Number 52 December 2002
Ascidiologists have been busy this year; there are 140 new publications listed at the end of this newsletter! We worked at the Roscoff laboratory in Brittany during July and August where Charlie looked into factors leading to the success of invasive ascidians and Gretchen continued her studies in ascidian systematics (see Work in Progress). This was our first visit to the laboratory in 5 years and it was a great pleasure to meet our long-term friends. Danielle Georges and Marie and Henri Goudeau are now retired and live elsewhere for most of the year but still spend their summers in Roscoff. After our stay at the labs we drove through Normandy and visited Claude and Françoise Monniot in Paris. We spent most of September at the Friday Harbor Labs giving us ample opportunity for ascidiological discussions with Dick Whittaker. In late September we conducted an ascidiological survey of most of the harbors in southern Vancouver Island with Cathy Carolsfeld from Victoria. In October we traveled to Nahant Massachusetts where Gretchen presented a workshop on ascidian identification to 33 participants. The workshop was organized by Robert Miller and largely funded by Massachusetts MIT Sea Grant headed by Judy Pederson. We collected ascidians for the workshop in several Mass. harbors and also got down to Woods Hole with Bob Miller. While in the Boston area we visited Bob Woollacott in his lab at Harvard. From Nahant we visted Larry Harris in NH and examined float ascidians with him. He showed us huge colonies of the cream colored Didemnum sp. which seems to be present everywhere in the Gulf of Maine. At the Darling Marine Center in Maine the director Kevin Eckelbarger gave us a most interesting and informative tour. The laboratory is much larger than we had envisioned with many new and refurbished buildings and superb housing. Gretchen identified a troublesome Molgula species there. From the Darling lab we traveled north to the Huntsman Marine Science Centre (named in honor of A.G. Huntsman a Canadian ichthyologist who did very fine work in the systematics of ascidians) in St. Andrews, New Brunswick, Canada. Gretchen presented a workshop there at the invitation of Gerhard Pohle. We greatly enjoyed the New England autumn tree colors which were at their peak. We plan to work at the Friday Harbor labs next summer and hope to see many of you there.
*Ascidian News is not part of the scientific literature and should not be cited as such.
NEWS AND VIEWS
1. Our congratulations to Dr. Hitoshi Sawada, who recently moved from the Graduate School of Pharmaceutical Sciences, Hokkaido University, to become professor and director of the Sugashima Marine Biological Laboratory, Graduate School of Science, Nagoya University. Dr. Sawada writes: Sugashima island in Toba-city (http://www.city.toba.mie.jp/eigo/brief/brief.htm) is located in the Ise-Shima National Park in Mie Prefecture, Japan, and it is a very beautiful place. Since the building of Sugashima Marine Biological Laboratory for the visitors was built about six years ago, and also since the old building was renovated at that time, I suppose that visiting investigators and students must be satisfied by nice accommodations and meals (handmade bread and cakes will also be served in the restaurant!). Although it is not so convenient to go shopping because of the isolated location, the accommodations and facilities are excellent. For instance, radioisotope tracer experiments using some marine animals are also permitted in this MBL. Please contact the URL of my laboratory (http://www.bio.nagoya-u.ac.jp:8001/%7Esaraki/MarineE.html). Lastly, I would like to express my hearty welcome to visitors to the Sugashima Marine Biological Laboratory, Nagoya University. Sincerely yours, Hitoshi Sawada, Director
2. Quentin Bone (email@example.com) writes: John Bishop and I are looking at possible sub-micron particle capture by ascidians and its efficiency. No problem with 0.3-0.45 micron particles, but we are at present trying smaller diameter. I would be interested to hear from others looking at ascidian particle capture.
3. From Hervé Bizot, Unité de Physicochimie des Macromolecules, Inst. Nat. Recherche Agronomique, Nantes, France: Our lab, involved in structural studies of plant cell wall polysaccharides, wishes to purchase tunicin or raw ascidian test, which may offer convenient possibilities to extract pure highly crystalline cellulosic microfibrils. We need to reach multiple gram quantities of purified cellulosic microfibrils to make various physical chemistry experiments including adsorption studies. Apprently the usual choice is Halocynthia roretzi but our attention was also drawn by different colleagues (i.e. Dr. Hirose) towards Microcosmus fulcatus (likely difficult to purify) or Oikopleura rufescens (likely with limited yields). If you have suggestions please contact us by e-mail : firstname.lastname@example.org .
4. Ascidians of the British Isles--a Colour Guide, by Bernard Picton, published in 1985, is now out of print. But the author has revised the text and species photographs and they are now available online at http://www.ulstermuseum.org.uk/marinelife/ Go to the section on Sea Squirts. There is also a page of literature references.
5. From Dr. Billie Swalla (email@example.com)
Dept. of Zool., Univ. of Washington, Seattle WA:
Summer Course Announcement 2003, Friday Harbor Laboratories (http://depts.washington.edu/fhl/classlist03.html)
Marine Invertebrate Zoology June 9- July 12, Instructors: Drs. Susie Balser and Bruno Pernet
Comparative Embryology June 9- July 12: Drs. Richard Strathmann and George von Dassow
Evolution and Development of the Metazoans July 14- August 15: Drs. Billie J. Swalla and Ken M. Halanych
6. Also from Dr. Swalla: I visited Rosaria de Santis’ lab
and gave a week-long workshop at the Stazione Zoologica Anton Dorhn in
Naples, Italy September 2 - 6, 2002: "Development and Evolution of Animal
Body Plans". The students read papers on the development and evolution
of body plans, then presented a research paper of their choosing on the
final day. The presentations were excellent, and showed the large amount
of work that the students had done. It was a great experience for me, and,
I hope, for the students.
The Italian government recently proposed assimilating the Stazione into the CNR (National Research Council), thus ending the 130 year independence of the Stazione. [See 16 August issue of Science, p. 1106-1107.] There was an immediate outcry by the scientific community. Dr. Giorgio Bernardi, President of the Stazione, writes: “Dear Colleagues and Friends, I am pleased to inform you that the Italian Ministry of Instruction, University, and Research has decided to maintain the present statute of independence and autonomy of the Stazione Zoologica Anton Dohrn. The prompt response to our circular plea was massive and impressive, and this certainly played a fundamental role in the final decision of the Ministry. Once more, the scientific community showed its solidarity when general research policies are involved. Furthermore, the decision of the Ministry reaches us at a very appropriate time because, as you may know, we will have our working space more than doubled in a few months (end of February). This means that we can now seriously consider our research priorities in view of a further improvement of our activities. Let me, therefore, express my deepest gratitude on behalf of the entire staff of the Stazione Zoologica for your crucial support.”
