Gretchen and Charles Lambert

12001 11th Ave. NW, Seattle, WA 98177

206-365-3734 or

home page:


Number 60                                                                                                                   April 2007


   Thanks to all of you who sent in contributions; this is the largest issue of AN we have ever put together!  There are 114 new publications listed at the end of this issue!  Keep up the good work and to assure that your publications will appear in AN please don’t forget to send us hard copy or PDFs; thank you.

     We heartily congratulate Dr. Xavier Turon who was chosen this year’s Paul Illg Distinguished Lecturer for the Friday Harbor Labs; we look forward to hearing about his latest findings.

   In December we spent a few days at the Friday Harbor Labs for the 90th Birthday of Charley’s Ph.D. advisor at the Univ. of Washington, Arthur Whiteley. Gretchen attended an invasive ascidian risk assessment meeting on Prince Edward Island (Canada) during March; mussel growers there are suffering major losses due to overgrowth by Ciona intestinalis, Styela clava and other species. We will attend the Intl. Bioinvasions meeting in Boston in May and participate in a pre-meeting rapid assessment survey around the Boston area, then will be at the Friday Harbor Labs for a few weeks of research and writing during June and July. Following Friday Harbor we will participate in a rapid assessment survey of New England from Maine to Cape Cod. In October we will attend the Second Intl. Invasive Sea Squirt Conference on Prince Edward Island (see announcement below). In the meantime Gretchen spends hours every day identifying ascidians collected for various governmental and academic agencies. 


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




1. Upcoming 4th Intl. Tunicata Conference June 23-27, 2007, Villefranche-sur-Mer, France, organized by Christian Sardet, Station Zoologique, Observatoire, CNRS, Univ PM Curie, Villefranche.   The meeting will be held in Residence Delcloy situated on St. Jean Cap Ferrat about a mile from the Marine Station.  To see the conference site:, click on France then Mediterranee then St. Jean Cap Ferrat and click on the 360 panoramics to see the facilities.  For meeting details:   We  have already more than 100 participants and have made the call for abstracts. The meeting will be limited to 120-130 people.


We have assembled a new archive of 29 ascidian films and embryos

It goes with a review  "From oocytes to 16 cell stage: the cytoplasmic and cortical reorganization which pattern the ascidian embryo" by our group which will be posted on-line in Developmental Dynamics.


2. The Biology, Biogeography, and Ecology of Invasive Ascidians. Oct. 1-5, 2007, Prince Edward Island, Canada.  Organized by Mary Carman, Woods Hole Oceanog. Inst. ( Sponsored by WHOI Ocean Life Institute.  View the conference website at for registration, details on the conference, and guidelines for abstract submission.  Arrival Monday Oct. 1 at Rodd-Brudenell River Hotel (30 min. from Charlottetown in the Brudenell River Provincial Park); Oct. 2, morning field trip/charter boat outing to view aquacultured mussel lines and walk to nearby floating docks, followed by afternoon taxonomic workshop given by Gretchen and Charles Lambert at Atlantic Veterinary College (ascidians collected during the field trip or brought from home will be welcome). Oct. 3-4 will include invited plenary talks, contributed research presentations, posters, and discussions at the hotel (banquet evening Oct. 3), with departure Oct. 5.  A special block of rooms has been reserved for us at the hotel; view their website at

3. From Dr. Ivan Goodbody : The Mona Institute of Applied Sciences (MIAS)  at the University of the West Indies, Mona Campus, Jamaica has released a CD-ROM featuring ‘An Introduction to Caribbean Sea Squirts (Ascidiacea)’ compiled by Emeritus Professor Ivan Goodbody and illustrated by photographs of most common species taken while alive in their natural environment. Each photograph is accompanied by both a vocal and a textual description to guide the viewer. The work is designed for use by students and researchers working in the Caribbean who may need assistance in identifying animals which they see in the marine environment. Further information on the CD and how to obtain copies can be found on the website: or by contacting the Marketing Manager, Mona Institute of Applied Sciences, 4 Belmopan Close, University of the West Indies, Kingston 7, Jamaica, West Indies, (876) 970-2021 or email:

Also from Ivan The Pelican Cays in Belize, Central America is a group of small mangrove islands situated at the southern end of the Barrier Reef. Many of the islands have a central lagoon surrounded by mangrove forest, the hanging roots of which dip into the water and provide substrate for communities of sessile marine invertebrates among which ascidians are a prominent element. A detailed scientific survey of the cays was undertaken by the Smithsonian Institution’s Caribbean Coral Reef Ecosystems Project (CCRE) directed by Dr.Klaus Rützler, Curator of Invertebrates at the National Museum of Natural History. Major outputs of this survey were published in March 2000 as a special volume of Atoll Research Bulletin edited by Ian Macintyre and Klaus Rützler. One of the findings of the survey is that the lagoons had an unusually high diversity of sessile invertebrates, especially sponges (Porifera) and ascidians, warranting the area being granted protection as an area of special scientific interest. In response to this the Belize Coastal Conservation Unit expanded the boundaries of an already protected area, the Southwater Marine Park, to encompass the Pelican Cays. Ivan Goodbody has documented the importance of the area for ascidian diversity in two papers, Atoll Research Bulletin No.480, March 2000 and Aquatic Conservation: Marine and Freshwater Ecosystems 5: 355-358 (1995).

    Recently, disturbing reports have been received that developers have moved into the area with the intention of creating a tourist resort. Ivan Goodbody considers that the Pelican Cays is one of the most important sites for ascidians in the entire Caribbean and notes that present reports indicate that within the archipelago the two most significant cays, Cat Cay and Manatee Cay, are now under threat from the development. Descriptions of these two sites can be found in a paper by MacIntyre et al. in Atoll Research Bulletin No. 467 (March 2000). At the time of writing it is too early to say how much damage has been done to ascidian populations but the presence of a dredger in the lagoon, pumping up sediment is likely to cause large-scale re-suspension of sediments in the water column. This may later settle on sessile communities resulting in smothering and death of delicate organisms, especially colony forming ascidians.

4. From Lucia Manni, Dept. Biology, University of Padova, via U. Bassi, 58/B, 35121 Padova, Italy.  A special meeting:

   More than 50 years ago, Prof. Armando Sabbadin, now professor emeritus at the University of Padova (87 and still working in the lab almost every day rearing his selected genotype), introduced Botryllus schlosseri in the lab as a model organism in various field of biology, beginning a series of researches which are still going on in our and in other Universities. The importance of B. schlosseri as a reference organism in the study of asexual and sexual reproduction, developmental biology, regeneration, allorecognition, immunobiology, stem cell biology, apoptosis, is now well established.
   As pupils of Prof. Sabbadin, in order to remember his lucky choice and to acknowledge him for his great passion for ascidian biology and scientific rigour, we wish to dedicate a one-day meeting to discuss, together with Prof. Sabbadin, the most recent results and views in compound ascidian biology. The meeting will be entitled:
    Fifty years of Botryllus schlosseri as a model organism in biology: results and perspectives
   The meeting will take place at the dept. of Biology, University of Padova, Friday, June 29, 2007, a couple of days after the end of the international Tunicate Meeting at Villefranche. It will have an informal character and will offer the occasion to meet and exchange opinions about botryllus with prof. Sabbadin (now 87 but always active in research).
Please, contact the organizers ( ; ; ; ; ) if interested.

