Gretchen and Charles Lambert

12001 11th Ave. NW, Seattle, WA 98177

206-365-3734 or

home page:


Number 62                                                                                                                  June 2008


   Thanks very much to the many AN readers who contributed to this issue; this is the largest issue we’ve ever done. In addition to a number of Work in Progress articles and meetings abstracts, there are several thesis abstracts with significant new information, and 124 new publications listed at the end of this issue.    

   In April we enjoyed a marvelous trip to Italy, beginning with seminars presented at the Univ. of Milan; many thanks to our hosts Drs. Fiorenza De Bernardi and Carmela Gissi. We finished with a memorable visit with Thomas and Rosmarie Honegger in Zürich. A week later we were in Alaska, to present a 2 day ascidian workshop at the Kachemak Bay Research Reserve in Homer for the Smithsonian. We will spend a few weeks of research and writing again at the Univ. of Washington Friday Harbor Labs during June and July. August 4-8 we will co-teach a 5 day ascidian workshop with Drs. John Bishop and John Ryland in northern Ireland.


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




1. From Mary Carman, Woods Hole Oceanog. Inst., Woods Hole MA. : The proceedings of the International Invasive Sea Squirt Conference-II held at Prince Edward Island, Canada, in October 2007 were peer reviewed for a special issue of the journal Aquatic Invasions.  Andrea Locke and Mary Carman are serving as guest co-editors for the special issue that will include 28 research papers.  Two papers of note will be “Adventures of a sea squirt sleuth: unraveling the identity of Didemnum vexillum, a global ascidian invader” by Gretchen Lambert and “Genetic conspecificity of the worldwide populations of Didemnum vexillum Kott, 2002” by Lauren Stefaniak, Gretchen Lambert, Adriaan Gittenberger, Huan Zhang, Senjie Lin, and Robert Whitlatch.  The results of these two papers indicate that Didemnum sp., Didemnum sp. A, Didemnum vexillum and Didemnum vestum are the same species.  The IISSC-III is planned for April 2010 in the Seattle area.  Please check the conference website for updates at


2. International Tunicate DNA Bank is being set up at  

   Since invasive tunicates spread on a global level, it is crucial that invasive species researchers have efficient access to invasive tunicate DNA from around the world.

   Who are we? Founded in 2007, the International Tunicate DNA Bank is housed in Dr. Jeff Davidson’s lab at the Atlantic Veterinary College at the University of Prince Edward Island, Can-ada. The DNA Bank maintains frozen tunicate DNA from samples collected throughout the world. Funding for the DNA Bank is currently through the Canadian Aquatic Invasive Species Network.

   Goals: To create a comprehensive collection of invasive tunicate DNA; to make this collection available to all researchers; to facilitate more efficient molecular research.

   How can you contribute?

The International Tunicate DNA Bank relies on contributions from researchers like you!

Please send tunicate samples to us from your area so that the collection becomes as comprehensive as possible.  Samples must be fixed in 70-95% ethanol (formalin fixed samples are not appropriate for DNA extractions). Samples can be sent to:

Sarah Stewart-Clark, UPEI, RM 311N AVC, 550 University Ave, Charlottetown, PEI , Canada

C1A 4P3

What can we do for you?

DNA samples will be sent to any researcher upon request. Please search through our online database for samples that may be useful for your research. This database is available at:  

Or contact Sarah Stewart-Clark,  for a list of available samples.  I have collection kits ready that I can send out to any lab willing to contribute to the collection. 

 Checklist for sending samples to the International Tunicate DNA Bank:

 1. Please ensure that there is enough ethanol in each vial to properly preserve samples.

 2. The species name, date, and collection location must be clearly marked in permanent marker on each vial.

 3. Send a form with each shipment including all information required for a complete record.

 4. Do not overfill vials.

 5. Do not put more than one species in a vial.

 For more information contact Sarah, or Dr. Jeff Davidson,


3. From Tatsuya Ueki, Hiroshima University, Japan  Mr. Masao Yoshihara has finished his doctorate course in March 2008, and received the degree (Dr. of Science) from Hiroshima University after supervision of Prof. Michibata. He is now employed in a private company in the biological field in Nagasaki. 

    Recent publication: T. Ueki, M. Satake, K. Kamino, H. Michibata.  Sequence variation of vanabin2-like vanadium-binding proteins in blood cells of the vanadium-accumulating ascidian Ascidia sydneiensis samea.  Biochim. Biophys. Acta, in press (2008). 




1. Cameron Moore and Jenn Dijkstra, Zool. Dept., Univ. of New Hampshire, Durham, NH.  Temperature effect on heart rate in introduced and invasive colonial ascidians (Botryllus schlosseri, Didemnum vexillum, Botrylloides violaceus and Diplosoma listerianum) in the Gulf of Maine.

   Invasive species often compete with native species for resources such as space, and can alter community structure. Temperature effects on the distribution of marine invasive species are often unknown yet are critical to determine their spread. We used heart rates of four colonial tunicate species (Botryllus schlosseri, Didemnum vexillum, Botrylloides violaceus and Diplosoma listerianum) as a proxy for health and growth. Heart rates and growth rates correlated with increasing temperature for Botryllus schlosseri, Botrylloides violaceus, and Diplosoma listerianum, but not Didemnum sp. Heart rate was determined to be a good indicator for health for all species, and was also a good indicator for growth within a species, but not between species. For example, Botryllus schlosseri had a higher heart rate, but slower growth than Botrylloides violaceus. The goal of this study was to determine if increasing temperatures could facilitate the spread of invasive colonial ascidians into colder waters.


2. Mary Carman and Stephan Bullard. In January 2008 we conducted exploratory surveys at the Pacific Panama Canal and on the Pacific coast of Panama between the islands of Isla Canales de Tierra (ICT) and Isla de Coiba (IC).  During these surveys, ascidian species were observed at the canal and in the island chain; species identifications are pending.  The apparent low diversity and abundance of ascidians in Pacific Panama waters is in stark contrast to the high diversity of ascidians in the Atlantic-Caribbean waters at Bocas del Toro, Panama (Rocha et al. 2005).  The unique nature of the Panama Canal and its potential role in the movement of invasive species warrants additional study of the ascidian fauna of the Pacific coast of Panama.  In this context, Carman, Bullard, Gretchen Lambert, Jennifer Dijkstra, and Rosana Rocha will return to Panama in January 2009 and address the specific questions: 1) How abundant and diverse are the ascidians occurring at the Pacific entrance to the Panama Canal and in the chain of islands from ICT to IC (Panama’s largest national preserve)?  2) Does the composition of the ascidian fauna differ with the degree of shoreline development? 3) Are the ascidians currently residing in Pacific Panama waters considered native or invasive species?