7. Arjan Gittenberger writes: A new Ascidians webpage is under construction on www.ascidians.com . If anyone has any suggestions (e.g. for better identifications of the species), please let me know. Within a few months photographs of ascidians from Palau and the Red Sea will be added. Ph.D. student, National Museum of Natural History, Naturalis, Leiden The Netherlands (firstname.lastname@example.org).
8. More information about Dr. R.H. Millar from Tracy-Elizabeth
Price, Secretary to Director Rupert Ormond, University Marine Biological
Station, Millport, Isle of Cumbrae, KA28 0EG. email@example.com.
I knew Dr R H Miller for 42 years. He was known to his friends as Robin. Born in 1916, he served for a time in the British Army, presumably during WWII but we have no information on this. He did his M.A. and Ph.D degrees at Glasgow University and joined the staff of the Scottish Marine Biological Association at its Millport Marine Station in 1947. Robin's first interest was ascidians and at first he studied the morphology and life histories of the local species in the Firth of Clyde. As you know, he became a leading expert on the group and during his career and much of his retirement he examined and reported on collections sent to him from all over the world. What most of his ascidian colleagues may not know is that when Robin came to Millport he also took on responsibility for a research programme designed to revive oyster fisheries on the West Coast of Scotland, which continued into the 1960's. He published a number of papers on this work. He became a fellow of the Royal Society of Edinburgh in 1955 and was awarded his D.Sc. from Glasgow University in 1963. In 1964 he was appointed Deputy Director of the Millport Marine Station. In the late 1960's the Scottish Marine Biological Association transferred its activities from Millport to Oban, on the mainland west coast of Scotland and Robin was Deputy Director for the new Dunstaffnage Marine Research Laboratory until he retired in 1978. Robin was a quiet unassuming man who was always very interesting to talk to. His friends and colleagues have many happy memories of him. He leaves a wife, Joan, who has been in poor health for a number of years.
WORK IN PROGRESS
1. Hemichordates of the Pacific Northwest
This is work Shannon completed as an undergraduate at the University of Washington. Shannon was a Hughes Undergraduate Researcher and a Mary Gates Scholar in Billie Swalla's lab. Last spring, she received her B.S. in Biology and B.A. in Zoology at UW. She also received the John and Dorothy Franco Award for excellent undergraduate research in the Biological Sciences for this manuscript. She is currently working on ascidian metamorphosis in the Swalla Lab.
Morphological and Molecular Identification of Saccoglossus (Hemichordata: Harrimaniidae) in the Pacific Northwest. Shannon E. Smith, Rob Douglas, Karen Burke da Silva, and Billie J. Swalla. Canadian J.of Zool. (in press). Zool. Dept., Univ. of Washington, Seattle, WA 98195.
Hemichordates, especially enteropneust worms, have become increasingly important in phylogenetic studies to test theories of chordate evolution. However, there are multiple populations of enteropneusts along the Pacific Northwest Coast of North America that have not been identified. Here we show that two common Pacific Northwest enteropneust species, Saccoglossus pusillus and S. bromophenolosus can be distinguished by both morphological and molecular characters, and we identify several populations of both species. We compare them to a closely related species, S. kowalevskii, from the Atlantic Coast of North America. We compile the morphological characters used to distinguish harrimaniid enteropneusts, and we describe a new staining method to examine the gill bars and proboscis skeletons of enteropneusts to aid in identification. Using 18S and 16S ribosomal DNA sequences, we determine that the range of Saccoglossus pusillus extends from Southern California, where the worm was first identified, to Southern Canada. This previously unknown large range shows a dramatic geographic cline in adult body size, with the smallest populations found in the south and the largest adults found near Vancouver Island. In contrast, S. bromophenolosus may be a Pacific Northwest species that was relatively recently introduced from the Atlantic.
2. From Gretchen and Charles Lambert: During our 6 week
stay in France during July and August at the Station Biologique in Roscoff,
Brittany, we found numerous specimens of Corella eumyota at two
marinas: Perros Guirec and Camaret-sur-Mer. This species is abundant
and widespread in the southern hemisphere but to our knowledge this is
the first record of this species in the northern hemisphere. Claude Monniot
at the Paris Museum National d’Histoire Naturelle confirmed the identification,
and GL also confirmed it by comparing them with some samples from New Zealand.
We plan to give a short talk about it at the next bioinvasions symposium,
at Scripps next March.
A second surprising find is that all the subtidal Cionas collected in the NE Pacific from Alaska and British Columbia are not C. intestinalis as they were originally identified; but are actually C. savignyi. Cathy and Yogi Carolsfeld from Victoria, BC recently collected several hundred Ciona from about 12-18m at several sites off mainland British Columbia, near Sechelt and Bella Bella, and all are C. savignyi. Old museum specimens from Alaska (1913) and BC (1937) were reexamined by Hoshino and Nishikawa (Hoshino, Z.-I. and Nishikawa, T. 1985. Taxonomic studies of Ciona intestinalis (L.) and its allies. Publ. Seto Mar. Biol. Lab. 30: 61-79) and found to be C. savignyi though they had originally been identified as C. intestinalis. GL has just finished examining several Ciona specimens in the Friday Harbor Laboratories voucher collection, all collected in the 1960’s around southern Vancouver Island, BC, and all are C. savignyi. Thus we urge our colleagues to be very careful when identifying Ciona species. The two characters used by Hoshino and Nishikawa are the presence of an endostylar appendage in C. intestinalis that is absent in C. savignyi, and the location of the paired pharyngeo-epicardiac openings. In addition, there is a difference in the follicle cells which is most obvious in living animals because the follicle cells fall off the eggs in preserved animals. See Byrd, J. and Lambert, C. C. 2000. Mechanism of the block to hybridization and selfing between the sympatric ascidians Ciona intestinalis and Ciona savignyi. Mol. Repro. Dev. 55: 109-116. This paper contains a photograph of the egg of these two species. There is a single large refringent droplet in each follicle cell in C. intestinalis, and several small droplets in each follicle cell in C. savignyi. There are often other differences as well. In many areas (though not all), C. intestinalis has a large red spot at the end of the sperm duct; this is always absent in C. savignyi.
