Gretchen and Charles Lambert

12001 11th Ave. NW, Seattle, WA 98177

206-365-3734 or

home page:


Number 66                                                                                        June 2010


Thanks to the many contributions, this is another packed issue, with 121 new publications listed at the end. We hope you will find it very useful and interesting.


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



1. From Cristian Cañestro: I'd like to share with the ascidian community that I have recently moved to the Department of Genetics at the University of Barcelona (Spain), where I'm setting up my own group working with Oikopleura, among other chordate models. See our new publication Cañestro et al. 2010 at the end of this newsletter. My new email address is

2. From Rosana Rocha: I have pdf’s of a large number of old and difficult to obtain ascidian papers which I am happy to share with anyone who needs them. You can contact me  ( and I will send you the website address where you can download them.


3. From from Changho Yi: Another ascidian recipe! This one for ascidian whiskey—sure to be a favorite! .

  I saw Korean foods 'mong-gae bibim bap' in AN65. We enjoy Styela clava too. It was very funny in the cultural view, at least to me. Because I like that much. And I enjoy the 'mong-gae ju', means ascidian whiskey. It's little maniac, but many Koreans like that too. It's very simple:

   We make that by pouring Soju (korean traditional whiskeys) into hollow tunics of Halocynthia roretzi.'removing inner part' but 'remaining body fluid' is important. It provides unique smells.

 Can you imagine its taste? I like that much! J

[Editor’s note: the website is all in Korean; a translation for the second recipe would be appreciated but the photos speak for themselves!]


4. More dining adventures with ascidians at these websites:

Dining Out: Korean fare so fresh it's not even dead! The fourth anniversary of the Gastronauts, an online club for adventurous eaters, with a Korean banquet at East Seafood Restaurant in Flushing New York.  “Getting closer to the main event, a selection of kimchi (spicy pickled vegetables) arrived, followed closely by an array of sashimi, including such exotica as sea squirt, sea cucumber, abalone and sea urchin, along with the more familiar things aquatic, like salmon and tuna. The sea squirt was the least accessible of the bunch, being extremely iodiney and bitter. Folks in the know say that those are the very qualities this creature is prized for, giving the mouth a tingling, numb sensation similar to cloves. Definitely not for everybody.”


And another website for a slightly different Korean bibimbap:


5. From Christian Sardet, BioMarCell group UMR 7009 BioDev CNRS / Univ P et M Curie Paris 6,
Observatoire, Villefranche sur Me.

   We posted on line episodes of our series of short films called "Plankton Chronicles/ Chroniques du Plancton" as part of a project I initiated in the context of the Tara Oceans expedition ( and the 125th anniversary of the Villefranche Marine station (this fall). Hope you enjoy these short (1-2 min ) films edited with Noe Sardet and Sharif Mirschak from sequences shot in our Villefranche lab and on the Tara. (password:planktonic for all.) They will soon be in English and released for the general public.

Thanks for your suggestions and comments.

1) Macroplancton gélatineux 

2) Ctenophores: orgies de couleurs 

3)Le plancton 


5)Ptéropodes, des mollusques qui nagent:

6)Larves et embryons:

7)Protistes, ambiance spatiale (no sound):


They will soon be in English and released for the general public. Thanks for your suggestions and comments.


6. From Gretchen Lambert: I have a softbound copy for sale for $65.00 of Van Name, W.G. 1945. The North and South American Ascidians. Bull. Amer. Mus. of Nat. History. 476 pp., 31 black and white plates. This was the personal copy of Dr. Patricia Dudley, who studied parasitic copepods of ascidians. The money will go toward a student scholarship at the Univ. of Washington Friday Harbor Labs. This volume is now available online as a pdf :  (   209mb), but Dr. Dudley’s copy is on long lasting museum quality high rag heavy paper. Even though published more than 60 years ago, this volume is still the most useful publication for ascidian taxonomy and I consider it “the bible” for N. & S. America. Contact me at


7. From Howard Reid, Executive Director, Mona Institute of Applied Sciences, 4 Belmopan Close, University of the West Indies, Mona Kingston 7, Jamaica, Telephone: (876) 970-2021, (876) 970-2042, Fax: (876) 970-0289, Email:, Website: .

   For sale: compact disc entitled: Caribbean Sea Squirts: The Goodbody Collection produced by the Mona Institute of Applied Sciences (MIAS) in collaboration with the Environmental Foundation of Jamaica.  The CD is based on over fifty years of research by author and zoologist, Professor Ivan Goodbody, on Caribbean Ascidiacea at the University of the West Indies.   

   Over 100 species of Sea Squirts (Ascidiacea) have been recorded from shallow water environments in the Caribbean. The compact disc illustrates 59 of these species by means of photographs and four others by diagrams. The photographs are of living animals most of them taken in their natural environment.  Most of the pictures have been selected from a collection of photographs taken by Ivan Goodbody during his research on the diversity of ascidians in the Caribbean.  The project illustrates almost 58% of known species at this time and certainly illustrates almost all of the common species likely to be encountered by students or others exploring the shallow-water environments of the Caribbean.  For each species illustrated, there is an accompanying text describing the pictures and highlighting the principal characters which guide identification in the field.  For most species the written text is supplemented by a vocal description recorded by Ivan Goodbody.

   The CDs are informative, interactive, colorful, and are tailored to attract scholars, researchers, students and marine enthusiasts.  The cost is US$75 plus shipping and handling and can be purchased online through our website:  Further information such as videos and reviews can also be viewed on the website.  For purchases of 3 CDs or more, the price is US$60 plus shipping and handling and can be accessed through our office using the information above.  


8. From Ken Hastings, Montreal Neurological Institute, McGill University, Montreal, Quebec,


    Please mark your calendars. The Sixth International Tunicate Meeting will be held in MontrealCanada, at McGill University, July 7-11, 2011 (NEXT summer). Music lovers take note - these dates overlap with the Montreal International Jazz Festival. Initial outline plans follow the model of the meeting in Okinawa in 2009. July 7 and 8 will be working days with morning and afternoon plenary sessions (registration on the morning of July 7, so plan to arrive in Montreal July 6). July 9 will be an excursion day and that night the Jazz Festival’s closing giant free street concert will also occur. July 10 and 11 will be working days with morning and afternoon sessions, and with a closing event in the evening of July 11. Departure date will be July 12. The program committee includes me, Robert Lauzon (Union College, Schenectady New York), Tom Meedel (Rhode Island College, Providence, Rhode Island), Ian Meinertzhagen (Dalhousie University, Nova Scotia) and may be expanded as appropriate.

   See you in Montreal in 2011!




1. From Serena Teo and Serina Lee, Tropical Marine Science Institute, National University of Singapore.

  Currently our laboratory is conducting surveys of marine fouling organisms in Singapore waters, with the aim to develop baseline knowledge of fouling species in Singapore's coastal waters. Tunicates are a significant but often neglected group in the fouling communities found in Singapore as very little information is known for the tropical Indo-Pacific region. We have been collecting ascidians extensively from around Singapore in the past year. These have been photographed, preserved, and taxonomic work is in progress. In May this year, Gretchen Lambert came to Singapore to conduct a 5 day ascidian taxonomy workshop. Through this workshop and subsequent work, we hope to publish a first checklist of ascidians from Singapore soon.


2. The hemolymph of the ascidian Styela plicata contains heparin in intracellular granules of basophil-like cells. Cintia Monteiro De Barros1,2; Giselle Cristina Ferreira Corrêa1; Silvana Allodi2; Mauro Sérgio Gonçalves Pavão1. 1Laboratório de Tecido Conjuntivo – IBqM – UFRJ –  2Laboratório de Biologia de Invertebrados – ICB – UFRJ –

   Introduction: In vertebrate chordates, heparin is synthesized on a specific protein core, forming the Serglycin proteoglycan (PG). This PG occurs in secretory granules of some immune cells, such as tissue mast cells and circulating basophils. In mast cells, the Serglycin PG is glycosylated with heparin, whereas in basophil, chondroitin sulfate E is the glycosaminoglycan (GAG) component of the serglycin protein [1]. Ascidians, invertebrate chordates, contain different types of immune cells, occurring either in the tissue or circulating in the hemolymph. These cells display morphological and biochemical characteristics that resemble vertebrate mast cells and basophils, respectively. In the ascidian S.plicata, a heparin GAG was shown to co-localize with histamine in intracellular granules of only one type of hemolymph hemocyte, named granulocyte [2].     

Objective: To characterize the PG of the ascidian granulocyte and their biological function.

