Number 50                                                                                                               December 2001

In May we moved to the Friday Harbor Labs where Charley worked on germinal vesicle breakdown in ascidian oocytes.  Shortly after getting settled in, Gretchen rushed off to California for the birth of our first grandchild (a boy, Alex), and she also identified several hundred ascidians at the California Academy of Sciences in San Francisco and taught an ascidian identification workshop there.  Back at the labs, Gretchen continued with taxonomy projects including a description of a new species of Trididemnum from the Friday Harbor region which she will name after her grandson; she hopes it will be an inspiration to him to become a biologist like his parents and grandparents.  Charley taught the summer comparative embryology course at FHL with Mark Martindale from Hawaii. We enjoyed talking to fellow ascidiologists Billie Swalla and Richard Whittaker who also spent the summer at the labs.  In August we spent a week in Kodiak, Alaska with the Smithsonian Environmental Research group (SERC) looking for invasive species.  Fourteen species of ascidians were collected but none were considered nonindigenous.  Among the ascidians we collected were specimens of Aplidium coei described by Ritter on the Harriman Alaska Expedition of 1899.  It was a real thrill to find Ritter’s species just about where he said it was after so many years.  We were also impressed by the huge numbers and size of the Molgula retortiformis we found on floats. We just returned from the SERC facility in Edgwater, Maryland where we spent a week giving workshops on how to identify the hundreds of ascidian samples from their various sampling sites around the U.S.
Gretchen spends many hours assembling and editing AN twice a year; she was wondering if it were worth while to continue with it after so many years.  Accordingly, she sent out an inquiry as to the usefulness of continuing with this effort.  Numerous replies were unanimous that AN continues to be very useful and interesting to many researchers; thus the newsletter will continue for a few more years at least!  We are grateful for past articles but need your continued input to insure that AN remains useful and informative.  Please send us a meeting abstract, thesis abstract, work in progress, or copy of your latest publications for the next issue.  There are 118 new publications listed in this issue, plus numerous additional ones in the Table of Contents for The Biology of Ascidians included near the end of this newsletter just before the New Publications.

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

1. The word ascidian is derived from the Greek askidion, a diminutive of askos, meaning wineskin or bladder. It was Aristotle who said, "For man once a leathern bottle was."

2. About 40 copies of "The Biology of Ascidians" (eds by H. Sawada, H. Yokosawa, and C.C.Lambert), Springer-Verlag Tokyo 2001, are still available in the secretariat of the First International Symposium on the Biology of Ascidians (c/o Department of Biochemistry, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, Japan).  Anyone interested in having this proceedings is requested to send a purchase order form, obtainable from the following URL, by FAX. http://www.hokudai.ac.jp/pharma/seika/ISOBA.htm  The Table of Contents is included below and can also be found on the ISOBA website.

3. New major monographs on ascidian taxonomy.  Drs. Françoise and Claude Monniot have published Ascidians from the tropical western Pacific. Zoosystema 23: 201-383 (2001). With so little known about the ascidians from this large part of the world, this excellent new work representing many years of collecting and identifications by the Monniots will be a standard reference for many years. We have used it extensively already and find it an invaluable addition to the literature.
   Dr. Patricia Kott has published the fourth and last volume of her set of monographs on the Australian ascidians: Kott, P. 2001. The Australian Ascidiacea part 4, Aplousobranchia (3) Didemnidae. Mem. Queensland Mus. 47: 1-407.  This monumental volume focuses entirely on the didemnids and is the product of many years of work by Dr. Kott.
   Both volumes contain descriptions of many new species.

4. Web site about Development of Ascidians, submitted by Dr. Christian Sardet.
Our new web site has a section on ascidians you may like to consult. Connect directly at http://www.obs-vlfr/ and click on Biomarcell. We will complete the site in the next few months and your comments will help us.  Christian Sardet, BioMarCell, UMR 7009 CNRS/UPMC, Station Zoologique, Villefranche sur Mer 06230. Email: sardet@obs-vlfr.fr

5. There will be a joint meeting of the British Soc. for Cell Biology (topic: Cell Regulation through Molecular Machines) and the genetics society and British Soc. for Developmental Biology (topic: Evolution of Developmental Mechanisms) at the Univ. of York, 20-23 March 2002. Further information is available at http://www.bscb.org . There will undoubtedly be some ascidian papers presented and we hope the presenters will send us their abstracts for the spring 2002 AN.

6. Dr. Teruaki Nishikawa contributed the following obituary of his deeply revered mentor and major professor.  We are greatly saddened by the death of this remarkable man.
   Dr. Takasi Tokioka, Professor Emeritus of Kyoto University, passed away on the 30th of September, 2001, at the age of almost 88. He was a distinguished taxonomist of sessile and pelagic tunicates, chaetognaths, ctenophorans, crustaceans (Argulus), etc. After his retirement in 1977, he continued to have good days with his beloved wife and daughter at his home near the Seto Marine Biological Laboratory, where he worked since 1962 (as the director from 1975 to 1977), in the town of Shirahama, Wakayama Pref., Japan. Though suffering from heart disease and poor sight in his later days, he retained a warm heart, a strong will, and a sharp brain. Unfortunately, a serious heart attack suddenly stopped his wide-ranging considerations on animal phylogeny and evolution. At his request, the body was donated to science, and a simple funeral ceremony was attended by his close relatives only.
   Dr. Tokioka published ca. 220 works from 1936 on, many appearing in the "Publications of the Seto Marine Biological Laboratory". His last ones were chapters on ctenophorans and chaetognaths contributed to a Japanese book, "Dobutsu Keito Bunruigaku [Systematic Zoology], Supplement" (Nakayama-shoten, 2000), where he gave original phylogenetic considerations of these two animal groups with a new classification of ctenophorans. He had a sincere hope to revise the chapters for English publication, but this was not to be. His tireless scientific activities with deep insights till his latest days were obviously made possible by his extensive and detailed biological knowledge, derived from his very long, various taxonomic and morphological experiences.
As may be well known by readers of "Ascidian News", his significant contributions to tunicate taxonomy are represented by ca. 60 works on ascidians and ca. 30 on pelagic tunicates with accurate descriptions and exact (also very artistic and beautiful) figures. Among others, "Ascidians of Sagami Bay" (Iwanami-shoten, 1953) and "Pacific Tunicates of the United States National Museum" (Smithsonian Institution, 1967) are really significant works. The specimens treated in the latter were examined during his stay at the museum in 1957, supported by the National Academy of Sciences, USA. Furthermore, his 1971 paper of "Phylogenetic speculations of the Tunicata" has been often cited in discussions of chordate phylogeny.
   From 1967 to 1969, Dr. Tokioka was one of the leaders of a grass-roots movement against the reclamation of land from a shallow inlet in Shirahama, although the result was unsuccessful. Nevertheless, his consistent interests in and activities for conserving the environment bore fruit in that an island in Tanabe Bay off Shirahama survived a plan to turn it into a pleasure park by being purchased by Kyoto University in 1968. Since then, the island has been protected as a reserve for marine biological studies, and he began an intertidal census of biota there.
In his private life, he was a famous philatelist, even publishing a book for stamp collectors probably in 1949. His huge world-wide collection of stamps, as well as old biological books, will probably be donated to the Yamaguchi Prefectural Museum, because he loved Yamaguchi where he was born (in 1913) and lived until entering into Kyoto Imperial University. He and his family were also famous in Shirahama for keeping many cats once discarded in front of his home by those who looked for their kind care. They truly loved the cats by giving them individual names, as he did for many ascidian species.
                                                                            Teruaki Nishikawa (The Nagoya University Museum)

