Gretchen and Charles Lambert

12001 11th Ave. NW, Seattle, WA 98177

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Number 63                                                                                                     December 2008


   Thanks very much to the many AN readers who contributed to this issue. In addition to a number of Work in Progress articles and meetings abstracts, there are two thesis abstracts with significant new information, and 113 new publications listed at the end of this issue.   

   It is with very great sadness that we report the death of Dr. Claude Monniot, world-renowned ascidian taxonomist at the Paris Museum national d’Histoire Naturelle, who with his wife and colleague Dr. Francoise Monniot vastly increased our knowledge of the world’s ascidian species with their publication of hundreds of monographs, especially from the tropical west Pacific, the deep sea, and the Antarctic. Francoise continues their valuable work, and has contributed a moving and very interesting biography of her remarkable husband Claude; see below. 

   August 4-8 Gretchen co-taught a 5 day ascidian workshop with Drs. John Bishop and John Ryland in Northern Ireland, at the Queens University marine lab at Portaferry, a dramatic and beautiful location on Strangford Lough. We thank the hard-working organizers and participants for a very successful workshop. The next scheduled ascidian workshop will be June 4-18, 2009 at the Smithsonian Tropical Research Institute, Bocas del Toro, Panama, taught by Rosana Rocha and Gretchen and Charles Lambert. Participation is limited to only 14 or 15 so please get your application in early to Rosana.


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




1. From Francoise Monniot, Museum national d’Histoire naturelle, Paris.

   Claude Monniot died suddenly of a stroke the 12th of July 2008. Born in 1936 at Fontenay/sous/Bois (Val de Marne), he went to school in Paris, and all his holidays at the sea shore in Brittany, collecting animals. His interest in marine life was due to an early reading:” La vie étrange des rivages marins” by Y. Le Danois. After 3 years at the university he obtained degrees in Botany, Geology and Zoology and undertook a specialisation in biological oceanography at the Banyuls marine station. There he had the opportunity to learn SCUBA diving with the military parachutists and acquired degrees in this sport, the first for civilians in the French federation.  He was much indebted to professor Claude Delamare-Deboutteville who encouraged him to begin a Master’s thesis about ascidians and afterwards gave him the possibility to teach zoology from 1961 to 1963 at Nancy university. There he married Francoise who also worked with ascidians at Banyuls, and they collaborated all his life. From the end of 1963 to 1964 he was sent to Brazzaville (French Congo) for military service as professor at the university. Back to France he was promoted maitre-assistant at the Museum national d’Histoire naturelle in Paris, where he achieved his doctoral thesis in 1965: “Etude systematique et évolutive de la famille des Pyuridae”. Assistant director in the laboratory of marine invertebrates in 1969, he became professor in 1992.

   Not only interested in ascidians, C. Monniot collected many ascidian parasitic copepods and studied them with many publications in parallel with other taxonomic works. During all his career, Claude participated in numerous oceanographic cruises on many ships: Pluteus, Noroit, Suroit, Thalassa, Coriolys, Jean Charcot, Marion Dufresne, Alis … and collected at many marine laboratories.

   Several events oriented his research in varied major directions, which resulted in a total of 163 publications, many of them with Francoise. The discovery of deep sea ascidians by the American cruises of the N.O Alvin was at the origin of a passionate interest for carnivorous ascidians, and this continued with the participation in French cruises to sample the deep Atlantic, south Indian and Pacific oceans.

   An occasional meeting with Prof. Newell, working at the time with mites and elaborating tabular keys instead of dichotomic ones, drove him to try this new system. So with his wife, C. Monniot established this kind of key to all ascidian families and genera, with the encouragement of R. Millar. The offer of the Smithsonian Institution to study the large American Antarctic collections was a new center of interest and allowed a biogeographic study. A travel to South Africa gave them the opportunity to study self collected and material given by colleagues along this very southern shore.

To increase their knowledge of the more diversified shallow tropical fauna, C. and F. Monniot using SCUBA dives collected ascidians in many places around the world: Bermuda, Guadeloupe, Martinique, Azores, Tahiti, New Caledonia, Mozambique, Socotra. Claude also studied with his wife the large collections from the western Pacific made by the American Coral Reef Research Foundation for medicinal purposes. The number of new species described is impressive. Numerous shorter studies were published in the interval.

   Aside his scientific research, C. Monniot assumed diverse responsibilities in committees including oceanography, administration, publications, museology, and was even treasurer of the Museum restaurant during some years. He created the CENTOB in Brest, in charge of sorting the material collected by the oceanographic cruises. He participated in large CNRS programs including “Ecoprophyce” and “GRECO Manche”. C. Monniot was not a sportsman but he liked long walks in the city and particularly outdoors in search of mushrooms that he knew very well. He liked to go fishing with his small boat in Corsica. He was a skilful handy-man with a particular interest in wood works. He read many history books and his memory was amazing in this field. His 2 grand-daughters were very fond of him as he was always ready to play with them. He is survived by his wife Francoise, daughter Nadine and his two grand-daughters



2. 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 28 research papers for the journal Aquatic Invasions and are now in press and will appear online before the end of this year ( ).  Andrea Locke and Mary Carman served as guest co-editors. Among the papers are several concerning the worldwide invasive Didemnum vexillum, including two papers showing that all the worldwide populations are the same species both morphologically and genetically. (See New Publications section: Lambert, G.; and Stefaniak, L. et al.)