Methods: The hemocytes were harvested from the pericardial cavity of the ascidians. The PGs of the hemocytes were extracted and purified in ion-exchange chromatography. The hemocytes were also prepared for immunoelectronmicroscopy and western blotting using primary anti-serglycin antibody. In addition, an in vivo inflammatory assay was performed with the injection of E.coli into the tunic. After 5 days, a fragment of the tunic was dissected, fixed for immunohistochemistry with anti-serglycin and anti-MCP-1 antibodies.

Results: The Immunoelectronmicroscopy and western blotting analyses with the anti-serglycin antibody indicate that a serglycin-like protein co-localize with heparin and histamine in the intracellular granules of the ascidian basophil-like cell (Figure 1). An initial biochemical analyses indicate that the granulocyte heparin is linked to a serglycin proteic core. Moreover, in vivo inflammatory assays confirm the involvement of granulocytes (Figure 2). Theses results suggest that in primitive basophil-like cells from invertebrate chordates, the serglycin PGs are glycosylated with heparin chains, different from basophils from vertebrate chordates. Supported by CNPq, CAPES, FAPERJ, FUJB.


[1] Iozzo, R.V. 1999. Marcel Dekker Inc. New York, NY.

[2] De Barros, C.M., Andrade, L.R., Allodi, S., Viskov, C., Mourier, P.A., Cavalcante, C.M., Straus, A.H., Takahashi, H.K., Pomim, V.H., Carvalho, V.F., Martins, M.A., Pavão, M.S.G. J. Biol. Chem. 2007. 282(3): 1615-1626.


3. From Anna Di Gregorio

Temporal regulation of the muscle gene cascade by Macho1 and Tbx6 transcription factors in Ciona intestinalis. In press, Journal of Cell Science.

 Kugler JE, Gazdoiu S, Oda-Ishii I, Passamaneck YJ, Erives AJ, and Di Gregorio, A., Dept. of Cell and Developmental Biol., Weill Medical College of Cornell Univ., New York, NY 10065, USA

    For over a century, muscle formation in the ascidian embryo has been representative of "mosaic" development.  The molecular basis of muscle fate predetermination has been partly elucidated with the discovery of Macho-1, a maternal zinc-finger transcription factor necessary and sufficient for primary muscle development, and of its transcriptional intermediaries Tbx6b and Tbx6c.  However, the molecular mechanisms by which the maternal information is decoded by cis-regulatory modules (CRMs) associated with muscle transcription factor and structural genes, and the ways by which a seamless transition from maternal to zygotic transcription is ensured, are still mostly unclear.  By combining misexpression assays with CRM analyses, we have identified the mechanisms through which Ciona Macho1 (Ci-Macho1) initiates expression of Ci-Tbx6b and Ci-Tbx6c, and we have unveiled the cross-regulatory interactions between the latter transcription factors.  Knowledge acquired from the analysis of the Ci-Tbx6b CRM facilitated both the identification of a related CRM in the Ci-Tbx6c locus and the characterization of two CRMs associated with the structural muscle gene fibrillar collagen 1 (CiFCol1).  We use these representative examples to reconstruct how compact CRMs orchestrate the muscle developmental program from pre-localized ooplasmic determinants to differentiated larval muscle in ascidian embryos.


4. Preparation of pickle from Herdmania pallida, simple ascidian (In Press - World Journal of Dairy and Food Science - IDOSI Publications)

M. Tamilselvi, V. Sivakumar, H. Abdul Jaffar Ali and R.D. Thilaga 1 2 3 4

Dept. of Zool. V.V. Vanniaperumal College for Women,Virudhunagar - 626 001, India 1

Dept. of Zool. V.O. Chidambaram College, Thoothukudi - 626 008, Tamilnadu, India 2

Dept. of Biotechnol. Islamiah College, Vaniyambadi - 635 752, Tamilnadu, India 3

Dept. of Zool. St. Marys College, Thoothukudi - 628 008, Tamilnadu, India 4

  Herdmania pallida, simple ascidian, belongs to the Subphylum Urochordata, Class Ascidiacea, Family Pyuridae and is commonly available in Tuticorin coast of India. Like other animals such as prawns, molluscs and fishes, ascidians are also consumed as food in various parts of the world due to its high nutritional and medicinal value. In the present study, biochemical components like carbohydrate, protein, lipids and amino acids were quantitatively estimated in H. pallida. As this species has high protein content in its body tissues, pickle was prepared from the mantle bodies and the product was packed in sterilized bottles and stored at ambient temperature. Lemon pieces, garlic, fenugreek etc., and vinegar were added to the blanched mantle bodies. It would have dissolved the spicules in H. pallida as we never felt the presence of spicules in the mantle bodies while tasting. The shelf life period was assessed for a period of six months. Quality indicators like pH, microbial load and sensory evaluation were analysed. Spoilage was not observed through out the study period. In conclusion, the prepared pickle from the mantle bodies of H. pallida is good and safe product for human consumption and can be stored for a period of six months.

  [Personal note added by author: After preparing the pickle, our family members were consuming it continuously for a month. We received appreciation whole heartedly from the groups of people who had the good opportunity to taste it. Really this pickle excelled in taste and flavour. To my knowledge, I have not enjoyed this taste and flavour in the pickles of molluscs, prawn and fish. This is the best one. As H. pallida contains calcium, protein, iron, etc this species could be recommended for anemic, osteoporesis patients. Further research is needed on these aspects.]


5. From Dr. H. A. Jaffar Ali: research activities carrying out by my students for their Ph.D programs.

a. Mrs. R..Radhalakshmi, Assistant Prof. in Zoology,VVV College, Virudhunagar is doing research for her Doctoral Degree on Nutritional value of marine ascidians in Indian coastal waters under the supervision of Dr.H.A.Jaffar Ali, Asst. Prof. of Biotechnology, Islamiah College, Vaniyambadi, India with Co-Guide Dr. V.Sivakumar, Associate Prof. of Zoology, V.O.C.College, Tuticorin. e-mail:

 This work focuses on the nutritive value such as protein, amino acid profile, carbohydrates, lipid, omega – 3 – fatty acids, vitamins and micro & macro minerals of both simple and colonial ascidians along the Indian coastal waters with the objective to pave the way for better utilization of marine food sources and meet out the nutritional requirements of the ever–increasing population in India.

b. Mr. A.Kallemullah Khan is doing his Ph.D. in the discipline Biotechnology Dravidian University, Kuppom, Andra under the guidance of Dr. Jaffar Ali. Title of the thesis is

Marine natural products from ascidians off Indian coastal waters and their biotechnological implications. This research work focuses on isolation of marine natural products from chosen ascidians along the Indian coastal waters and screening for their biological activities such as, antimicrobial, antifungal, cytotoxic and antifouling activities. This work also focuses on biosynthesis of silver/gold nanoparticles.




1.International Invasive Sea Squirt Conference-IISSC III, Woods Hole MA April 26-28, 2010. The complete program with links to abstracts can be accessed at . The proceedings will be published in the journal Aquatic Invasions.


Half day ascidian taxonomy workshop led by Gretchen Lambert.


Plenary talk: James T. Carlton, The Future Sea Squirt World: Impacts of Climate Change on Ascidian Invasions.

Biology, Systematics and Biogeography Session:

Linking physiology, biogeography and success of non-natives in subtidal communities in the Gulf of Maine Dijkstra, Jennifer A.; Westerman, E.L.; Brooks, C.; Harris, L.G.
Reproductive biology of Ciona intestinalis in Prince Edward Island, Canada Bourque, Daniel 
Spawning and culture techniques for the invasive ascidian Didemnum vexillum Fletcher, Lauren M.; Forrest, B.M.; Bell, J.J.
Monitoring ascidians on natural and anthropogenic habitats in Bocas del Toro, Panama
Rocha, Rosana M.
Abundance and diversity of ascidians in Pacific Panama: Differences with coastal development levels and distance from the mainland Bullard, Stephan G.; Carman, M.R.; Rocha, R.M.; Djikstra, J.A.; Goodwin, A.
Ascidians at the Pacific and Atlantic entrances to the Panama Canal Carman, Mary R.; Rocha, R.M.; Lambert, G.; Bullard, S.G.; Dijkstra, J.A.; Roper, J.J.; Goodwin, A.; Baker, E.M.
Persistence of colonies of the tunicate Didemnum vexillum over a 5-year period at specific sites on gravel habitats of Georges Bank, New England Valentine, Page C.; Gallea, L.; Guida, V.; Blackwood, D.S.

