Charles and Gretchen Lambert

12001 11th Ave. NW, Seattle, WA 98177

206-365-3734 or

home page:


Number 54                                                                                                                   December 2003


   There are 118 new publications listed at the end of this newsletter. This is a longer than usual newsletter and seemed to be a lot more work for GL than usual; thanks to all of you who sent contributions, and thanks also for the many letters of support. We are happy to know that AN seems to be a very useful resource for many of you; that makes it worth the effort. 
   During June and July we worked at the Friday Harbor Labs where Gretchen completed a manuscript on the southern hemisphere Corella eumyota that we found in 2 harbors in Brittany in 2002 (the paper will appear in the Jan.-Feb. 2004 issue of J. Mar. Biol. Assoc. UK; we ask that you keep your eyes open for this recent invader and let us know if you find any) and Charlie worked on germinal vesicle breakdown in Boltenia villosa oocytes.  We very much enjoyed interacting with John Bishop from Plymouth who presented the Illg memorial lectures.  The summer was greatly enlivened by spirited discussions on evolution and phylogeny by Klaus Nielsen, Billie Swalla, Doug Eernisse, Ken Halanych, Richard Strathmann and others. In August we surveyed the major harbors of 9 New England states in 7 days with a number of specialists of various taxa.  The survey was organized by Judy Pederson as part of her monitoring of east coast harbors for invasive species, and was sponsored by the Env’l. Protection Agency and Massachusetts Sea Grant.  We were especially on the lookout for invasive ascidians, but were amazed at the incredible biomass of Molgula manhattensis in many of the harbors. The other most common species were Ascidiella aspersa, Styela clava, Botrylloides violaceus and the newest invader Didemnum “vexillum” which has now been found not only in inshore areas but smothering up to 90% of the sea floor in a 6.5 square mile area of the Georges Bank at about 50 meters (P.C. Valentine and T. Frady pers. comm.). What is apparently the same species has therefore now invaded New England, northern California, New Zealand and Brittany (France), and has the ability to grow rapidly not only on man-made structures but also natural substrates.

   September found us in Ketchikan Alaska for a week with several workers from the Smithsonian bioinvasions group.  We surveyed harbors and intertidal areas and identified all the ascidians there.  On several settling plates suspended at 1m depth from floating docks we saw both Corella willmeriana and C. inflata, an unusual finding, as C. willmeriana is generally much deeper.  In January we will spend a week at the Smithsonian bioinvasions lab in Edgewater Maryland identifying hundreds of ascidian vouchers and later in the year will survey harbors in Florida, South Carolina and Nova Scotia with the same group.

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




1. From Dr. Hitoshi Sawada, Sugashima Marine Biological Lab., Graduate School of Science, Nagoya Univ., Toba 517-0004, JAPAN Phone: +81-599-34-2216, FAX:   +81-599-34-2456, email:

  I am organizing "The 4th International Symposium on the Molecular and Cell Biology of Egg- and Embryo-Coats", which will be held in Shima, Mie-Prefecture, Japan, November 8-12, 2004.  The organizing committee is now considering the program and speakers.  The first circular of the symposium (4th MCBEEC) will be published on-line by the end of this year at the following website.


2. From Jarrett Byrnes, Population Biology Graduate Group, Univ. of California, Davis, CA

     The diversity of ascidians around the world is truly stunning.  Here on the West Coast of North America, Light's Manual alone lists 46 species without including several recently introduced species.  As field ecologists, it is important to be able to rapidly identify species in the field.  Taking a cue from Arjan Gittenberger's, the Stachowicz lab at University of California, Davis, has begun collecting and identifying photographs of a variety of ascidians both in adult and settler stages from the North American West Coast and posting them on the web so that any scientist can use them as a reference.  This site, located at , allows visitors to add comments on photos, so that others may share their notes, tips, and tricks on identifications.  We recognize that the gallery is currently an incomplete catalog, and would welcome any outside contributors.  We are also open to hosting galleries for ascidians from other geographic locales, as well as other marine taxa.  For more information, please contact .


2. From Tito Monteiro da Cruz Lotufo ( and Rosana Moreira da Rocha (

    With deep sadness we must inform you that Dr. Sergio de Almeida Rodrigues passed away on October 14th, after a difficult battle with pneumonia. Dr. Rodrigues (Sergio to his friends and loved ones) was born in 1937, in São Paulo, Brazil.  He obtained his bachelor’s degree in Natural History and shortly thereafter was invited to be the first resident assistant in the new Marine Biology Center of the University of Sao Paulo.  He began his studies of ascidians at that time, publishing several of the few papers then available on this group.  Dr. Rodrigues then turned his attention to the study of thallassinid crustaceans, becoming one of the most renowned specialists worldwide on this group, describing many new species and publishing many papers.  During his crustacean years, Dr. Rodrigues never abandoned his interests in ascidians, deciding in the early 1990¹s to return with all his efforts to the study of these wonderful animals. Throughout this time, Dr. Rodrigues advised all the Brazilian students of ascidians. He retired as a Full Professor in the Department of Ecology at the University of Sao Paulo, yet continued his work.  As a result of his enthusiasm in the study of ascidians, with diving and collecting, photographing and describing in Brazil and elsewhere, Dr. Sergio Rodrigues fathered the only two research groups currently working on Brazilian ascidians, and his memory will be cherished by his former students and friends. Dr. Sergio Rodrigues is survived by his companion, Gisela Shimizu, and his two sons.


3.  From Hiroki Nishida: I will be moving to Osaka University to be a professor there next

March. Our lab will continue to study ascidian embryogenesis there and will also start research on larvacean Oikopleura development. The following address will be effective after mid March:

Dept. of Biology, Graduate School of Science, Osaka Univ., 1-1 Machikaneyama-cho, Toyonaka, Osaka 560-0043, Japan  Tel: +81-6-6850-5472    email:


4. From Gretchen Lambert: Did you know that you can search each issue of AN online using the Find command in the web browser? It is one of the choices in the Edit menu. 

   More and more websites are being created that are either devoted completely to underwater color photos of ascidians or include ascidians along with other marine invertebrates. Here are a few; if you know of others please contact me and I will include them in the next AN.

a)                  Bernard Picton's Encyclopedia of marine life of Britain & Ireland at

b)         U.S. west coast; described more fully at the top of this page

c)         developed by Arjan Gittenberger of the Natl. Mus. Of Nat. History in Leiden, the Netherlands: see AN#53 page 2 for more details.