5. 'It is really difficult to get some drama out of a sea squirt,' said Sir David Attenborough, in an interview by Jonathan Dimbleby at the Natural History Museum in London in October, while recounting highlights (and apparently lowlights) of his lifelong adventures. You can read more about the interview at

6. You will find lots of drama in the following: J. of Experimental Mar. Biol. and Ecology special issue vol. 342 (1), 2007: Proceedings of the 1st Intl. Invasive Sea Squirt Conference April 21-22, 2005 Woods Hole Oceanog. Institution:


Introduction to the Proceedings Pp. 1-2. Robert B. Whitlatch and Stephan G. Bullard

Invasive sea squirts: A growing global problem. Pp. 3-4.  Gretchen Lambert

The non-native solitary ascidian Ciona intestinalis (L.) depresses species richness.  Pp. 5-14. Julia C. Blum, Andrew L. Chang, Marcela Liljesthröm, Michelle E. Schenk, Mia K. Steinberg and Gregory M. Ruiz

Effects of small-scale disturbance on invasion success in marine communities. Pp. 15-29. Safra Altman and Robert B. Whitlatch

Spatial and temporal effects of pre-seeding plates with invasive ascidians: Growth, recruitment and community composition. Pp. 30-39. Brad P. Agius

Variation in the ability of Didemnum sp. to invade established communities. Pp. 40-53. Richard W. Osman and Robert B. Whitlatch

Do artificial substrates favor nonindigenous fouling species over native species?  Pp. 54-60. Megan C. Tyrrell and James E. Byers

Distribution and long-term temporal patterns of four invasive colonial ascidians in the Gulf of Maine. Pp. 61-68. Jennifer Dijkstra, Larry G. Harris and Erica Westerman

Invasion of the southern Gulf of St. Lawrence by the clubbed tunicate (Styela clava Herdman): Potential mechanisms for invasions of Prince Edward Island estuaries. Pp. 69-77. Andrea Locke, J. Mark Hanson, Karla M. Ellis, Jason Thompson and Rémy Rochette

Reproduction and early life history of an invasive ascidian Styela clava Herdman in Prince Edward Island, Canada. Pp. 78-84. Daniel Bourque, Jeffrey Davidson, Neil G. MacNair, Garth Arsenault, Angeline R. LeBlanc, Thomas Landry and Gilles Miron

Spatial and temporal patterns of recruitment of the tunicate Ciona intestinalis on a mussel farm in Nova Scotia, Canada. Pp. 85-92. S. Howes, C.M. Herbinger, P. Darnell and B. Vercaemer

Feeding preference of Strongylocentrotus droebachiensis (Echinoidea) for a dominant native ascidian, Aplidium glabrum, relative to the invasive ascidian Botrylloides violaceus. Pp. 93-98.Melissa Simoncini and Robert J. Miller

The colonial ascidian Didemnum sp. A: Current distribution, basic biology and potential threat to marine communities of the northeast and west coasts of North America. Pp. 99-108.  S.G. Bullard, G. Lambert, M.R. Carman, J. Byrnes, R.B. Whitlatch, G. Ruiz, R.J. Miller, L. Harris, P.C. Valentine, J.S. Collie, et al.

Ecological observations on the colonial ascidian Didemnum sp. in a New England tide pool habitat. Pp. 109-121. Page C. Valentine, Mary R. Carman, Dann S. Blackwood and Erin J. Heffron

Recent population expansions of non-native ascidians in The Netherlands. Pp. 122-126. Adriaan Gittenberger

Ascidian suspension feeding. Pp. 127-137. Jens Kjerulf Petersen

Phylogenetic diversity of bacteria associated with ascidians in Eel Pond (Woods Hole, Massachusetts). Pp. 138-146. Elia Tait, Mary Carman and Stefan M. Sievert

Benthic foraminifera associated with the invasive ascidian, Didemnum sp. A.  Pp. 147-153. Mary R. Carman

Development and application of tools for incursion response: Lessons learned from the management of the fouling pest Didemnum vexillum. Pp. 154-162. Ashley D.M. Coutts and Barrie M. Forrest

Competition among invading ascidians and a native mussel. Pp. 163-165. Rubi Rajbanshi and Judith Pederson

Fragmentation of colonial ascidians: Differences in reattachment capability among species.  Pp. 166-168. Stephan G. Bullard, Betsy Sedlack, James F. Reinhardt, Carolyn Litty, Keith Gareau and Robert B. Whitlatch

The role of colonial ascidians in altering biodiversity in marine fouling communities. Pp. 169-171. Jennifer Dijkstra, Hillary Sherman and Larry G. Harris

Effects of temperature on growth rates of colonial ascidians: A comparison of Didemnum sp. to Botryllus schlosseri and Botrylloides violaceus. Pp. 172-174. Anna McCarthy, Richard W. Osman and Robert B. Whitlatch

Water quality, nitrogen pollution, and ascidian diversity in coastal waters of southern Massachusetts. Pp. 175-178. Mary R. Carman, S.G. Bullard and J.P. Donnelly

The occurrence of the colonial ascidian Didemnum sp. on Georges Bank gravel habitat — Ecological observations and potential effects on groundfish and scallop fisheries. Pp. 179-181.Page C. Valentine, Jeremy S. Collie, Robert N. Reid, Rebecca G. Asch, Vince G. Guida and Dann S. Blackwood

The distribution of Styela clava (Tunicata, Ascidiacea) in European waters.  Pp. 182-184. Martin H. Davis and Mary E. Davis

Spread of Microcosmus squamiger (Ascidiacea: Pyuridae) in the Mediterranean Sea and adjacent waters.  Pp. 185-188. Xavier Turon, Teruaki Nishikawa and Marc Rius

Occurrence and distribution of ascidians in Vizhinjam Bay (south west coast of India). Pp. 189-190. Jaffar Ali H. Abdul and V. Sivakumar


7. From Erin Newman-Smith, Director, Ascidian Stock Center, MCD Biology, University of California, Santa Barbara, CA 93106-9610. 

   The Ascidian Stock Center is now available to take your requests.  The Stock Center, funded by a grant from the NIH to William Smith and Mike Levine, can supply wild type Ciona Intestinalis and Ciona savignyi. In addition, the Stock center is making stable Ciona lines carrying tissue specific transgenes.  These will be made available to all researchers. Specific stable transgenic lines can be made upon request. Please visit our web site for more information:




1. Anna Epelbaum (NSERC Visiting Fellow,, Chris Pearce, and Tom Therriault (Pacific Biological Station, Nanaimo, British Columbia, Canada) have a project in progress regarding abiotic and biotic factors affecting the growth, survivorship, and reproduction of non-native tunicate species in British Columbia (BC), Canada.  Currently, there are at least four species of tunicates in BC which are non-native and potentially invasive: the solitary tunicate Styela clava and the colonial tunicates Botrylloides violaceus, Botryllus schlosseri, and Didemnum sp.  While these species are present at many shellfish culture leases around Vancouver Island, they do not appear to have proliferated to the extent that they have in Atlantic Canada and the BC shellfish culture industry would like to minimize the potential impact of invasive tunicate species by better understanding the factors affecting their invasion success.  We have conducted preliminary lab experiments examining survivorship and growth of juvenile colonies of B. schlosseri and B. violaceus under 36 combinations of a wide range of temperatures and salinities (0-25ºC, 8-38 ppt).  Lab experiments aimed at assessing predation effects on survivorship and growth of B. schlosseri, B. violaceus, and S. clava are currently underway.  We are testing predators which can potentially be used to control tunicate fouling on aquaculture gear (e.g. sea urchins, skeleton shrimps), as well as those which would potentially control tunicate spread on natural substrates and limit their ability to invade new habitats (e.g. various species of crabs and sea stars).  We are also planning to perform a series of trials to assess the feasibility of various physical and chemical treatments to eradicate these non-native tunicate species.


2. From Dan Minchin, 3 Marina Village, Ballina, Killaloe, Co Clare, Ireland.

New tunicate records in Ireland.