1. Gian-Luigi Russo and Elisabetta Tosti (Stazione Zoologica “Anton Dohrn”, Naples, Italy; glrusso@alpha.szn.it) and colleagues M. Tosto, A. Cuomo and I. Castellano have a number of projects in progress regarding the molecular mechanisms regulating meiosis resumption in the ascidian Ciona intestinalis. Now, they have a paper in preparation describing the role of protein kinase CK-2 (formerly known as casein kinase II) at fertilization. In vertebrates, this enzyme is formed by two catalytic subunits (alpha and/or alpha’), and two regulatory subunits, beta. Tosti’s group cloned the CK2 beta subunit in C. intestinalis (Gene Bank accession number AF360544) and characterized the activity of the holoenzyme during meiosis resumption.  It is worthwhile to note that in Xenopus oocytes, CK-2 beta subunit interacts and blocks the activity of c-mos, the main component of the cytostatic factor leading to the hypothesis that CK-2 might play an important role in meiosis regulation.  The cloning and molecular characterization of CK-2 catalytic subunit, alpha, is in progress and will hopefully be completed by the end of the year.

2. Blood cell vanadium storage in Ascidia ceratodes shows ecological variations. An X-ray absorption spectroscopic study. Patrick Frank^a,b, Robert M. K. Carlson^c, Elaine J. Carlson^d and Keith O. Hodgson^a,b
a. Dept. of Chemistry, Stanford Univ., Stanford, CA 94305; b. Stanford Synchrotron Radiation Laboratory, SLAC, Stanford Univ., Stanford, CA 94309; c. Chevron Petroleum Technology Co., Richmond, CA 94802; d. Buck Institute, Novato, CA 94945.
     Vanadium storage in the blood cells of the tunicate Ascidia ceratodes resident in Bodega Bay, California was assessed using x-ray absorption spectroscopy (XAS). Six blood cell samples, representing between 1 and 6 animals, were prepared and frozen on the docks of the Spud Point Marina.  About 96% of the blood cell vanadium was found stored as V(III)-sulfate in aqueous solution, implying a high-acid, high sulfate environment.  No evidence was found for a systematic change in vanadium storage following admixture of blood cells from different animals, implying a lack of connection between vanadium distribution and immune response. A divergent distribution of V(III) complexes between individual specimens indicated that blood cell vanadium is not tightly regulated.  Comparison of the average vanadium distributions within blood cells from A. ceratodes from Monterey Bay, California (~ 200 km south) with those of Bodega Bay revealed a greater preponderance of V(III) in a pH~0 intracellular environment in the Bodega Bay ascidians.

3.  H. Abdul Jaffar Ali, Ph.D student under Dr. V. Sivakumar, V. O. Chidambaram College, Tuticorin 628 008, India and a junior research fellow working on marine biodiversity and taxonomy of Indian ascidians under Dr. V.K. Meenakshi. I am comparing population distribution, associated flora and fauna, microbial load, heavy metal accumulation, and proximate compositions of Phallusia nigra between the east and west coasts along the southern peninsula of India.  I am also analyzing bioactive compounds from P. nigra.  Any directions and suggestions are most welcome. zaphar2002@yahoo.co.in

1. Functional and biochemical characterization of cytostatic factor (CSF) in the ascidian Ciona intestinalis. Immacolata Castellano, M.S. thesis, Laboratory for Cell Biology, Stazione Zoologica “Anton Dohrn”, Naples, Italy. Advisor Dr. Elisabetta Tosti.
   In this thesis, we demonstrated the presence of an active CSF in extracts of C. intestinalis oocytes arrested in metaphase I, that, when microinjected into blastomers, was able to block their division. In addition, we presented evidence that a CSF mechanism mediated by protein kinase Mos is active in ascidian oocytes; in fact, an antibody raised against Xenopus Mos, was able to block the ascidian CSF. Our data are partially in agreement with the proposed role of Mos in oocyte maturation in vertebrate. In fact, in C. intestinalis a Mos-like protein seems to be expressed constitutively during the meiotic division as in Xenopus; however, in Vertebrate, Mos is degraded by the proteasome complex at the metaphase-anaphase II transition, i.e. after the removal of metaphase block at fertilization.  Same fate is due to MAP kinase activity, another component of CSF. On the other hand, in C. intestinalis oocytes, Mos expression is still detected after metaphase I-anaphase I transition, and MAP kinase activity is maximal after five minutes from fertilization. Finally, we demonstrated that the metaphase I block in C. intestinalis is independent from the protein synthesis, similarly to vertebrate, a situation that does not find similarity among other invertebrates, where the existence of short-lived-proteins seems to regulate meiotic arrest. This finding further confirms that meiosis regulation in ascidians resembles the vertebrate one better than the invertebrate one, in agreement with the position of these organisms in the evolutionary tree.