3. From Patrick Frank, Dept. of Chemistry, Stanford Univ., Stanford, CA 94305-5080 (Frank@ssrl.slac.stanford.edu): In work that includes Dr. Tony De Tomaso at Hopkins Marine Station, and Keith Hodgson and Britt Hedman at SSRL (Stanford Synchrotron Radiation Laboratory), we recently looked at the x-ray absorption spectra of blood cells and whole bodies of Perophora annectens. These colonials are mentioned in "Intertidal Invertebrates of California" (Robert Morris, Donald Abbott and Eugene Hadlerlie) as being particularly rich in blood cell vanadium.We have already characterized the vanadium in blood cells from Ascidia ceratodes and Phallusia nigra, and wanted to look at an organism that is as different as possible from those solitary species. However, to our surprise, when we did the experiment, the vanadium signal was almost non-existent. What we found instead was a very intense iron signal! This was totally unexpected. There is no doubt that the organism was correctly typed. The iron spectroscopically looks more like Fe(III) than Fe(II), and the same spectrum was observed both in whole blood cells and in the whole animal. We hope to look at a sample of P. annectens taken from another location to check this result. If these indeed contain vanadium, then it may be possible that this organism can switch between metals (or that there is a sub-species).
1. Molecular embryology of a larvacean urochordate, Oikopleura
dioica, and the origin of chordate innovations. Susie Bassham,
Dept. of Biology, Univ. of Oregon, June 2002. Advisor Dr. John Postlethwait,
Institute of Neuroscience. firstname.lastname@example.org
Phyla are recognized by unifying characters, some of which are unique evolutionary innovations, but how do new structures and body plans evolve? Defining characters of the phylum Chordata include the notochord, dorsal hollow nerve cord, and post-anal tail. Despite these commonalities, the chordate subphyla Vertebrata, Cephalochordata, and Urochordata span a broad range of morphological diversity. Vertebrates are distinguished from the other chordates by an elaborated brain, paired sense organs, and a skeleton. Two embryonic tissues, neural crest and placodes, are essential for these features. It became widely accepted that crest and placodes are vertebrate innovations because no clear homologues of these tissues or their derivatives had been observed in other chordates. Further speculation suggested that acquisition of neural crest and placodes permitted or propelled the evolution of raptorial vertebrates from a sessile, filter-feeding ancestor. By comparing ontogenies of extant chordates and their outgroups, we hope to reconstruct the timing and mechanism of the origin of chordate novelties.
Within the most basal chordate lineage, Urochordata, larvaceans uniquely allow us to analyze development of adult characters in a chordate body plan that does not become reconfigured by sweeping metamorphic events. This dissertation examines the evolutionary history of two chordate innovations, notochord and placodes. I present DNA sequences and expression patterns for larvacean homologues of several vertebrate genes important in notochord and placode development: T (Brachyury), Pitx, Eya, and members of the Pax and Six gene families. Larvacean T is expressed in notochord, but also in posterior endoderm. Because deuterostome and protostome outgroups display a similar pattern, chordate T may have a conserved role in endoderm patterning more ancient than its role in the notochord. I also show that several genes important for the development of a subset of vertebrate placodes are expressed in the developing ciliary funnel and ventral organ in larvaceans. Ultrastructure and topography had previously suggested that these organs are candidate homologues of pituitary and olfactory placodes. My results are consistent with an origin of these placodes in the common ancestor of modern chordates, predating the evolution of vertebrates.
2. Microcosmus squamiger, a solitary ascidian introduced to
southern California harbors and marinas: salinity tolerance and phylogenetic
analysis. Andrew Lowe, M.S. thesis, , Dept. of Biol. Sci., Calif. State
Univ. Fullerton, Fullerton, CA 92834. Advisor Dr. Roger Seapy.
Current address: Dept. of Biol., Calif. State Univ., San Marcos, CA 92096 email@example.com
The non-indigenous ascidian Microcosmus squamiger has become a dominant fouling organism in sheltered portions of southern California harbors where it appears to be increasing in abundance at the expense of Molgula manhattensis, a common estuarine ascidian [editors' comment: also non-indigenous]. I have compared mortality rates of M. squamiger and M. manhattensis based on laboratory experiments that tested the tolerances to salinities ranging from 150/00 to 330/00. Both species survived at 330/00 and 300/00, but at 250/00 the average percent survival of M. squamiger was much higher (80%) than that of M. manhattensis (50%). This difference was more pronounced at 200/00 (77% and 20%, respectively) and 150/00 (60% and 0%, respectively). These results support the hypothesis that differential survival allows M. squamiger to have an advantage needed to claim limited space over M. manhattensis.
In order to examine patterns of M. squamiger introduction into southern California and subsequent dispersal among southern California harbors, mitochondrial DNA sequences were analyzed to assess genetic variation at a series of sampling locations. The California grouping of sequences is paraphyletic with the Mediterranean sequences nested within it. Furthermore, these data suggest that Microcosmus squamiger was introduced from Australia to southern California, then later introduced from California to Spain. The average within population genetic diversity (Pi) was higher (1.28) for southern California populations than either the Spanish (0.61) or Queensland (0.50) populations, possibly suggesting multiple introductions. For the southern California sites, diversity between populations (PiT) was lowest (0.88) for Bahia Point and Mission Bay, suggesting high levels of gene flow. Within population diversity (Pi) was highest at Oceanside and Long Beach (1.91).
1. 5th International Larval Biology Meeting, Univ. of Vigo, Spain, Sept. 15-20, 2002
a. Intraspecific variation in larval size affects the length of the
planktonic period in lecithotrophic larvae. Marshall DJ, Keough MJ,
Dept. of Zool., Univ. of Melbourne, Parkville 3052, Australia firstname.lastname@example.org
For many species of marine invertebrate, variability in larval settlement behaviour appears to be the rule rather than the exception. This variability is important as it has the potential to affect larval dispersal. Despite the ubiquity and importance of this variability, relatively few sources of variation in larval settlement behaviour have been identified. One important factor that can affect larval settlement behaviour is the nutritional state of larvae. For a number of non-feeding larvae, as energetic reserves run low, larvae become less discriminating in their "choice" of settlement substrate, i.e. more desperate to settle. We tested whether variation in larval size (and presumably nutritional reserves) also affects the settlement behaviour of three species of colonial marine invertebrate larvae, the bryozoans Bugula neritina and Watersipora subtorquata and the ascidian Diplosoma listerianum. For all three species, larger larvae delayed settlement for longer in the absence of settlement cues, and settlement of Bugula neritina larvae was accelerated by the presence of settlement cues, independently of larval size. In the field, larger Watersipora larvae also took longer to settle than smaller larvae and were more discriminating towards settlement surfaces. These differences in settlement time are likely to result in differences the distance that larvae disperse in the field. We suggest that species that produce lecithotrophic larvae can indirectly affect the dispersal potential of their offspring by manipulating larval size and thus larval desperation.
b. Larval activity levels and prolonged swimming affect post-larval
performance in the colonial ascidian Diplosoma listerianum.