5.  From Teruaki Nishikawa, Nagoya University Museum, Chikusa-ku, Nagoya, Japan ( At the Phuket Marine Biological Center (PMBC) in Thailand, the "Internatioal Training Course and Workshop on Environmental Technology related to Taxonomy, Biology and Ecology of Ascidians in Thai Waters" was held 22-28 Nov. 2003. It was organized by PMBC and co-sponsored by PMBC, Southeast Asia START Regional Center (SEA START RC), and National Center for Genetic Engineering and Biotechnology (BIOTEC)), organized by Dr. Somchai Bussarawit (PMBC).  As the "resource person", I gave many lectures and practices about ascidian taxonomy, biology and ecology to about 20 participants (including 2 Malaysian MC students, other than Thai students and scientists).  Dr. Kanit Suwanarak of Chulalongkorn University gave a lecture on pharmaceutical aspects of ascidian bioreactive substances. For about a week prior to the workshop, I  prepared lectures and practices, mainly based on the materials collected from a pearl oyster farm in Phuket.  I was astonished to find 18 species of ascidians among the fouling organisms.


6.  From Dr. Patricia Kott ( ), to the Tunicata email listserv on June 5 of this year, on the precedence of subphylum Tunicata over Urochordata. (To subscribe, click on the link at the bottom of the AN home page.)  We felt it was important to re-run it in AN.

Dear list members,

   Quite correctly we are all subscribers to a tunicate list- not a urochordate list. If you are uncertain about this the following notes could help:

The correct name is TUNICATA Lamarck, 1816: The name was used by Lamarck to accommodate the related groups of organisms, ascidians, Pyrosoma and salps. Subsequently Milne Edwards (1843) added the Bryozoa to the Tunicata in the Class Molluscoidea; then Hancock (1850) added Brachiopoda to the Bryozoa and Tunicata in the Molluscoidea; and finally Huxley (1851) recognised the Tunicata (ascidians, salps, doliolids and Appendicularia) as a distinct phylogenetic entity separate from Mollusca, Bryozoa and Brachiopoda. This was later supported by Bronn (1862). Kowalewsky (1866) recognised a chordate affinity in the notochord -like cells in the larval tail and the group Tunicata was regarded as a subphylum of the Chordata. 

   The name Urochordata was not used until Balfour (1881), quite un-necessarily, created it as a replacement name for Tunicata, presumably to emphasise the chordate affinity. A perfectly good name already existed for a well defined entity and a replacement name was not required. The name Urochordata is a junior synonym of the name Tunicata. The use of the junior synonym for the phylogenetic entity originally established by Lamarck in 1816 is inappropriate. Balfour also unnecessarily introduced the names Perennichordata (for Appendicularia with a tail through life) and Caducichordata (Thaliacea, which occasionally have a tailed larva, and Ascidiacea which always do [with a few molgulid and styelid direct developer exceptions; GL]). There is no justification for the erection of replacement names based on a single character subjectively judged to be of greater significance than others. This practice causes ambiguity and certainly does not lead to the stability in nomenclature that is desirable. The name Tunicata is almost universally used to refer to this group of organisms in the major monographic works on any of its contained Classes, e.g Alder (1863), Herdman ( 1882, 1886 etc.), Alder and Hancock (1905-12), Harant and Vernieres (1933), Brien (1948), Van Name (1945), Berrill (1950), van Soest (1970s-1990s), Fenaux (1993), Bone (1998) and many others.

I hope this helps- see Fenaux(1993) and Brien (1948) for further clarification.

Patricia Kott


7.  From Charles and Gretchen Lambert. Further comments on classification within the Ascidiacea. 

   The Tunicata includes 4 classes of organisms: Ascidiacea, Sorberacea (considered part of the Ascidiacea by Dr. Patricia Kott), Appendicularia (formerly called Larvacea) and Thaliacea.  The first two are sessile and the second two are pelagic as adults, but most have swimming juveniles. Ascidians comprise the most numerous and widely known members of the subphylum or phylum (another issue in classification).  In most modern treatments the class is divided into the orders Enterogona and Pleurogona with the suborders Aplouosobranchia and Phlebobranchia in the Enterogona and a single suborder, the Stolidobranchia in the Pleurogona (Abbott et al. 1997, Kott 1985).  Originally the suborders were designated as the orders Aplousobranchia, Phlebobranchia and Stolidobranchia (Lahille 1886). This was based upon the complexity of the branchial sac. Subsequently, Perrier (1898) devised the orders Enterogona and Pleurogona based upon the position of the gonads and other morphological considerations. Garstang (1928) and others such as Huus (1937) combined the classification of Lahille and Perrier incorporating Lahille’s Aplousobranchia and Phlebobranchia as suborders of the Enterogona with the Pleurogona containing only the suborder Stolidobranchia (Berrill, 1950). However, this classification has not been universally accepted. Van Name (1945) retained the 3 orders of Lahille in his monumental treatise as did the Monniots in their 1991 book Coral Reef Ascidians of New Caledonia (which remains the most comprehensive account of all aspects of ascidian biology that we know of). In addition, Lahille’s 3 orders are listed as current designations in a recent textbook of invertebrate zoology (Ruppert and Barnes, 1994). 

   Thus, it is clear that the terminology of Lahille has precedence over that of Perrier and several prominent ascidiologists have recognized this. Moreover, uniting the suborders Aplousobranchia and Phlebobranchia into the order Enterogona implies an evolutionary relationship that may or may not be supported. Therefore we prefer Lahille’s 3 orders and advocate that this classification should be adhered to in all future accounts of the ascidians. We also commend Dr. Kott for her succinct review of the precedence of using subphylum Tunicata over Urochordata.


Abbott, D. P., Newberry, A. T., and Morris, K. M. (1997). Reef and Shore Fauna of Hawaii. 6B: Ascidians (Urochordata). Bishop Mus. Press, Honolulu.

Berrill, N. J. (1950). The Tunicata with an Account of the British Species. The Ray Society, London. 354 pp.

Garstang, W. (1928)  The morphology of the Tunicata, and its bearings on the phylogeny of the Chordata. Quart. Jour. Micr. Sci., 72: 51-187.

Huus, J. (1937) Tunicata: Ascidiaceae. Handb. Zool. Kukenthal und Krumbach, V, second half, pp543-672.

Kott, P. (1985). The Australian Ascidiacea part 1, Phlebobranchia and Stolidobranchia.  Mem. Queensland Mus. 23: 1-440.

Lahille, F. (1886). Sur la classification des tuniciers. CR Acad Sci Paris 102: 446-448.

Monniot, C., Monniot, F. and Laboute, P. (1991). Coral Reef Ascidians of New Caledonia. Orstom, Paris. 247 pp.

Perrier, J.O.E. (1898) Note sur la classification des Tuniciers. C.R. Acad.Sci. Paris 126: 1758-1762.