2. Reproductive strategies in colonial ascidians: relationships with other life-history traits and genetic structure. Isabel Tarjuelo. Ph. D. Thesis. University of Barcelona, X. Turon thesis advisor. isabelt@bio.ub.es
    Current theory on the investment in reproduction by invertebrates has been mainly developed for groups with both lecitotrophic and planktotrophic larval developmental modes, and this dichotomy explained a great deal of the diversity of strategies found. Colonial ascidians, in spite of a uniform (lecitotrophic) developmental mode and a prevalence of brooding, feature a wide range of fecundities, larval sizes, and reproductive investment. We wanted to quantify these parameters in a range of species and to look for correlates of the reproductive traits that may explain the variability found. We quantified fecundity, reproductive investment (in terms of larval production), and investment in tunic production in 11 colonial forms. There was a wide range (about one order of magnitude) of variation in all parameters. Tunic production was related to growth form, and was higher in encrusting/massive forms than in stoloniferous forms. It bears no relationship, however, with reproductive investment. The latter was positively related to the degree development of juvenile structures in the larva, and negatively to zooid weight, fecundity and absolute larval weight.
    For six of the species, we further studied the biological cycles, palatability and energy contents of tunic, zooids and larvae, growth rates, and male reproductive investment. No relationship was found between adult and larval palatability. Larvae of those species with lowest fecundity and largest larval sizes were better defended than small larvae. Energy content and the amount of inorganic material were related to palatability. Seasonal reproductive cycles, but with varying length and season of the reproductive period, were observed over three years for the six species. Species with high growth rate tended to have a short brooding period, low tunic production, and produced a high number of small, palatable larvae with a low degree of structural complexity. On the other hand, species with low growth rates tended to have long brooding periods and produced a low number of big, complex and well-defended larvae. Total reproductive investment was higher in the low-fecundity species. We chose two species representative of these two strategies, Clavelina lepadiformis and Pseudodistoma crucigaster, to analyze and compare dispersal capabilities and juvenile mortality. Our prediction was that both would be higher in the species with higher fecundity and a more opportunistic strategy (C. lepadiformis). Dispersal was indirectly estimated through population structure using COI partial sequences of several populations from the Spanish Mediterranean littoral. We found a high degree of genetic structure in both species. In C. lepadiformis this was related to the distinction, possibly at the species level, between the form inhabiting harbours and the population in the open shore habitats. In P. crucigaster, genetic differences, although not so important as to justify a species-level distinction, were found among color morphs. When comparable populations were studied, however, the degree of gene flow at a scale of tens of kilometres was ten times higher for C. lepadiformis. On the other hand, post-settlement mortality (during the first four weeks of benthic life) was three times higher in C. lepadiformis. It is concluded that there is a continuum of reproductive strategies in colonial ascidians, the extremes of which are the production of a high number of small larvae vs. the production of a single, complex larva per zooid. These extremes have important correlates in terms of allocation to reproduction and defense, growth rates, tunic production, dispersal capabilities and larval and post-metamorphic mortality.

3. Comparative biology of Clavelina lepadiformis (Ascidiacea) populations from inside and outside harbours in the western Mediterranean.  Sònia de Caralt, Master’s thesis, University of Barcelona, X. Turon thesis advisor. socabo@latinmail.com
    Clavelina lepadiformis is a colonial ascidian that inhabits both harbour environments and open sea rocky communities. Recent investigations have shown that the populations of these habitats exhibit a marked genetic divergence and a highly restricted genetic flow between them. However, no morphological differences between the two forms could be substantiated. In this work we compared the biology and heavy metal accumulation of populations of these two forms. Our goals were to investigate whether the genetic isolation is reflected by adaptive divergences in the two types of habitat, and to ascertain whether the two forms can survive in each other’s habitat. The abundance and seasonal cycles showed contrasting trends in the two types of habitat: the populations inside harbours reach densities of ca. 3000 zooids m-2, and active colonies were found all year round. In the open littoral habitat, abundance was an order of magnitude lower and showed a clear seasonal pattern, with disappearance of zooids during the summer season (aestivation). The reproductive cycles were also different, as larvae were present in the populations inside harbours from November through July, with several sexual cycles during this period, while in the outer habitats larval appearance was restricted to 2-3 months during winter-spring, with only one gonadal cycle per year. The zooids of the internal form were significantly bigger than those from the exterior populations, and so were the larvae produced, but the total reproductive effort and tunic production (in weight ratios), as well as the fecundity, were not significantly different between habitats. The populations from inside harbours accumulate significantly more Cu and Pb than the exterior populations, and the heavy metal concentrations showed a seasonal cycle with minima in summer. On the other hand, both varieties accumulate a similar amount of vanadium, a metal known to play a role in ascidian metabolism. Both the production of secondary metabolites and the toxicity of polar extracts were higher in the external populations than in those from the interior habitats. These results, however, did not correlate with the outcomes of palatability tests with specialist and generalist predators, where no preference could be substantiated. An experiment of juvenile transplantation between habitats showed that newly-settled individuals from the external habitat can survive in both habitats (with survival figures of 30-50% during the first four weeks), while juveniles from the internal form survive very poorly in the outside habitat (ca. 5% survival after 4 weeks). It appears that both varieties showed marked contrasts in most biological parameters studied. The variety inhabiting harbours reaches very high densities, produces several sexual generations per year and seems to withstand heavy metal pollution without deleterious effects. The variety inhabiting open rocky littoral shows more restricted growth, aestivation phenomena, and a single larval generation per year. On the other hand, the internal population produces less secondary chemistry, which may explain its inability to survive in the external habitat, where predation pressures are presumably higher. The lack of genetic flow between these two forms suggests that the differences may not be mere phenotypic adaptations to the different environments and may have a genetic basis.