Marshall DJ, Pechenik JA, Keough MJ. [also presented at the temperate reef
meeting, New Zealand.
It is becoming widely recognized that extending the larval period of marine invertebrates, especially of species with non-feeding larvae, can affect post-larval performance. As these carry-over effects are presumed to be caused by the depletion of larval energy reserves, we predicted that the level of larval activity would also affect post-larval performance. This prediction was tested with the cosmopolitan colonial ascidian Diplosoma listerianum, in field experiments in southern Australia. Diplosoma larvae, brooded in the parent colony, are competent to settle immediately after spawning, and remain competent to metamorphose for >15 h. Some larvae were induced to metamorphose 0-6 h after release whilst others were induced to swim actively by alternating light and dark periods for up to 3 h prior to metamorphosis. Juvenile colonies were then transplanted to a subtidal field site in Port Phillip Bay and left to grow for up to 3 weeks. Extending the larval period and increasing the amount of swimming both produced carryover effects on post-larval performance. Colonies survived at different rates among experiments, but larval experience did not affect survival rates. Delays in metamorphosis and increased swimming activity did, however, reduce colony growth rates dramatically, resulting in 50% fewer zooids per colony. Moreover, such colonies produced initial zooids with smaller feeding structures, with the width of branchial baskets reduced by 10-15%. These differences in branchial basket size persisted and were still apparent in newly budded zooids 3 weeks after metamorphosis. Our results suggest that, for Diplosoma, larval maintenance, swimming, and metamorphosis all use energy from a common pool, and increases in the allocation to maintenance or swimming come at the expense of post-larval performance. [Soon to be published in Marine Ecology Progress Series]
c. Relationships of defence mechanisms of larvae and adults of colonial
ascidians: patterns of palatability and toxicity. I. Tarjuelo,
S. Lopez-Legentil, M. Codina and X. Turon, Dept. of Animal Biology (Invertebrates),
Fac. of Biol., Univ. of Barcelona, 645 Diagonal Ave., 08028 Barcelona,
We studied the defence patterns of larvae and adults of six species of colonial ascidians from the Mediterranean Sea. We tested palatability with common sympatric predators (fish and crustaceans). The tests were performed separately for the larvae and for the main compartments of the adults (zooids and tunic). For the species that proved unpalatable, tests were repeated with chemical extracts. We also tested toxicity with the Microtox method. Total energy content, amount of inorganic material and pH were analysed for all compartments. We looked for patterns of defence among species, and for relationships of adult/larval palatability, of toxicity/palatability, pH/palatability and food quality/palatability. Overall, we found a high variability among species, compartments and predators, but unpalatability was found in all species in at least one test. Those species with low per zooid fecundity and large larvae (Cystodytes dellechiajei, Polysysncraton lacazei, Diplosoma spongiforme and Pseudodistoma crucigaster) had larvae that were unpalatable to at least two of the predators, while larvae of the two species with higher fecundity and smaller larvae (Clavelina lepadiformis and Ecteinascidia herdmanni) were the most palatable. There was no relationship between adult and larval palatability. Tunic material was the least palatable in general, and zooid material the most. Only one of the species studied had acidic tunic. Toxicity was in general low and not related to palatability, while energy content was positively related to the latter. Tests with extracts substantiated a chemical basis for unpalatability in only a few cases, and there was a pattern of increased palatability of tissues with higher energy content and lower amount of structural material. We conclude that the defence strategies of colonial ascidians are highly variable among species (even of the same family), and that allocation to defence varies between compartments and between ontogenetic states (larvae or adult). The pattern found indicates a higher protection against larval predation in species with low fecundity producing large and costly larvae.
2. Sixth International Conference of Systematic and Evolutionary Biology in Patras, Greece. September 9-16, 2002
Evolution of the chordates: worms or squirts? When, why and how?
Billie J. Swalla, Zool. Dept., Univ. of Washington, Seattle, WA 98195.
Presented in the Symposium "Life, the Universe and Everything" email@example.com
The Deuterostomia are a monophyletic group of animals that diverged from the Ecdysozoa and Lophotrochozoa some time before the Cambrian period, as all of the four major clades of deuterostomes are present in the Cambrian fauna. Molecular clocks provide varying estimates of the period of time in which this radiation occurred, from shortly before the Cambrian explosion to many millions of years prior to the appearance of these groups in the fossil record. The problems inherent in molecular clocks will be discussed and evaluated. One of the most debated questions about chordate ancestry is whether the ancestral group was sessile or a motile worm. Neither cladistics nor the fossil record allows unequivocal answers, so we have turned to developmental gene expression to examine the evolution of chordates. A strictly cladistic approach will be contrasted with an analysis that considers evolutionary developmental processes. The importance of distinguishing solitary and colonial species when examining body plan evolution will be highlighted. Using developmental characters to compare the four major deuterostome groups, echinoderms, hemichordates, urochordates and vertebrates, we will show that echinoderm and urochordate adult body plans appear to have been highly derived at the time of their divergences from the other major clades. Using a total data approach, we will show that the deuterostome ancestor was likely to have been a motile, benthic, filter-feeding worm with gill slits, an organized skeleton, coelom, and perhaps even neural crest cells. Ongoing experiments to test these hypotheses will be presented and critiqued.
3. European Life Scientist Organisation ELSO 2002 meeting, Nice, France, 29 June - 3 July 2002 (previously European Cell Biology Organisation, ECBO).