   A rapid assessment survey in Ireland during 2005-6 revealed the presence of three tunicates not previously recorded in Ireland. The surveys took place at marinas where pontoons were sampled. Several colour morphs of  Botrylloides violaceus forming small colonies overgrowing sessile species were found at two locations in the Irish Sea on the NE Irish coast at Malahide, near Dublin and in Carlingford Lough, where the border between Ireland and Northern Ireland occurs.  A form of Didemnum that could not be identified to species was found at the same localities, however, in Malahide there were extensive pendulous growths of this tunicate found attached to pontoons and some boat hulls.  Samples of material have been sent to Brent Copp in New Zealand for biochemical studies and to John Bishop who may be able to look at their genetic identity. The sub-Antarctic Corella eumyota was recovered from five sites ranging from Tralee Bay in south-west Ireland to Carrickfergus in Belfast Lough.  In Cork Harbour it was found attached to the hull of a small yacht. The survey method improved the knowledge of the distribution of tunicates, as well as other species, and the sampling can be carried out by a single person.

   In an earlier study the Asian tunicate was found on the south-west coast of Ireland in Tralee Bay and Dingle Bay, however the 2006 survey did not reveal its presence in Dingle Bay.  A further new locality was in a ferryport in Dublin Bay.

   The researchers in Ireland greatly appreciate the support provided by those who were approached and working on didemnids, as a fuller understanding of the significance of the Irish findings would otherwise not have been possible.

Minchin D, Davis MH, Davis ME 2006. Spread of the Asian tunicate Styela clava Herdman, 1882 to the east and south-west coasts of Ireland.  Aquatic Invasions 1(2): 91-96.

Minchin D, Sides E 2006. Appearance of a cryptogenic tunicate, a Didemnum sp. fouling marina pontoons and leisure craft in Ireland.  Aquatic Invasions 1(3): 143-147.

Minchin D 2007. Rapid coastal survey for targeted alien species associated with floating pontoons in Ireland Aquatic Invasions 2(1): 63-70.


3. Patrick Frank, Dept. of Chemistry, Stanford Univ., Stanford, CA 94305

  We are in the final presubmission stages of work that will include a hypothesis describing the biological reduction of inorganic vanadate to vanadyl and V(III) in ascidians. This work was done with Bob and Elaine Carlson, Britt Hedman, and Keith Hodgson. We used x-ray absorption spectroscopy to follow the fate and uptake of vanadyl ion in the blood cells of A. ceratodes. The results are fully consistent with previous work on Phallusia nigra with Bill Robinson and Ken Kustin (2001 J. Inorg. Biochem. 86, 635-648). All the detected intracellular forms of vanadyl ion varied in linear concert, implying biological regulation with active feedback. The enzymatic hypothesis, which proposes the existence of a vanadium reductase in ascidians, follows directly from the results of our study, and includes details of an active site mechanism that accomodates the varied structural requirements of all three vanadium oxidation states. We will also report new inorganic chemistry that validates this proposed mechanism. The brand-new and undoubtedly rich field of vanadium metalloenzyme redox biochemistry is indicated.


4.Maximilian Haeussler, Equipe INRA U1126 "Morphogenèse du système nerveux des Chordés", UPR 2197 DEPSN, CNRS, Institut de Neurosciences A. Fessard, Gif-sur –Yvette, France.

   In a side-activity from my thesis on Ciona intestinalis, I have built a local website that my group leader Jean-Stéphane Joly and I would like to make available to the Ciona community. At present, it contains a home page, a simple genome browser customized for ascidian studies (see below), a series of images of Ciona intestinalis tailbud stage embryos obtained by confocal microscopy that were used to refine the definition of stages, and links to the site of the Gif transgenesis platform (under construction)  and to other ascidian websites. Here is the rational for adding one more genome browser to the already excellent existing ones: I first spent a lot of time using them. What bugged me was that I had to switch all the time between the browsers made by Ensembl, UCSC, VISTA, JGI, Kyoto University's Ghost and Aniseed. The standard genome browser Ensembl and UCSC are good, but special data about Ciona is dispersed within different specialized browsers and the generic browser groups don't care that much about the relatively small ascidian community. Specialized sites that aim to this do exist (e.g. Aniseed).  This browser presents the same data in a different, more genome-centric way that researchers used to the UCSC browser may prefer. I've modified the UCSC genome browser into a new version that simply combines other researchers' work into one common interface. Thanks to all the labs that make their data available to download, this was quite simple. I have integrated so far: Gene models from Genebank (from papers), Ghost, JGI1, JGI2, Ensembl and TIGR, ESTs from Genebank and Kyoto, added links to Aniseed and Ghost, orthologous-gene information from Ensembl Compara and Savignyi-conservation display from the VISTA browser.

The idea is that you can type in any kind of gene number (even human symbols) and see where there is information in one of the various databases about it, to compare their intron/exon structure and 5' / 3' ends. As the browser is in a very preliminary stage (e.g. many gene models don't distinguish between UTR and coding sequence yet), I'm happy for any feedback on it or on the developmental table to  or .     The site will be online by about mid-April.




1. Intl. Council for the Exploration of the Sea (ICES) Symposium, Boston, Oct. 30-Nov. 2, 2006.


Reducing the Impact of the Invasive Ascidian Didemnum sp. on the Shellfish Industry in New England.  Mary R. Carman1 and Page C. Valentine2. 1Woods Hole Oceanographic Inst., Woods Hole, MA; 2U.S. Geological Survey, Woods Hole, MA

   Several species of ascidians have invaded the New England coast during the past 30 years.  Ascidians, also called tunicates or sea squirts, are marine invertebrate filter feeders that attach to artificial and natural substrates in subtidal zones.  The colonial ascidian Didemnum sp. was first recorded in Maine in the late 1980s and now occurs along the coasts of all New England states where it poses a threat to the shellfish industry.  It attaches to hard substrates, forming mats in agitated waters and long tendrils in quiet waters.  It overgrows other attached organisms, including bivalves, sponges and seaweeds.  When healthy, it is not eaten by other species, and other organisms do not grow on it.  Didemnum sp. spreads by sexual and asexual reproduction and by natural fragmentation of colonies.  The species also spreads by fragments of colonies generated from cleaning shellfish and aquaculture equipment, from trawling and dredging through colonies living on the seabed, and from disruption of the seabed by storms.  The growth of ascidians on aquaculture cages and bags can potentially impact shellfish by restricting water flow, by reducing food availability, and by promoting anoxia in extreme cases.  Preliminary observations indicate that Didemnum sp. causes harvested oysters (Crassostrea virginica) to smell like ammonia if the shells are not properly cleaned.  Moreover, the tissue of Didemnum sp. is highly acidic (pH 3) and may cause the shell margins of infested oysters to become brittle.  It is unclear whether Didemnum sp. causes death of shellfish, but handling and cleaning heavily fouled equipment and product are time-consuming, costly tasks.  Although, there is no proven practical technique for eradicating ascidians, several methods for controlling them in aquacultures have been tried and include:  power spraying of shellfish lines; transfer of shellfish to clean culture gear; scrubbing of individual bivalves by hand; dipping of shellfish in saline solution or freshwater, and exposing them for longer periods to lower salinity water.  Shellfish tolerate emersion and drying in air for longer periods of time than soft-bodied ascidians.  At suitable aquaculture sites, Didemnum sp. can be deterred by placing shellfish in the intertidal zone during the summer months when colony growth and reproduction are most vigorous.  Spread of the species can be slowed by preventing colony fragmentation from human activities, and by avoiding transport of colonies on fouled equipment and ship hulls. 