4. New Zealand ascidians: masters of amino acid-derived secondary metabolites.
Allison Norrie Pearce, Ph.D. thesis, Dept. of Chemistry, The Univ. of Auckland, Auckland, New Zealand. Thesis advisor, Dr. Brent Copp b.copp@auckland.ac.nz
   A survey of 34 New Zealand ascidians was performed for the purpose of investigating the biological and chemical properties of ascidians from New Zealand waters. The crude extracts were assayed for biological activity and were also analyzed by HPLC with photodiode array capability for comparison of absorption spectra. In this manner biological profiles of the cytotoxicity and antimicrobial activities of the crude extracts were obtained, as well as a UV metabolic profile. The (-)-enantiomer of the known compound 1,2,3-trithiane was isolated from the ascidian Hypsistozoa fasmeriana, and the (+)-enantiomer from the ascidian Distaplia stylifera, a member of the same family (Holozoidiae). The (+)-enantiomer was originally isolated from the New Zealand ascidian Aplidium sp. D and appears to be unique to New Zealand waters. A new class of compound, the fasmerianamines A and B, derivatives of 1,2,3-trithiane were isolated from a second collection of Hypsistozoa fasmeriana. Another new class of metabolite, the fluorescent, biologically inactive distomadines A and B were discovered in the ascidian Pseudodistoma aureum. A previously unknown trimethylated purine, 2,2,7-trimethylguanine as well as other known purine bases were detected and isolated.  Much of the biological activity of the ascidian extracts could be attributed to the presence of unsaturated long-chain amino alcohol-type compounds.  The presence of the pyridoacridine class of compound, a large class of aromatic polycyclic alkaloids of worldwide distribution, was detected in a New Zealand ascidian for the first time.
   The pyridoacridone alkaloid ascididemin possesses a wide range of biological activity and progressed as far as in vivo xenograft antitumoural assays at the NCI, revealing its value as a lead compound.   The antitumoural activity of ascididemin is attributed to the intercalative ability of its planar pentacyclic structure. The synthetic strategy for another natural pyridoacridone, kuanoniamine A was optimized, and the synthetic compound also showed good human solid tumour selectivity, progressing as far as in vivo hollow fibre testing at the NCI. Three other synthetic ring A-modified analogues were prepared. Two, combining either a furan or a thiophene A ring with a carboxylic acid methyl ester substituent in position two on ring A, were tested at the NCI and were found to be inactive. This indicated conclusively that the carboxylic acid methyl ester substituent modification had a detrimental effect on cytotoxicity. The disruption of the planarity of the heterocyclic ring system with subsequent inability to intercalate into DNA might be implicated. An isoxazole ring A modified analogue was found to be too unstable for evaluation purposes.

5. Three thesis titles submitted by Dr. Masaaki  Morisawa, Misaki Marine Station. morisawa@mmbs.s.u-tokyo.ac.jp
Studies on the ion channels regulating the activation of sperm motility in the ascidians Ciona intestinalis and C. savignyi. Hiroko Izumi, Ph. D. thesis, Univ. of Tokyo (1999).
Studies on the cell signaling for the activation of sperm motility in ascidian. Mamoru Nomura, Ph. D. thesis, Univ. of Tokyo (2000).
The study on the mechanism of metamorphosis in the ascidian, Ciona savignyi. Yikiko Kimura, MS. Thesis, Univ. of Tokyo(2000).

1. Annual Conference of the Italian Embryology Group, Fano, Italy, 7-9 June 2001

Serotonin localization in Phallusia mammillata larvae and during first events of metamorphosis. R. Pennati, S. Groppelli, C. Sotgia, U. Fascio, *M. Pestarino and F. De Bernardi, Dept. of Biology, Univ. of Milano, Italy.  *Dept. of Experimental, Environmental and Applied Biology, Univ. of Genova, Italy.
    Serotonin [5-Hydroxytryptamine (5-HT)] is a neurotransmitter which plays an important role in a wide range of non-neural processes, during embryonic development such as egg cleavage, gastrulation cell movements and morphogenesis of many invertebrate and vertebrate embryos and larvae. The presence of serotonin has been reported in the adults of a variety of ascidian species, in which was localized in the enterocromaffin cells of the gut, in the peripharyngeal band and in the endostyle (Georges, 1985. Cell Tissue Res. 242: 341-348; Pestarino, 1982. Cell Tissue Res. 226: 231-235) but the origin and the distribution of serotonin-containing cells during the ontogeny of ascidians are not known.
    We used an anti-serotonin antibody under a confocal microscope to localize endogenous serotonin by immunofluorescence in the swimming and metamorphosing larvae of the ascidian Phallusia mammillata. The serotonin-containing cells in the swimming larvae were detected in the central nervous system. Bright fluorescence is present in 11 pear-shaped cells surrounding the ocellus, corresponding to the retinal cells of the photoreceptor complex, in at least one of the elongated cells of each papilla and in the pericharia of two epidermal neurons of the rostral trunk, which are present on the axon-like fibers connecting the papillae with the sensory vesicle. In the tail, serotonin is evident in the pericharia of two groups of neurons in the ventral and in the dorsal epidermis, connected by immunopositive fibers. These neurons are often paired but their distribution and their number varied among the larvae. An immunofluorescent signal is found in some ventral endodermal cells of the anterior trunk. These cells may contribute to the formation of the posterior half of the adult endostyle.
    During all the swimming larva period the serotonin is detectable principally in the nervous system as described above, when larvae stop swimming and tail retraction begins, the immunofluorescent signal in the cells of photoreceptor complex progressively faints together with signal in the neuroepithelial cells of the rostral part of the trunk. At the end of tail retraction, five days after fertilization, when the ampullae definitively keep the larvae attached to the substrate, serotonin appears in the cells of the peripharingeal band, of the endostyle and of the gut.
    The serotonergic system may control the complex larval behaviour in response to light. In fact the larvae of the solitary ascidians show a positive phototaxis during the dispersal-phase of their life, but at the time of settlement, most ascidian larvae avoid light and prefer to settle on dark or shaded surfaces (Cloney, 1982. Amer. Zool. 22: 817-826)
    Metamorphosis transforms a swimming larva into a sessile juvenile.  The early photoreceptive function of the serotonergic system may be lost, together with the reduction of sense organs, and serotonin becomes restricted to other systems. In fact after metamorphosis we were not able to detect serotonin in the neural complex of the juveniles. Experiments carried out treating newly hatched swimming larvae with ritanserin, an antagonist of 5-HT2 receptor subtype, significantly shorten the swimming period. A 6 hour treatment with 10µM ritanserin induces attachment to the substrate of 70 % of larvae. After 24 hours of treatment all larvae are attached to the substrate by the papillae, while 80% of control larvae are still swimming. This suggests a photoreceptive or a photoneuroendocrine function of serotonin for ascidian larvae similar to that observed in amphioxus and in vertebrates (Terio, 1964. Atti Soc. Peloritana Sc. Fis., Mat. e Naturali 10: 111-125; Nunez et al., 1981. J. Histochem. Cytochem. 29: 1336-1346).

