes, and they correspond respectively to the Type I, Type II, and Type III cells described by Aoki et al. (1989). Tunic phagocytes and tunic granulocytes occasionally contain phagosomes and elongated bacteria of unique forms. These bacteria are also distributed outside the tunic cells. To identify the luminous cell (luminocyte) exactly, it was necessary to record luminescence in vitro. We succeeded in transferring the tunic cell onto a glass slide. Each of three types of tunic cells described above are also discriminable under in vitro condition.

K. Chiba1, M. Hoshi1, and E. Hirose2. 1Dept. Life Sci., Tokyo Inst. Technol., Yokohama; 2Col. Bioresource Sci., Nihon Univ., Fujisawa.
In Clavelina miniata, tunic is luminous tissue, where morula-like tunic cells, tunic phagocytes, and tunic granulocytes are distributed. Because cell density in the tunic was so high , it had been difficult to determine which type of cells were involved in bioluminescence. To solve this problem, we isolated tunic cells and transferred them onto glass slides. Tunic cells on glass slides responded to hypotonic seawater and showed luminescence as had been observed in the tunic. We monitored the luminescence with a photon-counting camera connected to a microscope and found that only the tunic phagocytes luminesced. Similar results were obtained when each cell was punctured with microneedle; tunic phagocytes luminesced immediately after destruction, but morula-like tunic cells and tunic granulocytes did not show any luminescence. Thus we conclude that the tunic phagocyte is the luminous cell, named luminocyte.

S. Kimura and T. Itoh. Wood Research Institute, Kyoto Univ.
Cellulose synthesizing enzyme complexes (= TCs) have been found in the plasma membrane of epidermal cells in the ascidians Metandrocarpa uedai, Halocynthia roretzi and Perophora japonica, by using freeze-fracture replication techniques for electron microscopy. In plant cells, celluloses are synthesized by TCs on their plasma membranes. The present results suggest that the participation of the TCs in cellulose synthesis is a universal phenomenon both in plant and animal kingdoms. The TCs of ascidians consist of two types of membrane subunits: large particles (14.5 nm in diameter) on the periphery and small subunit particles (7.2 nm) filling its inside. The TCs are the linear type (ca. 200 nm in length and 80 nm in width), They are often connected with the termini of microfibrils.

T. Nishikawa, Grad. School of Human Inform., Nagoya Univ.
In the Ascidiacea, the tunic is usually separable more or less easily from the mantle except in siphonal areas. Rarely, however, a small part of tunic is mingled complicatedly with adjoining area of mantle (sometimes involving gonads) to form a complex, as is known in the genus Seriocarpa. I made a detailed observation of serially thin-sectioned or whole specimens clearly assignable to this genus, collected from sandy bottoms of Omura Bay and the Seto Inland Sea, ca. 25m deep. In the specimens, up to 14 mm long, a small patch of tunic protruded inward to enclose thinly and almost completely ca. a dozen globular hermaphroditic gonads individually, situated in two rows beneath the endostyle. Each gonad projected from the mantle body outwards with enveloping thin mantle epidermis, and opened into the peribranchial cavity with short ducts. In the gonadal structure, the present specimens were rather similar to S. rhizoides Diehl, 1969 from the North Atlantic and the Banda Sea, of the known 3 congeners.

T.Okada1, Y.Okamura2,3. 1Ushimado Marine Lab., Univ.of Okayama; 2Lab of Cell Biochem., NIBH., Tsukuba; 3Univ.of Tokyo.
TuNaI is a neuronal sodium channel expressed in the larva of the ascidian, Halocynthia roretzi. We identified TuNaI positive neuronal cells by the combined method of whole-mount in situ hybridization with serial sectioning. TuNaI positive cells were found in: 1)brain vesicle and the related region, 2) neck neural tube, 3) papilla, 4) epidermal sensory neurons. Double staining with DAPI confirmed location of nucleus for respective TuNaI signal. Intense signals in the posterior part of 1) correspond to a pair of large neurons which lie at the ventral region. We studied the cell lineage of TuNaI positive cells by microinjecting cell lineage tracer, dextran biotin, into each blastomere at the 8 cell and 16 cell stage. Double staining of TuNaI in situ hybridization and HRP-staining showed TuNaI positive cells in 1) and anterior part of 4) were derived from a4-2 blastomere. On the other hand, TuNaI positive cells in 2) were derived from A5-2, probably corresponding to cell soma of motor neurons. We also compared the expression pattern of TuNaI with that of TuNaII, another putative ascidian sodium channel gene.