Ruppert,E.E, Barnes, R.D. (1994) Invertebrate Zoology 6th Edition. Saunders, New York, 1056 pp.

Van Name, W. G. (1945). The North and South American Ascidians. Bull. Amer. Mus. Nat. Hist. 84: 1-476.




1. Mary Carman, Geology & Geophysics, Woods Hole Oceanog. Inst., Woods Hole, MA 02543 (  I am studying the foraminifera that preferentially live on both native and non-native species of ascidians along the New England coast in the near shore, upper subtidal zone. The benthic forams that attach to ascidians living on rocks include calcitic and agglutinated forms.  The benthic forams that live on ascidians attached to floating docks are calcitic only.  Benthic forams have been previously documented as living on hydroids and calcareous algae, up to a few centimeters off the bottom.  However, they have rarely been documented living on ascidians, nor have they been previously documented as living up to nine meters above the bottom sediments. At the same location, forams living on the varied species of ascidians may be taking on different oxygen and carbon stable isotopes, than the forams living in the bottom sediments.  Therefore, I am comparing the stable isotopes of the infaunal and epifaunal benthic forams to the stable isotopes of the calcareous spicules in Didemnum.  I am documenting just exactly where the native and non-native species of ascidians, including Didemnum vexillum, are living in the near shore environment around Cape Cod.


2.  P. Frank, A. DeTomaso, B. Hedman and K. O. Hodgson: "Perophora surprise: a new structural motif for biological iron." Dept. of Chemistry; Hopkins Marine Station; and The Stanford Synchrotron Radiation Laboratory, SLAC; Stanford Univ., Palo Alto, Calif.

   At the 30th Annual Users' Meeting at SSRL in October 2002, we reported the preliminary results of our x-ray absorption study of iron in Perophora annectens, whole blood and whole body (noted in AN 52). We have now looked at 2 independent collections, and the iron is of a constant type and in very high abundance. Vanadium is in very low amounts. The iron looks to be an iron-oxo complex containing more than 2 iron atoms per unit and does not look like the biological iron in ferritin. The structure is similar in many respects to the Fe4O4 cube found in the mineral magnetite but with clear structural differences that show up in the x-ray spectra. This is a brand new motif for biological iron. In the near future, we intend to report the full details of the proposed new structure coming from EXAFS analysis. What is most clear is that blood cell iron in P. annectens is not Fe+2 or Fe+3 floating as the aqua ion in acid solution, as one finds for V+3 in many ascidians.  We do not know whether the iron in P. annectens is similar to biological iron in other iron-containing ascidians, or is unique to the species.


3.  Charles Lambert: Enzymatic removal of the ascidian egg vitelline coat at low pH.  Previous methods of enzymatic removal of the VC involved S-S reduction at high pH along with a proteolytic enzyme (Mita-Miyazawa, Ikegami and Satoh, 1985, J. Embryol. Exp. Morphol.87: 1-12; Byrd and Lambert 2000. Molecular Repro. and Dev. 55: 109-116).  For work on GVBD I needed VC-free oocytes that had never been exposed to pHs above 4. Tris(2 carboxyethyl)phosphine hydrochloride (TCEP) is reported to reduce S-S bonds irrespective of pH. To 10 ml of Boltenia villosa oocytes in pH 4 SW (10mM citrate) add 143 mg TCEP (Molecular Probes T-2556) and 10 mg pepsin (Sigma).  Incubate with shaking for 1 hour at 10-120C. and wash several times with pH 4 SW.  Most of the oocytes will be VC free.


4.  From Christian Sardet, Station Zoologique, Villefranche-sur-Mer, France ( ) : We had a great meeting in Carry le Rouet near marseille organized by the Lemaire team where we felt the ascidian developmental biology community was really taking off. Villefranche  had 8 abstracts at the Marseille meeting.  We have now 3 new Research Staff in Villefranche all recruited by the CNRS and  starting labs: Alex Mc Dougall (signalisation/ calcium/cell cycle control) and Hitoyoshi Yasuo (notochord formation) working with Clare Hudson (neural development). See our web site:    We will prepare a new edition of the site for 2004.  There are 2 multimedia documents (BioClips) where ascidian eggs and embryos are featured. They can be downloaded on our site devoted to this new way of exchanging informations via multimedia documents:

   Another project (in French for the moment) is our effort to present the history, patrimony and science of Villefranche on a site for the public ( Jean and Colette Febvre who are now retired are very active in this context.  Finally on a personnal level I am busy finishing my term as president of the French Cell Biology Society and organizing the next big Meeting in Nice (World Congress of Cell Biology: 25000 people expected) with a new and very dynamic European Society called ELSO (This will be an exciting international meeting.  See




1. International Urochordate Meeting 2003, Carry le Rouet, France, October 11 - 15th

Organized by Patrick Lemaire, Marseille (France)

Opening lecture: Let's move on ascidian biology with new ideas. Nori Satoh, Dept. of Zool., Kyoto Univ., Kyoto, Japan.       

Session 1.1: From fossil evidence to molecular phylogeny

Session 1.2: Evolution of developmental patterns 

Session 2: Characteristics of the tunicate genomes 

Session 3: Functional analysis of the ascidian genomes: Tools and approches

Session 4: Early embryonic patterning 

Session 5.1: Neural development and chordate evolution

Session 5.2: Neural function

Session 6.1: Oogenesis, fertilization and early development

Session 6.2: Metamorphosis and immunity

Click on link to see the complete list of speakers (with addresses, most with email), abstracts, and posters:


2. 9th Intl. Congress of the Intl. Soc. for Dev. and Comp. Immunology, Univ. of St. Andrews, Scotland, UK 29th June - 4th July 2003


a) Immunotoxicity of Cu(I) and Irgarol 1051 in ascidians. F. Cima, P. Burighel, L. Ballarin, Dept. of Biol., Univ. of Padova, Italy

   After the widespread ban of TBT due lo a severe impact to coastal biocenoses mainly related to its immunosuppressive effects on both invertebrate and vertebrates, alternative biocides like Cu(t) salts and the triazine Irgarol 1051 (previously used in agriculture as a herbicide) have been massively introduced in combined formulations of antifouling paints against a wide spectrum of fouling organisms. Our interest in the study of ascidian defence reactions led us to investigate the effects of Cu(l) and Irgarol on cultured phagocytes of the colonial ascidian Botryllus schlosseri, as previously done with TBT. We set up short-term haemocyte cultures (60 mìn) exposed to sublethal concentrations of these compounds (Cu(l) LC50 =281 µM; Irgarol LC50 > 500 µM). In contrast to TBT, both substances did not cause significant effects on cell morphology. Generally, Cu(I) appeared more toxic than Irgarol; it significantly inhibited(p < 0.05) yeast phagocytosis at 0.1 µM, and affected calcium homeostasis and the mìtochondrial cytochrome-c-oxidase activity at 0.01 µM. Both substances were able to change membrane permeability, induce apoptosis from concentrations of 0.1 µM and 200 µM for Cu(I) and Irgarol, respectively, and alter (with different mechanisms) the activity of hydrolases (acid phosphatase, esterases) and oxidases (phenoloxidase). Although both the xenobiotics are less toxic tram TBT and their LC50 values are lower than the concentrations in the aquatic environment, their impact on organisms must be considered as they can alter immune defences and consequently endanger the survival of the individual.


b) Morula cell behaviour in the rejection reaction between incompatible colonies of the ascidian Botryllus schlosseri.  L. Ballarin and F. Cima, Dept. of Biol., Univ. of Padova, Italy.