TBA York, Amber
A time-series of morphological development of the tunicate Didemnum vexillum from settled larva to colony in New England Valentine, Page C.; Carman, M.R.; Blackwood, D.S.

Ecology and Genetics Session:

Total genome analysis of Didemnum vexillum Kott, 2002, a model organism for the study of marine bio-invasions Gittenberger, Adriaan; Stefaniak, L.M.
High resolution temporal patterns of larval settlement: Do settlers avoid their spatial competitors? Hamilton, John F.; Whitlatch, R.B.; Osman, R.W.
Multiplex beads assay as a sensitive tool to detect tunicate aquatic invasive species in Prince Edward Island sea water Siah, Ahmed; Pariseau, J.; McKenna, P.; Sandjong, B.;Davidson, J.; Johnson, G.; Berthe, F.C.J.
Interspecific competition: Effect of colonial tunicate presence on Ciona intestinalis recruitment Paetzold, S. Christine; Davidson, John; Hill, J.; Davidson, Jeff
The vasa gene as a phylogenetic marker for identification of invasive botryllids Tankouo-Sandjong, Bertrand; Siah, A.
Ecology and management of invasive solitary ascidians in New Zealand Willis, Kate; Nutsford, S.; Floerl, O.
Investigations in Ciona intestinalis biofouling in Nova Scotia, Canada Vercaemer, Benedikte; Sephton, D.; Nicolas, J-M.; Keays J.
Biology, ecology and trials of potential methods for control of the introduced ascidian Eudistoma elongatum in Northland, New Zealand Page, Michael, J.; Morrisey, D.J.; Handley, S.J.
Global phylogeography of the solitary ascidian Styela plicata Pineda, Mari Carmen; López-Legentil, S.;Turon, X.

Impacts Session:

Ecosystem consequences of vase tunicate (Ciona intestinalis) infestation in mussel-producing estuaries of Prince Edward Island Locke, Andrea; Bourque, D.; Leger, C; Barkhouse, C.
An approach utilizing inhibitors of invertebrate settlement and metamorphosis in preventing fouling of aquaculture structures by ascidian pests Heasman, Kevin; Chautard, C.; Dodgshun, T.; Mountfort, D.O.
First occurrence of the invasive tunicates Ascidiella aspersa and Didemnum vexillum in eelgrass habitat Colarusso, Phil; Carman, M.R.; Grunden, D.W.; Chintala, M.M. Blackwood, D.S.
If You Build It, They Will Come: Settlement of tunicates on artificial seagrass Grunden, David W.; Carman, M.R.; Colarusso, P.; Chintala, M.M.; Blackwood, D.S.
Interspecific competition between the colonial tunicates Botryllus schlosseri, Botrylloides violaceus and the cultured mussel Mytilus edulis, and efficacy of pressurized seawater as an anti-fouling treatment Arens, Collin J.; Davidson, J.; Ramsay, A.
TBA Locke, Andrea

Risk Assessment and Management Session:

 Treatments to rid tunicates from cultured blue mussels Mytilus edulis Green-Beach, Emma; Carman, M.R.; Karney, R.C.; Bagnall, P.L.; Carman, M.M.
Rapid Assessment for Didemnum vexillum in southwest New Brunswick Martin, Jennifer L.; LeGresley, M.M.; Cooper, J.A.; Thorpe, B.; Locke, A.; Simard, N.; Sephton, D.; Bernier, R.; Bérubé, I.; Hill, B.2; Keays, J.; Knox, D.; Landry, T.; Lander, T.; Nadeau, A.; Watson, E.J.
Successes and failures: proof-of-concept trials with new mitigation treatments against invasive tunicates  Paetzold, S. Christine; Davidson, J.
Predicting the impacts of ocean warming on a California fouling community using stage-structured population modeling  Sorte, Cascade J.B.; Cockrell, M.L.
Tunicate monitoring in Nova Scotia 2005-2010: Program recap

2. Society for Integrative & Comparative Biology (SICB), Seattle, WA Jan. 3-7, 2010.


a) Comparative Proteomics: The Response of the ascidian congeners Ciona intestinalis and C. savignyi to acute temperature stress. SERAFINI, L., TOMANEK, L. Cal Poly, SLO.
    Ciona intestinalis and C. savignyi are congeneric tunicates that are commonly found along the Pacific coast of North America. In order to compare the thermal stress response between these two sister species, we studied global changes in protein expression in response to heat stress using proteomics. The ascidians’ phylogenetic position as urochordates, along with their sequenced genomes, make C. intestinalis and C. savignyi model organisms for proteomics research. To observe the effect of heat shock, animals of both species were exposed to either a 22˚C, 25˚C, or 28˚C temperature treatment for 6 hours, and then allowed to recover at 13˚C for 16 hours. Two-dimensional gel electrophoresis was employed to separate proteins and create protein expression profiles. We found that 32% of the total protein spots detected for C. intestinalis and 24% of those detected for C. savignyi underwent significant changes in their expression across treatment groups. In order to further characterize the thermal stress response, we used MALDI TOF/TOF mass spectrometry to identify proteins of interest and had a 90% success rate. After comparing the identified proteins across species, we were able to determine that different proteins showed changes in their expression across treatments. This suggests that the thermal stress response differs between these two species. For C. savignyi, the thermal stress response consisted of variations in the expression of cytoskeletal proteins; while C. intestinalis had a more complex response consisting of changes in a wide-range of proteins including chaperones, transport proteins, and metabolic proteins. These interspecific differences in the thermal stress response suggest possible biochemical differences that may lead to variation in thermal tolerance.


b) Proteomic analysis of acute salinity stress in the two ascidian species Ciona savignyi and C. intestinalis. KOMAN, James S., TOMANEK, Lars. Cal Poly SLO
   The ascidian species Ciona savignyi and C. intestinalis have been fully sequenced and thus allow analysis of their proteomic response to environmental stress. We have chosen to analyze their response to hyposaline conditions, which are known to occur during heavy winter rains in the coastal regions in which Ciona populations live. Such conditions often lead to population declines and it has been suggested that it may contribute to determining the life history of these species along the West coast of North America. Testing for their proteomic response to salinity may provide insights into the mechanisms that are responsible for setting tolerance limits to hyposaline conditions in these two congeneric species. Characterizing the proteomic response to acute salinity and other stresses also contributes to our understanding of the minimal stress proteome, which we are characterizing for these species. We exposed each species to decreasing salinities, 100%, 85% and 70% full-strength seawater, for 6 hours. After exposure to hyposaline conditions, the specimens were brought back to 100% seawater to recover for 4 hours. Organisms were dissected to remove the tunic, and 2D SDS-PAGE was performed to separate proteins and characterize changes in protein expression. Using a 2D gel image analysis software we identified 5% and 19% of the detected proteins to be differentially expressed in C. savignyi and C. intestinalis, respectively, in response to acute salinity stress in the treatment in comparison to the control group. Analysis of these proteins with MALDI TOF-TOF mass spectrometry has identified numerous cytoskeletal and general stress response proteins, providing insights into the intense cellular and cytoskeletal restructuring that occurs in response to hyposaline exposure.


c) Cellular Basis for ascidian neurulation. SHERRARD, K.*; ROBIN, F; CARVAJAL, D.; DANAHER, B.; DENG, W.; JOSHI, S.; MONTGOMERY, M.; SEMON, S.; WHITE, D.; MUNRO, E.; Center for Cell Dynamics, Univ. of Washington; Center for Cell Dynamics (U.W.) and Univ. of Chicago; Univ. de Antioquia, Columbia; Center for Cell Dynamics (U.W.); SARS Institute, Bergen, Norway; Univ. of Pittsburgh; Univ. of Washington; Univ. of Washington; Univ. of Alberta, Edmonton; Center for Cell Dynamics (U.W.) and Univ. of Chicago
    Ascidians form a neural tube of only a few hundred cells, providing an unparalleled opportunity to study this fundamental morphogenetic process at the cellular level. Several distinct subprocesses contribute to ascidian neurulation. First, primary invagination of the neural plate is followed by axial elongation accompanied by mediolateral cell-cell intercalation and oriented cell divisions. Second and concurrently, constriction of the blastopore and medially-directed crawling bring epidermal cells together at the posterior midline to initiate neural tube closure. Finally, neural tube closure proceeds through anterior to posterior zippering of both the neural tube and the overlying epidermal cells. Experiments with explants and laser ablation reveal that neural plate invagination proceeds normally in the absence of the posterior zipper-forming cells, but zippering fails. Conversely, zipper initiation and propagation proceed normally in the absence of the primary neural plate. Furthermore, unlike in vertebrates, formation and elongation of the neural tube does not require the presence of a notochord. A propagating zone of localized rho-kinase-dependent myosin activation is required for blastopore closure and for zippering, but not for invagination of the neural plate. Thus in ascidians, the complex process of neurulation can be decomposed into, and understood in terms of, a set of fundamental cellular mechanisms that are directly amenable to experimental analysis.