S. Kajiwara, Dept. Biol., Fac. Educ., Iwate Univ., Morioka.
Scanning and transmission EM studies were made on the cerebral ganglion of the solitary ascidian, Halocynthia roretzi. The cerebral ganglion of the adult H. roretzi (weights about 150g) has a cylindrical shape. Nerve fibers in the cortex of the ganglion run parallel to the long axis of the ganglion. I found many varicosities, 10 (m in diameter, of the fibers by TEM. They resemble cell bodies of the ganglion by SEM, but they have no nuclei and other organelles particular to the soma. On the other hand, the medulla consisted of nerve fibers which ranged from 0.3 to 1.0 (m diameter.

H.Koyama, Coll. Nurs., Yokohama City Univ.
The organization of the cerebral ganglion was studied in the solitary ascidian Halocynthia roretzi by the application of the neuronal tracers horseradish peroxidase, cobalt conjugated with wheat germ agglutinin (Co-WGA), and carbocyanine fluorescent dyes (DiI, DiA etc.). Of these, only Co-WGA and carbocyanine fluorescent dyes were effective. For the application of carbocyanine dyes, the cerebral ganglia were isolated with the four main nerve trunks attached and fixed in 4% paraformaldehyde in 0.1M PBS. Dyes were applied directly as powder, or dissolved in DMSO/EtOH. In about 4 weeks, dyes were transported to about half the length of the ganglion. When viewed with an excitation filter of 330-385 nm, the neuronal somata of the ganglion fluoresced brightly, but with a filter for DiI (520-550 nm), autofluorescence was negligible. In horizontal sections of the cortex, there were columns of soma arranged longitudinally. DiI-labeled fibers ran between these columns in longitudinal bundles.

T. Iwasa, T. Azuma, M. Ohkuma , and M. Tsuda. Dept. of Life Sci., Fac. of Sci., Himeji Inst. of Technol., Harima Science Garden City, Hyogo.
In order to clarify the signal transduction system of the ascidian larva, we investigated G-proteins expressed in the larva by PCR methods. Using degenerate primers corresponding to conserved amino acid sequences of vertebrate G protein a subunits, we successively cloned several classes of a subunit using a cDNA library of ascidian larva as a template. We screened the cDNA library with the PCR fragment which showed a similarity to Go-class. The deduced amino acids sequence was composed of 357 amino acids. Although the deduced sequence exhibits the highest similarity to Goa, the sequence lacks a consensus cysteine residue at C-terminal, which is a pertussis toxin target. The phylogenetic tree inferred from the deduced amino acid sequence shows the protein belongs to the Gia-class, and branched from Goa. The deduced amino acid sequence was different from the conserved amino acid sequences within Goa-class. These results indicate that the Ga protein obtained here is a novel one. Northern blot analysis showed that the message is expressed after the 64-cell stage, and the amount increases with developmental stage. In situ hybridization revealed that the message is expressed in a restricted number of cells of the brain region at the early tailbud stage of ascidian larva.

K. Terakado, M. Ogawa*, K. Inoue, K. Yamamoto** and S. Kikuyama**. Dept. of Regul. Biol. and *Molec. Biol. and Biochem., Fac. of Sci., Saitama Univ. Urawa and **Dept. Biol., Sch. of Educ., Waseda Univ. Tokyo.
There is growing evidence that the vertebrate adenohypophysis originates from neuroectoderm. This suggests that equivalents of pituitary cells, if any, in protochordates may exist in derivatives of neuroectoderm. The neural complex in ascidians is known to be derived from embryonic neural tube. Electron microscopical studies on the structure of these organs in Halocynthia roretzi were performed with particular attention to their secretory systems. We found that the cells scattered along the dorsal strands as well as neural cells in the cerebral ganglion contain electron-dense secretory granules of varying sizes. Immunoelectron microscopical studies of these granulated cells using antiserum against bullfrog prolactin revealed that some of these cells contain prolactin-like substance within the secretory granules of 100-250 nm in diameter. The cells belonging to the neural grand and dorsal strand neither contained electron-dense granules nor showed immunoreactivity. The results suggest that phylogenetic progenitors of the adenohypophyseal cells in ascidians may exist in the cerebral ganglion and along the dorsal strand.