3.  Dan Minchin, Marine Organism Investigations, Ballina, Killaloe, Co. Clare, Ireland.

    The European tunicate Clavelina lepadiformis was found at two marina sites in Hout Bay, west of Cape Town, South Africa as part of a rapid assessment of alien fouling species. Some hundreds of colonies were attached to the vertical sides of floating pontoons. It is almost certain it was moved to this harbour with small craft.


4. Honoo Satake, General Manager & Senior Researcher, Division of Biomolecular Research,

Suntory Inst. for Bioorg. Res., Shimamoto, Mishima, Osaka, Japan.

   My group is focusing on functional and molecular characterization of ascidian neuropeptides and peptide hormones. We have so far elucidated more than 20 novel ascidian neuropeptides and hormone peptides plus several of their receptors by comprehensive mass spec. analysis, and the following functional and physiological analyses, although we have not published most of them.

The mass spec.-based detection is extremely important for characterization of peptides, as BLAST-based homology search on the genomic database is virtually useless for detection of peptide genes due to sequence divergence of peptide genes.  Recent publications:

1. Aoyama, M., Kawada, T., Fujie, M., Hotta, K., Sakai, T., Sekiguchi, T., Oka, K., Satoh N. and Satake, H. A novel biological role of tachykinins as an upregulator of oocyte growth: identification of an evolutionary origin of tachykinergic functions in the ovary of the ascidian, Ciona intestinalis. Endocrinology, in press (2008).  

2. Kawada T., Sekiguchi, T., Itoh, Y., Ogasawara, M., and Satake, H. Characterization of a novel vasopressin/oxytocin superfamily peptide and its receptor from an ascidian, Ciona intestinalis.

Peptides, in press (2008).


5. Genesis Special Issue on Chordate Origins and Evolution 2008

a. Kugler, J.E., Passamaneck, Y.J., Feldman, T.G., Beh, J., Regnier, T.W. and Di Gregorio, A. : - Evolutionary conservation of vertebrate notochord genes in the ascidian Ciona intestinalis In press.

   To reconstruct a minimum complement of notochord genes evolutionarily conserved across chordates, we scanned the Ciona intestinalis genome using the sequences of 182 genes reported to be expressed in the notochord of different vertebrates and identified 139 candidate notochord genes.  For 66 of these Ciona genes expression data were already available, hence we analyzed the expression of the remaining 73 genes and found notochord expression for 20.  The predicted products of the newly identified notochord genes range from the transcription factors Ci-XBPa and Ci-miER1 to extracellular matrix proteins.  We examined the expression of the newly identified notochord genes in embryos ectopically expressing Ciona Brachyury (Ci-Bra) and in embryos expressing a repressor form of this transcription factor in the notochord, and we found that while a subset of the genes examined are clearly responsive to Ci-Bra, other genes are not affected by alterations in its levels.  We provide a first description of notochord genes that are not evidently influenced by the ectopic expression of Ci-Bra and we propose alternative regulatory mechanisms that might control their transcription.


b. Capellini, T.D.*, Dunn, M.P.*, Passamaneck, Y.J., Selleri, L. and Di Gregorio, A.: Conservation of notochord gene expression across chordates: insights from the Leprecan gene family. (*equal contribution)    In press.
   The notochord is a defining character of the chordates, and the T-box transcription factor Brachyury has been shown to be required for notochord development in all chordates examined.  In the ascidian Ciona intestinalis, at least 38 notochord genes have been identified as bona fide transcriptional targets of Brachyury.  We examined the embryonic expression of a subset of murine orthologs of Ciona Brachyury target genes in the notochord to assess its conservation throughout chordate evolution.  We focused on analyzing the Leprecan gene family, which in mouse is composed of three genes, as opposed to the single-copy Ciona gene.  We found that all three mouse Leprecan genes are expressed in the notochord. Additionally, while Leprecan expression in C. intestinalis is confined to the notochord, expression of its mouse orthologs includes dorsal root ganglia, limb buds, branchial arches, and developing kidneys.  These results have interesting implications for the evolution and development of chordates.


6. Nishiyama, A. and Fujiwara, S. 2008. RNA interference in Ciona embryos RNA interference by expressing short hairpin RNA in the Ciona intestinalis embryo. Develop. Growth Differ. 50 in press.

   We carried out RNA interference by expressing short hairpin RNA (shRNA) in the Ciona intestinalis embryo. For this purpose, we identified a gene encoding U6 small nuclear RNA (snRNA) in the C. intestinalis genome. The 1-kb sequence upstream of the U6 snRNA gene was sufficient for directing transcription of short RNA as revealed by Northern blot hybridization. A shRNA-expressing plasmid vector was constructed, in which shRNA-encoding oligonucleotides are inserted downstream of the U6 promoter. A shRNA that contained a sequence homologous to the C. intestinalis tyrosinase gene (Ci-tyrosinase) suppressed melanization of pigment cells in the brain of morphologically normal tailbud embryos. A shRNA that perfectly matched the translated sequence of enhanced green fluorescent protein (EGFP) (a mutant type of Aequorea victoria green fluorescent protein) suppressed the expression of the coelectroporated EGFP transgene. These results suggest that the expression of shRNA interferes with functions of both endogenous and exogenous genes. The shRNA-expressing plasmid constructed in the present study provides an easy and inexpensive alternative for the functional analysis of genes in ascidian embryos.


