ASCIDIAN NEWS^*

Gretchen and Charles Lambert

12001 11^th Ave. NW, Seattle, WA 98177

206-365-3734

glambert@fullerton.edu or clambert@fullerton.edu

home page: http://depts.washington.edu/ascidian/

Number 57 June 2005

In March and early April Charlie taught a course that dealt chiefly with ascidians at the Sugashima Marine Station of Nagoya University with Hitoshi Sawada and Yoshito Hirada. During this period he also had an opportunity to visit Teruaki Nishikawa in his lab at Nagoya University and learn about advances in ascidian systematics. In April we attended the Intl. Invasive Sea Squirt Conference at Woods Hole where Gretchen gave the keynote address. The meetings were a very exciting time where workers from throughout the world discussed the origins and problems associated with invasive tunicates. Gretchen is expanding the awareness of invasive tunicates locally by teaching dive clubs and other groups how to identify ascidians, a program sponsored by Washington Sea Grant and the Dept. of Fish and Wildlife. She also identified large collections for the Smithsonian and Moss Landing Marine Laboratory in California.

During June and July we will be in Santa Barbara, Calif. where Gretchen will work at the Santa Barbara Museum of Natural History on their ascidian collection and Charlie will work in the laboratory of Kathy Foltz at UCSB. We will attend the Intl. Tunicata meeting at UCSB in July and look forward to getting together with many of you there. In August Gretchen will take part in a rapid assessment survey for nonindigenous ascidians and other invertebrates in Maine. Late in August and early September we will join John Bishop and Frederique Viard on an assessment of invasive ascidians in French harbors, focusing on the English Channel and Atlantic ports around Brittany. There are 114 new publications in this issue of AN! Keep up the good work and please don’t forget to send us hard copies (if in color) or PDFs of your new papers.

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

NEWS AND VIEWS

1. The International Invasive Sea Squirt Conference at Woods Hole, Mass., April 21-22, was a huge success. Approximately 100 people attended. You can see the entire program at the website

http://www.whoi.edu/institutes/oli/activities/seasquirt-program.html

Just click on the individual titles to read the abstracts. The Proceedings will be published in the Journal of Experimental Marine Biology and Ecology, with Bob Whitlatch generously donating a great deal of his time to be the guest editor of this special edition. Attendees wishing to submit manuscripts must do so to Bob by July 1. robert.whitlatch@uconn.edu

2. Don’t forget the Third International Tunicata meeting, July 9-13, 2005 at Univ. of California Santa Barbara. Organized by Bill Smith (UCSB) w_smith@lifesci.lscf.ucsb.edu and Billie Swalla (Univ. of Washington Biol. Dept.) bjswalla@u.washington.edu . See the website: http://www.lifesci.ucsb.edu/urochordate-2005 for the Preliminary Program and other information, including visa information for attendees from outside the U.S.

3. The January 2005 special issue of Canadian Journal of Zoology on the Protochordata is now out (vol. 83 number 1): The issue is dedicated to Dr. Norman J. Berrill. This is the journal’s 4^th special review on various invertebrate groups; earlier ones are annelids (Feb. 2001), echinoderms (July 2001) and cnidarians (Nov. 2002). The editors K.G. Davey, A.S.M. Saleuddin and M.B. Fenton have provided a short Foreword. Titles of all the included papers are marked with an asterisk _* in the New Publications section at the end of this newsletter. You can order a copy of this issue for $35.00 (U.S. or Canadian); send an email to pubs@nrc-cnrc.gc.ca.

4. From Françoise and Claude Monniot: we continue our taxonomic work at the Museum National d’Histoire Naturelle in Paris, with most of our time devoted to tropical ascidians from the western Pacific and Indian Oceans. These specimens have significantly increased the MNHN collections. A new paper about the Polyclinidae and Polycitoridae from the western Indian Ocean will appear in 2006 in Zoosystema. In November we were happy to meet again Xavier Turon from Barcelona, visiting us to examine the Microcosmus collection. A Spanish student, Carmen Primo, stayed at the lab in November 2004 to ascertain her identifications of South African ascidians, and a Tunisian student will spend two weeks with us to learn how to identify her local ascidians. Maybe they are 2 future new ascidiologists!!