Y. Ohtsuka and T. Obinata. Dept. of Biol., Fac. of Sci., Chiba Univ.
Gelsolin is an actin filament severing and capping protein. It is detectable in a variety of tissues of higher vertebrates, particularly abundantly in platelets, macrophages and muscle tissues. Its activity is regulated by Ca2+ and phosphoinositides. Previously, we found that gelsolin is present abundantly in ascidian body wall muscle and determined its cDNA entire sequence. In this study, we examined expression of gelsolin during ascidian development to clarify its role in morphogenesis. Immunoblot analysis showed that gelsolin expressed maternally but its amount gradually increased after the neurula stage. When expression in embryos was examined by in situ hybridization and immunofluorescence staining with the whole mount specimens, gelsolin was detected in neural cells and unidentified cells beneath the epidermis in larva, and the cells in tunic during metamorphosis. When mantle was formed, gelsolin became detectable abundantly in body wall muscle.

N. Iwai, T. Iino, K. Sawaya and Y. Nakauchi. Dept. of Biol., Fac. of Sci., Yamagata Univ., Yamagata.
It has been reported that the mantle of the ascidian Halocynthia roretzi is constructed with multinucleated smooth muscle cells (Terakado, K. & Obinata, T., 1977, 1982, 1987; Shinohara,Y. & Konishi, K. 1982), and that these muscle cells contain vertebrate striated muscle-type proteins, e.g. troponin (Toyota, N. et al. 1979) and connectin (titin) (Nakauchi, Y. & Maruyama, K. 1992). Some high molecular (~ 800 kDa) protein bands are seen in SDS gel electrophoresis pattern of a total SDS extract of the mantle muscle cells of H. roretzi. It is known that a polyclonal antibody against chicken skeletal muscle nebulin does not cross react with these peptides. We applied the preparation methods of rabbit 1,200 kDa fragment of a-connectin (Matsuura, T. et al. 1991) and crayfish kettin-like protein (Maki, S. et al. 1995) to isolate the ~ 800 kDa proteins of H. roretzi smooth muscle cells. We investigated the relations between these proteins and 1,200 kDa fragment of rabbit or kettin-like protein of crayfish.

H. J. Yuasa and T. Takagi. Biol. Inst., Grad. Sch. of Sci., Tohoku University, Sendai.
Troponin C (TnC) is one of three components of troponin, a main regulator of striated muscle contraction, and two isoforms have been identified in mammalian and avian muscles. One is fast skeletal TnC (sTnC) which is expressed in fast skeletal muscle, and the other is slow/cardiac TnC (cTnC) which is present in heart muscle and slow skeletal muscle, and two isoforms were encoded by distinct genes. We have determined two distinct cDNAs of TnC isoforms from the ascidian, Halocynthia roretzi. These two isoforms did not correspond to vertebrate TnCs. One was expressed in larval striated muscle and the other in body-wall smooth muscle and heart muscle. From the results of Southern blot analysis and genomic sequencing, these isoforms were encoded by a single gene and expressed by alternative selection of a third exon. The localization of intron was identical to vertebrate cTnC except the 5th intron, corresponding to the 4th intron of vertebrate. We also determined the genomic structure of amphioxus, Brachiostoma lanceolatum TnC. In amphioxus, alternative splicing between larva and adult seemed not to be involved. The positions of introns were identical to vertebrate sTnC except 4th intron.

T. Kakuda and A. Hino. Dept. Biol. Sci., Kanagawa Univ. Hiratsuka.
Halocynthia roretzi is distributed around the coast of Hokkaido, Honshu Island and Korean Peninsula. Because of differences in the breeding season and the start time of spawning, H. roretzi is classified into three types A, B, C. Only type C occurs all around Honshu Island. Over 150 individuals of 10 populations of H. roretzi that were collected from the shores of Hokkaido and Honshu Island were studied by RFLP and restriction site analysis of mtDNA using 13 restriction enzymes. From these analyses, we found some individuals which showed polymorphic pattern after digestion by Hinc II, Hpa I, Ban II, Eco T22I and Xba I, and finally defined 17 halotypes. Sugashima (Ise Gulf) population had no polymorphism. In this area, oyster farming has existed for 10 years. It seems that a small population of H. roretzi was introduced with juvenile oysters and spread their habitat to the Sugashima area. Even if we calculated without this population, the frequencies of polymorphism of the Japan Sea population were higher than that of the Pacific Ocean populations.