7. Update on the development of the ANISEED model organism database.

Delphine Dauga1, Fabrice Daian1, Daniel Sobral1, Mike Gilchrist2 and Patrick Lemaire1

1: IBDML, UMR6216 CNRS-Université de la Méditerannée, Case 907, campus de Luminy, F-13288 Marseille cedex 9, France

2: The Wellcome Trust/Cancer Research UK Gurdon Institute,  Tennis court Road, Cambridge, UK

   Over the past year, data included in ANISEED v3.0 ( have been expanded and new pages created.  Transcript models from JGI v1.0, Kyotograil2005 and ENSEMBL were clustered by Mike Gilchrist (Cambridge, UK) to give rise to the Aniseed v3.0 gene models, which inherited all annotations from their transcript model components. A large annotation/curation effort was made to enter most of the molecular literature, thanks to the agreement and help of many people in the community. Aniseed now hosts 131 curated articles, totalling around 3000 ISH in experimentally manipulated embryos. An article page was created for each article entered, which summarises all molecular and phenotypic information included in the paper (eg Articles can be searched for by author's name, gene name, territory specified, keywords, etc… via a novel search interface (eg: More than 630 molecular tools were entered, mainly morpholino sequences, but also chemical inhibitors, etc… all linked to the genes they allow to study, the experiments they were used in, and the articles describing them. Over 200 cis-regulatory sequences have been described, annotated and linked to their activity pattern, to the genes they regulate, and, when known, to the genes that regulate them. For an example see ( Last, Delphine Dauga, the curator of ANISEED, has continued visiting labs in Europe and Japan to present the system and the data submission pipeline, and to ask for comments, suggestions for improvements, etc…

   Coming soon:

  • 550.000 new ESTs from Gateway compatible clones and a direct access for each gene to coordinates of the corresponding clones in the Gene collection release 1 (Nori Satoh's Lab) and in the Full ORF Gateway collection (Lemaire and Satou Labs).
  • A tab in the anatomy card of each territory that summarises the gene regulatory networks acting in the territory. A tab will further allow for each gene to recapitulate its upstream regulators in each territory of expression and, for regulatory genes, the genes they regulate in each territory.
  • A Cytoscape module to display the inferred regulatory network as it expands.
  • 3D virtual embryo reconstructions up to the early gastrula stage.

The system is now widely used by ascidian labs, with the number of independent connections up 3 fold since April 2007. We strongly encourage members to enter their own data either because they are of low interest to them (but may be highly valuable for others, and vice versa) and will remain unpublished, or after publication. Data can remain private and only seen by your lab members if you wish. Entering data into the system leads to an increase in the impact of published work, and allows to back up high resolution data including images, to organize data, to compare data with others. Delphine Dauga will be happy to visit your lab to give a tour of the system and its submission/curation tools. Please contact me at if you are interested.


8. A GATEWAY-compatible Ciona full ORF UNIGENE collection

Ute Rothbächer1, Mike Gilchrist2, Fabrice Daian1, Pierre Khoueiry1, Erika Lindquist4, Yutaka Satou3 and Patrick Lemaire1  

1: IBDML, UMR6216 CNRS-Univ. de la Méditerannée, campus de Luminy, Marseille, France

2: The Wellcome Trust/Cancer Research UK Gurdon Institute,  Tennis court Road, Cambridge, UK

3: Dept. of Zool., Graduate School of Sci., Kyoto Univ., Sakyo-ku, Kyoto, Japan

4: DOE Joint Genome Institute, 2800 Mitchell Drive Bldg 400-467, Walnut Creek, CA 94598, USA

   Successful functional analysis depends greatly on the quality of over-expressed cDNAs, in terms of integrity of the coding region (open reading frame, ORF) and easy transfer into electroporation or mRNA synthesis vectors. We have generated a GATEWAY compatible full ORF UNIGENE collection for Ciona intestinalis. This collection contains two independent clones for approximately 9000 independent Ciona intestinalis protein-coding genes, and thus covers around 70% of the estimated coding gene content of Ciona.

   This collection was generated from two cDNA libraries, one from French Atlantic coast Ciona intestinalis eggs and embryos of cleavage to neurula stages (U. Rothbächer, Lemaire lab), which was completed by a second library from adult Ciona from Japan (Y. Satou lab). Both libraries were made in the GATEWAY pDONR221 vector background to allow rapid cloning in destination expression vectors. A Ciona Community Sequencing Project (DOE Joint Genome Institute) generated 400.000 ESTs and 200.000 ESTs from the embryonic and adult libraries, respectively. These sequences were clustered with known Ciona ESTs by M. Gilchrist (Cambridge, UK) to refine the 15.000 Ciona intestinalis gene models (F. Daian, P. Khoueiry, Marseille and Y. Satou, Japan), and to select two full ORF clones for each gene from these two libraries. 19000 selected clones were subsequently arrayed in 384 well plates. The resulting 50 plates constitute the Ciona intestinalis full ORF UNIGENE collection.

   This full ORF UNIGENE collection is compatible with the Ciona GATEWAY vector collection (Roure et al. 2007) and will constitute an important functional genomics tool for the Ciona community. Systematic functional screening of diverse cellular processes is now possible. The distribution to Ciona labs is currently being organized. Please contact us as soon as possible ( if you wish to buy a copy of the collection. A manuscript, describing its generation is in preparation. The selected clones can be visualized on the ANISEED Genome Browser ( and will shortly be incorporated into Aniseed and linked to their corresponding gene.  Roure, A., Rothbächer, U., Robin, F., Kalmar, E., Lamy, C., Missero, C., Mueller, F. and Lemaire, P. (2007) A multicassette Gateway vector set for high throughput and comparative analyses in Ciona and vertebrate embryos. PLoS-ONE, 2:e916.


9. From Hitoshi Sawada, Director, Sugashima Marine Biological Lab., Graduate Sch. of Sci., Nagoya University, Sugashima, Toba, Japan.

    Since T. H. Morgan's published several papers on self-sterility in C. intestinalis in the early part of the last century, the molecular mechanism of self-sterility in ascidians has been a long-standing enigma.  Now, Harada and Sawada and their colleagues have revealed a molecular basis of self-sterility in C. intestinalis.  It was also discovered that the frameworks of the self-incompatibility system in this species is very similar to that of flowing plants. Harada, Y., Takagaki, Y., Sunagawa, M., Saito, T., Yamada, L., Taniguchi, H., Shoguchi, E. and Sawada, H. 2008. Mechanism of self-sterility in a hermaphroditic chordate. Science 320: 548-550.




1. IX Scientific Meeting of the Italian Association for Developmental and Comparative Immunology (IADCI), Varese (Italy), February, 27-29, 2008.


a.  The immune system of compound ascidians.

Loriano Ballarin, Dept. of Biology, Univ. of Padua, Padua, Italy.  