   Contact between genetically incompatible colonies of the ascidian Botryllus schlosseri result in a rejection reaction that is characterised by the appearance of a series of dark-brown necrotic spots along the touching borders of the facing vascular ampullae. Morula cells (MC), a common haemocyte-type in botryllid ascidians, are directly involved in this reaction as they contain and release the enzyme phenoloxidase which is responsible for the observed cytotoxicity. Since MC are known to undergo in vitro degranulation upon the recognition of incompatible blood plasma, we re-investigated the whole rejection process with particular reference to the behaviour of MC. MC were observed to crowd inside the ampullar tips in early stages of the rejection: their vacuoles share an equal size and an uniformly electron dense contents, which are yellow green in colour after aldehyde fixation and positive for phenoloxidase. As their migration into the common tunic begins, their vacuolar contents progressively flake off and are finally released into the ampullar lumen or in the tunic as the MC degranulate. Most of the degranulated MC are still observable inside the ampullae in advanced stages of the rejection process. Just before the beginning of degranulation, MC acquire immunopositivity lo anti-lL-1-a and anti-TNF-a, thus confirming their important immunomodulatory rote. In vitro experiments demonstrate that the synthesis of cytokine-like molecules is consequent to the recognition of humoral factors from incompatible, allogeneic blood plasma and is followed by an increase of nitrite concentration in the incubation medium.


c) External amoebocytes perform immunosurveillance of the pharynx entry in ascidians (Urochordata). P. Burighel, L. Ballarin, F. Gasparini, F. Caicci, F. Cima, Dip. Biol., Univ. di Padova, Italy

   In vertebrates, the mouth and gills are the principal targets of pathogen invasion and these are protected by motile, phagocytic and cytotoxic cells of the local lymphatic tissues. The body or lower chordate ascidians is covered by the tunic, which extends over the epidermis and into both the siphons. In botryllids, the tunic contains various cell types. particularly granular amoebocytes coming from blood, and forms a superficial cuticle bearing numerous papillae protrusions, An unusual feature that we observed was the presence of granular amoebocytes, over the tunic into both the siphons of Botryllus schlosseri, completely exposed to seawater These free amoebocytes in the oral siphon were especially accumulated at the base of the tentacles, and were in contact with the cuticle protrusions and their long filopodia. Electron microscopy revealed that the amoebocytes appear mononucleate and with numerous round granules, varying in content. We consider these cell represent “sentinel-cells” belonging to the phagocytic line of the immune system since they share with blood phagocytes the same hydrolytic enzyme pattern, and labelling by both n-mannose specific lectin and anti-CD39 antibody produced against mammalian macrophages. After exposing the filtering colonies lo bacterial spores the external amoebocytes were observed to contain bacteria inside heterophagic vacuoles, starting after 5 min exposure to the spores. Moreover, these cells seem to participate in cell signalling since they cross the siphonal epidermis triggering a cascade of events leading to morula cell degranulation inside the siphonal blood sinus and the progressive increase of circulating, bacteria-containing macrophages, which were finally discharged into the peribranchial chamber.


d) Complement mediated chemotaxis in the deuterostome invertebrate Ciona intestinalis. Maria Rosaria Pinto1, Cinzia M. Chinnici3, Yuko Kimura2, Rita Marino1, Daniela Melillo1, Rosaria De Santis1, Nicolò Parrinello3, John D. Lambris2  1Cell Biol. Lab., Stazione Zool. “A. Dohrn”, Napoli, Italy; 2Protein Chemistry Lab., Univ. of Pennsylvania, U.S.A.; 3Dept. of Animal Biol., Univ. of Palermo, Italy

  Some deuterostome invertebrates posses complement-like genes and in limited instances, complement mediated functions have been reported for invertebrate species. However, the organization of complement pathway(s) as well as the functions exerted by the cloned gene products is largely unknown. There is no evidence of the inflammatory and lytic pathways, which are key effector mechanisms of the mammalian complement cascade. To address this issue, we have initiated studies to characterize the structure and functions of Ciona intestinalis complement components and receptors. In a recent study, we have cloned two C3-like genes, CiC3-1 and CiC3-2, from C. intestinalis. Here we expressed the fragment of C3-1a (rCiC3-1a) that corresponds to mammalian C3a and assessed its chemotactic activity using C. intestinalis blood cells. The CiC3-1a was expressed in E. coli, purified using nickel chelating affinity chromatography and HPLC, and its identity verified by mass spectrometry. Migration of C. intestinalis coelomocytes towards rCiC3-1a was dose-dependent, peaking at 500 nM and was specific for rCiC3-1a as it was inhibited by an anti-rCiC3-1a specific antibody. Similarly to the mammalian C3a, the chemotactic activity of C. intestinalis C3-1a is localized at the C-terminus of the C3a molecule as a peptide representing the 18 C-terminus amino acids (CiC3-1a59-77). Ci-C3a promotes, similarly to the expressed molecule, coelomocyte chemotaxis. The C3a mediated chemotaxis was inhibited by pre-treatment of cells with pertussis toxin thus suggesting that the receptor molecule mediating the chemotactic effect is Gi protein-coupled. The possible role of complement in C. intestinalis inflammatory processes will be discussed.


e) Inflammation in ascidians. N. Parrinello, C. Chinnici, A. Vizzini, M. Cammarata, Dept. of Animal Biol., Univ. of Palermo, Italy.