d) A propagating zone of localized protrusive and contractile activity drives zippering and neural tube closure in ascidians. ROBIN, F.B.*; SHERRARD, K.M.; MONTGOMERY, M.; SEMON, S.; MUNRO, E.M.; Univ. of Washington & Univ. of Chicago; Univ. of Washington & Univ. of Chicago; Univ. of Washington; Univ. of Washington; Univ. of Washington & Univ. of Chicago
    The ascidian neural tube forms by neurectoderm folding, followed by fusion of the neural folds at the dorsal midline by a posterior-to-anterior "zippering" process. Using time-lapse fluorescence microscopy, we found that neural closure is accompanied by the constriction of a supra-cellular purse string-like actin cable that develops at the boundaries between presumptive neurectoderm and lateral epidermis. Significantly, constriction of the purse string is strongly biased to it’s posterior end where it is accompanied by highly localized protrusive activity and cortical contractility. Immuno-staining of fixed embryos and time-lapse microscopy of live embryos revealed that the active RhoA and active forms of Myosin are similarly localized to the highly contractile posterior zone, which propagates posterior to anterior and cell to cell as zippering proceeds. Blocking the Rho-pathway kinase ROCK with the inhibitor Y-27632 leads to loss of localized activated Myosin and specifically causes the zipper to collapse and the entire purse string to relax. Following fusion of opposed neural fold cells, midline cells are first dragged anteriorly as the zipper progresses further forward, then abruptly released as the apical domains of presumptive dermal and neural cells lose contact. The result of this process is to form two epithelia (one neural, one dermal) from a single initial epithelial layer. Our data highlight the existence of a "morphogenetic organizer" that coordinates supercellular force generation in time and space to achieve the completion of an essential step in chordate development.


e) Neural Tube Closure: Zipper Propagation in Ascidian Embryos. SEMON, Shelby N*; ROBIN, Francois; SHERRARD, Kristin; MUNRO, Edwin; Univ. of Washington; MGCB, Univ. of Chicago; Center for Cellular Dynamics, Univ. of Washington; MGCB, Univ. of Chicago
    Ascidians are a member of the chordate phylum and as such they undergo many of the same developmental processes that other chordates undergo. The difference is that Ascidians have a much smaller number of cells and larger cell size to embryo size ratio making them an ideal system in which to study the fundamental processes involved in neural tube closure. Focusing on the cellular processes involved in neural tube closure, we used confocal microscopes for fixed and time lapse imaging with fluorescent probes to carefully observe the cellular behaviors of control embryos. We found that neural tube closure proceeds unidirectionally from posterior to anterior in a behavior we call zippering. This behavior involves three main processes: cell crawling, adhesion and contraction. Focusing on the role of contractility, we found that there is a mechanically continuous actin purse string that has myosin localized in an area of high contractility in the boundary cells closest to the zipper. Using Y-27632, a drug that inhibits RhoKinase-activated myosin to inhibit contractility at different stages during zipper closure, we found that when Y-27632 was added near the beginning of the zippering process, zippering did not proceed and when added late in the zippering process, the zipper broke and the interior structures of the embryo extended out of the anterior half. However, invagination of the floor cells of the neural plate occurred normally in Y-27632 treated embryos. We conclude that contractility is essential for zipper initiation and propagation but not a driving force in the invagination that proceeds the zippering process.


f) Initiation of Neural Tube Closure in Ciona Intestinalis. MONTGOMERY, M.S.*; MUNRO, E.; SHERRARD, K.; ROBIN, F.; Univ. of Washington, Seattle
    Ascidian neural tube closure (zippering) is initiated by the assembling of cells and materials at a posteriorly located center we are calling the zipper origin. The zipper origin as a structure has not previously been described in the literature. To advance our understanding of the prospective identity and morphological change of cells in contact with the zipper origin, we created 3-D reconstructions through Amira and 3-D Virtual Embryo of early neurula we fixed and stained in phalloidin. We find that posterior-most muscle, neural plate, notochord, and epidermal cells meet as they extend towards the zipper origin. With time-lapse imaging and confocal microscopy, we show that initiation of neural tube closure occurs by three main events: 1) cinching of the apical ends of muscle cells lining the closing blastopore; 2) rolling-inward of the two posterior-most roof cells; and 3) crawling of posterior epithelial cells towards the zipper origin. We provide evidence that this gathering at the zipper origin is significantly driven by myosin contractility. We find the presence of monophosphorylated nonmuscle myosin II at the same time and location of blastopore closure and building of the zipper origin. We also find diphosphorylated nonmuscle myosin II concentrated at the zipper origin at earlier and later stages corresponding to the time of initiation of neural tube closure. We show further that inhibition of nonmuscle myosin II through the Rho kinase pathway causes a drastic disassembly of material already collected at the zipper origin and significant deformation of zipper-initiating cells. Our results show that activation of Rho kinase and myosin II is necessary to initiate tube closure. With reduced myosin contractility, formation of the neural tube does not occur, indicating the motor protein’s role as an indispensable biomechanical device.


g) Computational approach to neural tube closure. VELASQUEZ-CARVAJAL, D.*; SHERRARD, K.M.; ROBIN, F.B.; MUNRO , E.M.; Univ. of Washington and Univ. of Antioquia; Univ. of Washington; Univ. of Washington and Univ. of Chicago; Univ. of Washington and Univ. of Chicago
    Neural tube closure in ascidians occurs by unidirectional zippering of epithelial and neural tube cells, and involves several distinct cellular processes. On each side of the leading edge of the zipper, filopodia protrude, make contact and pull the edges together until the cells form new adhesions. At the same time, a “V” of localized acto-myosin contractility is progressively activated in just a few cells as they reach the zipper’s leading edge, and experiments inhibiting this contractile “V” demonstrate it is essential for closure. It is not clear how the filopodia, formation of adhesions, and localized contractility work together to generate neural tube closure. We used a two-dimensional computational model in which individual cells could exhibit these behaviors to varying degrees to explore their relative importance. We found that a combination of localized “V” contractility and filopodial action best reproduced the shape changes observed in real embryos. In contrast, uniform purse-string contractility failed to form a long tube, while filopodial action alone was ineffective at sealing the tube. Localized “V” contractility could drive normal closure kinematics, but only a for very restricted set of parameter choices. Further, the model was able to reproduce an experimental observation from partially ablated embryos, in which the “V” had a wider angle than under normal conditions and neural tube closure failed. A concomitance of different cell and tissue conditions are needed for a morphogenetic process to occur properly, and computional modeling provides a means, complementary to experiments, to separate key features essential to such processes.


h) Ascidian neural tube morphogenesis proceeds normally following ablation of the notochord DANAHER, B.*; MUNRO, E.; SHERRARD, K.; ROBIN, F.; Univ. of Washington.
    Ascidian neurulation proceeds through concurrent invagination and extension of a monolayer neural plate. The underlying notochord forms simultaneously through invagination and convergent extension of a monolayer plate into a cylindrical rod. The notochord is a likely candidate for effecting neural morphogenesis: it is the substrate for neural tissue throughout neurulation, and its robust convergent extension is a probable source of extensile force in the ascidian tailbud. Here we use laser ablation at the 64-cell stage and time lapse confocal microscopy to examine neural morphogenesis in the absence of a notochord. We show that neural plate organization, invagination, and neural tube closure all continue normally. Neural tube extension is slightly limited, but neural cells retain normal organization and significant elongation. Our results indicate that neural morphogenesis is a robust process, and suggest alternative driving forces for ascidian tailbud elongation.