2. Congress of the Italian Society of Neuroscience, Torino, Italy, 8-11 September 2001.

Localization and possible role of serotonin during the development of the ascidian Phallusia mammillata.  R. Pennati, S. Groppelli, C. Sotgia and F. De Bernardi, Dept. of Biology, Univ. of Milano, Italy.
    It is known that the neurotransmitter serotonin [5-hydroxytryptamine (5-HT)] plays an important role in a wide range of non-neural processes. Its activity is mediated by multiple receptors subtypes. 5-HT is present in early stages of sea urchin embryo development (Buznikov et al., 1964, 1972) and it has been demonstrated that it has a key role during the morphogenesis of chicken and mouse embryo (Gustafson and Toneby, 1970, Moiseiwitsch and Lauder, 1995). By immunofluorescence with an anti-serotonin antibody,we demonstrated the presence and localization of 5-HT in brain and in some neurons of the larval tail of Phallusia mamillata.
    To test the effects of  serotonin on ascidian development, we treated embryos with different pharmacological substances: fluoxetine, an inhibitor of 5-HT reuptake, and three antagonist for different serotonin receptor subtypes. The treatments were performed at 10 micromolar concentration, begun before cleavage or at late gastrula stage, and were terminated at swimming larva stage. Malformations induced by the treatments were analyzed successively (i) at the end of the treatment, (ii) 7 days after fertilization, corresponding to the beginning of metamorphosis, and (iii) 15 days after fertilization, corresponding to a completely metamorphosed juvenile. The early treatment of fluoxetine caused anomalies of the segmentation and malformations , consisting mainly in roundish cephalenteron and short tail, in 50% of larvae developed from treated embryos. Few larvae metamorphosed and 15 days after fertilization juveniles showed reduction of the neural ganglion size.
    Treatments with serotonin antagonists were more efficient from gastrula stage onwards. WAY, an antagonist for the 5-HT1A receptor subtype, caused extreme reduction of brain vesicle and of sensory organs; ondansetron, an antagonist for the 5-HT3 receptor subtype, induced high incidence of malformations of cephalenteron and short tails in later treatment. Juveniles developed almost normally. Ritanserin, a substance with a high affinity for the 5-HT2 receptor subtype, caused mainly truncation of the anterior end of larvae cephalenteron. The juveniles developed from these larvae had an abnormal cardio-circulatory system: no blood circulation, the heart contractions were no-rhythmic, and the blood cells were less numerous than in controls. We conclude that serotonin play an important role in the morphogenesis of the ascidian. An appropriate level of serotonin is necessary for a correct segmentation, since increased concentration caused by the inhibitor of 5-HT reuptake, fluoxetine, severely perturbed this early phase of embryo development. During morphogenesis the action of the serotonin is mediated by different receptor subtypes, among which 5-HT1A, 5-HT2 and 5-HT3 appear to play a key role in modulating the multiple morphogenetic effects of serotonin.

3. 4th Workshop "A. Dohrn" New Perspective in Tunicate Biology, Ischia, Italy, 29 September - 2 October 2001.

Sperm chemotaxis in ascidians. M. Morisawa and M. Yoshida, Misaki Marine Biol. Station, Graduate School of Sci., Univ. of Tokyo, 1024 Koajiro, Misaki, Miura, Kanagawa, 238-0225 Japan.
   Spermatozoa of the ascidians Ciona are immotile in seawater.  Motility is activated around the egg, and then the activated sperm are attracted towards the egg, suggesting that the factor for sperm activation and attraction is released from eggs at fertilization (Yoshida et al., Dev. Biol., 1993). The factor named as sperm-activating and -attracting factor (SAAF) in Ciona (Yoshida et al., DGD, 1997) was recently purified and identified as a steroid with molecular mass of 596 (Yoshida et al., submitted). Cell signaling for sperm activation has been clarified (Izumi et al., Dev. Biol., 1999; Nomura et al., DGD, 2000). Ca2+ introduced into the sperm cell through Ca channel activates calmodulin-calmodulin kinase system to hyperpolarize the plasma membrane, and the change in membrane potential synthesizes cAMP via activation of adenylyl cyclase.  The cAMP phosphorylates dynein light chain and a 26kDa protein, and the phosphorylation triggers the final step for the activation of sperm motility.
   Sperm chemotaxis does not require cAMP, and Ca is the only prerequisite second messenger (Yoshida et al., DGD, 1997), although cell signaling cascade underlying the sperm chemotaxis in ascidians as well as other species is unclear. Nifedipine, Flunarizine, omega-conotoxin had no effects on chemotaxis of Ciona sperm, but SK&F96365, Ni2+ and Zn2+, blockers of store-operated Ca2+ channel completely inhibited chemotactic behavior of sperm. Furthermore, depletion of internal Ca2+ store by thapsigargin, caused increase in intracellular Ca2+.  These suggest that store-operated calcium channel exists in the ascidian sperm and participates in the chemotactic behavior of the sperm.
   Quantitative analysis of sperm trajectories measuring four parameters: distance and orientation between SAAF and sperm head, radius of curvature of sperm trajectory; and sperm velocity and computer simulation proposed model shows that the change of SAAF concentration controls chemotactic behavior of ascidian sperm.

4. 11th Intl. Symp. On Environmental Pollution and its Impact on Life in the Mediterranean Region, 6-10 Oct. 2001.