   The next IISSC, #3, will be held at WHOI, April 27-29, 2010. Oral and poster presentations will be considered for the conference topics of Taxonomy and Genetics, Biogeography, Ecology, Risk Assessment and Management. There will be another taxonomic workshop presented by Gretchen and Charley Lambert and a field trip to Martha's Vineyard. More information will be in the next issue of AN.


3. Congratulations to Dr. Dustin Marshall, Univ. of Queensland School of Integrative Biology, who received $75,000 as one of the annual UQ Foundation Research Excellence Awards given September 23.  From the UQ News Online: “Dr. Marshall is using the humble sea squirt to look at how marine animals adapt to pollution. Dr Marshall will examine how events such as freshwater runoff affect the evolution of these organisms under field conditions, a first for marine research. By monitoring changes in growth rate and their resistance to other stresses, he will examine whether sea squirts evolve to develop a tolerance to pollution.”


4. From Ed Munro and Kristin Sherrard, Center for Cell Dynamics, Friday Harbor Labs, 620 University Road, Friday Harbor, WA 98250.

   We would like to announce a Spring 2009 research apprenticeship course at Friday Harbor Lab's Center for Cell Dynamics, titled Dynamics of Cellular Morphogenesis in ascidian embryos:  Experiments and Computer Modelling, taught by Ed Munro, Kristin Sherrard, and François Robin. The course will run March 30 - June 5 and comes with a stipend towards the cost of tuition and room and board at FHL. 

   This 10-week course will introduce students to a modern approach to the study of cellular dynamics and tissue morphogenesis, integrating molecular genetics, microscopy and computer simulations.   We will focus on ascidian embryos, because of their stereotyped early development, small cell numbers, optical clarity, and the ease with which they can be manipulated using molecular or physical methods. During the first part of the course, lectures will cover essential

background including: early development in ascidians,  fundamentals of cell and tissue mechanics, cytoskeletal dynamics and cell-cell adhesion, principles of light microscopy and live imaging.  Wet lab exercises will introduce students to basic culture and handling of ascidian embryos, microinjection and electroporation (to introduce fluorescently tagged proteins and function-perturbing molecules), fixation and immunocytochemistry and basics of light microscopy.  Dry lab exercises will introduce students to the use of computer simulations as a way to explore complex cell and tissue dynamics. During the second part of the course, student teams will work closely with faculty mentors on open-ended research projects, combining experimental and computational approaches.  Along the way, students will develop presentation skills through weekly journal clubs and research updates, culminating in a final public seminar presentation. Possible topics include:

*  Mechanisms of invagination and cell rearrangement during neurulation.

*  Dynamics of cell-cell adhesion during cleavage and morphogenesis.

*  Cytoskeletal dynamics and cytoplasmic flow in early embryos.

   Prospective apprentices should have at least a year of calculus, an introductory biology course, a willingness to immerse themselves in a fascinating scientific problem and an enthusiastic determination to learn. We expect students to come with a fundamental orientation towards either

experimental or computational biology and to get from their peers and instructors a strong dose of what they don’t yet know.

   Please see the FHL website ( ) for more information.  Deadline for applications is January 10.  Also see the Center for Cell Dynamics website ( for information about past CCD apprenticeships.




1. From 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?

2.  Projects from the laboratory of Zeti Hussein, Ph.D., Centre for Bioinformatics Research, Institute of Systems Biology (INBIOSIS), Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor, MALAYSIA 

a) Classification of protein sequences from Ciona savignyi.

   This project is focused on the protein classification of one hundred and four sequences from Ciona savignyi, a marine organism which is commonly known as tunicate. These sequences were classified into six classes, with reference to the SCOP classification system. Six classes identified are as follow: class α (twenty five sequences), class β (ten sequences), class α/β (sixteen sequences), class α+β (eleven sequences), membrane and cell surface proteins and peptides class (thirteen sequences) and small protein class with fifteen sequences. Two sequences have been chosen for structural modeling, in order to have a better insight on their biochemical function. These sequences were O15998 (which was an actin) and Q5QQ54 which was a type of enzyme known as xylosyltransferase. These two sequences were modelled by applying a comparative modeling and fold recognition approach, respectively. The knowledge generated from this project is very useful in order to have a better understanding on this unique organism.


b) Fold identification of protein sequences from Ciona intestinalis genome.