2. VIIIth scientific meeting of the Italian Association for Developmental and Comparative Immunology (IADCI), 1 and 2 March 2007, Area della Ricerca, CNR, Naples, Italy. ISJ 4: 24-36


a. A novel rhamnose-binding lectin from the compound ascidian Botryllus schlosseri. L Ballarin, N Franchi, B Spolaore, F Gasparini. Univ. of Padua, Padua, Italy

Animal lectins play a fundamental role in invertebrate immunity, as they are involved in the recognition of microbial molecular patterns which, in turn, triggers various effector responses, such as opsonisation, encapsulation, activation of the pro-PO activating system, phagocytosis. In a previous study, we purified by affinity chromatography and partially characterised a soluble Ca2+-independent lectin, with specificity for b-galactosides, from the blood of the colonial ascidian Botryllus schlosseri. The molecule can agglutinate rabbit erythrocytes, is secreted by haemocytes upon the recognition of foreign particles and behaves as an opsonins (Ballarin et al., 1999, 2000). Recently, we purified further this protein by RP-HPLC, obtaining 4 lightly different peaks, likely isoforms of the same molecule. The MWs estimated using mass spectrometry ranged between 10.7 and 11.1 Kda. The lectin was digested with trypsin and tryptic fragments were sequenced by mass spectrometry. Blast analysis of the main sequences obtained indicated a high degree of homology with rhamnose-binding proteins, a family of S-type lectins described in sea urchin and teleosts. The specificity for rhamnose (and the similar melibiose) was successively demonstrated in haemoagglutinating inhibition assays. We prepared a full length cDNA library from Botryllus colonies from which we obtained three full sequences of transcripts which, after BLAST analysis, resulted highly homologous to known genes for rhamnose-binding lectins. Their putative aminoacid sequences contained our tryptic peptide sequences.


b. Undifferenteed cells in the blood of the colonial ascidian Botryllus schlosseri: a morpho-functional charaterisation. F Cima, L Ballarin. Dept. of Biology, Univ. of Padua, Padua, Italy

Colonies of the ascidian Botryllus schlosseri undergo a periodic tissue renewal in the take-over stage of the colonial blastogenetic cycle, during which an extensive apoptosis occurs in the adult zooid tissues and the senescent cells are progressively removed by circulating phagocytes. The haemocytes which circulate in the common vascular system also die partly by apoptosis during this stage. These cells are replaced by new haemocytes, likely differentiating from stem cells. Up to now, haemopoiesis was observed only in solitary ascidians in which haematopoietic noduli were described in the branchial wall. Nothing is known on haemopoiesis in colonial species, in the blood circulation of which two cell types with the morphology of undifferentiated cells are recognizable: haemoblast and lymphocyte. We have studied the cytochemical and immunocytochemical properties of these haemocytes: results indicate the haemoblast as a pluripotent stem cell since it shows a basophilic nucleus labeled either with Hoechst 33342 for euchromatin or anti-Ki-67 and anti-PCNA antibodies specific markers of nuclear proteins involved in cell proliferation and its plasma membrane is labeled by anti-CD34 and anti-CD100 antibodies, specific for haemopoietic cells in vertebrates. Commercial antibodies for cytokine receptors, like interleukin 1 receptor I (IL-1RI) and stem cell factor receptor (SCF-R) label haemoblast plasma membrane, suggesting the presence of growth factor receptors. Both lymphocytes and haemoblasts during the colonial cycle show a significant increase in concentration during the blastogenetic replacement. However, mitosis figures were rarely observed in circulating haemocytes. In vitro assays of haemocyte exposure to colchicine showed the presence of mitosis figures, which significantly increase after exposure to bacteria indicating a proliferating capability in blood circulation mainly as an immune response as observed in other invertebrates like molluscs.


c. Toll-like receptors in haemocytes of the colonial ascidian Botryllus schlosseri: preliminary results. A Menin, L Ballarin, Dept. of Biology, Univ. of Padua, Padua, Italy

Toll-like receptors (TLRs) represent a well-known family of pattern recognition receptors, expressed by immunocytes, the importance of which in non-self recognition was demonstrated in both Vertebrates and invertebrates. In the colonial ascidian Botryllus schlosseri, we used commercial anti-TLR2 and anti-TLR4 antibodies to inquire into the presence of Toll-like receptors (TLR) in haemocytes lysates. After SDS-PAGE, the immunoblot analysis revealed single protein bands recognised by the two antibodies, of 34 kDa and 32 kDa for TLR2 and TLR4, respectively.

Immunocytochemical investigation on monolayers of fixed haemocytes, previously exposed to E. coli LPS and yeast cells, revealed the expression of molecules recognised by TLR2 on activated phagocytes, whereas no labelling was observed with TLR4. We also studied the role of NF-kB in the signal transduction pathway related to phagocytosis. Immunocytochemical analysis with anti-NF-kBp65 antibody revealed the labelling of the cytoplasm of untreated cells, whereas haemocytes exposed to yeast cells or Bacillus clausii spores showed a marked staining of the phagocyte nucleus. The NF-kB inhibitors Na-pyrrolidinedithiocarbamate and parthenolide, at sublethal concentrations, significantly inhibits both the ingestion of yeast cells by Botryllus phagocytes and the nuclear translocation of the activated factor. The same molecules have no effects on the morphology of haemocytes. On the whole, our data suggest that, in our species, TLR are involved in phagocytosis and act through the activation of NF-kB.


d. Signal transduction in phagocytosis of the colonial ascidian Botryllus schlosseri: a preliminary approach. A Menin, E Chemello, L Ballarin, Dept. of Biology, Univ. of Padua, Padua, Italy.

In the course of our study on the role of immunocytes of the colonial ascidian Botryllus schlosseri in immune responses, we began to investigate the signal transduction pathways involved in yeast cell phagocytosis. Both calphostin C, a specific inhibitor of protein kinase C (PKC), and H-89, a specific inhibitor of protein kinase A (PKA) significantly inhibit the increase in the phagocytic index. This indicates that both cyclic AMP, which activates PKA, and phospholipase C, which results in the production of IP3 and DAG (the former mobilising Ca2+ from intracellular stores, the latter activating PKC), are routinely required for phagocytosis. In addition, manumycin A, inhibiting Ras activation, PD98059, inhibitor of ERK activation, SP600125, preventing JNK activation, SB202190, inhibiting p38 kinase, significantly inhibit yeast phagocytosis by Botryllus phagocytes. This suggest that the main MAP kinase pathways are involved in the ingestion of foreign cells. The frequency of phagocytes expressing molecules recognised by anti-pan Ras antibody increase significantly when haemocytes were pre-incubated in the presence of foreign cells. Activated haemocytes also express molecules recognised by anti-p-ERK and anti-p38. Therefore, a complex network of intersecting pathways is emerging and future research will aim to a better clarification of the main steps of signal transduction in ascidian phagocytosis.


3. 48th Symp. of the Soc. for Histochemistry, Stresa, Lake Maggiore, Italy, 7-10 September, 2006. Histochemistry of Cell Damage and Death.


a. Recognition and clearance of apoptotic cells in colonial ascidians. L. Ballarin, Dipt. di Biol., Univ. di Padova, Italy.