Numerous splendid photos are sent to us from all parts of the world, but we are sorry to repeat that serious identifications cannot be given if the picture is not accompanied by the specimen.

4. Recipe for Styela clava!!! Available online (and copyrighted) from the Korea National Tourism organization. It’s called Mideodok-chim (steamed Styela clava). Mmmmmmm. Maybe it’s the garlic, shredded beef, clams, green pepper, onions, watercress, bean sprouts, etc. that make it sound so good (and some sake wouldn’t hurt). If anyone has other recipes for ascidians, please send them to us.

http://english.tour2korea.com/05food/LocalFood/gs_13.asp?kosm=m5_3&konum=5

WORK IN PROGRESS

Didemnum sp. update from Gretchen Lambert: I continue to receive reports of new sightings of this highly invasive species. We now know of 3 sites in British Columbia, large colonies covering vertical rock walls at 7-15 meter depth, near oyster and mussel farms though we do not know if this is significant. Personnel from the Vancouver Aquarium plan a survey this summer. At the one site that has been re-visited, Didemnum sp. is spreading alarmingly rapidly. For the latest photos and location information, see http://woodshole.er.usgs.gov/project-pages/stellwagen/didemnum/index.htm New evidence indicates that the earliest records for this species may be from Japan; this is being verified. This species has been referred to in various parts of Europe by 2 names: Didemnum lahillei and Didemnum helgolandicum. After a careful examination of the type specimens of these species, I now know that it is neither one. I luckily found larvae in the D. helgolandicum type specimen, kindly borrowed from the Hamburg museum, something that Michaelsen missed when he described the species. The larvae have only 2 adhesive papillae and this character, along with the spicule morphology and other characters, indicates that it is actually D. maculosum and thus the name D. helgolandicum is invalid. The name Didemnum lahillei has now been shown by Francoise Monniot, Xavier Turon and Elsa Vazquez to be valid only for Mediterranean and Spanish Atlantic specimens, not the more northern cool-water populations. Thus it appears that this Didemnum sp. is also introduced to Europe. We do not yet have any substantiated European records earlier than the 1990’s.

This information is unpublished, is for readers of Ascidian News only, and must not be cited in any publications!!! This is an ongoing investigation as people around the world struggle to cope with and understand this species.

THESIS ABSTRACTS

1. Yureiboya Comparative Genomics. (Yureiboya means "ghost ascidian" in Japanese, the common term for the Ciona genus.) Dave Johnson, Dept. of Genetics, Stanford Medical School.  Ph.D dissertation submitted March 2005.  dsj@stanford.edu

We use the ascidians Ciona savignyi and Ciona intestinalis, as model systems to investigate current issues in comparative genomics.  First, we develop genomics resources for Ciona savignyi, including a genome sequencing effort. We then characterize the relationship between noncoding sequence constraint and function. We find that the high level of sequence divergence, coupled with virtually identical embryology, makes noncoding sequence comparisons between the Ciona species uniquely sensitive. We then use computational and functional methods to characterize the Ciona muscle-specific regulatory module. We find that conserved clusters of muscle-specific motifs are sensitive predictors of function. We search the C. savignyi genome for conserved motif clusters and find new enhancers with complex expression patterns.  Finally, we use five exemplary gene families to examine evolutionary trends of 5¹ regions and their corresponding proteins (paralogues and orthologues).  We find that constraint in 5¹ regions and proteins sequences are highly correlated.  This work demonstrates that Ciona has strong potential to solve problems that are of interest to the greater genomics community.