   A fold relationship to an already characterized protein will often allow general information about function to be deduced. Methods of deducing function directly from structure without the use of evolutionary relationships are developing rapidly. In this project, one approach has been design to determine the folds adopt by the proteins in the genome of Ciona intestinalis. One thousand and seventy eight sequences have been identified from this genome, and these sequences were categorized into four groups; known proteins (677 sequences), putative proteins (78 sequences), hypothetical proteins (17 sequences), and fragments (306 sequences). Group of fragments was eliminated from this study. The sequences in the known and putative proteins were inspected carefully and were systematically classified into an appropriate protein class (all-α, all-β, α +β, α /β, multidomain proteins, membrane and cell surface proteins, and small proteins). The sequences in the known proteins category adopted one hundred folds, which were dominated in the all-α protein class. Protein in the putative category adopted forty-eight folds dispersed in all seven proteins; where these sequences were threaded into a fold library and the results were a list of suggested fold which likely to be adopted by one particular sequence. All seventeen hypothetical sequences were suggested to adopt different fold. It is often the case that although no sequence relationship can be detected between the query protein and any known structure,the fold will nevertheless turn out to have been seen before. Recognizing a homologous fold relationship does usually allow low or medium resolution function to be deduced.


c) Molecular modeling of selected protein sequences from Halocynthia roretzi. 

   This research was focused on the classification of proteins from Halocynthia roretzi, and structural modeling on its selected proteins. There were one hundred and ninety two sequences retrieved from SWISS-PROT (21 sequences) and TrEMBL (171 sequences) databases. Protein classification on these proteins was performed using SCOP classification as a reference. Excluding the five proteins that are fragments, the proteins were classified into seven categories as follow, α (62 sequences), β (46 sequences), α+β (27 sequences), α/β (16 sequences), multidomains (3 sequences), membranes and cell surfaces (15 sequences), and small proteins (11 sequences). Out of twenty one sequences from SWISS-PROT databases, ten sequences were modeled with a comparative modeling approach, three sequences were modeled by using fold recognition approach and six sequences were not modeled (P16589 was structurally determined as 1IW4 and Q10997 was a fragment and was eliminated from further analyses). In this research, the 3D models generated were for comparative modeling and fold recognition targets. Meanwhile, six sequences in the new fold dataset were analyzed in detail, where two domains of specific proteins for vertebrates were identified that were t-box and brachyury proteins. Detailed analyses on the results from this research should be performed in the future study, as these result were useful in finding the answers on chordate- urochordate scenario in tunicates.




1. Western Society of Naturalists annual meeting, Vancouver British Columbia, Canada Nov. 6-9, 2008.

a) Environment has a larger effect than interference competition on the population grown rate of the exotic ascidian, Botrylloides violaceus, in Washington, USA.

Erin K. Grey, Dept. of Ecology & Evolution, University of Chicago, Illinois.

To effectively manage marine exotic species we must understand how different factors influence their population growth. I used Life Table Response Experiments to tease apart the effects of environment and interference competition on the population growth of a ubiquitous exotic ascidian, Botrylloides violaceus, at four sites in coastal Washington. For two years I tracked colonies in 4 different environments in two competition treatments: colony perimeter in contact with a competitor or not in contact with a competitor. For each treatment I estimated growth rate, survival rate and fecundity and then used a matrix model to calculate the population growth rate. I found that environment explained more of the variation in population growth rate than did competition, and that populations grew faster at sites with warmer temperatures because of increased growth and survival rates. Competition decreased the survival of colonies but, unexpectedly, increased their growth rate thereby resulting in an overall small and variable effect of interference competition on population growth. These results highlight the need to consider how different factors influence overall population growth of exotic species and suggest that environmental factors, especially temperature, determine the success of exotic ascidians in non-native ranges.


b) Growth rate comparison of the invasive colonial ascidians Botrylloides and Botryllus spp. between a coastal and an estuarine environment.

J. L. Donald, Romberg Tiburon Center for Environmental Studies, San Francisco State Univ., CA.

Colonial tunicates Botryllus and Botrylloides spp. of the protochordate family Botryllidae are invasive invertebrates found worldwide in fouling communities. They grow as flat irregularly shaped colonies composed of clonally-replicated zooid systems. Both species are used in a variety of studies concerning developmental biology, evolutionary ecology and allorecognition studies. At two San Francisco Bay area locations (one outer coast site and one inner bay), growth rates of adult individuals were measured over 3-4 weeks in October and November of 2007. Fouling panels were groomed to remove any organisms that could compete directly for space and food with the experimental subjects.  Preliminary analysis of data compares growth patterns including zooid counts and colony spatial extent between these two separate habitats where both genera are commonly found. Use of outer coast and inner bay environments provides two dramatically different environments in terms of salinity, temperature, food abundance, composition and diversity and abundance of local and other invasive fauna. Information about growth rates of Botryllus and Botrylloides spp. will provide important information about the relative abilities of these organisms to successfully compete for space and invade new habitats.


2. 54th Meeting of the Italian Embryology Group, Rome, June, 4-7, 2008

Hovering between life and death: the blastogenetic cycle of the colonial ascidian Botryllus schlosseri. Loriano Ballarin and Adams Menin,  Dept. of Biol., Univ. of Padua, Italy

   The colonial ascidian Botryllus schlosseri forms new zooids by blastogenesis, through the formation of palleal buds which progressively grow and mature until an adult is formed. At a temperature of 19°C, adult zooids remain active for about one week; then they contract, close their siphons and are gradually resorbed, being replaced by buds which reach functional maturity, open their siphons and begin their filtering activity as adult zooids. This recurrent generation change, known as regression or take-over, is characterised by the occurrence of diffuse programmed cell death by apoptosis, as indicated by the increasing activity of various caspases. In addition, the pattern of expression of Bcl-2, Bax, Fas and FasL also changes with respect to mid-cycle stages, suggesting the involvement of both the extrinsic and the intrinsic pathway in natural apoptosis of Botryllus. During the take-over, colonies do not eat, but morphogenetic processes continue. The death of adult zooids puts a quantity of material at the colony disposal. Circulating phagocytes engulf apoptotic cells: in this way, nutrients derived from the demolition of adult tissues are recycled and used for the development of new tissues allowing both bud growing to maturity and haemocyte proliferation to replace senescent blood cells. Therefore, recurrent apoptosis in Botryllus appear as an efficient strategy for a cyclical rejuvenation of the colony involving a cross-talk between old tissues, phagocytes and developing buds and tissues.