  Inflammatory responses in solitary ascidians include cell migration, phagocytosis, encapsulation of larger particles, tissue injury, and wound repair. In encapsulation responses in the tunic of Ciona intestinalis, an increased expression of type IV-like collagen and elastin-like molecules have been found, apparently produced by the epidermis. Inflammatory cells have been identified as amoebocytes, univacuolar cells, unigranular refringent cells (URG) and morula cells. We show the involvement of a large amount of URGs following LPS injections. These cells contain polyphenols and, in vitro, showed a phenoloxidase-dependent cytotoxic activity. Probably, URGs migrate through the epithelium from tissue lining the lacunae under the tunic. Chemotactic stimuli, that induce migration into the inflamed area, could be due to a C3-like molecule while an immunohistochemical study shows that molecules containing interleukin-1-like epitopes are expressed (2-4 hours) by endothelial tissue lining the pharyngeal wall. An IL-1-like functional activity may be indicated by the increased number in the lacunae as a result of the cell proliferation response. Accordingly, we found IL-1-receptor epitopes in cell nodules of the pharyngeal bars ansae. The recently elucidated genome of Ciona intestinalis did not reveal IL-1-like genes whereas an IL-1-receptor was found. However, human IL-1 traits can be observed by examining the Ciona genome sequence. Finally, the expression of a phenoloxidase component could be stimulated by inflammatory stimuli. Inflammation in ascidians presents invertebrate and vertebrate characteristics.


3. IBMANT-ANDEEP (Interactions between the Magellan Region & the Antarctic-Antarctic Benthic Deep-sea Bioiversity) Intl. Symposium & Workshop 20-24 October 2003 Ushuaia, Argentina


a) Genetic differentiation between populations of the ascidian Aplidium falklandicum from South Georgia and South Orkney Islands. Sahade Ricardo1, Demarchi Milagros1, Chiappero Marina2, Tatián Marcos1 & Gardenal Noemí2 1- Ecología Marina, F.C.E.F. y Nat. U.N.C., Av. Vélez Sársfield 299, CP 5000, Córdoba, Argentina. 2- Cátedra de Genética de Poblaciones y Evolución, F.C.E.F. y Nat. U.N.C., Av. Vélez Sársfield 299, CP 5000, Córdoba.

   The genetic structure of populations of sessile marine animals depends largely on the dispersal abilities of the larval stages. It is expected that species with higher dispersal capabilities will present less genetic structure than those with larvae that disperse only relative short distances, which in turn would present small scale genetic differentiation. Among the factors that could affect the dispersion of the free-living stages, and therefore the gene flow between populations are the variable spawning and recruitment success, habitat availability, oceanographic conditions and physical barriers.

The Polar Front and the abissal depths that surround Antarctica can be considered as the main physical barrier in isolating the Antarctic system, particularly effective for shelf benthic species. It has been hypothesized that the Scotia Arc Islands could act as a bridge, or step stones, between the Magellan and Antarctic Regions. In this work we will test this hypothesis on Aplidium falklandicum, an ascidian species that due to its reproductive behaviour, short lived and lecithotrophic larvae, common to all the group, can be a good model for testing possible gene flow  throught the Scotia Arc in sessile benthic species. Genetic population structure of two populations of A. falklandicum at South Georgia and South Orkney Islands were determined using simple sequence repeats (ISSR). ISSR are semiarbitrary markers amplified by PCR. Three primers were used: MARA: [(AC)10 AA]; DO: [(AC)10 AG] and NA: [(GACA)5] and amplified a total of 93 bands. AMOVA and multivariate analysis clearly separated both populations (FST = 0.43 P < 0.01) and Nucleotide Diversity Indices indicated that South Orkney were more diverse than South Georgias population (SG: p = 0.0056; SO: p = 0.010). This pattern could be produced by serial bottle-neck processes suffered by SG population or even by local extinction and recolonization processes. Then alleles are eliminated from the population by genetic drift. These ideas are consistent with the environmental instability caused by the suggested variable position of the Polar Front in this area. While the separation of the populations suggest the absence of gene flow between them, is also true that even existing some actual conection this could be hindered by bottle-neck and genetic drift processes in SG population determining that alleles reaching the SO population would not be present any more in the source population and little more can be said with only two populations sampled. These results even fragmentary show the potential of molecular methods for a better understanding of ecological and evolutionary processes in the Antarctic system.  


b)  Reproductive seasonality of five Antarctic ascidians species at Potter Cove, South Shetland Islands.  Sahade Ricardo, Botta Vanina & Tatián Marcos  Ecología Marina, F.C.E.F. y Nat. U.N.C., Av. Vélez Sársfield 299, CP 5000, Córdoba, Argentina.

   Primary production pulses and temperature have been signed among the most important factors in determine reproduction traits, especially in benthic fauna. Although temperature changes are slight year-round it has been argued that due to the evolutionary history of Antarctic organisms even such variations are detectable and could regulate reproductive cycles. On the other hand, the strong seasonal nature of energy input to the system has also been signed as the driving force behind all seasonal processes in the Southern Ocean, particularly important in organisms situates in the first levels of the food chain. It has also been suggested that different development strategies would determine reproductive cycles in benthic organisms. Thus, reproduction in animals with planktotrophic larvae should be coupled to primary production pulses, while those with lecithotrophic larvae or direct development should be released from those pulses and can reproduce aseasonally. Ascidians are common members of the Antarctic benthic communities and reproduce via a lecithotrophic larvae. This study intends to answer the question whether reproduction of ascidians at Potter Cove is continuous, as to be expected from the lecithotrophic nature of ascidian larvae and/or the low annual temperature amplitude, or whether it is limited to the summer, as to be expected from the distinct seasonality in primary production. Five ascidian species, Ascidia challengeri, Cnemidocarpa verrucosa, Corella eumyota, Molgula pedunculata and Pyura setosa were sampled at Potter Cove over a ca 15 months period during 1996/1997. The reproductive cycles were examined by histological analysis of the gonads. Temperature and chlorophyll-a data were obtained in the water column between 20 and 30 m depth from a long term monitoring programme running at Potter Cove. Reproduction of these suspension feeders seems to be decoupled from the pulses of primary production characteristic of Antarctic systems, except for P. setosa which showed their reproductive peaks coincident with chlorophyll-a pulses. While none of the reproductive cycles studied were related to temperature changes.  Although reproduction in A. challengeri and C. eumyota did not show a significant relation to chlorophyll-a levels the vitellogenesis in these species took place during the austral summer. Different were the case of C. verrucosa and M. pedunculata which reproduced during the austral winter and showed a marked vitellogenic period previous to spawning. These results were striking and somehow unexpected, in first term because these are phylogenetically very close organisms and are living under the same environmental pressures. And in second term because, at least at first sight, to reproduce during the Antarctic winter could appears as energetically disadvantageous especially for filter feeders.  However, C. verrucosa and M. pedunculata are two of the dominant species of macrobenthic communities at Potter Cove. Whether these reproduction strategies are phyllogenetically fixed or are local ecological adaptations is still an open question. Energetic implications of these cycles as well as their possible relation to small-scale distribution patterns are discussed.


c) Report on the trophic ecology of the macrophagous ascidian Cibacapsa gulosa Monniot & Monniot, 1983. Lescano M. N. 1, Tatián M. 1, Sahade R. 1 & Fuentes V.L. 2 1Ecología Marina, FCEFyNat, UNC-CONICET. 2 Dept. de Ciencias Biológicas, FCEyN, UBA-CONICET.