Cell-mediated immune responses in the colonial ascidian Botryllus
schlosseri: the role of morula cells. Ballarin L., Cima F., Sabbadin
A., Dept. of Biol., Univ. of Padova, Italy. firstname.lastname@example.org
The mulberry-shaped vacuolated haemocytes called morula cells (MC) are a common cell-type in the ascidian blood. In the compound ascidian Botryllus schlosseri, their number ranging from 20 to 60% of the total haemocyte number. This fits with the important role of MC in immunomodulation which results from the observation that they can produce IL-1-alpha- and TNF-alpha-like molecules when haemocyte cultures are incubated with the microbial polysaccharide mannan or the protein kinase C activator phorbol myristate acetate. Besides, MC are the effectors of the rejection reaction between contacting, genetically incompatible colonies which represents the typical outcome of colony specificity. In the early stages of this reaction, MC crowd inside the outgrowths of the marginal vessel known as ampullae, which contact the opposite colony. Then, they degranulate and release their vacuolar content (mainly phenoloxidase and its polyphenol substrata), cross the fenestrated epithelium of the ampullar tips and reach the tunic where they contribute to the formation of the necrotic masses which characterise the rejection reaction. Degranulation of MC is preceded by their acquisition of immunoresponsiveness to anti-human-IL-1-alpha which indicate a role of this cytokine-like molecule in the rejection reaction. In addition, previous reports, showing an increase in phagocytosis by ascidian phagocytes when haemocytes were incubated in the presence of MC extracts, suggest the involvement of MC in the process through a sort of cell cooperation mediated by the production of soluble factors. In our opinion these factors correspond to cytokine-like molecules synthesised and released by MC upon their recognition of foreign molecules. Our hypothesis is supported by the observation that anti-human-IL-1-alpha antibodies significantly inhibit the ACTH-induced ingestion of yeast cells by Botryllus phagocytes.
4. 37th European Marine Biology Symposium, Reykjavik, Iceland, 5-9 August 2002
An Atlantic clade of Clavelina lepadiformis (Ascidiacea) may
have recently colonised Mediterranean marinas by ship transport.
*I. Tarjuelo, 1D. Posada, 1K.
A. Crandall, **M. Pascual, S. Duran & *X. Turon. *Dept. of Animal
Biol. and **Dept. of Genetics, Fac. of Biol., Univ. of Barcelona. 645,
Diagonal Ave. 08028 Barcelona. Spain. 1Dept. of Zool.,
Brigham Young Univ., Provo. UT 84602-5255 email@example.com
We studied the genetic structure of populations of the Atlanto-Mediterranean ascidian Clavelina lepadiformis. A 500 bp segment of the COI mitochondrial gene was sequenced in Mediterranean and Atlantic populations from inside and outside harbours and marinas (interior and exterior form, respectively). We found a marked genetic differentiation between C. lepadiformis inhabiting the Mediterranean harbours and those from the Mediterranean rocky littoral, with a genetic divergence of 5%. Gene flow values among these forms were unappreciable and of the same order as the values found among both forms and other congeneric species. The lack of gene flow and the genetic divergence suggests that the interior and exterior forms of Mediterranean C. lepadiformis are in fact cryptic species. Levels of gene flow were higher among interior habitats than among exterior habitats, a pattern likely maintained by genetic exchange through ships. When compared with four Atlantic populations (one inside and three outside harbours), we found that these were closely related to the interior clade in the Mediterranean. We put forward the hypothesis that both clades evolved separately at both seas, and that a recent colonisation of Mediterranean marinas from the Atlantic has occurred via ship-hull transport.
5. The 10th Pacific Congress on Marine Science and Technology (PACON2002), July 21-26, 2002, Chiba, Japan.
Accumulation and reduction of vanadium by ascidians. H.
Michibata, Mukaishima Mar. Biol. Sta., Hiroshima Univ., Mukaishima-cho,
Hiroshima 722, Japan (firstname.lastname@example.org)
Ascidians, so-called seasquirts, are known to accumulate high levels of vanadium. The highest concentration of vanadium, 350 mM, was found in the blood cells of Ascidia gemmata belonging to the suborder Phlebobranchia. This concentration is 107 times higher than that in seawater. Vanadium accumulated in the blood cells is reduced to V(III) via V(IV) and stored in vacuoles of vanadocytes (vanadium-containing blood cells). The contents of vacuoles are maintained in an extremely low pH of 1.9 by vacuolar-type H+-ATPase. Recently, we have found out at least two types of vanadium-binding protein, 12.5 kDa and 15 kDa, and cloned cDNAs encoding them. Both novel proteins, designated as Vanabins, have been disclosed to consist of about 120 amino acids in which the content of cysteine residues is very high and occurrence of cysteine residues at regular intervals is characteristic and common in both novel proteins. Recombinant Vanabin was found to bind about 10 to 20 atoms of vanadium and its binding constant is 10-6 to 10-7M, which might have a clue to resolve the mechanism of vanadium accumulation. Concerning the reduction of vanadium in vanadocytes, we have localized four enzymes involved in the pentose phosphate pathway that is known to produce a reducing agent, NADPH. In fact, NADPH was revealed to conjugate the reduction of V(V) to V(IV) in vitro. Furthermore, the other types of protein such as a metal-ATPase and cDNAs involved in the accumulation and reduction of vanadium will be reported.
6. The 73rd annual meeting of the Zool. Soc. of Japan, Kanazawa, Japan, Sept. 24-27, 2002.
The ultrastructure of the sensory cells associated with a branchial
tentacular tunic in the ascidian, Polyandrocarpa misakiensis.
H.Koyama, Coll. Nurs., Yokohama City Univ., Yokohama. email@example.com
In Polyandrocarpa misakiensis, I studied the ultrastructure of the ciliated intraepithelial sensory cells in the basal part of branchial tentacles. The pear-shaped sensory cells form small clusters, and are supported by thickened epithelial cells of the descending or ascending epithelium of siphons. The supporting epithelial cells cover the sensory cells except for their apical part, which has microvilli surrounding a cilium. The basal cytoplasm of the sensory cells is dense with rough endoplasmic reticulum, mitochondria, lipid droplets, and dense bodies. The apical cytoplasm, about 1/3 of the entire cell height, is electron-translucent, and has a few organelles. Since some of the sensory cells have a basal process with the ultrastructural features of an axon, these cells are regarded as primary sensory neurons, like many other sensory cells of protochordates. The apicolateral parts of the sensory cells are joined together by two types of junctions (gap junctions? and adherent junctions). The supporting cells and sensory cells are connected by adherent junctions. Certain sensory cells are coupled to what seem to be neurosecretory cells.
7. American Society for Cell Biology annual meeting, San Francisco CA, Dec. 14-18, 2002.
(a) The role of integrins in sperm activation in the tunicate,
Ascidia ceratodes. Janice Soratorio and Robert A. Koch.