3. II scientific meeting of the Italian Ascidiologists, 30 June–1 July 2008, Dept. of Animal Biology, Univ. of Palermo, Palermo, Italy.

Morula cells, phenoloxidase and DOPA-containing proteins in the compound ascidian Botryllus schlosseri. L. Ballarin1, S. Scippa2, F. Cima1     1Dept. of Biol., Univ. of Padua, Italy, 2Dept. of Biol. Sci., Section of Genetics and Molec. Biol., Univ. of Naples, ”Federico II”, Italy

    Morula cells (mcs) represent the most abundant circulating haemocyte type in the compound ascidian Botryllus schlosseri. They are involved in defence reactions as they: i) can recognise alien substances and cells and induce cytotoxicity; ii) are the effectors of the cytotoxic rejection reaction which occurs between contacting, genetically incompatible colonies. A main role in MC-related cytotoxicity is exerted by the enzyme phenoloxidase (PO) which converts polyphenol substrata to quinones; the latter, in turn, polymerise to form melanins. In the present research, we carried out new spectrophotometrical and cytochemical analysis to investigate further the behaviour of PO and the nature of its substrates. Results confirm that PO is located inside MC vacuoles. In addition, immunocytochemical analysis indicate that mcs contain quinones which probably represent ready-to-use cytotoxic molecules, likely deriving from the oxidation, by PO, of DOPA-containing proteins. In addition, small DOPA-containing peptides, called tunichromes, are likely present inside mcs.


4. 25th Congress of the new European Society of Comparative Biochemistry and Physiology, Ravenna, Italy, September 7–11, 2008

Signal transduction in phagocytosis of the compound ascidian Botryllus schlosseri.

A. Menin (Univ. of Padua, Italy); M. Betti (Univ. of Urbino, Italy); L. Ballarin (Univ. of Padua)

   The compound ascidian Botryllus schlosseri is an emerging model organism for the study of immunobiology of invertebrate chordates. Recently, we started to investigate the signal transduction pathways involved in phagocytosis. Both H89, inhibitor of PKA, and calphostin C, inhibitor of PKC, significantly inhibit in vitro yeast ingestion by Botryllus phagocytes. This suggests the involvement of the pathways activated by cAMP and inositol-triphosphate-diacylglycerol, respectively. Manumicin, an inhibitor of the monomeric G protein Ras also inhibit the ingestion of yeast cells. The involvement of Ras in phagocytosis is supported by the expression, by yeast-matched haemocytes, of molecules recognised by anti-pan-Ras antibodies, as demonstrated by immunohistochemistry and immunoblot analysis. In addition, MAPK inhibitors, such as PD98059 (ERK pathway), SP600125 (JNK pathway), SB202190 (p38 pathway) can decrease the frequency of yeast-ingesting haemocytes in vitro and an increase in the expression of activated MAPKs, such as p38, ERK, SAPK/JNK, was observed in immunoblot analysis of lysates of yeast-matched haemocytes. PI3K is also involved in phagocytosis and its role seems  to be related to the cytoskeletal re-organisation required for pseudopod formation. Immunopositivity to anti-NF-kB antibodies, is located in the cytoplasm of unstimulated phagocytes, whereas it migrates into the nucleus of yeast-activated cells.


5. Society for Integrative and Comparative Biology (SICB), January 2009, Boston.

Palatability and anti-predatory chemical defenses in a suite of ascidians from the Western Antarctic Peninsula. Gil Koplovitz, James B. McClintock , Charles D. Amsler (Univ. of Alabama at Birmingham) and Bill J. Baker, Univ. of South Florida.

   Fourteen species of colonial and solitary ascidians were collected from hard and soft bottom habitats at depths ranging from 0 to 40 m near Palmer Station, Antarctica (64° 46’ S, 64° 03’ W) on the Western Antarctic Peninsula.   Palatability was evaluated in laboratory bioassays using the sympatric omnivorous fish Notothenia coriiceps and the omnivorous sea star Odontaster validus.   When compared to a control food, small bite-sized pieces of outer body tissue of all 14 ascidian species were unpalatable to fish (spit out), while 9 of the 14 species tested were unpalatable (rejected from the ambulacral feeding groove) to sea stars.  Eleven ascidian species were collected in sufficient quantity to prepare lipophilic and hydrophilic extracts and incorporate these at tissue level concentration into alginate food pellets.  Control (no extract) and experimental alginate food pellets tested in fish and sea star bioassays indicated that only the lipophilic extract of the colonial ascidian Distaplia colligans was deterrent, and in this case to both predators.  Similarly, the omnivorous sympatric amphipod Gondogeneia antarctica was fed ascidian extracts (10 species) in alginate food pellets and only one species (the colonial Distaplia cylindrica) deterred feeding, while extracts in 8 species actually stimulated feeding.  Thus, while Antarctic ascidians are unpalatable to sympatric consumers, their lack of palatability appears to be rarely based on organic chemical defenses, but rather be related to one or more of the following: 1) inorganic acids, 2) structural defenses (e.g., tunic toughness), or 3) low nutritional value.  This research was supported by NSF grants # OPP-0442769 and OPP-0442857.