   To know the trophic ecology of the macrophagous ascidian Cibacapsa gulosa Monniot & Monniot, 1983 (Ascidiacea, Octacnemidae), microscopical analyses were performed both, on stomach contents and on the wall of the postpharyngeal digestive tract. Octacnemids represent a different pathway in the evolution of the typical suspension-feeding strategy in ascidians. A total of three specimens were collected during the LAMPOS cruise in the area of the South Sandwich Islands at depth of 590 m. Specimens were immediately fixed in buffered formaldehyde 2.5% in sea water. The different prey items found in the gut contents were identified and counted under stereo-microscope using a Bogorov 10 ml counting chamber. Microscopical observations were also performed on different sections of the gut (oesophagus, stomach and intestine). The macrophagia in Cibacapsa gulosa was confirmed, but it is more diversified than previously supposed. Gut contents were constituted by harpacticoid and calanoid copepods, lumbrineriform polychaetes, halacarids, eusirid and gamaroid amphipods, ophiuroids, jelly-fishes, gastropods, crustacean parts and fish scams. All these items had a wide range size: from 100 µm in the case of small calanoid copepods up to 8 mm of some polychaetes. High quantities of orange lipid drops, were also observed in the contents. The wall of the post-pharyngeal digestive tract was lined by cylindrical mono-stratified epithelium, which reposes on a wide mesenchyme with blood sinus and extra-vascular blood cells. The external epithelium along the whole post-pharyngeal digestive tract was mainly formed by cubic cells. At the level of the oesophagus, the inner epithelium was ciliated and showed an intense basophilia in the apical region. Ciliated mucous cell was the main cell type identified; zymogenic cells were also scattered along the epithelium. Several cell types were observed in the gastric wall: ciliated mucous cells (with large supra-nuclear vacuoles); zymogenic cells; undifferentiated cells and concretion cells, these only identified previously in the class Sorberacea (Gaill, 1979). The most abundant cell type present in the intestinal epithelium was the ciliated mucous cell; other two cell types present in this region were zymogenic and undifferentiated cells. Capture of this wide variety of preys (some of these having a great mobility) by C. gulosa, suppose a special behaviour in this sessile species. Although prey items have a benthic and pelagic origin, the presence of components from the zooplankton could explain the high quantities of oil drops found in the contents: lipid storage as been stressed in amphipods and copepods living in polar regions, reaching the lipids an important percentage of its dry weight during the year-round. The presence of zymogenic cells in the whole post-pharyngeal digestive tract indicates that an intense enzyme secretion should be necessary to digest this diet, composed by organisms provided with hard parts. Intracellular concretions were not observed previously in other ascidians. Their presence in Sorberacea and in C. gulosa may be explained by homology or, by an independent acquisition in both groups (adaptation to the macrophagia). This special feeding ecology (based in the capture of a very energetic, but probably occasional preys) should be a successful strategy, regarding the scarcity of particles (i.e., products derived from the primary production) that reach this benthic ambient. 


d) Ascidians (Tunicata, Ascidiacea): biogeography along the Scotia Arc.  Tatián M., Antacli J., & Sahade R.  Ecología Marina, CONICET-FCEFyNat., UNC,

   In Southern Ocean, the Polar Front determines, as in many organisms, strong barriers for the ascidian distribution. The Northern Magellan and Southern Antarctic regions separated by this barrier, could be linked by the Scotia Arc, as was pointed out by Monniot & Monniot (1983) who found a faunal gradient between these regions through the Scotia Arc. The knowledge on the species composition in these areas is, nevertheless, fragmentary. During the LAMPOS cruise (ANT XIX/5, RV “Polarstern” April-May 2002), ascidians were collected on different stations along the Scotia Arc. Our objectives are to extend the knowledge on species composition and to establish affinities between the ascidian fauna at the different stations performed during the cruise, analyzing the influence of the Polar Front on the ascidian distribution. Material was collected by Agassiz (AGT) and bottom (GSN) trawls, at depths between 250-600 m, on different substrate types. Animals were relaxed in current seawater and later fixed in buffered formalin seawater 4%. Morphological features were analyzed under binocular and microscope, to identify the different species. The reproductive status of colonial species was taken in account, recording the presence of tadpoles. Photographs were taken on living animals using a digital camera, to document the coloration, which is usually lost after fixation. Affinities between the different stations/localities were performed using cluster analysis (Bray-Curtis similarity, UPGMA) and ordination (multidimensional scaling, MDS). Analyses were also performed pooling previous data on ascidian distribution (Monniot & Monniot, 1983) and the present results.

A total of 25 species were found along the different stations sampled during the cruise, being solitary species slightly more abundant. Two species are new for the science, whereas 7 species were collected in new localities, extending their known area of distribution. Ascidians were present in more than 80% of the captures. Muddy bottoms supported higher species richness than hard bottoms, like gravel, pebbles and volcanic stones.

Stations from South Georgia, South Orkney and South Shetland Islands (soft substrates) were related, while South Sandwich stations (hard substrates) were clearly distant. Burdwood, Herdman and Discovery Banks (hard substrates) were also related. Affinities between localities considering also previous data revealed a gradient from Magellan to South Shetland Islands South Georgia and South Sandwich are more related to Antarctica than to Magellan.  New collection of samples is still important to improve our knowledge on ascidian biodiversity from the Southern Ocean and particularly of the South American shelf. Substrate type explains affinities between the different stations sampled. Magellan and Antarctic fauna show stronger affinities than those previously considered. Although the Polar Front is a limit for the ascidian dispersion, its position might fluctuate between the north and south of South Georgia. Which are the northern limit for the distribution of the Antarctic species and, the southern limit for the distribution of Magellan species. The affinity gradient of ascidian fauna along the Scotia Arc indicates a connection between Magellan and Antarctic regions through these Islands according to a stepping stone pattern.