i) From Genome to Development in Amphioxus. HOLLAND, L.Z.*; SHORT, S.; Univ. of California San Diego; Portsmouth Univ, U.K.
    Although gnathostome genomes (mammals, fish) were the first deuterostome genomes sequenced, genomes of most major deuterostome groups have now been sequenced. These include an agnathan (lamprey), ascidian and appendicularian tunicates, a cephalochordate (amphioxus) an echinoderm (sea urchin) and hemichordate. Comparative genomics has answered many questions (e.g. the timing of whole genome duplications) but has also raised awareness of how little is known about how genes direct formation of a functional organism. Heritable traits not directly encoded by the genome (epigenetics) have assumed increased importance in this regard. One such epigenetic phenomenon is alternative splicing. Paired box (Pax) transcription factors are involved in development of such endocrine organs as the thyroid and its homolog, the endostyle (in larval lampreys, tunicates and amphioxus) [Pax2/5/8], the thymus [Pax3/7], pharyngeal endoderm [Pax1/9] and adenohypophysis [Pax4/6]. Alternative splicing can affect the DNA binding domains of Pax factors and/or the c-terminal transactivation and repression domains. Some splice forms are evolutionarily conserved, suggesting vital functions, while others are not. Pax splice forms are differentially expressed during development in both amphioxus and vertebrates. To investigate the importance of Pax isoforms in development, we used antisense morpholino-oligonucleotides (MOs) to knock down function of all isoforms of amphioxus Pax2/5/8 and Pax1/9 as well as that of specific isoforms. Our results show that knockdown of all isoforms of these two genes with an MO complementary to the ATG start codon gives the same phenotype as using a splice-blocking MO to convert the major isoform expressed in early development to a minor one. These results suggest that the minor isoforms may function to modulate function of the major ones and underscore the importance of post-transcriptional mechanisms for gene function.


j) Amphioxus thyroid hormone signaling pathway and the evolution of metamorphosis in chordates. PARIS, M*; ESCRIVA, H; SCHUBERT, M; BRUNET, F; BRTKO, J; CIESIELSKI, F; JAMIN, E; CRAVEDI, JP; RENAUD, JP; SCANLAN, TS; HOLLAND, ND; LAUDET, V; Univ. of California, Berkeley; Lab. Arago, Banyuls sur mer; Ecole Normale Superieure de Lyon; Ecole Normale Superieure de Lyon; Inst. of Experimental Endocrinol.; AliX, Illkirch; INRA, Toulouse; INRA, Toulouse; AliX, Illkirch; Oregon Health & Science Univ., Portland; Scripps Inst. of Oceanography, La Jolla; Ecole Normale Superieure de Lyon
    Metamorphosis is a spectacular post-embryonic developmental stage, allowing a larva to become a juvenile. In the chordate lineage, that comprises vertebrates, urochordates like tunicates and cephalochordates like amphioxus, the morphological changes during larva-to-adult transitions vary extensively from one species to another, suggesting that metamorphosis may have arisen several times in the chordate lineage. Does the molecular determinism of metamorphosis in this group reflect this morphological diversity? In the well-studied vertebrates, metamorphosis is triggered by thyroid hormones (THs) binding to their receptor TR, member of the nuclear hormone receptor superfamily. In order to get better insight into the evolution of the molecular determinism of metamorphosis in chordates, we focused on the most basal chordate amphioxus. Combined biochemical and phylogenetic approaches allowed us to establish that amphioxus produces various THs through metabolic pathways homologous to vertebrate ones. Then we showed that TH production as well as TH-dependent TR activation are essential for metamorphosis induction in amphioxus, like in vertebrates, with the slight difference that the active TH is not T3, the classical vertebrate TH, but possibly its derivative TRIAC. Consequently the homology of metamorphosis in chordates is revealed by the conservation of its triggering mechanism. This suggests that the evolution of metamorphosis in chordates is marked by the conservation of the couple TH/TR whereas other parts of the regulatory network may change to underlie the morphological diversity observed nowadays.


k) Tunicate genomics: a window into chordate development and evolution ZELLER, R.W.; San Diego State Univ.
    Tunicates, particularly ascidians, are an excellent biological system in which to study gene regulatory mechanisms important during embryological development. Because of their phylogenetic position within the chordates, tunicates will provide insight into the evolution of chordate gene regulation. Three tunicate genomes have been sequenced to date: two ascidians – Ciona intestinalis and C. savignyi, and one larvacean – Oikopleura dioica – possessing the smallest known chordate genome. This wealth of genomic information, coupled with extensive EST collections from all three species, has allowed researchers to initiate genome-level investigations of tunicate development. The Ciona genome encodes about 15,000 genes, but is one twentieth the size of typical vertebrate genomes. Tunicate genomes lack extensive gene duplication events thus gene regulatory mechanisms are believed to be simplified compared to vertebrates. A review of the current Ciona genome assembly will be presented and examples from genome-scale research efforts will be discussed.


l) Genomics of Amphioxus and Tunicates: Tracing the Evolution of the Endocrine System. SHERWOOD, Nancy M.*; ROCH, Graeme J.; TELLO, Javier A.; Univ. of Victoria
    The sequencing of the genomes for amphioxus and tunicates has allowed us to examine the foundation of the endocrine system in species that evolved at the transition between invertebrates and vertebrates. In regard to reproduction, the neuroendocrine system in these early chordates is vertebrate-like, but the pituitary and gonadal endocrine hormones and receptors are clearly lacking a number of vertebrate components. In tunicates (Ciona intestinalis and C. savignyi), six gonadotropin-releasing hormones (GnRH) and four receptors were cloned. The six GnRH peptides selectively activated recombinant forms of their receptors and in vivo spawning. In amphioxus (Brachiostoma floridae), four GnRH receptors were cloned and expressed in COS7 cells; their pattern of response provides evidence that both invertebrate-type and vertebrate-type GnRH receptors exist in amphioxus. In contrast, functioning neuroendocrine systems in basal chordates do not appear to activate specific pituitary hormones, as neither FSH nor LH hormones/receptors are identified in the amphioxus or tunicate genomes. Rather it is likely that the GnRH peptides act directly on the gonads. Only amphioxus and not Ciona has the necessary enzymes for biosynthesis of sex steroids. Also, both chordate groups have a small number of nuclear receptors but only amphioxus has steroid-type nuclear receptors. Outside of reproduction, the insulin-IGF family and receptors, essential for growth, development and metabolism, are present in both amphioxus and tunicates along with other hormones including the osmoregulatory hormone stanniocalcin. The endocrine components are homologous to those in vertebrates and reveal the foundation of the chordate endocrine system before the genomic duplications in early vertebrates.


m) A Dual Origin of the Pituitary Primordium in the Ascidian. KANO, S*; SATOU, Y; DESCHET, K; MARTIN, P; HAEUSSLER, M; JOLY, JS; CNRS, Gif-sur-Yvette, France; Kyoto University, Kyoto, Japan; CNRS, Gif-sur-Yvette, France; INRA. Jouy-en-Josas, France; CNRS, Gif-sur-Yvette, France; CNRS, Gif-sur-Yvette, France
    The pituitary is an essential endocrine organ in the vertebrates. While the urochordate possesses no distinct pituitary organ, it has been long proposed that the ascidian neural complex (NC) composes homologous organs to the brain and the pituitary. Recent molecular data suggests that the ciliated funnel (CF), one of the NC components, is the most probable candidate as several placodal genes including CiPitx are expressed at the ectodermal stomodaeum and the CF, respectively, in embryonic and the adult stages (Boorman and Shimeld, 2002; Christiaen et al., 2002). To reinforce this hypothesis with insights from the developmental process, we conducted lineage analyses mainly using a photo-convertible fluorescent protein Kaede driven with promoters recapitulating endogenous expressions of CiPitx and CiNut (the neural tube marker). We validated that a part of the ectodermal stomodeaum invaginates, separates from the major part and gives rise to the distal tip of the CF. Furthermore, we revealed that the proximal domain of the CF, which is adjacent to the distal tip, is derived from the neuroectodermal "dorsal duct". Thus, the ascidian CF has a dual origin and its organogenesis resembles the vertebrate pituitary. We then investigated whether the ectodermal CF expresses genes relevant to an endocrine activity like the vertebrate pituitary. However, we could not confirm it. It is likely that a pituitary primordium is present in the ascidian, but that the endocrine activity of the pituitary is a vertebrate innovation presumably with evolutions of adenohypophyseal transcription factors which terminally differentiate hormone-secreting cells.