6. 2nd Intl. Symposium on Environment, Athens, Greece

Potential Estuarine Water Quality Improvement Via Marine Invertebrate Bioremediation.

Lisa Draughon1, John Scarpa2, Patricia Keating1, and James X. Hartmann1.  1Florida Atlantic University, Boca Raton, Florida; 2Harbor Branch Oceanographic Institution, Ft. Pierce, Florida

   Estuarine water quality has declined during the past 100 years due to the development of land and harbors by mankind.  Increasing nitrogen and phosphorus from fertilizer run-off has enabled algal blooms with subsequent increases in chlorophyll a and turbidity.  Shoreline modification has altered water circulation in some bodies of water, resulting in unhealthy bacterial concentrations.  These factors have caused a reduction in water clarity.  Regulations have had a favorable impact in reducing the input of nutrients.  However, the utilization of native filter-feeding invertebrates for the bioremediation of estuarine waters contaminated with pathogenic bacteria, unwanted algae, and general turbidity has not been thoroughly evaluated.  Tunicates, commonly found in estuarine waters throughout the world, are capable of indiscriminate filtration of organic and inorganic particles, making them potential bioremediators.  Therefore, the subtropical rough tunicate Styela plicata was examined for its prospective reduction of bacterial particle concentration.  Laboratory tests reveal one average size (~ 40 g) S. plicata, exposed to 105 and 106 bacteria ml-1, can filter as much as 4.7 L hr-1 with 100% efficiency.  From these results it is estimated that 200 rough tunicates could fully remove 105 bacteria ml-1 from 22,600 L each day.  The size and concentration of suspended particulates, water flow, and temperature would affect the rate of bacterial removal.  Controlled concentrations of filter-feeders, such as the rough tunicate, strategically placed in contaminated areas could substantially reduce unwanted bacteria and algae, thus improving water quality.  Other tunicates or filter-feeders common to problematic areas warrant further investigation in bioremediation.


7. 1st Euro-Mediterranean Conference on Marine Natural Products, Pyramisa Resort & Villas, Sharm El Sheikh, Egypt October 31-November 4, 2008.

Sequestering Seasquirts - Native metal chelation in Eudistoma gilboviride. Marcel Jaspars, Marine Biodiscovery Centre, Dept. of Chemistry, Univ. of Aberdeen, Old Aberdeen, Scotland UK

   The marine ecosystem presents an extremely broad spectrum of dissolved metal ions but contains very low levels of physiologically important ones except sodium and potassium. For this reason it is believed that marine organisms have developed unique capabilities for the acquisition, sequestration and utilisation of these important trace metal ions. It is speculated that for marine invertebrates these processes may involve secondary metabolites, many of which have structural features such as polar functional groups in chelate-like arrangements and sometimes macrocyclic cavities which suggest potential metal chelating properties. Amongst the marine invertebrates ascidians (seasquirts) are generally known to hyperaccumulate metals from the surrounding seawater, with high concentrations of V, Ti, Cr, Mn, Fe, Co, Cu, Zn, Rb, Zr, Nb, Mo, Cs, Ta and Sn having been found. There has been much speculation regarding the organic complexation of these metals in the ascidians, but little research has been conducted in this area. Ascidians are therefore an important source in the search for metal complexing compounds from marine sources.

   We have been developing methodology to allow parallel on-line element specific and molecular specific detection. This is achieved by coupling the outflow of an LC system to an inductively coupled plasma mass spectrometer (ICP-MS) and an electrospray mass spectrometer (ES-MS) in parallel. Using this approach we were able to identify an intact metal-ligand complex present in a crude organism extract. This is the first time that an intact metal-ligand complex has been recovered from an ascidian extract, which has significant implications for the elucidation of the ecological function of these ligands and the metal accumulation abilities of these organisms. Potential uses of these complexes are as selective complexation agents for diseases of metal homeostasis, as biomedical research tools, and as many carry fluorophores, as fluorescent metal chemosensors.


8. American Soc. For Cell Biology annual meeting, San Francisco, CA. December 13-17, 2008.

Rho kinase (ROCK) and G α12/13 function as key proteins in crosstalk between integrin- and G protein coupled receptor-dependent signaling events in ascidian sperm activation.

Jose Corleto and Dr. Robert Koch, Dept. of Biol. Science, California State Univ., Fullerton, CA.