   The colonial ascidian Botryllus schlosseri forms new zooids by blastogenesis, through the formation of palleal buds which progressively grow and mature until an adult is formed. At a temperature of 19°C, adult zooids remain active for about one week; then they contract, close their siphons and are gradually resorbed, being replaced by buds which reach functional maturity, open their siphons and begin their filtering activity as adult zooids. This recurrent generation change, known as regression or take-over, is characterised by the occurrence of diffuse programmed cell death by apoptosis. During the take-over, circulating phagocytes infiltrate in zooid tissues and engulf apoptotic cells; in addition, the frequency of haemocytes showing nuclear condensation and annexin-V labelling significantly increases. Moreover, the number of circulating phagocytes showing a globular morphology and containing ingested cells or cell debris significantly rises whereas the frequency of hyaline amoebocytes, which represent mobile, active phagocytes decreases. Phagocytes, both professional and occasional, actively recognise senescent cells and ingest them. As regards the eat-me signals, PS seems to be involved in the recognition of effete cells, as the addition of phospho-L-serine, a soluble analogue of PS, inhibits in vitro phagocytosis of apoptotic cells. CD36, a part of the receptorial complex binding thrombospondin which acts as a bridging molecule between phagocyte surface and apoptotic cells, is expressed on Botryllus phagocytes: the frequency of cells recognised by anti-CD36 antibodies significantly increases during the take- over and the expression pattern changes from a patchy distribution to a uniform staining of the phagocyte surface during the take-over. Anti-CD36 antibodies significantly decreases the phagocytosis of effete cells suggesting that similarly to that described in Vertebrates the thrombospondin receptor play a role in apoptotic cell removal by phagocytes.

b. Cyclic apoptosis in the digestive tract of a protochordate. F. Cima. Dipt. di Biol., Univ. of Padova, Italy.

   Tissue degeneration which occurs during development of organisms is often of morphogenetic importance as well as proliferation and differentiation. Cyclic apoptosis of organs was progressively lost in Chordates. Botryllus schlosseri is a colonial ascidian continuously forming new zooids by blastogenesis, through the recurrent formation of palleal buds, which grow and mature until an adult is formed. Three blastogenic generations are commonly co-present: adult, filtering zooids, their buds and budlets on buds. At a temperature of 19°C, adult zooids remain active for about one week (mid-cycle stages); then they contract, close their siphons and are gradually resorbed, being replaced by a new generation of adult zooids, represented by buds which reach functional maturity, open their siphons and begin their filtering activity (regression or take-over stage). This stage is characterised by the occurrence of diffuse programmed cell death by apoptosis in zooid tissues, as evidenced by TUNEL reaction for chromatin fragmentation and annexin V labelling for detection of exposed phosphatidylserine, whereas infiltration of circulating phagocytes, which appear engulfed with apoptotic cells, is observed. With these characteristics, colonial tunicates are suitable subjects for studies on cyclical involution and resorption of tissues. In residual zooids remaining for a long time in the centre of each colony, melanin and lipofuscins accumulate as detected with Masson-Fontana, Ziehl-Nielsen and H2O2 bleaching methods. Immunocytochemical assays to detect pro- and antiapoptotic factors reveal an opposite expression which progressively extends in tissues of adult zooids with an organ gradient starting from the branchial basket. Results support the idea that fundamental mechanisms for the induction of apoptosis are well conserved throughout chordate evolution.


4. Marine Genomics International Conference. Sorrento (Naples)  28 Oct.- 1 Nov., 2006.

a. Regulation of the meiotic cell cycle in ascidian oocytes: a comparison with vertebrate models

Russo, G.L., Bilotto, S., Cuomo, A., Silvestre, F., and Tosti, E., Stazione Zoologica ‘Anton Dohrn’, Napoli, Italy, and Institute of Food Sciences, CNR, Avellino, Italy.

   In all vertebrate, mature oocytes arrest at the metaphase of the II meiotic division, while some invertebrates arrest at metaphase I, others at pronucleus (G1) stage. Fertilization induces completion of meiosis and entry into the first mitotic division. How the different mechanisms underlying meiotic regulation evolved is very far from being clarified. In the past decades, several experimental models have been considered from both vertebrates and invertebrates in order to shed light on the peculiar aspects of meiotic division, such as the molecular mechanisms responsible for lack of S phase between the two meiotic divisions, and the regulation of the cytostatic factor (CSF) in metaphase I or II. Some of these questions remain elusive and could be approached by the introduction of new experimental models. For these reasons, few years ago, we proposed ascidian Ciona intestinalis oocytes as a new model to study the meiotic division both at the physiological and molecular level. Recently, this model has been strengthened by the publication of the draft copy of C. intestinalis genome. In the present communication, we are reporting data on the role of protein kinase CK2 and MAP kinase in regulating meiosis completion in C. intestinalis oocytes. These two kinases are physically interacting in arrested oocytes. After fertilization, degradation of CK2 regulatory subunits and enzymatic inactivation of MAP kinase drive fertilized oocytes to metaphase-anaphase I transition. We believe that this mechanism might be responsible for CSF inactivation. The results obtained indicate that meiotic regulation in C. intestinalis resembles that of vertebrates, such as Xenopus, more than that of other invertebrates. Finally, CK2 catalytic subunits (CK2-alpha) has been expressed in E. coli and purified, in order to study its biochemical and functional properties. In fact, we previously demonstrated that, in fertilized oocytes, C. intestinalis CK2-alpha is enzymatically active independently from its regulatory subunit (CK2-beta). A comparative biochemical analysis of CK2 enzyme from different sources will be presented.


b. HAP/APEX/Ref-1, apurinic/apyrimidic endonuclease mRNA is expressed in the oocytes of ascidian Ciona intestinalis. Elisabetta Tosti1, Stefania Bilotto2, Said El-Mouatassim3, Yves Menezo3, Gian Luigi Russo1,4  1Lab Biologia Cellulare, Stazione Zoologica “Anton Dohrn”, Napoli; 2Dipartimento delle Scienze Biologiche, Università degli Studi di Napoli Federico II; 3Lab. Marcel Mérieux, Ave. Tony Garnier LYON, France; 4Ist. di Scienze dell’Alimentazione, Consiglio Nazionale delle Ricerche, Avellino.

   DNA repair is probably one of the most important processes to be performed in the oocytes and zygote, at the time of fertilization and immediately after, in order to allow complete embryonic development. APEX/Ref1, Apurinic/apyrimidinic endonuclease-Red-Ox factor1 is capable of initiating the repair of apurinic/apyrimidic (AP) sites, the most common decay, in damaged DNA. In vertebrate models, the enzyme is also supposed to play an important role in response to oxidative stress. The DNA-binding activity of APEX is modulated by a post translational mechanism involving reduction oxidation and so at least partly mediated by ROS. Preliminary data indicate that APEX mRNA is expressed in human spermatozoa and oocytes and in pre-implantation embryos. In order to investigate if the level of expression of APEX was evolutionarily conserved, we extended our study to oocytes ad spermatozoa of ascidian Ciona intestinalis (sea squirt), an organism intensively studied in developmental biology and, more recently, proposed as a model to study meiotic regulation. From an evolutionary point of view, tunicates (appendicularians, salps and sea squirts) have very recently been re-evaluated as the closest relatives of vertebrates, more than cephalochordates, like amphioxus. This important discovery has been made possible since the advent of genomics that actually provide the opportunity for phylogenetics to resolve a number of outstanding evolutionary questions. In this respect, the draft copy of the C. intestinalis genome became publicly available providing new insights into origin and evolution of chordates. Based on this evidence, we used C. intestinalis gametes as comparative model to study the conservativity of APEX function. Our data indicate that APEX transcripts were detected in oocytes and embryos, but not in spermatozoa, of C. intestinalis. Of philogenetic significance is also the observation that ascidian APEX lacks redox transcriptional activity.


5. NW Developmental Biol. Conference at  Friday Harbor Laboratories, March 14-17, 2007.


a. Whole body regeneration in a colonial ascidian, Botrylloides violaceus. Anna D. Le, Federico D. Brown and Billie J. Swalla, Dept. of Biology, Univ. of Washington, Seattle, WA and Friday Harbor Laboratories, Univ. of Washington, Friday Harbor, WA.