Differently from vertebrates, having an adaptive immunity, ascidians are chordates with a germ-line based innate immunity in which phagocytosis and cytotoxicity are the main effector systems. In recent years, the comprehension of the immune system of compound ascidian has greatly improved. Most of the research was carried out in species of the genus Botryllus and Botrylloides. In these organisms, immunocytes constitute a considerable fraction of circulating haemocytes and are represented by phagocytes and phenoloxidase (PO)-containing, cytotoxic cells.

Phagocytes include hyaline amoebocytes (HA) and macrophage-like cells (MLC). The former are wandering cells which quickly recognise and engulf foreign particles or cells: after the ingestion, they withdraw their cytoplasmic projections and acquire a globular shape, turning to MLC. Phagocytosis requires the recognition of molecular patterns on the surface of target particles by receptors on phagocytes and is greatly influenced by the nature of the particle. The recognition triggers signal transduction pathways which lead to the activation of the MAP-kinase cascade and of NF-kB. Soluble lectins can increase the phagocytosis of foreign particles acting as opsonins.

Phagocytes play an important role during the colonial generation change (take-over), when the old adult zooids stop their filtering activity and are progressively resorbed. During this period, tissues of adult zooids undergo diffuse apoptosis and are rapidly infiltrated by blood phagocytes which rapidly and massively engulf senescent cells. Cytotoxic morula cells are characterised by the presence of vacuoles containing inactive pro-PO and its polyphenol substrata. They are the effectors of the rejection reaction between contacting, genetically incompatible colonies. These cells crowd inside the ampullae contacting the alien colony, cross the epithelium of the ampullar tips and enter the tunic where they degranulate and release the content of their vacuoles. The activated PO acts on substrata and induce necrotic death through the induction of an oxidative stress. There is an interesting cross-talk between cytotoxic an phagocytic cells: i) phagocytosis can be modulated by soluble molecules (cytokines) released upon the recognition of foreign particles (yeast cells and bacterial spores) by the cytotoxic cells; ii) some HA are exposed to seawater, on the internal side of the siphons, where they act as guard cells. Once recognised foreign materials, they activate morula cells in the tentacular lacunae which, in turn alert the whole immune system towards the potentially dangerous particles that can be phagocytosed or degraded.


b. Apoptosis signalling pathways in the compound ascidian Botryllus schlosseri during the colonial blastogenetic cycle. 

A Menin, L Comini, L Ballarin, Dept. of Biology, Univ. of Padua, Padua, Italy

In the colony of the ascidian Botryllus schlosseri, three blastogenetic generations are usually present: adult zooids, primary buds on zooids and secondary buds on primary buds. Colonies undergo recurrent generation changes in which adult zooids are gradually resorbed and replaced by new blastogenetic generations. It is possible, therefore, to define a colonial blastogenetic cycle that begins with the appearance of a new generation, and ends with the generation change, during the take-over phase, in which programmed cell death by apoptosis is largely diffuse.

Using the haemocytes as reference tissue we investigate the extent of cell death during the colonial blastogenetic cycle . Our results confirm the expression, on cells surfaces, of Fas receptors and theirs ligands Fas-L. Moreover, we showed the presence of members of the caspase family: the initiator caspases 8 and 9 and the executioner caspases 3 and 7. The activated executioner caspases can subsequently cleave distinct cellular proteins such as PARP: using immunoblot assay we observed the cleavage of proteins recognised by anti-PARP. In Vertebrates, intrinsically mediated initiation begins with mitochondrial membrane disruption resulting in cytochrome c (cyt c) release. We observed an increase of H2O2 in cytoplasmatic contents and a different expression of cyt c during the take-over. These results confirm Botryllus as an interesting model organism for the study of apoptosis.


c. A novel rhamnose-binding lectin from the colonial ascidian Botryllus schlosseri.

N Franchi, F Gasparini, B Spolaore1, L Ballarin, Dept. of Biology, Univ. of Padua, Italy; 1CRIBI, Univ. of Padua, Italy.

Lectins are carbohydrate-binding proteins which agglutinate cells and/or precipitate glycoconjugates. The family of rhamnose binding lectins (RBLs) includes various proteins, previously classified as galectins, with common sugar specificity and one to three homolougous carbohydrate-recognition domains (CRDs), about 100-aminoacids long and characterised by eight highly conserved cysteine residues. From a full-length cDNA library from the compound ascidian Botryllus schlosseri we identified five complete transcripts homologous to known RBLs. Comparisons of the predicted amino acid sequences (118 aa) suggest that they represent different isoforms of a novel RBL, called BsRBL-1-5 with only one CRD. Reverse-phase HPLC and mass spectrometry of the affinity-purified material confirmed the presence of four of these isolectins in Botryllus homogenate. Analysis of both molecular masses and tryptic digests of BsRBLs indicated that the N-terminal sequence of the purified proteins starts from residue 22 of the putative amino acid sequence, so that residues 1-21 represent a signal peptide. Analysis by mass spectrometry of V8-protease digests confirmed the presence and alignments of the eight cysteines involved in the disulphide bridges characterising RBLs. Functional studies confirmed the enhancing effect on phagocytosis of the affinity-purified material. The phylogenetic relationship of Bs-RBLs with orthologous molecules from protostomes and deuterostomes was also studied.


2. Marine Genomics Europe Final General Assembly, Faro (Portugal), May, 13-16, 2008.

a. MGE contribution to the colonial ascidian B. schlosseri genomic studies: insight into immunobiology and muscle development.

Fabio Gasparini, Paolo Burighel, Valentina Degasperi, Lucia Manni, Nicola Franchi, Loriano Ballarin.  Dept. of Biology, Univ. of Padua, Italy.

   Botryllus schlosseri, a colonial ascidian reared in our laboratory for more than 50 years, is used as a model organism for studying a variety of biological problems. Our group is involved in the study of development and immunity of B. schlosseri and is acquiring a know-how in genomics and molecular biology. During the MGE course at Berlin MPI “Generation of cDNA Libraries by Primer Extension” (20 Nov - 1 Dec, 2006) we have prepared an enriched full-length cDNA library from B. schlosseri colonies. From this library a pool of clones was selected from which transcripts of interest for immunobiology and muscle development was identified. As regards immunobiology we have characterised five transcripts homologous to known rhamnose-binding lectins (BsRBLs) which plays a key role in immune responses acting as opsonins and improving phagocytosis. Four of these isolectins were found in Botryllus homogenate after purification by affinity chromatography on acid-treated Sepharose, analysis by reverse-phase HPLC and mass spectrometry. Analysis by mass spectrometry of V8-protease digests confirmed the presence and alignments of the eight cysteines involved in the disulphide bridges which characterise RBLs.  We have also isolated two transcripts homologous to genes involved in muscle development: a muscle-type actin (BsMA2) and a troponin T (BsTnT). We analysed the expression of the two genes by in situ hybridization (ISH), in order to follow the muscle development throughout the asexual development of B. schlosseri, beginning from the early bud to adult up to the regression stages. ISH showed that the first diffuse signal of muscle differentiation appears in the intersiphonal area of young buds. Then, the muscle fibers differentiate in the body-wall, while an intense expression of BsMA2 marks the myocardium just when it begins contractions.