2. Ascidians Associated With Coral Reef in Tuticorin Coast. S. Senthamarai, Manonmaniam Sundaranar University, Tirunelveli. M. Phil Thesis. Thesis Supervisor: Dr. V.K. Meenakshi

Studies on the different habitats of ascidians in the Tuticorin sea has not been carried out earlier. Hence a preliminary attempt has been made to study the ascidians associated with the coral reef of Tuticorin coast. This survey carried out for a period of one year showed the presence of 34 species of ascidians belonging to 9 families and 20 genera. The number of genera of ascidians reported from India could be raised from twenty six to twenty seven by the report of Botryllus Gaertner, 1774 for the first time from Indian waters. Of the 34 species of ascidians associated with coral reef, 25 species have already been reported by earlier workers from other habitats and 9 are new records to India. So the present survey could raise the total number or ascidians reported from Indian water from 85 to 94.

3. Multidisciplinary studies of the genus Cystodytes (Ascidiacea): from molecules to species. Susanna López-Legentil, Fac. of Biol., Univ. of Barcelona, Spain. slopez@bio.ub.es Ph.D. advisors Dr. Xavier Turon (Univ. of Barcelona) and Dr. Bernard Banaigs (Univ. of Perpignan, France).

Intraspecies variability is widespread in marine invertebrates. Previous reports of Cystodytes (Ascidiacea, Polycitoridae) in the Mediterranean have been routinely attributed to the nominal species Cystodytes dellechiajei. In the Mediterranean, even though zooid anatomy is remarkably uniform, the general morphology of the colonies varies greatly, especially in terms of color and spicular composition (observed under scanning microscopy). Two different chemotypes, according to their variability in alkaloid chemistry, were defined for 4 different color morphs using both HPLC and RNM techniques and MALDI-TOF analyses. The purple morph displayed a chemotype based on the sulfur-containing pyridoacridines shermilamine B, kuanoniamine D, and their deacetylated forms: deacetylkuanoniamine D and deacetylshermilamine B (new natural product). The blue and green morphs displayed a chemotype based on the C9-unsubstituted pyridoacridines ascididemin and 11-hydroxyascididemin. No major alkaloid was found in the brown form. All of these alkaloids were present in both the tunic and zooids, with the exception of the purple morph, where shermilamine B and kuanoniamine D were found in the tunic, whilst their deacetylated forms were found in both tunic and zooids. Cellular localization of these compounds and ultra structural study of the tunic of the blue, purple and green morphs were attempted by electron microscopy and X-ray microanalysis. The main cell types identified were bladder cells, pigment cells, amebocytes, phagocytes, and morula cells. The sulfur-containing pyridoacridines appeared to be stored in the pigment cells. In addition, the anti-predatory properties of crude extracts, tunic acidity, and spicular contents were assayed in the blue and purple Mediterranean forms, and a purple morph of Guam (USA), using three generalist predators (damselfishes, puffer fishes and sea urchins). Toxicity of crude extracts was also measured by the Microtox bioassay. All crude extracts, as well as ascididemin, were toxic and significantly deterred fish but not sea urchin predation. In contrast, acidity by itself, and spicular shape and concentration did not deter feeding. To determine if the observed variation in the Mediterranean had a genetic basis, a fragment of the mitochondrial gene Cytochrome Oxidase I was sequenced and phylogenetic and population genetic analyses were performed. The results pointed towards the existence of at least 2 species within the 67 samples of 15 colors analyzed. The first well-defined group contained mainly blue colonies with the disc-shaped spicules typical of the genus. The second grouped all samples of a purple morph with sphere-shaped spicules in addition to the disc-shaped ones. When genetic variability was partitioned between color morphs and between geographic locations, the former component explained most of the variance. A study of the biological cycles of the blue and purple Mediterranean morphs was carried out from July 2002 to February 2004. Both the reproductive and growth periods, although partially overlapping, showed significant temporal lags, reinforcing previous genetic and chemical results indicating that these morphs are reproductively isolated and represent distinct species. To determine whether there is a seasonal pattern in the production of chemical and physical defenses, colonies of the blue Mediterranean morph were sampled for a year. Ascididemin was quantified by HPLC and the ash content was calculated as an estimate of the physical defenses. The results showed that when the ascidian invested in reproduction, the energy allocated to other life cycle parameters, such as growth, chemical and physical defenses, was significantly reduced. The multidisciplinary approach used to study the ascidian genus Cystodytes provided an interesting case study through which to develop a better understanding of the biology, ecology and secondary chemistry of marine invertebrates.