   Most organisms have the capacity to regenerate organs or tissues through the proliferation and differentiation of stem cells, but the phenomenon of whole body regeneration id s rare event in highly 

complex animals. Stem cells are self-renewing and unspecialized, but their daughter cells develop into specialized cells. There have been many studies documenting regeneration by stem cells throughout 

metazoans.  Some classically described stem cell lineages are the hematopoietic blood cell lineage and  primary germ cells (PGCs) that populate the gonads. However, there is little known about the other 

stem cell lineages that are not as easily localized and characterized. Colonial ascidians offer a remarkable advantage to studying these stem cell lineages in chordates. These animals are 

capable of budding, or growing an entire individual from the interaction of the epithelial bud with a pluripotent stem cell circulating in the blood. Thus these complex colonial ascidians offer 

the possibility of studying cell fate, maintenance, and proliferation in an invertebrate chordate, with a less complex genome than the vertebrates. Here we study the phenomenon of whole body regeneration 

in a colonial ascidian, revealing the capability of regenerating the entire body from stem cells circulating throughout the vasculature.

b. The evolution of cartilage and the deuterostome body plan. Amanda L. Rychel and Billie J. Swalla. Dept. of Biology, Univ. of Washington, Seattle, WA and Friday Harbor Laboratories, Univ. of Washington, Friday Harbor, WA.

   Deuterostomes are a monophyletic group of animals from which the vertebrates evolved. The invertebrate deuterostomes are Cephalochordata, Tunicata, Hemichordata, Echinodermata, and 

Xenoturbellida. Hemichordates and cephalochordates have pharyngeal skeletons morphologically similar to vertebrates, despite not having neural crest. Presently, only vertebrates are known to have neural crest cells, and they give rise to multiple cell types including the chondrocytes of the pharyngeal skeleton, although tunicates have placodes, and may have neural crest cells. We have shown that 

invertebrate pharyngeal cartilages are acellular and appear to be secreted by the pharyngeal endoderm in hemichordates and a combination of pharyngeal endoderm, ectoderm, and mesoderm in cephalochordates. We also examined the expression of soxE in hemichordates and found it expressed in the pharyngeal endoderm, suggesting that this vertebrate developmental program may be traced back to the deuterostome ancestor. Preliminary results suggest that ptychoderid hemichordates have cellular cartilages in their proboscis skeleton, so we are now conducting experiments to examine the source of this cartilage. We are particularly interested in the development of the proboscis skeleton, stomochord, and heart/kidney complex as possible homologues to vertebrate structures.


c. An in-depth look at invagination mechanisms in ascidian gastrulation. Kristin M. Sherrard , Ed Munro, Center for Cell Dynamics, Univ. of Washington Friday Harbor Labs, Friday Harbor, WA.

Dr. Sherrard won the second prize for best talk by a post-doc.

   Invagination is a fundamental mode of morphogenesis whose mechanical basis remains poorly understood.  We have been studying the process in gastrulating ascidians, whose ten endodermal cells (each the size of a C. elegans egg) invaginate in a simplified cellular context of a hundred-cell, monolayer blastula lacking extracellular structures.  Time-lapse videomicroscopy, immunofluorescence of mono- and di-phosphoyrlated myosin II regulatory light chain, explants, and experiments with inhibitors to myosin phosphorylation support a novel, 2-step mechanism of invagination whereby cells first columnarize and subsequently actively shorten and round up while maintaining tight apical contacts.  Apical constriction of the endoderm cells appears to drive the columnarization but does not operate during invagination proper, which is instead driven by contraction of the basolateral surfaces. Mono-phosphorylated MRLC is implicated in both apical constriction and shortening, but di-phosphorylated MRLC may be instrumental in maintaining tight apical collars during the second step.  Preliminary modeling simulations of invagination in two-dimensional cross-section demonstrate that such “collared rounding” of columnar cells can indeed drive an invagination.


6. Third Asian Biological Inorg. Chemistry Conference, NanjingChina, Oct. 30 – Nov. 3, 2006.


a. Accumulation mechanism of vanadium by ascidians based on genes and proteins involved in the accumulation. H. Michibata, M. Yoshinaga, M. Yoshihara, N. Kawakami, N. Yamaguchi and T. Ueki. Mol. Physiol. Lab, Dept. of Biol. Sci., Hiroshima Univ. Kagamiyama 1-3-1, Higashi-Hiroshima 739-8526, Japan.   

   The intracellular vanadium concentration of some species in the family Ascidiidae can be as high as 350 mM, which is 10(^7) times the concentration in seawater. Vanadium ions, thought to be present in 

the +5 oxidation state in seawater, are reduced to the +3 oxidation state via the +4 oxidation state and are stored in the vacuole of vanadocytes, the vanadium-containing cells, where high levels of 

protons and sulfate are also contained. To investigate this unusual phenomenon, we have isolated many proteins and genes that might be involved in the accumulation and reduction of vanadium. To date, more than five types of vanadium-binding protein, designated as Vanabin family, have been isolated from vanadocytes. In addition, four types of enzyme related to the pentose phosphate pathway that produces NADPH were revealed to be located in vanadocytes, and NADPH produced by the pentose phosphate pathway participates in the reduction of vanadium(V) to vanadium(IV). Vacuolar-type H(^+)-ATPase (V-ATPase) maintains the low pH in the vacuole and is thought to provide the 

energy for vanadium accumulation. Using an immobilized metal-affinity chromatography (IMAC) and gene homology cloning, we have further obtained a Vanabin homologue in blood plasma, metal-ATPase, glutathione-S-transferase and SO(_4)(^-2) transporter. Now, it becomes important to elucidate not only physiological roles of these proteins but also to resolve how these proteins share their roles to accumulate vanadium in ascidians.


b. Metal ion selectivity and affinity of wild type and mutant Vanabins. T. Ueki, H. Michibata

   Ascidians, tunicates or sea squirts, are well known to accumulate high levels of vanadium ions in the vacuole of one or more type(s) of blood cells. We previously identified five low molecular weight 

vanadium-binding proteins, designated Vanabin1, Vanabin2, Vanabin3, Vanabin4 and VanabinP, from a vanadium-rich ascidian Ascidia sydneiensis samea.  All of these Vanabins are about 100 amino acid in length and contain 18 cysteine residues, whose intervals were conserved very well. Among them, Vanabin2 is most predominant vanadium-binding protein expressed in the cytoplasm of vanadium- accumulating cells, or vanadocytes.  EPR and NMR analyses suggested that lysines and arginines in Vanabin1 and Vanabin2 mainly contribute as coordination sites for vanadium(IV) ions. We performed in vitro mutagenesis of Vanabin2 to modify lysines, arginines and some amino acid residues in possible binding sites, and assessed the metal binding ability of mutants by immobilized metal ion affinity chromatography. Mutation of some of lysines and arginines affected the vanadium binding ability of Vanabin2. Removal of all three histidines apparently did not affect the binding of vanadium.




1. Study on AsGSTs, vanadium-binding glutathione transferases, identified from the vanadium-rich ascidian, Ascidia sydneiensis samea. M. Yoshinaga, Mol. Physiol. Lab, Dept. of Biol. Sci., Hiroshima Univ. Kagamiyama 1-3-1, Higashi-Hiroshima 739-8526, Japan.   [No abstract available]


2. Analysis of the allorecognition mechanism of Halocynthia roretzi hemocytes. Yuko Doi, Dept. of Biosci. and Informatics, Fac. of Sci. and Technol,. Keio Univ., Japan. Master’s thesis. Advisors Motonori Hoshi and Midori Matsumoto.