   Phylogenetic analysis using the amino acid sequences of BsRBLs, BsMA2 and BsTnT and orthologous molecules from metazoans showed a close relationship between these tunicate  and vertebrate genes. Therefore, these results in our study of immunobiology and development of B. schlosseri have been reached thanks to the acquisition, with the contribution of MGE network, of experience in biomolecular techniques as well as adequate know-how in young researchers.


3. Amer. Soc. of Limnology and Oceanography, St. John's Newfoundland, June 8-13, 2008.


The viability of fragments of the invasive colonial tunicate Didemnum  vexillum in suspension and implications for long distance transport of the species.

Mary R. Carman, Woods Hole Oceanog. Inst., Woods Hole, Mass.

    Fouling by invasive species of tunicates, including Didemnum vexillum, is causing economic hardship for commercial shellfishers.  D. vexillum attaches to hard substrates and economically important shellfish, including blue mussels (Mytilus edulis), green mussels (Perna canaliculus), oysters (Crassostrea virginica), bay scallops (Argopecten irradians irradians), and sea scallops (Placopecten magellanicus). The first known record of D. vexillum in Atlantic North American waters is the early 1980’s at Damariscotta, Maine; it has subsequently spread along the coast from Eastport, Maine to eastern Long Island, New York, and is present offshore on Georges Bank.  It is expected to spread into Atlantic Canada waters.  Didemnid species can propagate by fragmenting and dividing into daughter colonies that grow by asexual reproduction.  Artificially cut pieces of D. vexillum reattach in the wild in New England waters and in the laboratory.  When a fragment of D. vexillum settles on a suitable substrate, it is capable of reattaching within one or two days.  Colonies are fragmented when they are removed from boats hulls, floating docks, and aquaculture equipment.  Fish and scallop trawls pulled through areas infested with D. vexillum (as occurs at Georges Bank in water depths of 45m to 60m) likely fragment the colonies and suspend them in the water column.  If viable fragments can survive in suspension for sufficient time, they may be transported via ocean currents into uninhabited habitats.  However, the amount of time that a fragment can survive without attaching was previously unknown.  We conducted an experiment to suspend fragments of D. vexillum for up to one month to test their survival in water temperatures similar to benthic Georges Bank.  At the start of the suspension period, fragments were flat and square, measuring about 2cm x 1.5cm.  During suspension, the fragments adapted to the water habitat by changing their gross morphology into spheres.  Smaller, lightweight fragments took longer to settle in a one-meter vertical tube of seawater than larger, heavier fragments.  We found that 60% of fragments were capable of surviving suspension for 18 days; and 15% survived in suspension for 30 days.  Thus, fragments are viable for a considerable amount of time and may tolerate being transported great distances before settling and reattaching.  These results can assist resource managers in developing strategies for reducing the spread of the species.  


4. Titles of recent talks from Yasushi Okamura. Dept. Dev. Neurophysiol., Okazaki Natl. Res. Inst., Okazaki, Japan.

Okamura,Y. :Mechanisms of voltage-sensor domain proteins and insights into physiological significance. Plenary Lecture, Sept. 27, Los Andes, Chile.

Okamura,Y. :Voltage sensor domain proteins. Ion channel, structure and function, European Biophyics Congress, July 16th, London, UK.

Okamura,Y.: Voltage-gated proton channels. Voltage-dependent proton channels come of age, 52nd Biophys. Soc. Annual Meeting, 16th IUPAB Intl. Biophysics Congress, Feb. 6, Long Beach, USA.

Okamura,Y., Sasaki, M., Kurokawa,T., Okochi,Y., Hossain,M.I., Iwasaki,H., Murata,Y. & Higashijima,S.: How do animals utilize signals of membrane potentials?  lessons from two voltage-sensing proteins. 6th Okazaki Biology Conference: "Marine Biology", 2007.12.3-8, Okazaki, Japan.

Hossain, M.I., Sakata, S., Murata, Y. & Okamura, Y.: Voltage range for tuning of phosphatase of Ci-VSP as measured by two PIP2-sensors. 52th Biophysical Soc. Annual Meeting, Feb. 5, Long Beach, USA.

Koch, H.P., Kurokawa, T., Sasaki, M., Okamura, Y. & Larsson, P. :Multimeric nature of voltage-gated proton channels. 52nd Biophysical Soc. Annual Meeting, Feb. 5, Long Beach, USA.


5. Michibata, H. and Ueki, T.   Hiroshima University, Japan.

Proteins involved in accumulation and reduction of vanadium in ascidians.

2nd East Asian Marine Bioscience Symposium, Sapporo, Japan. Dec. 4, 2007.  (No abstract)




1. Invasive potential of Didemnum perlucidum in a mussel farm.

Laura Pioli Kremer, Universidade Federal do Paraná, Brazil. Master’s thesis advisor: Dr. Rosana Moreira da Rocha.