   During fertilization, sperm cells are activated by binding to egg follicle cells (FC).  Upon activation, ascidian sperm cells undergo a characteristic morphological change involving mitochondrial rounding and translocation.  Activation is triggered by species-selective binding to FC N-acetylglucosamine, sperm integrin-dependent adhesion to FC surface and signaling via Rho GTPases to actin polymerization, and FC ligand activation of a G protein-coupled receptor (GPCR) that signals to myosin activation via myosin light chain kinase (MLCK).  Here we characterize crosstalk between the integrin- and GPCR-dependent signaling events responsible for MTL.  Sperm cells were diluted, treated, fixed and scored for activation by use of light microscopy.  Activation down the various pathways was achieved by using mAb 12G10 (integrin activator) and Mas-7 (GPCR activator).  Sperm cells treated with Clostridium difficile toxin A (CdtxA, 125 nM) showed a decrease in both integrin and GPCR-dependent activation from 62.36±3.7% to 4.03±2.5% and 62.36±3.7% to 5.01±2.51%, respectively.  Because the GPCR-dependent pathway showed some influence on the Rho family GTPases, the action of U-46619, which activates Ga12/13 the only G protein subunit that has a direct role on Rho family activation, was tested.  It activated sperm (50.5±6.01), an action blocked by CdtxA (7.67±3.2%).  To study the potential role of ROCK, we used the inhibitor Y-27632 (10µM), which blocked integrin-dependent activation (62.36±3.7% to 9.7±2.6%) but had no effect on GPCR-depndent activation.  Activation by U-46619 was inhibited by Y-27632 (50.5±6.01 to 9.17%), suggesting that Ga12/13 is upstream of Rho-dependent ROCK activation.  Lastly, we tested the placement of MLCK in the integrin- and GPCR- by using ML-9 (25 mM); ML-9 inhibited both pathways (62.36±3.7% to 10.36±5.03% and 62.36±3.7% to17.03±4.04%, respectively).  This study supports the presence of crosstalk between the integrin- and GPCR-dependent pathways with ROCK activating MLCK and Ga12/13 activating RhoGTPases, respectively.


9. 1st Intl. Congress on Invertebrate Morphology, Copenhagen,17-21 August 2008.

Origin and migration of GnRH neurons in ascidians. Kiyoshi Terakado, Saitama Univ., Innovative Research Organization, Japan

   In spite of the close similarity of embryonic development of urochordate ascidians and vertebrates, post-metamorphic development and morphology of ascidians are often difficult to correlate to those of vertebrates. However, important morphological and functional events that are similar to those of vertebrates occur in juvenile and adult stages, especially in certain ascidian species. Using the big solitary ascidian Halocynthia roretzi, I will show for the first time the formation of an elaborate neurogenic placode in the thickened regions of neural gland epithelium (termed dorsal strand placode) from where the dorsal strand is formed by invagination. GnRH neurons are born in the dorsal strand in two modes: one is within the epithelium and the other is in  cell masses lying besides the epithelium in contact with mature GnRH neurons. Some of these neurons migrate into the cerebral ganglion along the visceral nerve. Such GnRH neurogenesis in the peripheral organ and subsequent migration into the brain along the nerve fibers are very similar to vertebrate GnRH-1 neurogenesis in the olfactory placode and subsequent migration into the brain along the olfactory nerve. After delamination of GnRH neurons and young cells from the dorsal

strand epithelium, the epithelial cells may originate from the placode. Together with the previous demonstration of the occurrence of some types of adenohypophysial-like cells, it is hypothesized that the dorsal strand of ascidians is homologous to the vertebrate olfactory placode/adenohypophysis.


10. Amer. Soc. For Limnology & Oceanography (ASLO) June 10-13, 2008, St. John's, Newfoundland, Canada.

The viability of fragments of the invasive colonial tunicate Didemnum sp. in suspension and implications for long distance transport of the species. Mary R. Carman, Woods Hole Oceanog. Institution, Woods Hole, MA.

   Fouling by invasive species of tunicates, including Didemnum sp., is causing economic hardship for commercial shellfishers.  Didemnum sp. 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).  Didemnum sp. first appeared in Atlantic North American waters in the 1980’s at Damariscotta, Maine and 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 Didemnum sp. reattach in the wild in New England waters and in the laboratory.  When a fragment of Didemnum sp. settles on a suitable substrate, it is capable of reattaching within one or two days.  Colonies of Didemnum sp. are fragmented when they are removed from boats hulls, floating docks, and aquaculture equipment.  Fish and scallop trawls pulled through areas infested with Didemnum sp. (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 of Didemnum sp. 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 of Didemnum sp. can survive without attaching was previously unknown.  We conducted an experiment to suspend fragments of Didemnum sp. 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.  


11. Managing Alien Species for Sustainable Development of Aquaculture and Fisheries (MALIAF), Nov 5-7, 2008, Florence, Italy.

Invasive tunicates identified at shellfish aquaculture sites in the New England and Mid-Atlantic states, USA. Mary Carman1, Richard Karney2, James Morris3, Porter Hoagland1, David Grunden4.  1Woods Hole Oceanog. Inst.,  Woods Hole, MA; 2Martha’s Vineyard Shellfish Group, Inc., Oak Bluffs, MA; 3NOAA Natl. Ocean Service, National Centers for Coastal Ocean Science, Beaufort, NC; 4Oak Bluffs Shellfish Dept.,Oak Bluffs, MA.