Influence of TBT on the activity of the detoxicant enzymes, GST and GPX, from haemocytes of the colonial ascidian Botryllus schlosseri.  F. Cima, D. Dominici, L. Ballarin and P. Burighel, Dept. of Biology, Univ. of Padova, Italy.
   In the last decade, some authors have hypothesized that a considerable part of the immunotoxic effects described for tributyltin (TBT) in mammals was due to Ca2+-independent mechanisms involving changes in sulphydryl groups of both proteins and reduced glutathione (GSH).  Among the detoxicant enzymes, which play a multiple role in the metabolism of many xenobiotics of invertebrates and vertebrates, there are glutathione-transferase (GST) and glutathione-peroxidase (GPX).  GST catalyzes the conjugation reactions of GSH with electrophilic xenobiotics or their derivatives.  It has been described in the antioxidant defense systems mainly of the gill of bivalves and as a mediator of xenobiotic detoxification in the liver and kidney of marine fish.  In mammals, the activation of GST has been proposed to occur after TBT treatment, and the presence of mercapturic acid supports this hypothesis.  However, this derivative was never found in molluscs, suggesting that invertebrates possess a different detoxicant system of organotins.
   GPX is an antioxidant enzyme, which protects from the effects of reactive oxygen species production, requiring GSH consumption.  In marine fish, since the inhibition of GPX by various organotin compounds had been recently described, this enzyme can be used as an effective biomarker of these pollutants.  High concentrations of GPX were found in the blood of many invertebrates.  The GPX activity detected inside the gill of bivalves is associated with GST and is catalyzed by this enzyme.
   In cultured haemocytes of the colonial ascidian Botryllus schlosseri, we have observed by immunocytochemistry that positivity to both GST and Se-dependent GPX antibodies disappeared after incubation for 60 min at 25^o C, in the presence of 0.1 µM and 1 µM TBT, respectively. This suggests that conformational changes occur, probably due to direct interaction of the xenobiotic with these antioxidant enzymes.  Moreover, the spectrophotometric assays on the haemocytic lysate have shown that TBT was able to significantly inhibit the GST activity (µmoles/min/mg protein) 2.6 times at 0.01 µM and 4.8 times at 0.1 µM, whereas the Se-dependent GPX activity was inhibited 1.5 times at 0.05 µM TBT.  Our data suggest that in ascidian blood cells, enzymes involved in detoxification metabolic processes are present and that GST is the most important and sensitive among them.

Table of contents for: The Biology of Ascidians, edited by H. Sawada, H. Yokosawa and C. C. Lambert, the symposium volume published 2001 by Springer-Verlag Tokyo resulting from The First Intl. Symposium on the Biology of Ascidians, Hokkaido University, Sapporo, Japan, June 26–30, 2000.  If you have not yet purchased a copy, please go to the website http://www.hokudai.ac.jp/pharma/seika/ISOBA.htm.   Please request that your institution library also acquire a copy.