n) Multiple roles of retinoic acid in the pharyngeal endoderm development of amphioxus. KOOP, D.*; HOLLAND, L.Z.; Univ. of California, San Diego
    In the cephalochordate, amphioxus, the pharyngeal endoderm gives rise to Hatschek’s pit (homologous to the vertebrate adenohypophysis), endostyle (homologous to the vertebrate thyroid) and gills. However, in vertebrates, pharyngeal structures receive a major contribution from neural crest cells, whereas amphioxus lacks neural crest facilitating the study of the role of the endoderm in pharyngeal patterning. In both amphioxus and vertebrates homologous suites of genes pattern the pharyngeal endoderm. These include Tbx1/10, Eyes absent, Six genes and Pax genes. Our previous studies have demonstrated that retinoic acid (RA) signalling, mediated by Hox established the posterior limit of the pharyngeal endoderm. Here we present a second role for RA signalling in the development of the gills. To investigate the role of RA signalling during pharyngeal endoderm morphogenesis, we examined the effects of late RA treatments on the genes that pattern the gill pimordia such as Six and Pax genes. Our results show that at later stages in development RA does not inhibit gill primordia formation but does plays an important role in regulating gene expression and apoptosis in the forming gill slits. These results suggest that in amphioxus, as in vertebrates, pharyngeal development involves a complex interaction of signalling pathways, often playing multiple roles.


o) Potential resistance of Antarctic ascidians to sympatric bacterial epibiosis. KOPLOVITZ, G*; MCCLINTOCK, J.B.; AMSLER, C.D.; BAKER, B.J.; Univ. of Alabama at Birmingham; Univ. of Alabama at Birmingham; Univ. of Alabama at Birmingham; Univ. of S. Florida
    Both biotic and abiotic surfaces in benthic marine environments are subject to fouling by bacteria, protists and macroinvertebrate larvae. Although in certain circumstances epibiosis may be beneficial to the basibiont (the organism being fouled), for example, through the provision of vitamins and nitrogenous compounds, by in large, fouling is more harmful than beneficial. A large variety of fouling organisms are commonly found on the surfaces (tunic) of ascidians. These include bacteria, algae (such as diatoms), bryozoans and hydroids, as well as other macroinvertebrates. The focus of this study was to assess the incidence of antimicrobial activity in both lipophilic and hydrophilic crude organic extracts of a suite of solitary and colonial ascidians from the western Antarctic Peninsula. The bacterial strains tested were isolated from both the water-column and from the surfaces of sympatric benthic marine invertebrates. Antimicrobial activity of extracts from 13 species of Antarctic ascidians was evaluated using 20 strains of sympatric marine bacteria. Bacterial strains were tested using standard microbial growth inhibition techniques. Paper antimicrobial assay disks treated with ascidian solubilized extract were placed onto inoculated marine agar plates. Each plate was incubated at 4°C for several days until bacterial growth was visible and zones of inhibition could be measured. Zones of growth inhibition were compared to solvent control disks in order to evaluate antimicrobial activity of ascidian extracts. The results of this analysis will be interpreted in light of the prospective impacts of fouling in ascidians. This research was supported by NSF grants # OPP-0442769 and OPP-0442857.


p) Does temperature affect Whole Body Regeneration (WBR) rate in Botrylloides spp? PINNICK, G.L.*; COHEN, C.S.; San Francisco State Univ.
    Botryllid ascidians, colonial marine chordates, possess unique modes of propagating and regenerating that make robust models for studying the biological pathways for tissue regeneration. Here, we test the effect of temperature on regeneration ability and rate, as mediated by the retinoic acid (RA) pathway that is common in chordate embryonic development. Colonies of Botrylloides sp from 2 sites in San Francisco Bay with differing seasonal salinity and temperature regimes were compared. A total of 10 genotypes were used to create 33 ampullar clumps (1-6 clumps per genotype) that consisted solely of ampullar tissue with all zooids removed. Ampullar fragments were treated with retinoic acid and regeneration was observed at 3 temperatures. WBR (regeneration of an entire functional zooid) occurred rapidly, within 1-4 days. This temperature also accounted for the majority of zooids produced across the 10 day observation period. At 13 and 23 degrees, multiple zooids (1-4 zooids) were produced within a single ampullar clump. The second most productive temperature was 13 degC, which produced WBR of zooids within 5-8 days. The intermediate temperature had the highest rate of non-regeneration across all ampullar clumps, together with the highest mortality rate. WBR of botryllid zooids from ampullar fragments occurred at all 3 temperatures within 5 days with 2 out of 4 genotypes successfully regenerating in all treatments. Temperature may have an effect on WBR in this species, with the warmest temperature producing the most zooids in the shortest time in this series of experiments.


q) Bloody Whole Body Regeneration! BROWN, FD*; KEELING, EL; LE, AD; SWALLA, BJ; Univ. of Washington; Univ. de los Andes, Bogotá; Univ. of Washington; Calif. Polytechnic State Univ. San Luis Obispo; Univ. of Washington
    Colonial ascidians exhibit one of the most extreme cases of regeneration. Upon removal of all individuals in a colony of Botrylloides violaceus, the vascular network and blood left within the tunic of the colony reorganizes, and aggregates of blood differentiate into buds. One of these buds continues to complete regeneration of a whole individual. Using phase contrast microscopy, time-lapse video recording and detailed histological studies of regenerating colonies, we describe the earliest events of regeneration. We examine the pattern of cell proliferation by immunohistology using proliferating cell nuclear antigen (PCNA) antibodies. Next, we report the expression of piwi, a stem cell maintenance marker involved in microRNA processing and stem cell maintenance, in hemocytes surrounding the early regenerates. We rarely found Piwi or PCNA in differentiating tissues during vascular budding, suggesting that cells that form the epithelial tissues during budding and regeneration originate mostly from circulatory hemocyte precursors, and likely include stem cell progenitors. First attempts to establish lineage tracing are now in progress. Preliminary results using the nuclear stain DAPI (<360 nm excitation range to avoid endogenous autofluorescence) show that it can be reliably followed in the colony up to a month after labeled hemocyte injection. We find DAPI labeled cells integrate into tissue epithelia of different germ layers. Taken together, we propose that multiple stem cell types occur within the hemocytes, and that they undergo proliferation in the vasculature before differentiating into epithelial tissues.


r) Colony fusion common in a colonial ascidian. WESTERMAN, E.L.*; DIJKSTRA, J.A.; HARRIS, L.G.; Yale Univ.; Wells National Estuarine Research Reserve; Univ. of New Hampshire
    Many benthic colonial invertebrates have the ability to fuse and form chimeras with compatible colonies. Botryllid ascidians fusion rates have been determined for different populations and species by random sampling and fusion testing individuals. However, natural fusion rates over time nor their influence on colony size have not been documented. We deployed six settlement panels to monitor the growth of single genotypes of ,Botrylloides violaceus and Botryllus schlosseri from July to September 2006 in Salem Harbor, Massachusetts, USA, and nine settlement panels to monitor settlement, growth, and fusion of Botrylloides violaceus from July to mid-August 2007. Eighty percent of the recruits observed fused with at least one other colony, while six percent neither fused nor were over grown. Growth rates of the fused colonies did not differ from those previously reported for single colonies. However, fused colonies were considerably larger than single genotype colonies at the end of the study period, and colonies in the 2006 study were significantly smaller than fused entities from 2007, though not smaller than single genotype entities from 2007. These results suggest that larvae are settling in clumps of compatible individuals, and that large subtidal colonies may be the result of high numbers of fusions between compatible colonies.


s) Dispersal limitation and post-settlement survival of an introduced ascidian (Botrylloides violaceus) in San Juan Islands, WA. CAHILL, A.E.*; CRICKENBERGER, S.; CRIM, R.N.; SELDEN, R.L.; Stony Brook Univ.; Clemson Univ.; Univ. of British Columbia; Univ. of Calif. Santa Barbara
    Distributions of introduced species are often patchy; however, the mechanisms regulating these patchy distributions are poorly understood. Sessile organisms with short-lived larvae provide an opportunity to test whether these distributions are dispersal-limited or determined through post-metamorphic survival and growth. Here we used the non-native colonial tunicate Botrylloides violaceus to examine the roles of dispersal and predation in determining its distribution by transplanting recently settled juveniles to locations with and without adult B. violaceus colonies. Survival and growth were not different between caged and uncaged treatments at any site, suggesting that predation is not controlling the distribution of this species. However, survival and growth were different among locations. One site without established colonies had significantly lower growth and survival than all others, suggesting the importance of abiotic factors at this site. The other site where adults were absent had similar growth and survival to sites with established colonies, indicating that dispersal limitation is more important at this site than abiotic factors. Our study suggests that the distribution of B. violaceus is limited by both dispersal and environmental conditions that affect juveniles after settlement.