ABSTRACTS FROM RECENT MEETINGS

1. Benthic Ecology Meeting, Williamsburg, VA, April 2005. Diurnal, tidal, and lunar patterns of settlement in invertebrate larvae. Stephan Bullard, J. Hamilton, and R.B. Whitlatch. bullard@hartford.edu

Gross patterns of settlement have been documented for numerous species, however, few studies have examined fine-scale patterns of settlement. We used automated in situ sampling devices to monitor diurnal, lunar, and tidal patterns of settlement by invertebrate larvae in Long Island Sound, USA. From July-October 2003, we assessed diurnal differences in larval settlement in seven separate week-long assays; diurnal periods were defined as morning (0300-0900 h), day (0900-1500 h), evening (1500-2100 h), and night (2100-0300 h). To determine if lunar or tidal components affected settlement, we consistently sampled during the weeks of the full or last quarter moon; these lunar phases corresponded to morning high or morning low tides respectively. Different invetebrate species exhibited different diurnal patterns of settlement. The colonial tunicates Botryllus schlosseri and Botrylloides violaceus, the ploychaete Spirorbis sp., and the bryozoan Cryptosula pallasiana had the highest levels of settlement during the day and lowest levels at night. The colonial tunicate Didemnum sp. had high levels of settlement during the day and evening, but significantly lower levels during the morning and night. The solitary tunicate Ciona intestinalis, and the bryozoans Bugula spp. and Electra crustulenta had the highest level of settlement during the morning. The solitary tunicates Ascidiella aspersa and Molgula manhattensis had no significant differences in settlement across diurnal periods. Settlement of invertebrate larvae did not appear to be affected by lunar or tidal factors. Differences in settlement patterns appear to reflect differences in life history strategies between colonial and solitary tunicates.

2. American Academy of Underwater Scientists Meeting, Mystic, CT, March 2005. The invasive colonial ascidian Didemnum sp.: current distribution, basic biology, and potential threat to marine communities of the northeast and west coasts of North America. Bullard S., Lambert G, Carman MR, Byrnes J, Whitlatch RB, Ruiz G, Miller RJ, Harris L, Valentine PC, Collie JS, Pederson J, McNaught DC, Cohen AN, Asch RG, Dijkstra J, Heinonen K. bullard@hartford.edu

Didemnum sp. is a rapidly spreading invasive colonial ascidian that has recently become established on the east and west coasts of North America. It appeared on the west coast during the 1990s and probably arrived earlier on the east coast. Since its introduction, the species has undergone a massive population explosion and is now a dominant member of many subtidal communities on both coasts. To determine Didemnum sp.▓s current distribution, we conducted surveys from Maine to Virginia on the east coast and from British Columbia to southern California on the west coast of the U.S. between 1998 and 2004. In nearshore locations Didemnum sp. currently ranges from Eastport, Maine to Shinnecock Bay, New York on the east coast. On the west coast it has been recorded from Humboldt Bay to Port San Luis, California, several sites in Puget Sound, Washington, including a heavily fouled mussel culture facility, and several sites in southwestern British Columbia. The species also occurs at deeper subtidal sites (> 30 m) off New England, including Georges, Stellwagen, and Tillies Banks. On Georges Bank numerous sites within a 70 km² area are 50-90% covered by Didemnum sp.; large colonies cement the pebble gravel into nearly solid mats that may smother infaunal organisms. These observations suggest that Didemnum sp. has the potential to alter marine communities and affect economically important activities such as fishing and aquaculture.