    The invasion of nonindigenous species is one of the greatest threats to the marine ecosystem.  Didemnum perlucidum is a colonial ascidian very common in fouling communities on shellfish cultures in southern Brazil. It has a widespread distribution in the world and it was introduced in many regions. Here in Brazil it has a discontinuous distribution and it is better classified as a cryptogenic species. The main goal of this study was to evaluate the invasive potential of D. perlucidum in a shellfish environment where most of the species are nonindigenous. Two criteria were used: (i) the colonization ability of new habitats, and (ii) the competitive capacity and the consequent influence of this species on the sessile fauna. To test these premises experimental plates were exposed over a 14 month period (March 2006 – May 2007) in Penha, Santa Catarina. The experiment consisted of an experimental unit of four plates fixed by a steel rack at four depths. Among the four plates in a rack, one was an extra plate and the others were assigned to one of three treatments: bare plates that were replaced monthly; D. perlucidum removal plates, and unmanipulated control plates. The experimental unit was replicated four times at each depth. To assess the ability of colonization, larvae production was evaluated as well as recruitment and the growth of this species on the bare and D. perlucidum removal plates. To assess the competitive capacity and the influence of this species on the community, the richness and percent cover of all species that colonized the D. perlucidum removal and unmanipulated control plates were evaluated. D. perlucidum presented continuous breeding with the greatest larvae production between December 2006 and May 2007. Even though the greatest recruitment occurred on the bare plates, this species was able to colonize areas with a developed community, and it was benefited by small gaps in the community. Thus D. perlucidum was able to grow on the community plates as well as on the bare plates. Taken together the results demonstrate that the main barrier caused by the community was the lack of free space for the recruitment. Furthermore, D. perlucidum does not affect the richness of species, does not inhibit any species and at the population level the effects of this species were sporadic, showing a low competitive ability. The low effect of D. perlucidum on the community is explained by the low coverage of this species on the unmanipulated control plate and the great occupation of secondary substrate, although without smothering the covered species. There are strong evidences that the local population of D. perlucidum has been controlled by predators, so more information is needed about aspects of the basic biology of D. perlucidum to prevent economic and environmental threats if its populations are not naturally controlled.

2. Ecological aspects of the ascidian community along the Israeli coasts.

Noa Shenkar, Dept. of Zool., George S. Wise Faculty of Life Sci., Tel-Aviv Univ., Israel.  Ph.D. thesis advisor Dr. Yossi Loya.

   Ascidians (Phylum: Chordata, Class: Ascidiacea) are the largest and most diverse class of the sub-phylum Tunicata (also known as Urochordata). They comprise approximately 3000 species found in all marine habitats from shallow water to the deep sea. Despite the enormous progress in ascidian research worldwide, only a few studies have focused on the ascidians of the Red Sea and the Eastern Mediterranean. This study is the first to investigate this unique group of invertebrates from the Mediterranean and Red Sea coasts of Israel, from ecological, taxonomical and biological aspects. I first ascertained the dominant species found in the region, and determined whether these include any non-indigenous species. I then assessed the annual appearance and spatial distribution of these ascidians and how this appearance may be related to different abiotic factors. Finally, I studied the possible impact of anthropogenic activities on ascidian ecology in both coral reef and Mediterranean shore environments.

   Section I: The ascidian fauna along the Israeli coasts-- the dominant ascidian species found along the Red Sea coast of Israel, including a morphological description of a new species Botryllus eilatensis (Shenkar & Monniot 2006, Zootaxa 1256: 11-19) and a list of non-indigenous ascidians along the Mediterranean coast of Israel. In addition, two solitary species (Rhopalaea sp. and Boltenia sp.), found in Eilat, are also suspected to be new species. Six non-indigenous species (Phallusia nigra, Herdmania momus, Microcosmus exasperatus, Rhodosoma turcicum, Ascidia cannelata, and Symplegma brakenhielmi) were recorded from the Mediterranean shores of Israel. These species probably reached the Mediterranean via the Suez Canal, since they have an extra-tropical Indo-Pacific distribution and a restricted distribution in the Eastern Mediterranean.

   Section II: The solitary ascidian Herdmania momus: native (Red Sea) vs. non-indigenous (Mediterranean) populations--a comparative study of the spatial distribution, reproduction strategy and physiological parameters of the solitary ascidian Herdmania momus (published in Biological Invasion online 30 January 2008), including molecular data analysis of Mediterranean and Red Sea populations. In order to obtain a broader view of the ecology of non-indigenous species along the Mediterranean coast of Israel, I chose to focus solely on the study of comparative aspects of the ecology of the solitary ascidian H. momus, in its native (Red Sea) environment versus its introduced (Mediterranean) environment. During 2005 monthly samples of H. momus were collected along the Mediterranean and Red Sea coasts of Israel, in order to investigate possible differences in life history strategies of the two populations. The samples were preserved, dissected, and measurements were taken of length, total weight, gonad weight and oocyte diameter, and the occurrence of symbionts was recorded. In the Mediterranean H. momus exclusively inhabits artificial substrates and is common at greater depths than in Eilat (Red Sea). Individuals in Eilat reproduced year round. Although Mediterranean specimens were significantly larger than those collected in Eilat, their gonad indices and oocyte diameter measurements indicate that they have a short reproductive season. Copepods were found in 50% of the samples from both sites, while a pontoniine shrimp was found in 14% of the samples from Eilat only. The marked differences between the native vs. non-indigenous populations of H. momus are attributed to differential food availability, water temperature, currents and wave exposure.

   Section III: Ascidian recruitment patterns on an artificial reef in Eilat (Red Sea)-- the effects of season and spatial distribution on ascidian recruitment to artificial marine structures in a coral reef environment (Shenkar et al. 2008, Biofouling 24(2): 119-128). The location and timing of one species’ appearance may be the result of seasonality of reproduction, larval preferences, and effects of different a-biotic factors such as food availability, light intensity, water temperature and more. By identifying the ascidians to species level, we were able to distinguish between the specific recruitment patterns of the different species, rather than generalizing patterns among groups as done in most studies. The major contributions of the data presented in this section are: 1) two dominant species, Herdmania momus and Didemnum granulatum, established the pattern for the entire group; and 2) the epifaunal assemblage developing on an artificial substrate in a tropical environment is influenced by the season of deployment. These results should be taken into consideration when investigating community development on such structures, especially since in coral reefs, possibly due to the less dramatic changes between seasons, this aspect has not been considered to date.

   Section IV: Population dynamics of a coral reef ascidian in a deteriorating environment--the seasonal appearance, reproductive strategy and effect of high nutrient levels on the population of the newly-described colonial ascidian Botryllus eilatensis in the coral reefs of Eilat (accepted by Marine Ecology Progress Series).  A surprising phenomenonon observed in the coral reefs of Eilat during the study was the seasonal appearance of a new colonial ascidian, Botryllus eilatensis. In order to study overgrowth dynamics and the relationship between coral morphology, size, and orientation and ascidian overgrowth, I conducted monthly monitoring of 50 - 70 tagged coral colonies interacting with B. eilatensis. This study is the first to investigate coral-ascidian competitive interactions and is of major importance since the environmental conditions that are deleterious for corals (e.g. eutrophication) may create a favorable environment for filter-feeding organisms, such as ascidians, accelerating their growth and spread. The rate of B. eilatensis overgrowth on corals was influenced by the coral’s morphology and orientation combined with a seasonal appearance of the ascidian. This seasonality was linked to the vertical mixing phenomenon that occurs in Eilat every winter and results in higher nutrient levels in the water column, and thus in higher food availability. B. eilatensis fragments translocated to a nutrient-rich site increased three-fold in size and exhibited higher survivorship rates compared with intact fragments in a nutrient-poor area.