   Several species of invasive tunicates have spread throughout the marine coastal waters of the New England and the Mid-Atlantic states since the 1980s.  They have become dominant fouling organisms on docks, boat hulls, and mooring lines and pests at aquaculture sites and shellfishing areas.  For example, large mats of the invasive colonial tunicate Didemnum vexillum cover hundreds of kilometers of rocky seafloor on the commercially important sea scallop Plactopecten magellanicus shellfishing grounds of Georges Bank.  These mats block fish from accessing their polychaete prey, smother adult scallops, and result in an inhospitable, acidic substrate for scallop larval settlement.  When heavy biomasses of the invasive solitary tunicates Ciona intestinalis and Styela clava are attached to a cage or line of cultured blue mussels, they add weight; block exchanges of water, nutrients, and wastes; and compete with the shellfish for food.  While the presence of invasive tunicates has been well documented at piers, harbors, marinas, and docks, there are few published reports of invasive tunicates at aquaculture farms in the New England and the Mid-Atlantic states.  We conducted assessment surveys of shellfish aquaculture operations in Massachusetts (as representative of the region) and queried commercial shellfishers, requesting information on the presence of any tunicates at their sites.  We found that shellfish aquaculture operations for bay scallops (Argopecten irradians irradians), Eastern oysters (Crassostrea virginica), hard clams (Mercenaria mercenaria), and blue mussels (Mytilus edulis) in Massachusetts, Rhode Island, Maine, Virginia, North Carolina, New York, New Jersey and Maryland exhibited fouling problems with 10 alien species of tunicates.  These invasive species include Ascidiella aspersa, Botrylloides violaceus, Botryllus schlosseri, D. vexillum, Diplosoma listerianum, Molgula manhattensis, S. clava, Styela plicata, and cryptogenic C. intestinalis and Molgula citrina.  All surveyed growers reported fouling by one or more of these invasive tunicates.  Although we have not surveyed all of the aquaculture operations in these two regions, to date, no native tunicate species have been reported.  Our findings point out the importance of considering invasive species of tunicates when discussing the economic impact of fouling organisms on the aquaculture industry.  


12. Intl. Conference on Shellfish Restoration (ICSR), Nov 19-22, 2008, Charleston, South Carolina.

Invasive tunicates at shellfish restoration and aquaculture sites on Martha’s Vineyard, MA.

Richard Karney, David Grunden, Mary Carman, James Morris, Porter Hoagland. (addresses above)

   Shellfish restoration efforts on the island of Martha’s Vineyard, Massachusetts are impacted by invasive tunicate species.  Several species of non-endemic tunicates invaded New England marine coastal and offshore habitats in the 1980s and 1990s.  While the presence of invasive tunicates is well documented at harbors and marinas in New England, there are few published reports on tunicates at shellfish restoration areas and aquaculture farms.  Species such as bay scallops Argopecten irradians irradians, Eastern oysters Crassostrea virginica, quahogs Mercenaria mercenaria and soft shelled clams Mya arenaria are being cultured and stocked on Martha’s Vineyard in Lagoon Pond, Lake Tashmoo, Menemsha Pond, Sengekontacket Pond, Katama Bay, Poacha Pond, Edgartown Great Pond and Tisbury Great Pond.  Our surveys of shellfish restoration areas indicate that the invasive colonial tunicates Botrylloides violaceus, Botryllus schlosseri, Didemnum vexillum, Diplosoma listerianum, and solitary tunicates Ascidiella aspersa, Ciona intestinalis, Styela clava were common.  The native tunicates Aplidium constellatum, Didemnum albidum, Molgula manhattensis were found to be less common among the fouling organisms on cultured shellfish and shellfish equipment.  Untreated aquaculture equipment and cultured shellfish were infested with tunicates in areas with salinities of 28.0 to 33.5.  Shellfish in bottom sediments were not fouled with tunicates.  Treatment methods such as the spraying of aquaculture gear and shellfish with freshwater, shellfish dipping in salt brine and air-drying were found to be effective biofouling control practices.  


13. 43rd European Marine Biology Symposium, September 2008, Ponta Delgada, Azores, Portugal.

Pérez-Portela R., Bishop JDD and Turon X. Temporal evolution of the genetic diversity in invasive populations: the ascidian invader Perophora japonica.

   Invasive species have been described as one of the most devastating impacts of human activity on natural ecosystems. Coastal waters are especially susceptible to invasion representing one of the most invaded systems on the planet. Both harbours and marinas are the principal ways used by non-native species to colonize new habitats. Perophora japonica is one of the multiple invasive ascidian species found along the European marine environments. The European invasion of Perophora japonica, detected in 1982 from France, is spreading along the Atlantic European coast and it reached the South of England in 1999, when five colonies were discovered on the pontoons of Queen Anne's Battery (QAB) marina in Plymouth (UK, English Channel). Since that moment a

temporal monitoring of the invasive population was carried on the locality and samples were collected every summer from 1999 to 2007. To analyze the temporal evolution of the genetic diversity of the population a fragment of the mitochondrial gene cytochrome c oxidase subunit I (coI) was amplified and sequenced. Our results showed a dramatic change in the haplotype frequencies during the monitoring time matching with a linear erosion of genetic diversity (haplotype diversity) on the ascidian invasive population. Such events may result either from a natural selection or a strong genetic drift suffered by the QAB population together with a genetic isolation from other European invasive populations of the studied species.