California State University Fullerton, Fullerton, CA firstname.lastname@example.org
Mitochondrial translocation is an early event during ascidian fertilization in which the sperm mitochondrion binds to the egg outer surface, undergoes an actin:myosin-dependent translocation from the head of the sperm down to the tail. Due to integrin’s known association with actin filaments, we hypothesize that integrins mediate adhesion of the sperm cell to the egg outer surface and signal the formation of focal adhesion-like complexes (FC). Thus, we predict that one or more integrin family members will span the membrane and interact with FC-specific proteins in the cytosol, e.g., talin, paxillin and FAK. Using anti-integrin alpha Hr1 and beta Hr prepared against the ascidian Halocynthia roretzi protein and anti-integrin beta 1, integrins were detected on the mitochondrial region of ascidian sperm heads. Integrin beta 2, beta 3 and beta 4 were not detected in the sperm. The head also labeled positively for the cytoskeletal proteins, talin, paxillin and FAK, a tyrosine kinase that is known to associate with integrins in focal adhesion sites. It is known that FC formation depends on integrin receptor occupancy and clustering. To study the latter, sperm were exposed to an anti-integrin antibody known to induce integrin clustering, mAb 12G10. We found that mAb 12G10 triggered sperm activation in a manner similar to positive controls. To determine integrin’s function during fertilization, dose-dependent inhibition by echistatin was tested. In the presence of echistatin (1.5 and 15 µM), fertilization was inhibited and decreased to 60% and 47%, respectively. These data demonstrate: (1) the presence of integrins on the ascidian sperm cell surface, (2) the presence of protein characteristic of focal complexes, and (3) suggest the importance of integrins as sperm-surface egg receptors in ascidian fertilization. (Funded by NIH R25GM56820, R25GM56625 and R15HD36500).
(b) Cell polarity and asymmetric divisions in ascidian and
sea urchin embryos. G. Prulière, S. Patalano, C. Sardet,
J. M. Chenevert; Laboratoire de biologie du développement, UMR 7009-CNRS-Université
Paris 6, Villefranche sur Mer, France
The cortical sets of proteins PAR3-PAR6-aPKC-CDC42 and PAR1 control many events crucial for the establishment of cell polarity during oogenesis or development. We are exploring the roles of these complexes in ascidian and sea urchin embryos. In these animals starting at the 8-16 cell stage series of asymmetric cell divisions produce large and small cells (micromeres) and asymmetrically segregate determinants. At the 16 cell stage, in the sea urchin 4 vegetal blastomeres and in the ascidian 2 posterior blastomeres are generated. We have cloned the cDNAs encoding cell polarity proteins and find that PAR1, PAR6, CDC42, and aPKC are present as maternal transcripts in both sea urchins and ascidians remarkable feature of the ascidian system is the presence of a macroscopic structure responsible for the asymmetric divisions, the Centrosome Attracting Body ("CAB"). Labellings with heterologous antibodies indicate that PAR-3 protein is present in the ascidian CAB and also in the vegetal cortex of sea urchin micromeres. In ascidian embryos, aPKC protein colocalizes with PAR3 enriched in the CAB and is present on the cortex of all blastomeres. We are generating antibodies and dominant negative tools to determine the localization and function of these and other proteins involved in the definition of cell polarity. In addition we have produced an antibody to a 100kD ascidian protein which specifically recognizes the CAB and the sea urchin vegetal cortex. We are currently developing an in vitro assay for cell polarity using peeled cortices which retain microtubule attachment properties.Our results suggest that the cell polarity complexes Par-3/Par-1 participate in the establishment of the embryo polarity in these two experimentally tractable models and that the sea urchin vegetal cortex contains a structure similar to the CAB responsible for attraction of a centrosome.
(c) Polarization of the egg cortex and mRNA localization during
early development of ascidians. 1C. Sardet,
Prodon, 2H. Nishida, 2K. Sawada
; 1Station zoologique, Villefranche sur mer, France;
2TiTech, Tokyo, Japan.
The ascidian egg cortex is involved in calcium signalling, contractions, translocations subcellular domains, cleavages and the localisation of maternal determinants. The periphery of the mature oocyte is a polarized basket made of 2 juxtaposed domains: a mitochondria rich sub-cortical domain (myoplasm) and an ER rich cortical domain (cER)(1). These two domains are distributed as a gradient along the animal-vegetal axis (a-v). A class of cortical maternal mRNAs (postplasmic RNAs) including the muscle determinant macho1 are initially polarized along the (a-v) axis (2). These move vegetally and then posteriorly after fertilization to accumulate in the cortex of the 2 vegetal posterior blastomeres at the 8 cell stage. Postplasmic RNAs are concentrated in a cortical ER-rich macromolecular structure called the Centrosome Attracting Body (CAB)(2). The CAB is responsible for three successive asymmetric cleavages (8-64 cell stages) giving primary muscle cells and endodermal cells(2). Postplasmic RNAs can be isolated with the cortex made of the membrane to which a network of microfilaments, a network of cER and microtubules adhere (3). High resolution fluorescent in situ hybridization indicates that two postplasmic RNAs, macho-1 and Hr PEM are associated with the cortical rough ER network before fertilization, concentrate in the vegetal cortex with the cER after fertilization and accumulate in the CAB as patches at the 8 cell stage together with the cortical ER network. In conclusion, the polarized cER network organized along an (a-v) gradient in the egg is a major precursor of the CAB and plays an important role in the localization and segregation of maternal mRNAs determinants (download bioclip “Polarity inside the egg cortex” at www. bioclips.com) (1) Roegiers et al., 1999, Development, 126:3101-17; (2) Nishida, 2002, Int Rev Cytol 217:227-76 (3) Sardet, 2002, Dev.Biol. 241:1-23
7. 3rd Intl. Marine Bioinvasions Conference, La Jolla, California March 16-19, 2003.
a) Trans-hemisphere invasion of the south-temperate and subantarctic
ascidian Corella eumyota Traustedt, 1882, into two harbors in Brittany,
France. Lambert, G., Univ. of Washington Friday Harbor Laboratories,
Friday Harbor, WA. Monniot, C. and F., Mus. Nat. d’Hist. Nat., Biologie
des Invertebres marins, 55 rue Buffon, 75005 Paris, France; and Lambert,C.C.,
Univ. of Washington Friday Harbor Laboratories, Friday Harbor, WA.
Corella parallelogramma is the only Corella species recorded from the English Channel region of France. However, during July and August 2002 we found numerous large clumps of C. eumyota on ropes and floats at the marinas at Perros Guirec and Camaret-sur-Mer in western Brittany on the English Channel. Our identifications are based on a careful comparison with specimens from southern New Zealand and South Africa of the adult and egg morphology, and the fact that it is a brooder. Several thousand embryos are ovulated directly into the peribranchial cavity where they stick together in a cohesive gelatinous mass. The tadpoles are retained for several hours after hatching and are not released until they are competent to settle. Larval life outside the adult is therefore very short, perhaps only a few minutes; as a result the larvae settle near or on the adults and large heavy clumps result. The individuals are very tightly cemented together and often impossible to separate without tearing the tunics. C. eumyota’s previously known range has been limited to the southern hemisphere, where it is abundant and widespread in south temperate, subantarctic, and Antarctic waters: South Africa, S. America, south Australia, Tasmania, and New Zealand from Aukland to the south tip of the south island. This species was not present 5 years ago (our last visit to Brittany) at the 2 marinas where we found it abundantly this year. It has the potential to become a serious pest for the mussel culture industry in Normandy. We do not know how widespread it is in Europe but colleagues in the UK have not seen it. This may be the first record of a southern to northern hemisphere range extension for an ascidian, although several species are known to have been transported from Europe to the southern hemisphere.
b) Exotic organisms in southern California bays and harbors.