   The data collected in the above studies demonstrate the significant impact of human activities on ascidian ecology. Since ascidians are able to filter even minute particulate matter, any rise in nutrient levels and organic material in coastal waters will have a direct influence on their appearance. This was evident in the large size of H. momus found in the Mediterranean and the seasonal outbreaks of B. eilatensis in the coral reefs of Eilat. In addition, being a major component of the fouling community, ascidians play an important role in marine bioinvasion, as demonstrated by the appearance of six non-indigenous ascidian species with an Indo-Pacific origin. These species were particularly abundant on artificial marine structures, which are known to facilitate the spread of non-indigenous species. Thus, another human activity that may contribute to the spread of non-indigenous ascidians is the proliferation of artificial structures along the coast line. Ascidians can be strong spatial competitors, and once established they often experience population explosions that can develop into dense mats that overgrow and cover available surfaces. Although there is a growing awareness that ascidians can become of economic and ecological concern, relatively little is known about the ecology of many of these ascidians, unlike those taxa with a direct economic value. I hope this work will create a renewed interest in ascidian ecology, encourage future studies in this field and help in the development of tools for controlling the negative impacts of human activities on coastal regions.   


 3. Developmental change in germline and stem cell lineages in ascidian life history evolution. Federico D. Brown. Dept. of Biol., Univ. of Washington, Seattle, WA. Ph.D. advisor Dr. Billie Swalla.

   I studied the mechanistic evolution of coloniality in ascidians by comparing embryonic, larval and adult development of solitary and colonial ascidians.  I propose that flexibility in the specification of progenitor stem cell lineages and cell numbers in late embryonic and pre-metamorphic larval stages are necessary for life history transitions between solitary and colonial species. First, I studied expression of vasa, a DEAD box RNA helicase gene found in germ cells across the metazoans by in situ hybridization. I found that vasa mRNAs in the colonial ascidian Botryllus schlosseri are localized and segregated early in development to a posterior lineage of cells, resembling that of the germline of strictly sexual solitary ascidians. In mature colonies of botryllid ascidians, bot-vasa mRNA was expressed in putative spermatogonia, in oocytes of zooids, and unexpectedly in some circulating cells in the zooids and differentiating buds. Contrary to our predictions, vasa knockdown assays by siRNA in sexually mature colonies of Botryllus schlosseri  did not have obvious effects on germ cells, but resulted in heterochronic shifts in the development of buds during adult asexual cycles. These results suggest a novel and previously undescribed role of vasa in the regulation of budding cycles for Botryllus schlosseri. I hypothesize that circulating vasa-positive cells may be important for communication between the developing buds and the regressing mature zooid at the end of every blastogenic generation. I described the formation of early buds in the vasculature after inducing whole body regeneration in Botrylloides violaceus to characterize and identify circulating progenitor germline and somatic stem cells. I reported the expression of Piwi protein, involved in microRNA processing and stem cell maintenance, in hemocytes surrounding the regenerates, some of which were proliferating, as examined by proliferating cell nuclear antigen (PCNA) expression. In conclusion, I propose that multiple stem cell types are found in hemocytes of Botrylloides violaceus and that they undergo proliferation in the peripheral vasculature before differentiating into epithelial tissues for all three germ layers during whole body regeneration.


4. Looking for the deuterostome ancestor: hemichordate phylogeny, cartilage development, and regeneration.  Amanda Rychel, Dept. of Biol., Univ. of Washington, Seattle, WA.

Ph.D. advisor Dr. Billie Swalla.

    Hemichordates are deuterostomes, a group of animals that also includes xenoturbellids, echinoderms, tunicates, cephalochordates, and vertebrates. As the name suggests, hemichordates have some, but not all chordate characteristics, the most recognized being pharyngeal gill slits. This work investigates the origin of deuterostomes by studying evolutionary developmental transitions that occurred in the morphology between hemichordates, cephalochordates, tunicates and vertebrates. First, I characterized the structure and development of the pharyngeal slits and associated cartilages in a solitary hemichordate worm. Developmental origin and gene expression in the pharyngeal endoderm suggest that pharyngeal slits are homologous across deuterostomes, and hence likely present in the deuterostome ancestor. The slits and the cartilaginous skeleton that lies between are strikingly similar in hemichordates and cephalochordates.  Like vertebrate cartilage, the hemichordate and cephalochordate structures contain fibrillar collagen; however, they are entirely or partially secreted by the pharyngeal endoderm, rather than neural crest-derived chondrocytes that secrete pharyngeal cartilage in vertebrates. To further study evolution of deuterostome and chordate characteristics, I constructed a phylogeny of hemichordates using molecular markers to identify whether the ancestral form of hemichordates was solitary or colonial. In this phylogeny, colonial pterobranchs are not ancestral as traditionally placed. Instead, they are sister to the solitary, direct-developing harrimaniids. This relationship suggests that the deuterostome ancestor was a solitary, filter-feeding worm.  In addition to pharyngeal slits, some anterior structures in hemichordates may be homologous to chordate features. Anterior regeneration following amputation can be used to examine the development and homology of these structures. First, however, the basic cellular properties of hemichordate regeneration must be established. To this end, I examined the process of regeneration in the hemichordate Ptychodera flava. The process involved wound healing, followed by programmed cell death primarily in the endoderm, and proliferation of putative stem cells in the mesenchyme and cells in the ectoderm and endoderm as new anterior structures are elaborated. This study of Ptychodera flava regeneration provides a foundation for further development of a potentially powerful new stem cell and regeneration model in a close relative to vertebrates.


5. Influence of biotic interactions on the fitness, abundance and defense of colonial ascidians.
Gustavo Muniz Dias
, Departamento de Zoologia, IB, UNICAMP, Campinas - SP Brazil.    Ph.D. advisors Drs. Luiz Francisco Lembo Duarte and José Roberto Trigo.