1. Fertilization and Egg Activation
Structural and Molecular Investigations on the Egg Coat in Phallusia mammillata. Thomas G. Honegger and Monika          Füglister 3
Analysis of the Self-sterility in Halocynthia roretzi. Naoyuki Murabe and Motonori Hoshi 9
Further Observations on the Molecular Bases of Gamete Self-discrimination in Ciona intestinalis: Seasonal Variation of Self-sterility Rate. Rosaria De Santis, Rita Marino, and Maria Rosaria Pinto 14
Self-nonself Recognition and Lysin System in Fertilization of the Ascidian Halocynthia roretzi. Hitoshi Sawada and Hideyoshi Yokosawa 18
Fertilization-induced Glycosidase Release and Interspecific Sperm Competition in Ascidians. Charles C. Lambert 24
Cell Signaling in Ascidian Sperm: Upstream and Downstream of Internal Calcium Release. Robert A. Koch, Kathleen Allen, Ju Kim, and Ali Lotfizadeh 30
Sperm-triggered Calcium Oscillations at Fertilization. Alex McDougall 36
Diversity of Calcium Channels Involved in Meiosis Resumption of Ascidian Oocytes. Mireille Albrieux, Christophe Arnoult, Didier Grunwald, Marie-Jo Moutin, and Michel Villaz 47
Ascidian Sperm Acrosin and Spermosin: Structures and Roles in Fertilization. Eri Kodama, Tadashi Baba, Hideyoshi Yokosawa, and Hitoshi Sawada 54
Acrosome Differentiation in Ciona intestinalis Spermatozoa and Some Speculations on Ascidian Fertilization. Makoto Fukumoto 60
Follicle Cells of Styela plicata Eggs (Ascidiacea). Luisanna Villa and Eleonora Patricolo 67
High Level of Protein Ubiquitination in Ascidian Sperm. Kazuo Inaba 74
Calcium Transients Signal Ooplasmic Segregation through the Small GTPase rho in Ascidian Eggs. Manabu Yoshida, Yuji Horiuchi, and Masaaki Morisawa 81
Cell Signalings for Activation of Motility and Chemotaxis in the Sperm of Ciona. Masaaki Morisawa, Hiroko Izumi, Manabu Yoshida, and Yoshitaka Oka 86
Roles of MLCK and PI3 Kinase on Deformation and Ooplasmic Segregation at Fertilization in the Egg of Ciona savignyi. Noburu Sensui, Manabu Yoshida, and Masaaki Morisawa 92
Identification of Phallusia mammillata Egg ?-N-Acetylhexosaminidase with a Potential Role in Prevention of Polyspermy. Markus Eisenhut and Thomas G. Honegger 97
The “Complex” Ascidiosperm of Aplousobranchs. GianBruno Martinucci, Mila Della Barbera, Francesco Boldrin, and Paolo Burighel 102
2. Reproductive Biology and Neuroscience
The Origin of Germ Cells in Ciona intestinalis. Katsumi Takamura 109
Oocyte Maturation and Self-sterility by Treatment with Ovary Extracts of the Ascidian, Halocynthia roretzi. Takaharu Numakunai 117
Induction of Gamete Release by Gonadotropin-Releasing Hormone (GnRH) in Ciona intestinalis. Kiyoshi Terakado 125
Light Regulated GnRH Neurons in Biological Clock for Reproduction in the Ascidian, Halocynthia roretzi. Motoyuki Tsuda, Mahito Ohkuma, Masashi Nakagawa, and Yasuo Katagiri 131
Tailbud Embryogenesis and the Development of the Neurohypophysis in the Ascidian Ciona intestinalis. Alison G. Cole and Ian A. Meinertzhagen 137
The Peripheral Nervous System of an Ascidian Revealed by AChE Activity. Lucia Manni, Marina Sorrentino, Giovanna Zaniolo, and Paolo Burighel 142
Heterotrimeric G Protein ??and ??Subunit Genes of the Ascidian, Halocynthia roretzi. Tatsuo Iwasa, Kazue Kanehara, Ayako Watari, Mahito Ohkuma, Masashi Nakagawa, and Motoyuki Tsuda 147
Photoresponse and Habituation of Swimming Behavior of Ascidian Larvae, Ciona intestinalis. Motoyuki Tsuda, Isao Kawakami, Takayuki Miyamoto, Masashi Nakagawa, Shuhei Shiraishi, and Muneki Gouda 153
Multiple cis-Regulatory Regions Control Neuronal Gene Expression of Synaptotagmin in Ascidian Embryos. Jun Matsumoto, You Katsuyama, and Yasushi Okamura 158
3. Development, Differentiation, and Evolution
Maternal Genetic Information Stored in Fertilized Eggs of the Ascidian, Halocynthia roretzi. Kazuhiro W. Makabe, Takeshi Kawashima, Shuichi Kawashima, Yasunori Sasakura, Hisayoshi Ishikawa, Hiroshi Kawamura, Minoru Kanehisa, Takahito Nishikata, and Hiroki Nishida 165
RNA-binding Proteins in Ascidian Development. Takahito Nishikata, Michiko R. Wada, and Kimio J. Tanaka 178
Functional Analysis of Ciona intestinalis Y-Box Protein. Kimio J. Tanaka and Takahito Nishikata 186
Ci-sna cis-Regulation of Ascidian Tail Muscle Genes. Albert Erives and Michael Levine 193
T-box Genes and the Development of Axial Tissues in Ciona intestinalis. Anna Di Gregorio and Michael Levine 202
Cloning and Embryonic Expression of HrzicN, a Zic Family Gene of the Ascidian Halocynthia roretzi. Shuichi Wada and Hidetoshi Saiga 206
Analysis of a cis-Regulatory Element of Hroth, the Ascidian Homologue of the otx Genes, That Drives Its Transcription in the Anterior Larval Central Nervous System of the Ascidian, Halocynthia roretzi. Izumi Oda and Hidetoshi Saiga 211
Comparison of the Structure and Expression of otx Genes between Ciona intestinalis and Halocynthia roretzi. Nanami Utsumi and Hidetoshi Saiga 215
Phylogeny of the Urochordates: Implications for Chordate Evolution. Billie J. Swalla 219
Evolution of Anural Developmemt in Ascidians: Roles of Muscle-Specific Differentiation Genes. Takehiro Kusakabe 225
Maximum Direct Development and the Ascidiotypic Stage. William R. Bates 230
The Origin of the Neural Crest and Insights into Evolution of the Vertebrate Face. Hiroshi Wada 235
Participation of Neurotransmitters and Adrenergic Receptor in the Metamorphosis of Ascidian Larvae. Yukiko Kimura, Manabu Yoshida, and Masaaki Morisawa 241
4. Taxonomy and Ecology
A Global Overview of Ascidian Introductions and Their Possible Impact on the Endemic Fauna. Gretchen Lambert 249
Settlement and Metamorphosis of the Tropical Ascidian Herdmania curvata.  [now synonymized under H. momus--GL] Bernard M. Degnan 258
Ascidians in Brazil: The State of the art of Research in Taxonomy, Ecology and Natural Products. Rosana M. Rocha and Roberto G. S. Berlinck 264
The Biological Substratum Eudistoma carolinense Van Name, 1945 in the Beach Itapema do Norte, Santa Catarina, Brazil. Tatiane R. Moreno and Rosana M. Rocha 271
Ascidians of South Africa: A Historical Perspective. Shirley Parker-Nance 278
Mitochondrial DNA Analysis of Boltenia echinata iburi (Oka, 1934). Tsuneo Kakuda 283
5. Colonial Ascidians
Molecular and Cellular Advantage of Transdifferentiation System for Asexual Reproduction of the Tunicate, Polyandrocarpa misakiensis. Kazuo Kawamura 293
Molecular Bases of Bud Development in Ascidians. Shigeki Fujiwara, Mika Kamimura, Mitsuko Ohashi, and Kazuo Kawamura 300
Laboratory Studies of Mating in the Aplousobranch Diplosoma listerianum. John D. D. Bishop, Andrew J. Pemberton, A. Dorothea Sommerfeldt, and Christine A. Wood 305
Environmental Effect on the Reproductive Effort of Botryllus schlosseri. J. Stewart-Savage, Anne Stires, and Philip O. Yund 311
Phylogeny of Botryllid Ascidians. Yasunori Saito, Maki Shirae, Makiko Okuyama, and Sarah Cohen 315
Epithelial Differentiation in the Dorsal Strand of a Budding Ascidian, Polyandrocarpa misakiensis (Protochordata, Ascidiacea). Hiromichi Koyama 321
6. Biologically Active Substances
Lumichrome Is a Putative Intrinsic Substance Inducing Larval Metamorphosis in the Ascidian Halocynthia roretzi. Sachiko Tsukamoto, Haruko Kato, Hiroshi Hirota, and Nobuhiro Fusetani 335
Biological Activity and Chemistry of the Compound Ascidian Eusynstyela tincta. S. K. Chithra Lekha Devi, K. N. Rajasekharan, K. Padmakumar, Jun'ichi Tanaka, and Tatsuo Higa 341
Aquaculture of Ecteinascidia turbinata Herdman, 1880 as Source of Marine Anticancer Agents. S. A. Naranjo, H. B. Kukurtçu, C. Barbero, S. Martin, and J. L. Carballo 355
7. Heavy Metals
The Mechanism of Accumulation and Reduction of Vanadium by Ascidians. Hitoshi Michibata, Taro Uyama, Tatsuya Ueki, and Kan Kanamori 363
Immunotoxicity in Ascidians: the Case of Organotin Compounds. Loriano Ballarin and Francesca Cima 374
8. Host Defense Mechanisms
Immunodefense in Tunicates: Cells and Molecules. Edwin L. Cooper and Nicolò Parrinello 383
Immunological Activity of Ascidian Hemocytes. Nicolò Parrinello, Matteo Cammarata, Mirella Vazzana, Vincenzo Arizza, Aiti Vizzini, and Edwin L. Cooper 395
Identification of Type I and IX Collagens in the Ascidian Ciona intestinalis. Aiti Vizzini, Vincenzo Arizza, Melchiorre Cervello, Cinzia Chinnici, Matteo Cammarata, Roberto Gambino, Eleonora Patricolo, and Nicolò Parrinello 402
Primitive Complement System of the Solitary Ascidian, Halocynthia roretzi. Seita Miyazawa, Kaoru Azumi, and Masaru Nonaka 408
Aggregation, Tyrosine Phosphorylation, and Gene Expression in Hemocytes of the Ascidian Halocynthia roretzi. Kaoru Azumi and Hideyoshi Yokosawa 414
Common Cell Surface Ligands Functioning in Allogeneic Cytotoxic Reaction and Fertilization in Halocynthia roretzi. Makoto Arai, Shin-Ichi Ohtake, Hiroyoshi Ohba, Kunio Tanaka, and Joe Chiba 419
Allorecognition and Microsatellite Allele Polymorphism of Botryllus schlosseri from the Adriatic Sea. Baruch Rinkevich, Guy Paz, Jacob Douek, and Rachel Ben-Shlomo 426
Isolation of Marine Birnavirus from Sea Squirts Halocynthia roretzi. Sung-Ju Jung, Myung-Joo Oh, Tatsuya Date, and Satoru Suzuki 436
Colony Specificity in Botrylloides leachi (Savigny): Preliminary Reports. Giovanna Zaniolo and Loriano Ballarin 442
The Viriform Cell of Halocynthia roretzi: Fine Structure, Distribution, and Appearance. Shin-Ichi Ohtake, Teruhisa Ishii, Makoto Arai, Takeyuki Abe, Fumio Shishikura, Joe Chiba, and Kunio Tanaka 445
Hemopoiesis in Solitary Ascidians. Tomoo Sawada, Teruhisa Ishii, and Shin-Ichi Ohtake 450
9. Food Science
Antioxidant Activity of Quinone-derivatives from Freeze-dried Powder of the Ascidians. Osamu Inanami, Tohru Yamamori, Haruhisa Shionoya, and Mikinori Kuwabara 457
Gastroprotective Effect of Ascidian, Halocynthia aurantium (Akaboya), Extract on Acute Gastric Hemorrhagic Lesions in Rats. Hideyuki Chiji, Chizuko Hayashi, and Megumi Matsumoto 463