3. 108th annual meeting of Texas Academy of Science, Univ. of Texas Pan American, Edinburg, Texas, 3-5 March 2005.

Oxidative stress affects late, but not early, embryogenesis in Ascidia interrupta. Anna Stwora, Zen Faulkes,* Univ. of Texas Pan American, Edinburg, TX, and Virginia L. Scofield, Univ. of Texas M.D. Anderson Cancer Center Sci. Park Research Division, Smithville, TX

Ascidians are classic model organisms for the study of development, and sensitive bioindicators of habitat degradation. Oxidative stress is a common source of cell damage that can be caused by many environmental agents. To identify toxic effects of environmental oxidants on ascidian embryogenesis, we examined the effects of the oxidant hydrogen peroxide on the development of tadpole larvae of Ascidia interrupta. A. interrupta were collected from the waters around South Padre Island, Texas. Gametes were fertilized in vitro. After first cleavage, embryos were subjected to oxidative stress by bath applying hydrogen peroxide in concentrations ranging from 0.002 µM to 2 mM. Hydrogen peroxide affected development in a dose dependent fashion: no gross defects or developmental delays were observed at concentrations < 20 µM, whereas embryos in concentrations > 20 µM developed normally until they reached gastrulation, at which point development halted. These data are consistent with recent research indicating that there are potent defense mechanisms that protect embryos’ DNA from external stresses during very early development.

4. VIIth scientific meeting of the Italian Association for Developmental and Comparative Immunology (IADCI), 10-11 February 2005, Consorzio Universitario Provincia di Trapani, Trapani, Italy.

A. Morula cells and non-fusion reaction in the compound ascidian Botryllus schlosseri

L. Ballarin, F. Parisotto, F. Cima, Dipartimento di Biologia, Università di Padova, Padova, Italy.

Morula cells (MC) are a common haemocyte-type in the compound ascidian Botryllus schlosseri, their frequency ranging between 40 and 60% of the circulating blood cells. They are the effector cells of the non-fusion reaction, characterised by the appearance of necrotic foci along the contact border, which occurs when genetically incompatible colonies contact each other. We previously demonstrated that, in the course of this reaction, MC acquire immunopositivity to anti-cytokine (IL-1-and TNF-) antibodies, degranulate and release the enzyme phenoloxidase which is responsible of the cytotoxicity observed both in vitro (when haemocytes are incubated with blood plasma (BP) from incompatible colonies) and in vivo (non-fusion reaction). Subsequently, MC leave the facing marginal ampullae (sausage-like, blind endings of the colonial marginal vessels to reach the tunic, apparently attracted by soluble, diffusing factors, where they degenerate and contribute to the formation of the cytotoxic spots. In the present work, we focussed on the chemotactic recruitment of MC in the course of the nonfusion reaction. As a first approach, we studied the distribution of MC inside the facing marginal ampullae of both contacting colonies (either non-fusible or fusible) and solitary ones. Results clearly indicates a significantly higher concentration of MC inside facing marginal ampullae of incompatible colonies with respect to compatible colonies; the latter concentration is, however, significantly higher than that inside ampullae from solitary colonies. In addition, we used Transwell chambers to evaluate whether incompatible BP has chemotactic properties. We put haemocyte suspensions in filtered sea water (FSW) in the upper wells and BP from either incompatible or compatible colonies in the lower wells. We observed a significant increase migration of haemocytes, and of MC in particular, in the presence of incompatible BP. This migration was significantly decreased by the addition of anti-cytokine (IL-1- α, TNF- α or IL-8) antibodies, suggesting that molecules recognised by these antibodies can be responsible for the chemotaxis observed.

B. Responses of Botryllus schlosseri immunocytes to exogenous cytokines: results and perspectives. A. Menin, L. Ballarin. Dipartimento di Biologia, Università di Padova, Padova, Italy.