   Biological interactions are a very important factor influencing the fitness of sessile organisms in rocky shores.  This study was carried out in southeastern Brazil. The effects of competition for space on the ascidian Didemnum perlucidum were assessed by experimental removal of potential competitors.  During six months, colonies free of competitors and colonies competing for space had their area and perimeter measured every two weeks, and the percentage of the colony perimeter in contact with each one of the most abundant taxonomic groups (mussels, barnacles, algae, ascidians and bryozoans).  The obtained results demonstrated that colonies free of competitors presented daily growth rate nine times larger than colonies competing for space.  Analyses of gonads showed that besides limiting asexual reproduction, competition also reduced the production of female gonads.  In addition, different competitors resulted in different interactions at the border of D. perlucidum colonies:  mussels and barnacles were overgrown by D. perlucidum, while interactions with ascidians (the interaction occurred with a great number of species but mainly Didemnidae and Styelidade species: Trididemnum orbiculatum  and Symplegma rubra were very frequent) resulted, in most cases, in stand-off of both competitors.  Histological analyses revealed morphological differences in the border of colonies free of competitors from those competing for space.  In colonies competing for space, morula cells were observed more frequently in the contact zone.  This indicates that, even in stand-off situations, competition can induce a response similar to that presented in immune processes, since morula cells are considered the most important cell type involved in defense of ascidians.  Experiments using the chemical extract of seven ascidian species demonstrated that, similar to tunic acidity, these extracts, in general, do NOT work as an effective defensive mechanism against predation and epibiosis.  Only calcareous spicules inhibited predation, which suggests that for the species of ascidians studied, the only defense is structural.  The effect of predation on ascidians in the post-recruitment period was availed through recruitment plates. Half of these plates were protected against predators by a cage while the other half were exposed to predation.  The results corroborate the susceptibility of ascidians to predation by fishes, as the abundance of these tunicates was ten-fold smaller in plates exposed to predators than in plates protected from predation.

  Part of the thesis is in press in Marine Ecology Progress Series.


6. Antimicrobial activity of marine ascidians from Vizhinjam Bay.

P. Mohideen Askar Nawas, Bharathidasan University, Tiruchirappalli, India. M. Phil. Advisor H.Abdul Jaffar Ali.

   In the present study, two colonial ascidians Didemnum sp. and Aplidium multiplicatum were screened for antimicrobial activity against total heterotrophic bacteria and five bacterial strains Bacillus subilis, Escherichia coli, Vibrio cholerae, Salmonella typhii and Pseudomonas aureginosa following the disc diffusion method. Crude methanol extracts of the two species showed that the strongest antimicrobial activity was found in Didemnum sp., which was significantly higher than that displayed by Aplidium multiplicatum (p< 0.005). Furthermore, Didemnum sp. showed maximum activity and featured bacterial inhibition against all the bacteria used and so the extract was further fractionated with different solvent system using column chromatography. Pseudomonas was more susceptible to treatment with extracts of the species than the other four stains. Fraction 4 revealed potent antibacterial activity, indicated by zone of inhibition, against all species of bacteria tested at higher concentration (0.5 mg/ml) and its effects decreased with decreased concentrations. The zone of inhibition was narrow at a concentration of 0.3mg/ml of fractions 1, 2 and 5 whereas, no inhibitory activity was observed at 0.1 mg/ml of fraction 1 and 4. Of the five strains, Pseudomonas was the susceptible bacteria after treatment with all fractions but most susceptible to the fraction 4 with the inhibitory zone of 8.5 ± 0.2 mm dia at a concentration of 0.5 mg/ml and followed by Bacillus subilis with 5.9 mm inhibitory zone. Hence, the present study indicates that Didemnum sp. showing antimicrobial activity could be used for future pharmacological research to cure tumor, viral and bacterial infections.


7. Nutritional value and antimicrobial activity of marine ascidian species.

M. Rajesh,  Bharathidasan University, Tiruchirappalli, India. M. Phil. advisor H.Abdul Jaffar Ali.

The present study was aimed to understand the economic importance of the Indian ascidian Phallusia nigra collected from Vizhinjam Bay. For this, the test and the mantle body of P. nigra was analysed for the presence of carbohydrate, protein, lipid, minerals such phosphorous and calcium, amino acid profiles and certain vitamins. The results of the amount of carbohydrate, protein, lipid and minerals such phosphorous and calcium showed that the concentration of total carbohydrate was higher in the test as compared to the mantle body. This corresponds to the higher amount of cellulose and crude fibre. The protein recorded the maximum level of all the biochemical components in the mantle body (23%) which is followed by lipid, cellulose, carbohydrate and fibre. A total of eight amino acids alanine, arginine, aspartic acid, glycine, leucine, lysine, methionine and tyrosine were found to occur. A high concentration of free amino acids has been found in the mantle body of the study animals as compared to the test. In addition to the amino acid profile, some B – complex vitamins such as riboflavin and thiamin were also estimated to understand the nutritive value. Very low quantity of both vitamins was recorded in the test. The people in the far East and certain parts of the Mediterranean have realised and appreciate the food value of ascidians because of its low calorific value and high content of proteins.  Based on the available literature, the present investigation also supports this view as P. nigra was found to contain high food value and thus surpass many marine food sources in terms of value added marine food.  Hence, in the scenario of growing demands for protein rich food, P. nigra may prove to be a timely alternative, if it is cultured in quality water. 

   The test and the mantle body of the study animal were further analysed for their potent biological potentiality following the disc diffusion method against total heterotrophic bacteria and three bacterial strains Bacillus subilis,  Escherichia coli and Pseudomonas aureginosa. The test showed higher activity than the mantle body against THB and the three bacterial strains tested, so the test was further fractionated with a different solvent system using column chromatography which afforded four different fractions. Fraction III (Methanol 100%) revealed potent antibacterial activity against THB tested at higher concentration (0.5 mg/ml) and its effects decreased with decreased concentrations. Further it showed potent antibacterial activity against all species of bacteria tested at higher concentration and its effects decreased with decreased concentrations. Of the three strains, Pseudomonas was the susceptible bacteria after treatment with all fractions but most susceptible to the fraction III. Today people prefer to use pharmaceutical products of natural origin because of their less side effects and so the study animal may be studied further to use in pharmacology.




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