Adachi, Y., Nagao, T., Saiga, H. and Furukubo-Tokunaga, K. 2001. Cross-phylum regulatory potential of the ascidian Otx gene in brain development in Drosophila melanogaster. Dev. Genes Evol. 211: 269-280.

Armsworthy, S. L., MacDonald, B. A. and Ward, J. E. 2001. Feeding activity, absorption efficiency and suspension feeding processes in the ascidian, Halocynthia pyriformis (Stolidobranchia: Ascidiacea): responses to variations in diet quantity and quality. J. Exp. Mar. Biol. Ecol. 260: 41-69.

Ballarin, L. 2001. Morula cells as the major immunomodulatory hemocytes in ascidians: evidences from the colonial species Botryllus schlosseri. Biol. Bull. 201: 59-64.

Baylies, M. K. and Michelson, A. M. 2001. Invertebrate myogenesis: looking back to the future of muscle development. Curr. Opin. Genet. Dev. 11: 431-439.

Beaulieu, S. E. 2001. Life on glass houses: sponge stalk communities in the deep sea. Mar. Biol. 138: 803-817.

Bellas, J., Vazquez, E. and Beiras, R. 2001. Toxicity of Hg, Cu, Cd, and Cr on early developmental stages of Ciona intestinalis (Chordata, Ascidiacea) with potential application in marine water quality assessment. Water Res. 35: 2905-2912.

Bishop, C. D., Bates, W. R. and Brandhorst, B. P. 2001. Regulation of metamorphosis in ascidians involves NO/cGMP signaling and HSP90. J. Exp. Zool. 289: 374-84.

Bonetta, L. 2001. Anticancer squirt. Nat. Med. 7: 891.

Burighel, P., Brena, C., Martinucci, G. B. and Cima, F. 2001. Gut ultrastructure of the appendicularian Oikopleura dioica (Tunicata). Invert. Biol. 120: 278-293.

Burighel, P., Sorrentino, M., Zaniolo, G., Thorndyke, M. C. and Manni, L. 2001. The peripheral nervous system of an ascidian, Botryllus schlosseri, as revealed by cholinesterase activity. Invert. Biol. 120: 185-198.

Carballo, J. L., Naranjo, S., Kukurtzu, B., de la Calle, F. and Hernandez-Zanuy, A. 2000. Production of Ecteinascidia turbinata (Ascidiacea: Perophoridae) for obtaining anticancer compounds. J. World Aquacult. Soc. 31: 481-490.

Cima, F., Perin, A., Burighel, P. and Ballarin, L. 2001. Morpho-functional characterization of haemocytes of the compound ascidian Botrylloides leachi (Tunicata, Ascidiacea). Acta Zool. 82: 261-274.

Coma, R., Ribes, M., Gili, J.-M. and Hughes, R. N. 2001. The ultimate opportunists: consumers of seston. Mar. Ecol. Prog. Ser. 219: 305-308.

Connell, S. D. 2001. Predatory fish do not always affect the early development of epibiotic assemblages. J. Exp. Mar. Biol. Ecol. 260: 1-12.

Connell, S. D. 2001. Urban structures as marine habitats: an experimental comparison of the composition and abundance of subtidal epibiota among pilings, pontoons and rocky reefs. Mar. Env. Res. 52: 115-125.

Corbo, J. C., Di Gregorio, A. and Levine, M. 2001. The ascidian as a model organism in developmental and evolutionary biology. Cell 106: 535-538.

Darras, S. and Nishida, H. 2001. The BMP signaling pathway is required together with the FGF pathway for notochord induction in the ascidian embryo. Development 128: 2629-2638.

Darras, S. and Nishida, H. 2001. The BMP/CHORDIN antagonism controls sensory pigment cell specification and differentiation in the ascidian embryo. Dev. Biol. 236: 271-288.

Dumollard, D. and Sardet, C. 2001. Three different calcium wave pacemakers in ascidian eggs. J. Cell Science 114: 2471-2481.

Exposito, A., Fernandez-Suarez, M., Iglesias, T., Munoz, L. and Riguera, R. 2001. Total synthesis and absolute configuration of minalemine A, a guanidine peptide from the marine tunicate Didemnum rodriguesi. J. Org. Chem. 66: 4206-4213.

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Glasby, T. M. 2001. Development of sessile marine assemblages on fixed versus moving substrata. Mar. Ecol. Prog. Ser. 215: 37-47.

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