We studied the effects of recombinant TNF-α, IL-1-α and IL-8 and of antibodies raised against mammalian cytokines (polyclonal anti- TNF-α and anti- IL-1-α, monoclonal anti-IL-8 and anti-IL-12) on phagocytes of the colonial ascidian Botryllus schlosseri. In particular, we analysed the ability of phagocytes to assume an amoeboid shape (expressed as amoebocytic index) and to phagocyte yeast cells (expressed as phagocytic index). rTNF- α and rIL-1- α have no effects on both the amoebocytic and the phagocytic index, whereas rIL-8 significantly increases both the indexes. The observed increase in the phagocytic index was absent in the presence of calphostin C 0.1 µM, an inhibitor of PKC, and of H89 1 µM, an inhibitor of PKA, indicating the involvement of both the cAMP and IP3 pathways in signal transduction required for phagocytosis to occur. The IL-8- induced increase in amoebocytic and phagocytic index was not observed when haemocytes were pre-incubated in the presence of suramin 0.7 mM, a protein G inhibitor. Anti- TNF-α, anti-IL-1-α and anti-IL-8 antibodies significantly reduce the above indexes; no effects were observed in the presence of anti-IL-12. Our results suggest the presence of molecules able to cross-react with mammalian antibodies, involved in the regulation of phagocyte behaviour.

5. XI International Symposium of Marine Natural Products. Sorrento, Italy, 04-09 Sept. 2004.

A. Direct rapid chemotyping of minute samples from tunicates, marine sponges and associated bacteria with MALDI-TOF Mass Spectrometry. Dieckmann, R., López-Legentil, S., Turon, X., von Döhren, H. Fac. of Biol., Univ. of Barcelona, Spain.

Sessile marine animals coexisting with and harboring specific microbial populations show variable morphologies and compositions depending on colony structure, environmental conditions and genotypes. Probing chemical composition with minimal invasive techniques with respect to both
metabolites and macromolecules can be accomplished with several highly sensitive techniques including MALDI-TOF mass spectrometry. In addition to the characterization of minute environmental samples from different sources, this technique permits also the rapid dereplication of large numbers of bacteria with respect to diversity and biosynthetic activities. We have used a mass spectrometric approach for rapid characterization of metabolites in ascidians and sponges. In ascidians, we have determined the presence of e.g. didemnines in Trididemnum solidum and Aplidium albicans,
rubrolides in Synoicum blochmanni and Ecteinascidins in Ecteinascidia turbinata. For the species Cystodytes dellechiajei, we have detected the existence of two chemotypes containing the sulfur-containing pyridoacridines shermilamine B, kuanoniamine D and their deacetylated forms, or the C9-unsubstituted pyridoacridines ascididemine and 11-hydroxyascididemin.
In sponges, chemotype determination of the sponges Axinella damicornis and A. verrucosa yielded identical Maldi-tof spectra, which suggests the presence of similar microbial  populations or a lack of chemical differentiation of these two species. In an analysis of complex populations of boreal sponges from the European Lophelia reefs prominent metabolites have been directly identified e.g. from Geodia baretti (barettin) and Spongosorites (topsentins). Intact-Cell mass spectrometric profiles (ICM-MS) of metabolites and macromolecules in the range up to 20 kDa also provide rapid essential information to dereplicate bacterial isolates from marine sources with respect to phylogenetic differences and biosynthetic activities. The ease and speed of the method makes the mass spectral analysis the method of choice for the screening of large numbers of isolates and to identify new or rare groups.

B. Intraspecific variability in the pyridoacridine composition of the ascidian Cystodytes dellechiajei. Bontemps-Subiélos, N., López-Legentil, S., Jamme, F., Long, C., Banaigs, B. Fac. of Biol., Univ. of Barcelona, Spain.