t) Form, function and flow in the plankton: Jet wake structure and swimming performance of pelagic tunicates. SUTHERLAND, K. R.*; MADIN, L. P.; MIT/WHOI Joint Program in Oceanography; Woods Hole Oceanographic Institution
    Salps are barrel-shaped marine invertebrates that swim by jet propulsion. Morphological variations among species and life-cycle stages are accompanied by differences in swimming mode. The goal of this investigation was to compare propulsive jet wakes and swimming performance variables among morphologically distinct salp species (Pegea confoederata, Weelia (Salpa) cylindrica, Cyclosalpa affinis) and relate swimming patterns to ecological function. Using a combination of in situ dye visualization and particle image velocimetry (PIV) measurements, we described properties of the jet wake and swimming performance variables including thrust, drag and propulsive efficiency. Locomotion by all species investigated was achieved via vortex ring propulsion. We found that the slow-swimming P. confoederata produced the highest weight-specific thrust (T= 53 N kg-1) and swam with the highest whole-cycle propulsive efficiency (ηwc= 55%). The fast-swimming W. cylindrica had the most streamlined body shape but produced an intermediate weight-specific thrust (T= 30 N kg-1) and swam with an intermediate whole-cycle propulsive efficiency (ηwc= 52%). Weak swimming performance variables in the slow-swimming C. affinis, including the lowest weight-specific thrust (T= 25 N kg-1) and lowest whole-cycle propulsive efficiency (ηwc= 52%), may be compensated by low energetic requirements. Swimming performance variables will be considered in the context of ecological roles and evolutionary relationships.


3. 5th Intl. Tunicate Meeting, Naha-city, Okinawa, Japan, 21- 25 June 2009. See the complete program at

a. Differentiation of rostral sensory neurons in the larva of the ascidia Botryllus schlosseri.

Federico Caicci, Giovanna Zaniolo, Valentina Degasperi, Fabio Gasparini, Paolo Burighel, Lucia Manni, Dept. of Biology, Univ. of Padova, Italy.

   The larva of the colonial ascidian Botryllus schlosseri bears three anterior papillae for selection and settlement on the substrate. They are defined “ganglionated” for the presence of sensory neurons grouped in a ganglion-like structure at their base. We investigated at morphological (ultrastructural and immunocytochemical) level the differentiation of the rostral epidermis during late embryogenesis, larval swimming stage and onset of metamorphosis. The rudiments of papillae appear in early tail-bud stage as protrusions bearing at apex bipolar neurons. These neurons differentiate into two types of ganglionic neurons (central and peripheral), with long, rod-like dendritic terminations, which in larval stage become exposed to the sea-water. At the base of papillae and in the adjacent epidermis, other scattered sensory neurons extend into the tunic long-sinuous dendrites. We identified these neurons as equivalent to rostral trunk epidermal neurons described in Ciona intestinalis (Takamura, 1998). In B. schlosseri,  at onset of metamorphosis, the papillae retract and neurons undergo involution by apoptosis. On the base of dendrite features and larva behavior, we propose that ganglionic neurons are mechanoreceptors, whereas rostral trunk epidermal neurons are chemoreceptors. During embryonic development the epidermis among papillae acquires progressively secretory features culminating in apocrine secretion at settlement. We suggest that this interpapillary epidermis is involved in production of sticky substances which render the tunic suitable for temporary adhesion of the larva to the substrate.


b.Gene expression during body muscle differentiation in the colonial ascidian Botryllus schlosseri. V. Degasperi*, S.M. Shimeld**, F. Gasparini*, L. Manni*, P. Burighel*.

*Dept. of Biology, Univ. of Padova, Italy; **Dept. of Zoology, Univ. of Oxford, UK

   Ascidians display three types of muscles during their life: striated in the larval tail and heart, and smooth in the post-metamorphic sessile phase. The larval and cardiac muscles have an arrangement of myofilaments like the striated muscle of vertebrates. Instead, the smooth body-wall musculature has intermediate characters between smooth and striated muscle of vertebrates. A great number of ascidians form colonies constituted of clonal individuals (blastozooids) originated by asexual reproduction. We studied the musculature in Botryllus schlosseri analysing its organization, differentiation with electron microscopy. Moreover, we followed gene expression of muscle actin and troponin T in the larva and during the development of blastozooids, beginning from the early bud stage to adult and regression stage. We isolated and characterised two transcripts from colonies that resulted homologous to muscle genes of solitary ascidians: a muscle-type actin (BsMA2), a cytoplasmic-type actin (BsCA1) and a troponin T (BsTnT-c). Moreover, we obtained also the genomic sequences coding for BsMA2 and BsCA1. Phylogenetic analyses showed a close relationship between urochordates and vertebrates muscle genes. The BsMA2 and BsCA1 genomic sequences were compared in the exon-intron organization with other muscle and cytoplasmic–type actin genes of both invertebrates and vertebrates. Our data revealed that intron positions are conserved in ascidians and in the other deuterostomes. We detected the expression of the two genes by in situ hybridization on section (ISH) throughout the blastogenetic cycle of B. schlosseri. The ISH, in parallel with phalloidin staining experiments, showed that the first diffuse signal of BsMA2 and BsTnT-c labels differentiating muscles which appear in the intersiphonal area of young buds. Then, the muscle fibres differentiate into the body-wall, while an intense expression only of BsMA2 marks the myocardium just when it begins contractions. Thus, all the phases of muscle development and regression were documented by an integrated study showing the close correspondence of in situ expression of BsMA2 and BsTnT-c, phalloidin signal and ultrastructure.


4. 12th Congress of the European Soc. for Evolutionary Biology, Torino, Italy, 24-29 August 2009.

Characterization of a MuSashI-like transcript in a colonial chordate, phylogenetic analysis of the protein group and differential expression patterns in sexual versus asexual development.

F. Gasparini, V. Degasperi, E. Ruffoni, P. Burighel, L. Manni. Dipartimento di Biologia, Università degli Studi di Padova

   Tunicates are the unique chordates to possess species reproducing sexually and asexually. Between them, Botryllus schlosseri forms similar organisms (oozooids and blastozooids) trough embryogenetic and blastogenetic pathways respectively. We here illustrate the characterization and expression pattern, during both pathways, of a transcript for a gene that we named BsMSI. We demonstrate that BsMSI falls into MuSashI-like (MSI-like) group, formed by MSI1, MSI2 and DAZAP1 genes. They encode for RNA-binding proteins phylogenetically related to other protein groups: the TARDBP and several hnRNPs. These proteins share same modular domain structure: a 2xRRM motif. Analyses show that MSIs-like and 2xRRM-hnRNPs had a common origin from a TARDBP type, and that 2xRRM-hnRNPs form a monophyletic group. A unique MSI-like, orthologous to DAZAP1, was recognized in tunicates. We hypothesize that, as MSIs are widely present in metazoans, tunicates have lost them during their evolution. In vertebrates MSI1 is considered a stem cells marker, but also MSI2 and DAZAP1 are involved in differentiative processes. BsMSI is expressed in all the tissues of developing buds. This ubiquitous blastogenetic expression differs from the embryogenesis one, which varies spatio-temporally according to the differentiation of embryonic, larval or oozooid tissues. Interestingly, BsMSI expression ends in differentiated or regressing structures (adult blastozooids, differentiated larval structures). Because the BsMSI expression pattern doesn’t overlap between blastogenesis and embryogenesis, we speculate it operates substitutive functions of the vertebrates orthologues DAZAP, MSI1 and MSI2, and that the function, related to differentiation or tissue maintaining, varies during the B. schlosseri life cycle.


5. XIth sci. meeting of the Italian Assoc. of Developmental and Comparative Immunobiology 24-26 February 2010, Univ. of Modena & Reggio Emilia, Modena, Italy.

Natural apoptosis during the blastogenetic cycle of the colonial ascidian Botryllus schlosseri. F. Schiavon, L. Manni, M.Del Favero, L. Ballarin. Dipartimento di Biologia, Università di Padova, Padova, Italy

   Colonies of the compound ascidian Botryllus schlosseri undergo regular generation changes during which adult zooids are progressively resorbed and replaced by growing buds. The generation change, or take-over, is characterized by massive cell death by apoptosis, changes in the expression of surface molecules by senescent cells of zooid tissues and recruitment of circulating phagocytes in zooid tissues which assure the complete clearing of the dying cells. The entire process lasts 24-26 h at 20°C and has been subdivided, on the basis of the degree of contraction of old zooids, in four substages. It has an antero-posterior progression, at least in the digestive tube, as trace of apoptosis can be found in the epidermis, peribranchial epithelium, and heart in the late take-over, whereas they are easily found in the branchial basket after 2-4 h from the beginning of the generation change. During the take-over, massive recruitment of phagocytes, which ingest senescent cells, occurs: this suggests the release of diffusible chemoattracting factors by effete cells, able to attract phagocytes toward them. We are going to investigate the expression of genes related to apoptosis, such as IAP (inhibitor of apoptosis) and HSP70 during the various phases of the colonial blastogenetic cycle. Published in ISJ 7: 107-118.