4.  74th Annual Meeting of the Zoological Society of Japan Sept. 17-19, 2003, Hakodate, Japan


a) Analysis of metal-related genes in the vanadium-rich ascidian, Ascidia sydneiensis samea

N. Yamaguchi, K. Kamino, T. Ueki, T. Uyama and H. Michibata.  Marine Biological Laboratory, Graduate School of Science, Hiroshima University, Japan

   Several ascidian species are known to accumulate high levels of vanadium in their vanadocytes.  The highest level observed in Ascidia gemmata corresponds to about 107 times the levels in seawater.  To investigate the phenomenon, we carried out an expressed sequence tag analysis (EST) of blood cells of A. sydneiensis samea, in which 13 mM vanadium is accumulated.  We obtained randomly selected 1,300 ESTs from the vandocytes and whole blood cells cDNA libraries.  In this study, 62 metal-related genes were identified.  In particular, ferritin H-subunit, which is known as an iron storage protein, and novel vanabins were found.  Vanabins have been extracted from A. sydneiensis samea blood cells, cloned and identified as low molecular weight vanadium-binding proteins.  Vanadium binding ability of these proteins was confirmed by immobilized metal affinity chromatography and gel filtration column chromatography.


b) Analysis of metal binding activity of vanadium-binding proteins (vanabins) from an ascidian Ascidia sydneiensis samea.  T. Ueki, K. Fukui and H. Michibata

   We have previously identified several vanadium-binding proteins (vanabins) expressed abundantly in the cytoplasm of vanadium-accumulating cells, vanadocytes, of a vanadium-rich ascidian Ascidia sydneiensis samea. We have cloned cDNAs for the two vanabins, vanabin1 and vanabin2, and examined their metal binding ability using recombinant proteins. Vanabin1 and vanabin2 can bind 10 or 20 vanadium ions, respectively, at +4 oxidation state (VO2+) at a dissociation constant of around 2 X 10-5 M. In this study, we found that by an EPR study the coordination environment of vanabin2 against VO2+ ions is N2O2 type, and amino residue of lysines contribute to the coordination. In addition, by a metal-chelating column method, we found that vanabins can bind to copper (II) and iron (III) ions.


c) Cloning of cDNAs for sulfate transporters in the vanadocytes of Ascidia sydneiensis samea.  H. Kawamichi, N. Yamaguchi, T. Ueki and H. Michibata.

   A considerable amount of sulfate is always found in association with vanadium in ascidian blood cells. It is suggested that sulfate might be involved in the biological function and/or the accumulation and reduction of vanadium. In the case of Ascidia gemmata, 350 mM vanadium and 500mM sulfate ions were contained in vacuoles of vanadocytes, thus, the content ratio of sulfate to vanadium was estimated to be approximately 1.5, as would be predicted if sulfate ions are present as the counter ions of vanadium(III). As the first step towards an analysis of the possible correlation of vanadium and sulfate, we did plan to isolate the sulfate transporter gene and analyze its function. Since STAS sequence are conserved in the C-terminal of sulfate transporters, we aligned the amino acid sequences of sulfate transporter genes derived from Genebank, with those of putative Ciona sulfate transporter derived from EST project database. Based on the alignment, we have constructed some degenerate primers and amplified cDNAs by PCR from cDNA library of blood cells of Ascidia sydneiensis samea.


5.  6th International Marine Biotechnology Conference Sept. 21-25, 2003, Chiba, Japan


a) Vanadium-binding proteins (vanabins) of an ascidian Ascidia sydneiensis samea. T. Ueki, Y. Sakamoto, T. Watanabe and H. Michibata

   Ascidians, tunicates or sea squirts, are well known to accumulate high levels of vanadium ion in the vacuole of one or more type(s) of blood cells.  We previously identified several low molecular weight vanadium-binding proteins, designated vanabins, from the cytoplasm fraction of vanadium-containing cells or from the coelomic fluid.  We have cloned cDNAs for vanabin1 (12.5 kDa) and vanabin2 (15 kDa) from cytoplasmic fraction and vanabin-P from the ceolomic fluid.  We examined the activities of the recombinant vanabins to bind metal ions including vanadium (IV) and vanadium (V) by Hummel-Dreyer's method.  Recombinant proteins of the two vanabins, vanabin1 and vanabin2, bound to 10 and 20 vanadium(IV) ions with dissociation constants of 2.1 x 10-5 M and 2.3 x 10-5 M, respectively.  The binding of vanadium(IV) to these vanabins was inhibited by the addition of copper(II) ions, but not by magnesium(II) or molybdate(V) ions.  Vanabin2 also bound to 5 copper (II) ions, but not to iron (III) ions.  By expressing fusion proteins of vanabins in E. coli, the cells obtained ability for copper accumulation but not for vanadium.  Vanabins are the first proteins reported to show specific binding to vanadium ions; this should provide a clue to resolving the problem regarding the selective accumulation of vanadium in ascidians.


b)  Novel vanadium-binding protein (vanabin) from cDNA library of the ascidian, Ciona intestinalis.  Subrata Trivedi, Nobuo Yamaguchi, Tatsuya Ueki, and Hitoshi Michibata

   Some ascidians, particularly those belonging to the suborder Phlebobranchia are known to accumulate high levels of vanadium. Vanadium binding proteins (Vanabins) were first isolated from the  vanadium-rich ascidian, Ascidia sydneiensis samea. Data base search revealed five groups of vanabin-like genes in another ascidian Ciona intestinalis. Here we report the cDNA and genome sequence analysis of Ciona vanabins.  The predicted amino acid sequences of the five groups of vanabins were highly conserved and related to Ascidia vanabins.  The genes encoding each of Ciona vanabins were clustered in 8.4-kb genomic region.  We also report the functional assay of one group of the Ciona vanabins (Group 0). The recombinant protein was produced in E. coli, and vanadium binding experiment was done using metal-chelating column chromatography (batch method). The Group 0 vanabin bound to the vanadium (IV) ions immobilized on the resin, and was eluted by elution buffer containing different concentrations of NaCl. This is the first vanadium binding protein to be reported other than those found in Ascidia sydneiensis samea.


c) Marine biotechnological approaches to the accumulation of metals by ascidians.  Hitoshi Michibata, Tatsuya Ueki, Nobuo Yamaguchi and Hozumi Kawamichi