T. Kakuda1, A. Hino1, T. Numakunai2 and T. Nishikawa3. 1Dept. of Biol. Sci., Kanagawa Univ., Hiratsuka; 2Mar. Biol. Stn., Fac. of Sci., Tohoku Univ., Asamushi; 3Grad. Sch. Human Informatics, Nagoya Univ.
In the Japanese population of Halocynthia hispida, two forms are recognizable based on the difference in density of tunic spines. Although these forms have been considered only an individual variation, our present results strongly suggest their genetic differentiation. We extracted and purified mtDNA from both forms, which were collected at Shimokita Peninsula, and digested by 8 restriction endonucleases. After fragment pattern analysis, we estimated the genetic divergence (Nei & Li 1979) between the two forms to be 4.2/100. This value is far larger than the value within the reproductively isolated three types of H. roretzi, but almost equal to the value between H. roretzi and H. aurantium (Kakuda et al., 1995).

R. Kusakabe1, T. Kusakabe2, N. Satoh1, N. D. Holland3, L. Z. Holland3 and N. Suzuki2, 1Div. of Biol. Sci., Grad. Sch. of Sci., Kyoto Univ.; 2Div. of Biol. Sci., Grad. Sch. of Sci., Hokkaido Univ., Sapporo; 3Scripps Inst. of Oceanog., UC San Diego, California, USA.
To investigate the evolution of muscle differentiation mechanisms in chordates, we isolated actin cDNAs from amphioxus and medaka and examined their expression patterns and phylogenetic relationships. It was revealed that amphioxus has a muscle actin similar to the vertebrate muscle actin. However, the amphioxus muscle actin has characteristic amino acid residues that are not conserved in other vertebrate-type muscle actin genes, suggesting that the amphioxus muscle actin gene diverged early in evolution. Transcripts of this muscle actin gene appears in the somites before the notochord formation and the myotomal expression continues throughout larval development. The amphioxus muscle actin gene is also expressed in smooth muscle cells associated with gill slits. Medaka was also found to possess an actin gene that resembles the mammalian skeletal muscle actin gene. In contrast to the amphioxus muscle actin gene, medaka muscle actin gene begins to be expressed when CNS development proceeds considerably. The expression is restricted to somites destined to differentiate to skeletal muscle. Since this medaka actin gene was not expressed in heart, we predict the existence of a family of multiple muscle actin genes in medaka, including a cardiac muscle actin gene. Further analysis on differential expression of multiple actin genes during embryogenesis will provide insights into the evolution of developmental mechanisms in chordates.

K. Kubokawa1, M. Watanabe1, N. Azuma2 and M. Tomiyama3. 1 Ocean Res. Inst., Univ. of Tokyo, 2 Toyohashi Univ. of Tech. & Sci., Arch. 3
Japanese amphioxus (Branchiostoma bercheri) is on the endangered species list, its preferred habitat lost by environmental pollution; its population gradually decreases in Japan. However, the population and its environment has not been surveyed at all in Japan. We collected amphioxus in various seasons and characterized its natural environment. Since we found a high population density of amphioxus in the fields around Mikawa Bay, we analyzed their growth and gonadal development by collecting them once a month from May to November in 1995. In May, 1996, precise studies were performed on the population density of amphioxus and other benthos, submarine sediment and topography, water quality and a video recording at the same area by using the research boat, Tansei-maru, belonging to the Ocean Research Institute. The distribution of body length in every month showed two peaks which described more than two years' life. The testis and ovary matured in July and disappeared in September. The number of mature males was always higher than females. Habitat selection was severely dependent on sand diameter. However, the water quality, chlorophyll and plankton quantities were not different between a habitat and a non-habitat plot.

K. F. Tagawa1, T. Humphreys2, N. Satoh1. 1Dept. of. Zool., Kyoto Univ.; 2Kewalo Marine Lab., Univ. of Hawaii, Honolulu.
The phylogenetic status of hemichordates (enteropneusts and pterobranchs ) has long been disputed. This animal has recently attracted attention with relation to the origin of chordates. We discuss deuterostome evolution towards chordates by examining the ontogeny and phylogeny of hemichordates. As the first approach, we describe the early development of the Hawaiian acon worm Ptychodera flava.