   Recognition strategies play an essential role in the immune system, which faces thousands of foreign substances. They are classified into two groups; recognition of non-self and recognition of 

self. The latter has been important topic as the possible explanation for the transplantation-immunity-like reaction that can be seen throughout the animal kingdom. However, the molecular entity of self-marker is hardly elucidated with the only exception of the major histocompatibility complex in vertebrates. Ascidians belong to the phylum Chordata and hold the phylogenetic key position to discuss the evolution of the vertebrate allorecognition system. Hemocytes of the solitary ascidian, Halocynthia roretzi undergo the contact reaction (CR) against allogeneic hemocytes, indicating that they have the ability of allorecognition. Hemocytes of H. roretzi are morphologically classified into nine types and every type of hemocyte undergoes CR. The missing-self recognition model is proposed for the recognition mechanism of H. roretzi hemocytes. In this study, I analyzed the allorecognition mechanism of H. roretzi hemocytes using the moloclonal antibody ku-4-96, which inhibits the 

releasing of phenoloxidase (PO) in CR. The antigen(s) of ku-4-96 exists on the cell surface of hemocytes and therefore, is thought to be involved in the primary steps of cell-cell recognition. Among the nine cell types, vacuolated cells, fusogenic phagocytes and phago- amoebocytes were suggested to have the ku-4-96 antigen(s). To elucidate the relation of effector and target in CR, it is important to clarify which cell function ku-4-96 inhibits. First, I focused on the releasing of PO to foreign substances as the effector function and examined whether ku-4-96 can inhibit it. As a result, 

ku-4-96 inhibited the releasing of PO to Escherichia coli and zymosan. Secondly, I investigated the effect of ku-4-96 on the target cell; the susceptibility to cytotoxicity. The cytotoxicity was 

measured by the releasing of the cytoplasmic fluorescent viability dye calcein AM and the releasing of calcein was suppressed in the cells treated with ku-4-96. These two results suggested that the 

function of ku-4-96 antigen(s) is different for each cell type. To identify the ku-4-96 antigen(s), I purified plasma membrane fraction by chromatography on phenyl-Sepharose. Among the three 

molecules recognized by ku-4-96, the molecules of 180kDa (ku496P-1) and 160 kDa (ku496P-2) were isolated and each N-terminal amino acid sequence was determined. As for ku496P-1, partial degradation by the Cleaveland method was carried out and the fragment containing N- terminal was obtained.


3. Search for self-marker molecules of ascidian hemocytes in Halocynthia roretzi. Masaki Ema, Dept. of Biosci. and Informatics, Fac. of Sci. and Technol., Keio Univ., Japan. Master’s thesis. Advisors Motonori Hoshi and Midori Matsumoto.

   Many metazoan cells have a system to recognize alien substances as non-self (for example microorganisms and chemical substances etc.).  And, vertebrate and their close relatives have a system to distinguish their own cells from other cells in the same species. The former system is called xenogenic recognition and the latter system is called allogenic recognition. It is thought that the difference of these recognition systems is decided with self-marker molecules, which is indispensable to identify self from non-self. It is the major histocompatibility complex (MHC) that is well known as a self-marker in the allo-recognition mechanism of vertebrates. The transplant rejection happens because the type of MHC is not suitable. The feature of MHC is having a wide variety of alleles in the locus. By this combination, MHC can invent advanced diversity between individuals. Recently, MHC was identified in Chondrichthyes, and it was suggested that all vertebrates except the cyclostomes, the most primitive vertebrates, retained MHC.

   Self-marker molecules in allo-recognition mechanism of invertebrates are not well known. Recently, FuHC, which are polymorphic gene loci to determine colony fusion or rejection, was identified in one kind of ascidian, which is closely related to vertebrates, Botryllus schlosseri. But this is the only report that describes self-marker molecules in allogenic recognition mechanism of invertebrates.

In a solitary ascidian, Halocynthia roretzi, their hemocytes have a capacity for allogenic recognition. When hemocytes from two individuals are mixed in vitro, they immediately start to agglutinate 

each other and emit their cellular vacuoles. This reaction was named the Contact Reaction (CR). It is thought that self-marker molecules exist in Halocynthia roretzi hemocyte, because CR never happens in the contact of their own cells. To find the origin of self-marker molecules in CR, I designed the experimental system to detect the unique hemocyte proteins in each individual. The cellular 

extract from each ascidian hemocyte was labeled by fluorescents and separated by 2 D electrophoresis. By this system, a group of protein spots, which expressed differently between individuals, was 

discovered. They are candidates for self-marker molecules. These spots were excised, and then PMF and MS/MS were taken with MALDI-TOF MS. Then, those candidate self-marker molecules, amino acid sequences of the fragments, were sequenced.


4. Impacts of changing water temperatures on the life histories of two invasive ascidians in the Gulf of Maine: Botryllus schlosseri and Botrylloides violaceus. Erica Westerman, Dept. of Zoology, Univ. of New Hampshire. Masters Thesis. Thesis advisor Dr. Larry Harris.

   Temperature and its effects on life cycles in organisms have received great attention recently due to heightened awareness of global warming.  Relative growth and reproductive development were monitored in two dominant species of ascidians from the Gulf of Maine to compare effects of rising ocean temperatures on established versus recently arrived species. (Botrylloides violaceus is a recent invader that was first noticed in the Gulf of Maine in the early 1980s while Botryllus schlosseri, an established non-native species, has been in the Gulf of Maine since the late 1800s.)  Studies were conducted by deploying Plexiglas® settlement panels at three sites with different temperature regimes (Damariscotta, ME, Newcastle, NH, and Salem, MA) during two separate growing seasons, June to December 2005, and February to December 2006.  Settlement panels deployed at the same sites supported a recruitment study from May to December 2006.  Breeding season was elongated for both species in Salem, MA relative to Newcastle, NH and Damariscotta, ME.  The period of elongation matched that of longer warmer water temperatures at Salem, MA. Botryllus schlosseri colonies settled two weeks earlier than Botrylloides violaceus at all three sites, however this delay in settlement can be attributed to the longer brooding period of Botrylloides violaceus larvae, and not a lower tolerance of cold water temperatures. While increased periods of warm water temperatures result in longer breeding seasons, timing of initial settlement may not be as indicative of temperature tolerances as timing of initial egg production in brooding animals. 


5. Phylogeny, population genetics and biology of the genus Pycnoclavella (Ascidiacea). Rocío Pérez-Portela, Dept. of Animal Biol. (Invertebrates), Fac. of Biology, Univ. of Barcelona, 08028 Barcelona, Spain.  Ph.D. Supervisors Drs. Xavier Turon & Cruz Palacín.

   The taxonomy and phylogeny of the genera Pycnoclavella and Clavelina, and their inclusion in one or two different families within the order Aplousobranchia, have been controversial since the description of both genera. The genus Pycnoclavella has been widely studied in tropical areas but its diversity was scarcely known in Atlanto-Mediterranean waters, where the species Pycnoclavella aurilucens had been described as featuring high morphological variability. Moreover, its possible synonymy with another species Clavelina nana, has been a matter of debate not yet settled. Combining morphological, molecular and biological data we have explored the intra- and inter-specific variability of the genus Pycnoclavella, as well as its relationship with Clavelina, and its abundance and distribution in Mediterranean waters. Phylogenetic analyses were performed under different criteria (Maximum Likelihood and Bayesian Inference) on sequences of the cytochrome c oxidase I (COI) mitochondrial gene, of 21 species of Pycnoclavella, Clavelina and Nephtheis fascicularis collected in the Atlantic, Indian and Pacific Oceans and the Mediterranean Sea, together with 20 species of other Aplousobranchia and outgroups. The phylogenetic trees obtained confirmed that Pycnoclavella and Clavelina are valid genera rather than synonyms, as it had been suggested by some authors. Both genera, Clavelina and Pycnoclavella, formed a monophyletic clade with a common ancestor more recent than the ones with representatives of other families of Aplousobranchia. Molecular results, therefore, indicated that these genera are closely related and, although they feature differences in asexual replication mode and morphology of the larvae, these do not necessarily imply that the two groups stem from distant ancestors. Based on molecular results the monotypic genus Nephtheis should be included in the genus Clavelina. Our results support the view that the family Clavelinidae should be retained as classically considered, with the genus Pycnoclavella included, as a monophyletic family separated from other families of Aplousobranchia. The recently described family Pycnoclavellidae should therefore be abandoned.  A detailed analysis based on morphological and molecular data within the clade of Pycnoclavella led us to discover a complex of six different species of the genus on S. European coasts, with more than 10% divergence in the COI gene. Pigment distribution, larval type and incubation mode were found to be the most useful morphological characters separating species of Pycnoclavella. 