Intraspecies variability has been a long-standing source of taxonomic and biological controversy. Species having a large range of distribution (or cosmopolitans) usually show some morphological differences, usually related to their geographical or bathymetric distribution. Color variation is perhaps the difference most frequently reported, but texture, general shape and other morphological characters may also change from one individual to another. However, the possible implications of these variations in secondary chemistry remain unknown. Cystodytes dellechiajei (Della Valle, 1877) (Aplousobranchiata, Polycitoridae) is a colonial soft-bodied ascidian widely distributed around the world in tropical and temperate waters. Although some spicular and color differences exist, so far there is little basis to split C. dellechiajei into several species mainly because the morphology of zooids is remarkably uniform. Several pyridoacridines, a group of highly colored polycyclic aromatic alkaloids, were reported in C. dellechiajei, among these ascididemin, 11-hydroxyascididemin, cystodytins A-I, shermilamine B, kuanoniamine D and sebastianines A and B.

To determine the relationship between secondary chemistry and morphotype variation, the pyridoacridine distribution in 4 color morphs (green, blue, purple and brown) of C. dellechiajei from the western Mediterranean was assessed. More than 10 compounds were detected, among them: styelsamines and deacetylkuanoniamine D, never reported before in this genus, and 3 new compounds.

Besides, two major chemotypes were found: the first presented the sulfur-containing pyridoacridines shermilamine B and kuanoniamine D, corresponding to the purple morph. The second, presented the C₁₀-unsubstituted pyridoacridines: ascididemin and 11-hydroxyascididemin, corresponding to the blue and green morphs. No alkaloid was observed in the brown form. The pyridoacridine alkaloids identified in this study were characterized by a combination of mass spectrometry and one and two-dimensional NMR techniques on purified compounds. The structure elucidation of the 3 new compounds, deacetylshermilamine B from the purple morph and cystodimines A and B from the blue-green morphs is described together with the 2 chemotypes characterized by high performance liquid chromatography coupled with photodiode array detection or mass spectrometry.

6. XIII Iberian Symposium on Marine Benthos. Canary Islands, Spain, - 21-24 September 2004. The usefulness of genetics and secondary chemistry in assessing intraspecies variability: the colonial ascidian Cystodytes dellechiajei. López-Legentil, S., Dieckmann, R., Turon, X. Fac. of Biol., Univ. of Barcelona, Spain.

Intraspecies variability is widespread in marine invertebrates. Size, colour, texture, general shape and secondary chemistry can differ quite drastically from one individual or population to another. We chose as case study the colonial ascidian Cystodytes dellechiajei (Polycitoridae), which presents several morphotypes differing mainly in color and spicular composition. New molecular and chemical tools enable us to assess the taxonomic status of these morphotypes. To determine whether variation observed in Mediterranean C. dellechiajei has a genetic basis, we sequenced 45 specimens from eight locations of the western Mediterranean and obtained a 617 bp fragment of the mitochondrial gene COI. In addition, the alkaloid distribution in the most abundant color morphs (green, blue and purple) was assessed by MALDI-TOF MS. 15 different color morphs were recorded and 3 kinds of spicules were found: disk-shaped, typical of the genus, sphere-shaped and star-shaped. Zooid morphology was remarkably uniform in the whole sample set. Different tree reconstruction methods (distance-based, parsimony-based, and maximum-likelihood-based) yielded consistent results, and 6 major clades were recognized, which had no correspondence with spicule shape and were only partially consistent with color morphs. Two major chemotypes were found: that of the sulfur-containing pyridoacridines, shermilamine B and kuanoniamine D, for the purple morph; and that of the C₉-unsubstituted pyridoacridines, ascididemin and 11-hydroxyascididemin, for the blue and green morphs. MALDI-TOF MS analyses required a few mg of material and were rapid and sensitive in detecting targeted substances and determining chemotypes. Both genetic and chemical results showed that color and spicular shape were not consistent enough to be used to differentiate between Cystodytes species. We point out the importance of genetics and secondary chemistry in assessing the taxonomic status of species with variable morphology.

NEW PUBLICATIONS

Titles of all the papers in the special Protochordate issue of the Canadian Journal of Zoology are marked with an asterisk _*.