6. Upcoming symposium: The 7th Intl. Symp. on the Chemistry and Biol. Chemistry of Vanadium, October 6 - 9, 2010, Toyama City, Japan.

   Vanadium chemistry and biochemistry have become more attractive in part because of the synthetic utility of several vanadium complexes and in part because of new findings that vanadium compounds have insulin-mimic and anti-tumor properties. In addition, the discovery of several vanadium enzymes and proteins from marine organisms (including ascidians) are the subject of intensive research. Organizing Committee: Hitoshi Michibata (Chair), Hiroshima Univ.; Kan Kanamori (Co-Chair), Univ. of Toyama; Toshikazu Hirao (Co-Chair), Osaka Univ.; Tatsuya Ueki (Secretariat), Hiroshima Univ.; Honoh Suzuki (Secretariat), Univ. of Toyama.


7. ABCD meeting; Stem cells, development and regenerative medicine, Parma, Italy 9-10 April 2010.

Role of the alternative transcripts and targets of the Ci-POU IV gene in the development of the peripheral nervous system of the tunicate Ciona intestinalis.

Raoul Manenti*, Giuliana Zega*, Simona Candiani§, Mario Pestarino§, Fiorenza De Bernardi*, Andrea Pasini°, Roberta Pennati*, °Instit. de Biol. du Développement de Marseille Luminy (IBDML), UMR6216, CNRS/Univ. de la Méditerranée, Marseille, France ; * Dipto. di Biol., Univ. degli Studi di Milano, Milano, Italy; §Dipto. di Biol., Univ. di Genova, Italy.

    Some members of the POU gene family regulate the neural patterning and differentiation in both vertebrate and invertebrate embryos. The tunicate Ciona intestinalis has only three genes encoding for POU transcription factors and the gene Ci-POU-IV is specifically expressed in all the peripheral nervous system (PNS) territories and in some cells of the central nervous system during development. Within chordates, tunicates represent the sister group of vertebrates and their larvae have a typical chordate body plan. Notably larval nervous system is formed by few cells whose organization mirrors that of vertebrates. The aim of this work was to study the role played in neural differentiation by the two alternative transcripts of Ci-POU-IV, we discovered, and to identify the targets of this gene.

We designed Morpholino oligos to perform gene knock-down experiments for the different isoforms, a short and a long one. Preliminary results from these experiments revealed that the expression of the serotonin rate-limiting synthesis enzyme, tryptophane hydroxylase (TPH), and glutamate transporter (vGlut) could be regulated by the long transcript. To verify if the alternative transcripts are expressed in different neuron populations, we synthesized a probe selective for the short isoform. The in situ hybridizations, compared to the whole expression profile of Ci-POU-IV, showed a lack of expression of the “short” form in the sensory epidermal neurons of the trunk.  Furthermore, we look for the Ci-POU-IV targets by a bioinformatic approach. The possible consensus sequences were obtained by bibliographic research of those known for the POU IV family in both invertebrates and vertebrates. We used these sequences to build a matrix that was employed to perform a bioinformatic research in the whole C. intestinalis genome with a software elaborated by the Lemaire team of the IBDML of Marseille. We identified 19 possible targets of Ci-POU-IV and we preliminary selected 8 regions corresponding to 6 genes, including TPH. The activity of the selected regions is being evaluated. Results from this work allows us to hypothesize that the two isoforms play different roles during nervous system differentiation. and will help us to delineate the signal cascade of Ci-POU-IV during PNS differentiation.




1. The effects of Didemnum vexillum overgrowth on Mytilus edulis biology and ecology.
Linda A. Auker, Dept. of Biological Sci., Univ. of New Hampshire, Durham, NH USA. Ph.D. dissertation, thesis advisor Larry Harris.

   Didemnum vexillum is an invasive colonial ascidian in the Gulf of Maine that readily colonizes hard substrates. These substrates include hard-shelled organisms, such as the common blue mussel Mytilus edulis. Preliminary observations and short-term studies showed potential effects of epibiosis on M. edulis growth, specifically lip thickness and tissue index. This dissertation study further examined the effects of D. vexillum on growth and reproduction of, and predation on, M. edulis. Shell thickness index, tissue index, shell mass to tissue mass ratio, lip thickness and mussel length were measured throughout a 12- month period in control and overgrown mussels. Additionally, histological preparations of the mussels were used to determine reproductive condition of the mussels in each of these treatments. These variables were measured every three months (November 2008, February 2009, May 2009, and August 2009). Laboratory choice and consumption experiments examined the effects of overgrowth of the ascidian on predation by Carcinus maenas. Finally, mussel primary settlement was measured from summer 2008 through summer 2009 and compared to historical data. This settlement was also correlated with the abundance of neighboring D. vexillum.

   Overgrowth had a negative impact on mussel growth. Tissue index and lip thickness were negatively affected as the mussel lip margin was overgrown. Overall mussel growth was significantly higher in control mussels by the end of the experiment. The pattern of spawning and gonad development was reversed in overgrown male mussels from the control mussels. Predation studies showed a potential positive effect for the mussel, as crabs consumed more control mussels than overgrown mussels in both a choice study and a consumption study. Finally, winter mussel settlement in 2008-2009 was lower than historical 1980-1981 settlement, and there was a decrease in mussel plantigrades with D. vexillum recruits. These studies show tradeoffs in the effects of overgrowth by D. vexillum; growth and reproduction are inhibited, while predation is decreased. As mussels are an important source of food and habitat for other Gulf of Maine organisms, overgrowth by D. vexillum has the potential to change ecosystem dynamics.


2. Localization and identification of protein kinase C subtypes in ascidian sperm activation. Senior Thesis by Rolando Rui, Dept. of Biol. Sci., California State Univ., Fullerton. Thesis Advisor, Robert A. Koch, Laboratory for Sperm Cell Biology.

    In Ascidia ceratodes, sperm activation is characterized by a morphological change known as mitochondrial translocation.  This event is an actin:myosin-driven process and is dependent on internal calcium ion (Ca2+i) release by the ER.  Ca2+i release is dependent on the action of inositol 1,4,5-trisphosphate via signaling of the G-protein coupled receptor.  Ca2+i activates protein kinase C (PKC), which facilitates signal transduction leading to mitochondrial translocation.  Based on Rim-1 fluorescence labeling, we propose that in activated but not unactivated sperm cells, the elevation of Ca2+ concentration from ER stores stimulates PKC8 to relocate from mitochondrion-associated sites to the plasma membrane where it triggers a pathway leading to Ca2+ entry and myosin activation.  Myosin, in turn, powers mitochondrial translocation.  We also hypothesized that conventional PKCα subtype is present in ascidian sperm.  To test these hypotheses, cytosolic-, and mitochondrial-rich fractions were prepared and checked for purity using succinate dehydrogenase (SDH) and lactate dehydrogenase (LDH) enzymatic assays.  PKC activity was tested in the two fractions with a PKC assay kit.  Various PKC pseuodsubstrates (gift of Dr. Driss Zoukhri, Tufts University, Boston, MA) were used as inhibitors to identify the PKC subtype.  SDH activity was >85% in mitochondrial fractions and LDH activity was >85% in cytosolic fractions.  Nearly 100% of PKC activity was associated with cytosolic fractions in both activated and unactivated sperm cells.  PKCα and a novel PKC, PKCδ, were both present in ascidian sperm cells.  We concluded that PKC is not associated with the mitochondrion and both conventional and novel PKC are present in ascidian sperm.




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Kawamura, K. and Sunanaga, T. 2010. Hemoblasts in colonial tunicates: are they stem cells or tissue-restricted progenitor cells? Development Growth & Differentiation 52: 69-76.

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Khalaman, V. V. 2001. Fouling communities of mussel aquaculture installations in the White Sea. Russian Journal of Marine Biology 27: 227–237.

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Satake, H. and Sasaki, N. 2010. Comparative overview of toll-like receptors in lower animals. Zoological Science 27: 154 –161.

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