   About 90 years ago, Henze discovered high levels of vanadium in the blood (coelomic) cells of an ascidian collected from the Bay of Naples.  His discovery attracted the interdisciplinary attention of chemists, physiologists, and biochemists.  Two decades ago, we quantified the vanadium levels in several ascidian tissues definitively using neutron-activation analysis and revealed that some species in the family Ascidiidae accumulate vanadium at concentrations in excess of 350 mM, corresponding to about 107 times that found in seawater. Vanadium accumulated is reduced to the +3 oxidation state via the +4 oxidation state and stored in vacuoles of vanadocytes (vanadium-containing blood cells) where high levels of protons and sulfate are also contained.  To investigate this unusual phenomenon, we isolated several proteins and genes that are expressed in vanadocytes.  To date, two types of vanadium-binding protein, designated as vanabin1 and vanabin2, have been isolated, with molecular masses of 12.5 and 15, along with the cDNAs encoding these proteins.  Vanabins are rich in charged residues and a conserved motif in both vanabins can be described using the consensus sequence {C}-{X 2-5}-{C}. Using recombinant proteins of these two distinct vanabins, we revealed that they bind to 10 or 20 vanadium(IV) [VO 2+] ions with dissociation constants of 2.1 x 10 -5 and 2.3x10 -5 M, respectively.  An EPR study showed that vanadin2 can bind up to ~23.9 vanadium ions per molecule and most of the vanadium ions are in a mononuclear state and coordinated by amine nitrogen.  Since much more genes and proteins expressed in the blood cells are needed to clarify the entire mechanism involved in the accumulation and reduction, we have performed systematically an expressed sequence tag (EST) analysis of blood cells.



The role of integrins in Ascidia ceratodes sperm activation and fertilization
. Janice Soratorio, Master’s thesis, Calif. State Univ. Fullerton Dept. of Biol. Sci. Advisor Dr. Robert A. Koch. 

   Mitochondrial translocation is an early event during ascidian fertilization in which the sperm mitochondrion binds to the outer surface of the egg complex, undergoes a morphological change and translocates from the head of the sperm down to the tail.  This process is dependent on actin cytoskeletal reorganization and driven by myosin. Due to integrin’s known association with actin filaments, we hypothesize that integrins mediate adhesion of the sperm cell to the outer surface of the egg and signal the formation of focal adhesion-like complexes.  Thus, we predict that one or more integrin family members will span the membrane and interact with focal adhesion-like complex-specific proteins in the cytosol, e.g., talin, paxillin and FAK. Using anti-integrin aHr1 and bHr prepared against the ascidian Halocynthia roretzi protein and anti-integrin b1, integrins were detected on the mitochondrial region of ascidian sperm heads. Integrin b2, b3 and b4 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 focal adhesion complex formation depends on integrin receptor occupancy and clustering. To study these, sperm were exposed to an anti-integrin antibody known to induce integrin clustering, mAb 12G10, and an antibody known to have no effect on integrin activation and clustering, K20. We found that mAb 12G10 triggered sperm activation in a manner similar to positive controls. K20 also activated the sperm with less response. LY294002, a PI3 kinase inhibitor, was used to determine if integrin clustering is involved in the signaling cascade during MTL.  When sperm was pretreated with the inhibitor then exposed to the clustering antibody, induced activation by mAb12G10 was blocked. To determine integrin’s function during fertilization, dose-dependent inhibition by echistatin and anti-bHr was tested.  In the presence of echistatin (1.5 and 15 µM), fertilization was inhibited and decreased to 60% and 47%, respectively.  Similar results were observed in the presence of anti-bHr, with more inhibition with higher concentration from 50% to 30%.  These data demonstrate: (1) the presence of integrins on the ascidian sperm cell surface, (2) the presence of protein characteristic of focal complexes, (3) integrin clustering, found upstream of PI3 kinase, is not necessary for MTL, and (4) suggest the importance of integrins as sperm-surface egg receptors in ascidian fertilization.



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Addadi, L., Raz, S. and Weiner, S. 2003. Taking advantage of disorder: amorphous calcium carbonate and its roles in biomineralization. Adv. Mat. 15: 959-970.

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Burighel, P., Lane, N. J., Fabio, G., Stefano, T., Zaniolo, G., Carnevali, M. D. and Manni, L. 2003. Novel, secondary sensory cell organ in ascidians: in search of the ancestor of the vertebrate lateral line. J. Comp. Neurobiol. 461: 236-249.

Cañestro, C., Albalat, R. and Gonzàlez-Duarte, R. 2003. Isolation and characterization of the first non-autonomous transposable element in amphioxus, ATE-1. Gene 318: 69-73.

Carman, M. R. and Roscoe, L. S. 2003. The didemnid mystery. Massachusetts Wildlife 53: 2-7.

Carroll, M., Levasseur, M., Wood, C., Whitaker, M., Jones, K. T. and McDougall, A. 2003. Exploring the mechanism of action of the sperm-triggered calcium-wave pacemaker in ascidian zygotes. J. Cell Sci. 116: 4997-5004.

Chadwick-Furman, N. E. and Weissman, I. L. 2003. Effects of allogeneic contact on life-history traits of the colonial ascidian Botryllus schlosseri in Monterey Bay. Biol. Bull. 205: 133-143.

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Cima, F., Basso, G. and Ballarin, L. 2003. Apoptosis and phosphatidylserine-mediated recognition during the take-over phase of the colonial life-cycle in the ascidian Botryllus schlosseri. Cell Tiss. Res. 312: 369–376.

Cleto, C. L., Vandenberghe, A. E., MacLean, D. W., Pannunzio, P., Tortorelli, C., Meedel, T. H., Satou, Y., Satoh, N. and Hastings, K. E. 2003. Ascidian larva reveals ancient origin of vertebrate-skeletal-muscle troponin I characteristics in chordate locomotory muscle. Mol. Biol. Evol. 29: 29.

Copp, B. R., Kayser, O., Brun, R. and Kiderlen, A. F. 2003. Antiparasitic activity of marine pyridoacridone alkaloids related to the ascididemins. Planta Med. 69: 527-531.

D'Aniello, A., Spinelli, P., De Simone, A., D'Aniello, S., Branno, M., Aniello, F., Fisher, G. H., Di Fiore, M. M. and Rastogi, R. K. 2003. Occurrence and neuroendocrine role of D-aspartic acid and N-methyl-D-aspartic acid in Ciona intestinalis. FEBS Lett. 552: 193-198.

Davidson, B. and Levine, M. 2003. Evolutionary origins of the vertebrate heart: specification of the cardiac lineage in Ciona intestinalis. Proc. Nat. Acad. Sci. 100: 11469-11473.

Davidson, B., Smith Wallace, S. E., Howsmon, R. A. and Swalla, B. J. 2003. A morphological and genetic characterization of metamorphosis in the ascidian Boltenia villosa. Dev. Genes & Evol. 13: 13.

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Fanelli, A., Lania, G., Spagnuolo, A. and Di Lauro, R. 2003. Interplay of negative and positive signals controls endoderm-specific expression of the ascidian Cititf1 gene promoter. Dev. Biol. 263: 12-23.

Flood, P. R. 2003. House formation and feeding behaviour of Fritillaria borealis (Appendicularia: Tunicata). 143: 467 - 475.

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