Aiello, A., Fattorusso, E., Luciano, P., Macho, A., Menna, M. and Munoz, E. 2005. Antitumor effects of two novel naturally occurring terpene qinones isolated from the Mediterranean ascidian Aplidium conicum. J. Med. Chem. 48: 3410-3416.

Ballarin, L., Menin, A., Franchi, N., Bertoloni, G. and Cima, F. 2005. Morula cells and non-self recognition in the compound ascidian Botryllus schlosseri. Invert. Survival J. 2: 1-5.

Barenbrock, J. S. and Kock, M. 2005. Screening enzyme-inhibitory activity in several ascidian species from Orkney Islands using protein tyrosine kinase (PTK) bioassay-guided fractionation. J. Biotechnol. 117: 225-32.

*Bates, W. R. 2005. Environmental factors affecting reproduction and development in ascidians and other protochordates. Can. J. Zool. 83: 51-61.

Bellas, J., Beiras, R., Marino-Balsa, J. and Fernández, N. 2005. Toxicity of organic compounds to marine invertebrate embryos and larvae: A comparison between the sea urchin embryogenesis bioassay and alternative test species. Ecotoxicology 14: 337-353.

Bennett, C. E. and Marshall, D. J. 2005. The relative energetic costs of the larval period, larval swimming and metamorphosis for the ascidian Diplosoma listerianum. Mar. Freshw. Behav. Physiol. 38: 21-29.

Blunt, J. W., Copp, B. R., Munro, M. H., Northcote, P. T. and Prinsep, M. R. 2005. Marine natural products. Nat. Prod. Rep. 22: 15-61.

Braithwaite, R. A. and McEvoy, L. A. 2005. Marine biofouling on fish farms and its remediation. Adv. Mar. Biol. 47: 215-252.

Bullard, S. G. and Whitlatch, R. B. 2004. A guide to the larval and juvenile stages of common Long Island Sound ascidians and bryozoans. Connecticut Sea Grant Communications Office, Univ. of CT, 1080 Shennecossett Road, Groton, CT 06340, Groton, CT.

*Cameron, C. B. 2005. A phylogeny of the hemichordates based on morphological characters. Can. J. Zool. 83: 196-215.

Castilla, J. C., Uribe, M., Bahamonde, N., Clarke, M., Desqueyroux-Faúndez, R., Kong, I., Moyano, H., Rozbaczylo, N., Santelices, B., Valdovinos, C. and Zavala, P. 2005. Down under the southeastern Pacific: marine non-indigenous species in Chile. Biol. Invasions 7: 213-232.

Christiaen, L., Bourrat, F. and Joly, J. S. 2005. A modular cis-regulatory system controls isoform-specific pitx expression in ascidian stomodaeum. Dev. Biol. 277: 557-566.

*Cone, A. C. and Zeller, R. W. 2005. Using ascidian embryos to study the evolution of developmental gene regulatory networks. Can. J. Zool. 83: 75-89.

Davis, H. H. and Davis, M. E. 2004. The distribution limits of Styela clava (Tunicata, Ascidiacea) in European waters. Porcupine Mar. Nat. Hist. Soc. Newsletter 15: 35-43.

Davis, M. H. and Davis, M. E. 2005. Styela clava (Tunicata: Ascidiacea) -- a new addition to the fauna of the Portuguese coast. J. Mar. Biol. Ass. U.K. 85: 403-404.

Dolcemascolo, G., Gianguzza, P., Pellerito, C., Pellerito, L. and Gianguzza, M. 2005. Effects of tri-n-butyltin(IV) chloride on neurulation of Ciona intestinalis (Tunicata, Ascidiacea): an ultrastructural study. Applied Organometal. Chem. 19: 11-22.

Du Pasquier, L. 2004. Speculations on the origin of the vertebrate immune system. Immunol. Lett. 92: 3-9.

Duckworth, A. R., Samples, G. A., Wright, A. E. et al. 2004. In vitro culture of the ascidian Ecteinascidia turbinata to supply the antitumor compounds ecteinascidins. Aquaculture 241: 427-439.

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