Cohen, A.N., San Francisco Estuary Institute, Oakland, CA (email@example.com)
and 11 co-authors.
In recent decades, the world has witnessed an array of harmful invasions by exotic marine organisms. To provide the public and policymakers with better information on the status of exotic species in Southern California waters, the California Department of Fish and Game and the California State Water Resources Control Board, with supplemental funding from the National Fish and Wildlife Foundation, commissioned a Rapid Assessment Survey of selected sheltered waters between San Diego and Oxnard in the summer of 2000. The primary objective of the survey was to assess the status of exotic invasions within certain habitat types in the region. Secondary objectives included obtaining data for comparisons between habitats and regions and for comparisons with past surveys; obtaining baseline data for future assessments of changes in invasion status and the effectiveness of prevention or control efforts; detecting new invasions and documenting significant range extensions; and identifying new species. Twenty-two primary sampling sites and three secondary sites were selected to represent the three major commercial port areas in southern California, important marina areas and lagoon sites. Sampling was primarily of dock fouling along with adjacent soft-bottom benthos, nearby intertidal sites, and selected subtidal lagoon habitats, using a variety of manual techniques. Sixty-seven of the species collected were identified as exotic, with an average of 16.7 exotic species collected at each site. These included representatives from two algal divisions and seven invertebrate phyla. Ascidians were especially well-represented (15 exotic species) and widely occurring, and certain polychaetes, bivalves, isopods and amphipods also occurred widely.
c) "Le genie humain reunit les oceans": the movement of species
between the tropical eastern Pacific and western Atlantic via the Panama
Canal. Cohen, A.N., San Francisco Estuary Institute, Oakland,
CA, (firstname.lastname@example.org) and 10 co-authors.
The Panama Canal was completed in 1914, enabling ships to pass between the Atlantic and Pacific Oceans with typical transit times of less than half a day. The Canal may have affected the anthropogenic dispersal of marine organisms in several ways: by allowing transfers of organisms between the Eastern Tropical Pacific and Western Tropical Atlantic regions; by enabling more transfers or more rapid transfers between other parts of the world; by affecting the size and the operation of ports and other ship facilities located near either end of the Canal; and by limiting the size of a substantial part of the world's cargo fleet to "Panamax" ships. Possible mechanisms for the movement of marine organisms through the Canal include gradual dispersal through the locks and waters of the Canal over more than one organism's lifetime; the migration of fish or other strong swimmers, along with their internal or external parasites, through the Canal in less than one host organism's lifetime; and the transport of organisms in external hull fouling, in boreholes or crevices in wooden hulls, and in the ballast tanks or other components of ships' seawater systems in under a day. To assess the number of species moved through the Canal we reviewed records of species in both tropical regions on either side of the Canal, and conducted a Rapid Assessment Survey of selected habitats near the Pacific and Atlantic ends of the Canal in the spring of 2002.
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Chambon, J. P., Soule, J., Pomies, P., Fort, P., Sahuquet, A., Alexandre, D., Mangeat, P. H. and Baghdiguian, S. 2002. Tail regression in Ciona intestinalis (Prochordate) involves a Caspase- dependent apoptosis event associated with ERK activation. Development 129: 3105-3114.
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Cima, F., Brena, C. and Burighel, P. 2002. Multifarious activities of gut epithelium in an appendicularian (Tunicata). Mar. Biol. 141: 479-490.
Cima, F., Dominici, D., Ballarin, L. and Burighel, P. 2002. Influence of TBT on activity of detoxifying enzymes from haemocytes of a colonial ascidian. Fresenius Environ. Bull. 11: 573-577.
Davidson, B. and Swalla, B. J. 2002. A molecular analysis of ascidian metamorphosis reveals elements of an innate immune response. Development 129: 4739-4751.
Davis, R. A., Aalbersberg, W., Meo, S., Rocha, R. M. and Ireland, C. M. 2002. The isolation and synthesis of polyandrocarpamines a and b. Two new 2-aminoimidazolone compounds from the Fijian ascidian, Polyandrocarpa sp. Tetrahedron 58: 3263-3269.
Devine, C., Hinman, V. F. and Degnan, B. M. 2002. Evolution and developmental expression of nuclear receptor genes in the ascidian Herdmania. Intl. J. Dev. Biol. 46: 687-692.
Di Gregorio, A. and Levine, M. 2002. Analyzing gene regulation in ascidian embryos: new tools for new perspectives. Differentiation 70: 132-139.
Dumollard, R., Carroll, J., Dupont, G. and Sardet, C. 2002. Calcium wave pacemakers in eggs. J. Cell Sci. 115: 3557-3564.
Erba, E., Bassano, L., Di Liberti, G., Muradore, I., Chiorino, G., Ubezio, P., Vignati, S., Codegoni, A., Desiderio, M. A., Faircloth, G., Jimeno, J. and D'-Incalci, M. 2002. Cell cycle phase perturbations and apoptosis in tumour cells induced by aplidine. British J. Cancer 86: 1510-1517.
Etani, K. and Nishikata, T. 2002. Novel G-protein-coupled receptor gene expressed specifically in the entire neural tube of the ascidian Ciona intestinalis. Dev. Genes Evol. 212: 447-451.
Ferrier, D. E. and Shimeld, S. M. 2002. Evolution of developmental mechanisms. Genome Biol. 3: 4020.
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Green, K. M., Russell, B. D., Clark, R. J., Jones, M. K., Garson, M. J., Skilleter, G. A. and Degnan, B. M. 2002. A sponge allelochemical induces ascidian settlement but inhibits metamorphosis. Mar. Biol. 140: 355-363.
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im Gefolge der Befruchtung. Mikrokosmos 82: 129-135.
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Groepler, W. 2002. Ovulation in Diplosoma (Tunicata, Ascidiacea, Didemnidae): a light microscopical study. Helgoland Mar. Res. 52: 102-111.