   Four new species (P. atlantica, P. brava, P.communis and P. martae) were formally described and holotypes erected. Three of them were described from Atlantic and Mediterranean localities and one from the Indian Ocean. One Mediterranean species of Pycnoclavella remains unnamed (Pycnoclavella sp.) since insufficiently clear-cut characters did not allow the description. We demonstrate that species previously attributed to Clavelina and Archidistoma (C. detorta, C. flava, C. nana, A. productum) should be included in the genus Pycnoclavella and, therefore, other names should be abandoned. We also provided re-descriptions and neotypes of P. aurilucens and P. nana. Molecular data show that the genus Pycnoclavella is split into two different subclades corresponding to the two principal colony forms of the species, the Stanleyi group with basal stolons and the Aurilucens group with a common basal mass of test.  Besides morphological and genetic differences, the Mediterranean species of Pycnoclavella also show distinct biological traits. Life cycles of Pycnoclavella aurilucens, P. brava and P. communis were studied in two Mediterranean localities over a two and a half year period in order to understand their biological features and level of reproductive isolation. The three species revealed different life strategies, confirming the validity of the taxonomic rearrangement proposed here for species of Pycnoclavella on the basis of molecular clades and morphological features. The differences were especially significant in reproductive timing, as spawning asynchrony results in reproductive isolation. Gonad maturation and larval release took place during autumn and early winter for P. communis, during spring for P. aurilucens and over winter and spring for P. brava. Although some overlap in gonad maturation times was therefore observed between P. brava and the other two species, the overall reproductive patterns of the three species were clearly different. Differences in abundance were also recorded during the year. P. communis and P. brava showed similar dynamics, with a clear seasonal peak of abundance in winter and aestivation in summer. In contrast, although P. aurilucens showed strong changes in density over the year, active individuals were found throughout the year. Summer was found to be the unfavorable season for all species and all biological parameters studied showed a negative correlation with temperature, the high temperature and low rainfall in summer produces strong stratification and a decrease in nutrients in the NW Mediterranean waters, which may explain the aestivation period found for two of the species. The time courses of reproductive activity and growth rates for P. communis displayed a temporal lag that suggested partitioning of resources to either reproduction or growth, as described for other species of ascidians.

   The genetic variability of Pycnoclavella communis, studied from sequences of COI of W. Mediterranean Sea and Atlantic Ocean populations, pointed out the existence of two different mitochondrial lineages within the Mediterranean basin. Phylogenetic and network analyses raised concerns about the possible existence of cryptic species corresponding to these two lineages. The main mitochondrial (mt) lineage of P. communis was split into a Mediterranean and an Atlantic clade, indicating a genetic discontinuity between each side of the Strait of Gibraltar, as previously reported in other marine invertebrates. Population genetics analyses combining sequences of COI and eight microsatellite loci were applied to five Mediterranean populations of the main mt lineage. The mitochondrial gene as well as the microsatellite loci displayed high levels of polymorphism in P. communis. A strong genetic differentiation between populations was found with both markers. In addition, there were high numbers of private haplotypes (COI) and alleles (microsatellites) in the populations studied. Although asexual reproduction is intense in this species, it did not seem one of the main factors controlling the genetic structure of the populations in P. communis. We deduced from our results that this species features restricted dispersal (in agreement with the biological characteristics of its short-lived larvae), and that populations of P. communis are maintained through local recruitment, whereby each population is independently evolving because the infrequent genetic exchange is insufficient to homogenize differences among populations. Our results suggest that we may be witnessing in Pycnoclavella communis an instance of multiple ongoing speciation, possibly realized to a varying degree in the different parts of its range of distribution. Heterozygote deficiency was observed for the microsatellite loci in all populations analyzed. Presence of non-amplifying alleles with overestimates of homozygotes, mating among relatives due to the low dispersal capability, selfing and Wahlund effects, as a result of breeding subunits within each sampled locality, may explain such deficiency. The results from microsatellite data should be taken with caution, as the loci showed a strong incidence of failed amplifications, which we attribute to the marked intraspecies variability that hampered the application of these highly specific markers. The results of this work, therefore, question the applicability of microsatellite data in species of high genetic variability. The levels of variation detected for the mitochondrial COI gene, on the other hand, have enough resolution for studies at several levels (phylogenetic, phylogeographic and population genetics) in Pycnoclavella spp. and Clavelina. The application of different molecular tools, together with morphological and biological analyses, allowed us to clarify the main aspects of the phylogeny, taxonomy and population dynamics of Pycnoclavella spp.  [Pérez-Portela, R., Duran, S., Palacín, C. and Turon, X. The genus Pycnoclavella (Ascidiacea) in the Atlanto-Mediterranean region: a combined molecular and morphological approach. Invertebr. Syst. in press.]


6. Contribución al conocimiento de las ascidias coloniales (Chordata, Tunicata) de la Antartida Occidental y Región Magallánica.

Contribution to the knowledge of colonial ascidians (Chordata, Tunicata) from western Antarctica and Magellan region. Mª Mercedes Varela, Ciencias del Mar y Biología Aplicada, Universidad de Alicante, Spain. Ph.D. Thesis advisor Alfonso Ramos Esplá.

   Research over the past few decades has established that the Antarctic benthos, especially on the shelf, is characterized by high biomass and abundance levels on both hard and soft substate (White, 1984;  Clarke and Crame, 1989; Clarke, 1990;  Knox, 1994). These bottoms reveal a rich and diverse fauna of suspension feeders (Dell, 1972; Gallardo, 1987; Arntz et al., 1994; Starmans et al., 1999; Gili et al., 2001). One of the most important taxa in numerical and biomass abundances is ascidians in the epibenthic communities on the Antarctic continental shelf (Monniot & Monniot, 1983; Gallardo, 1987; Kirkwood & Burton, 1988; Galéron et al., 1992; Arnaud et al., 1998). Also, about 90% of the ascidians found in antarctic and subantarctic waters  have a high percentage of endemisms. (Monniot & Monniot, 1983). 

   The focus of this study was to enlarge our knowledge about the ascidian fauna in Scotia Arc, Western Antarctica (Antarctic Peninsula to Bellingshausen Sea) and the Magellan Region and their bionomical distribution in this area. Ascidians were collected in the Antarctic Spanish surveys: ANTÁRTIDA-8611, BENTART-94/95/03. In addition, I worked with the collections of the most important museums from Argentina that had not been studied previously. I examined more than 2000 ascidians belonging to 63 species and 10 families and three new species. Tabular keys are presented as well. An exhaustive analysis of the communities of colonial ascidians was made showing their importance. Finally, a biogeographical relationship of Antarctic, Subantarctic and Magellan regions was studied.




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