Hemmi, H., Yoshida, T., Kumazaki, T., Nemoto, N., Hasegawa, J., Nishioka, F., Kyogoku, Y., Yokosawa, H. and Kobayashi, Y. 2002. Solution structure of ascidian trypsin inhibitor determined by nuclear magnetic resonance spectroscopy. Biochemistry 41: 10657-10664.
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Holloway, M. G. and Connell, S. D. 2002. Why do floating structures create novel habitats for subtidal epibiota? Mar. Ecol. Prog. Ser. 235: 43-52.
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Imai, K. S., Satoh, N. and Satou, Y. 2002. An essential role of a FoxD gene in notochord induction in Ciona embryos. Development 129: 3441-3453.
Imai, K. S., Satou, Y. and Satoh, N. 2002. Multiple functions of a Zic-like gene in the differentiation of notochord, central nervous system and muscle in Ciona savignyi embryos. Development 129: 2723-2732.
Inaba, K., Padma, P., Satouh, Y., Shin, I. T., Kohara, Y., Satoh, N. and Satou, Y. 2002. EST analysis of gene expression in testis of the ascidian Ciona intestinalis. Mol. Reprod. Dev. 62: 431-445.
Inoue, I., Tsutsui, I. and Bone, Q. 2002. Excitation-contraction coupling in isolated locomotor muscle fibres from the pelagic tunicate Doliolum which lack both sarcoplasmic reticulum and transverse tubular system. J. Comp. Physiol. B 172: 541-546.
Jackson, D., Leys, S. P., Hinman, V. F., Woods, R., Lavin, M. F. and Degnan, B. M. 2002. Ecological regulation of development: induction of marine invertebrate metamorphosis. Intl. J. Dev. Biol. 46: 679-686.
Jang, W. S., Kim, K. N., Lee, Y. S., Nam, M. H. and Lee, I. H. 2002. Halocidin: a new antimicrobial peptide from hemocytes of the solitary tunicate, Halocynthia aurantium. FEBS Lett. 521: 81-86.
Jeffery, W. R. 2002. Programmed cell death in the ascidian embryo: modulation by FoxA5 and Manx and roles in the evolution of larval development. Mech. Dev. 118: 111-124.
Jeffery, W. R. 2002. Ascidian gene expression profiles. Genome Biol. 3: 10301.1-1030.4.
Jiang, D. and Smith, W. C. 2002. An ascidian engrailed gene. Dev. Genes Evol. 212: 399-402.
Jimeno, J. M. 2002. A clinical armamentarium of marine-derived anti-cancer compounds. Anticancer Drugs 13 Suppl 1: S15-19.
Kamer, I. and Rinkevich, B. 2002. In vitro application of the comet assay for aquatic genotoxicity: considering a primary culture versus a cell line. Toxicol. in Vitro 16: 177-184; 327 (correction).
Kawahara, G., Terakado, K., Sekiguchi, T., Inoue, K. and Kikuyama, S. 2002. Adrenocorticotropin-like immunoreactivity in the granules of neural complex cells of the ascidian Halocynthia roretzi. Zool. Sci. 19: 1061-1065.
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Kiyota, H., Dixon, D. J., Luscombe, C. K., Hettstedt, S. and Ley, S. V. 2002. Synthesis, structure revision, and absolute configuration of (+)- didemniserinolipid B, a serinol marine natural product from a tunicate Didemnum sp. Org. Lett. 4: 3223-3226.
Kobayashi, M., Matsuda, M., Asakawa, S., Shimizu, N., Nagahama, Y., Satou, Y. and Satoh, N. 2002. Construction of BAC libraries derived from the ascidian Ciona intestinalis. Genes Genet. Syst. 77: 283-285.
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Lambert, G. 2002. Nonindigenous ascidians in tropical waters: a review. Pacific Sci. 56: 291-298.
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Lee, I. H., Zhao, C., Nguyen, T., Menzel, L., Waring, A. J., Sherman, M. A. and Lehrer, R. I. 2001. Clavaspirin, an antibacterial and haemolytic peptide from Styela clava. J. Pept. Res. 58: 445-456.
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Marshall, D. J. 2002. In situ measures of spawning synchrony and fertilization success in an intertidal, free-spawning invertebrate [Pyura stolonifera]. Mar. Ecol. Prog. Ser. 236: 113-119.
Marshall, D. J., Styan, C. A. and Keough, M. J. 2000. Intraspecific co-variation between egg and body size affects fertilisation kinetics of free-spawning marine invertebrates. Mar. Ecol. Prog. Ser. 195: 305-309.
Marshall, D. J., Styan, C. A. and Keough, M. J. 2002. Sperm environment affects offspring quality in broadcast spawning marine invertebrates. Ecology Letters 5: 173-176.
Meenakshi, V. K. 2002. Occurrence of a new species of colonial ascidian - Eudistoma kaverium sp. nov. and four new records of Eudistoma to Indian coastal waters. Indian J. Mar. Sci. 31: 201-206.
Menzel, L. P., Lee, I. H., Sjostrand, B. and Lehrer, R. I. 2002. Immunolocalization of clavanins in Styela clava hemocytes. Dev. Comp. Immunol. 26: 505-515.
Mikhailov, A. T. and Gilbert, S. F. 2002. From development to evolution: the re-establishment of the "Alexander Kowalevsky Medal". Intl. J. Dev. Biol. 46: 693-698.
Mizuta, S., Isobe, S. and Yoshinaka, R. 2002. Existence of two molecular species of collagen in the muscle layer of the ascidian (Halocynthia roretzi). Food Chemistry 79: 9-13.
Monniot, C. 2002. Stolidobranch ascidians from the tropical western Indian Ocean. Zool. J. Linn. Soc. 135: 65-120.
Monteiro, S. M., Chapman, M. G. and Underwood, A. J. 2002. Erratum to "Patches of the ascidian Pyura stolonifera (Heller, 1878): structure of habitat and associated intertidal assemblages". J. Exp. Mar. Biol. Ecol. 275: 83-85.
Monteiro, S. M., Chapman, M. G. and Underwood, A. J. 2002. Patches of the ascidian Pyura stolonifera (Heller, 1878): structure of habitat and associated intertidal assemblages. J. Exp. Mar. Biol. Ecol. 275: 83-85.
Morokuma, J., Ueno, M., Kawanishi, H., Saiga, H. and Nishida, H. 2002. HrNodal, the ascidian nodal-related gene, is expressed in the left side of the epidermis, and lies upstream of HrPitx. Dev. Genes Evol. 212: 439-446.
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