1. Biology and population genetics of the invasive ascidian Microcosmus squamiger.

Marc Rius, Dept. of Animal Biology, Univ. of Barcelona, Spain.   

Ph.D. supervisors: Xavier Turon and Marta Pascual

In the last few decades there has been an increase in environmental awareness concerning biological invasions. The impacts created by invasive species are today considered one of the main threats to global biodiversity. Marine organisms have predominantly been moved around the world’s oceans since people first began navigating the seas, and the increase in transoceanic travel during the last century has seen a concurrent rise in the rate of introductions of alien marine species, especially along coastal areas. Ascidians are a common component of rocky shore communities worldwide, where they live attached to either natural or artificial substrata. Although most ascidians have a very limited larval dispersal and a short-lived planktonic larval stage, they can often be caught in ballast pumps and settle within the ship. Alternatively, adults can be transported on hulls and sea chests of ships whereby they spread their larvae in the locations where these ships stop. Genetic markers have been proposed as a very useful tool for monitoring and tracking the distribution of invasive species, and have been implemented particularly in phylogeographic studies. Two common markers used to study intraspecific structure in the context of phylogeography, population genetics and connectivity are mitochondrial DNA and microsatellites.

This Ph.D. thesis aims to study the biology and population genetics of Microcosmus squamiger, an ascidian species that has been introduced in several locations around the world and that has become invasive in some regions. The multidisciplinary approach used in this dissertation aims to create a broad study framework for this organism. The most relevant results of this dissertation are:

1. Taxonomic studies have misidentified M. squamiger and Microcosmus exasperatus. The present study highlights the importance of taxonomy for the study of invasive species.

2. M. squamiger has been introduced into, and has successfully colonized many regions around the world, mostly of Mediterranean climate.

3. The most probable method of transoceanic dispersal of M. squamiger is through shipping.

4. Non-independent colonizations have shaped the present composition of the introduced populations, which are as genetically diverse as the native populations.

5. Different source areas from Australia, predominantly the eastern and western regions, are the most likely origin of M. squamiger introduced populations.

6. The M. squamiger populations of the Atlanto-Mediterranean region generally show little genetic differentiation and no particular genetic structure associated with the Strait of Gibraltar. The low genetic differentiation found among populations is likely to be a result of the combined effect of high population connectivity and the short amount of time which has elapsed since M. squamiger’s introduction into the region.

7. M. squamiger is found exclusively in the western region of the Mediterranean Sea. It is present all year around and shows strong seasonal population dynamics with a 2-year life cycle, which indicates that this species has adapted well to the new environment.

8. High densities of M. squamiger are maintained over time in natural habitats in the Mediterranean Sea, where this species can form a monospecific crust covering most of the available substratum.

9. The reproductive cycle of M. squamiger populations in the Mediterranean Sea is seasonal, with gonad building taking place in spring and with one main spawning season in summer.

10. Marine invasive species have the potential to affect settlement and post-settlement interactions with native species and therefore alter the native population dynamics, as shown in experimental work involving M. squamiger and Styela plicata.

11. There is a need to control M. squamiger as this species colonizes natural environments and forms large aggregates that are a threat to native communities as well as human economic activities.

[ed. note: 4 papers and a book chapter have been published from this thesis work; see the New Publications section of this and the preceding issue of AN.]


2. Membrane microdomains (lipid rafts) in Ascidia ceratodes sperm cells.

 Daniel Diaz. Dept. of Biological Sci., Calif. State University, Fullerton, CA 92834.  Master’s Thesis, Dr. Robert Koch, Research Adviser.

   After sperm-egg binding, several molecules become involved in events such as adhesion, signaling, and cytoskeletal remodeling.  Ascidians are commonly used to study signaling cascades leading to sperm activation, and N-acetylglucosaminidase, G protein coupled receptors, and integrins are known to initiate these events.  Membrane platforms, called lipid rafts, are known to concentrate sphingolipids, cholesterol and signaling proteins; a role for such rafts in ascidian sperm activation and fertilization is the focus of this study.  To locate such structures, sperm cells were labeled with filipin (cholesterol specific) and a fluorescent cholera toxin B (CTB) conjugate (sphingolipid specific).  Both agents labeled membrane microdomains in unactivated sperm cells, but their number, intensity and distribution were different.  In sperm cells activated by pH 9.4 artificial seawater, the G protein activator mas7, or the integrin activator mAb 12G10, the number

of and concentration of cholesterol in microdomains were higher than in unactivated sperm.  Also, the distribution of these structures changed during activation placing them over the mitochondrial regions, where sperm-egg adhesion is known to occur.  If cholesterol was removed by exposing the sperm to methyl-β-cyclodextrin, the microdomains were significantly diminished in number and fluorescence intensity and sperm were unable to fertilize eggs.  Further studies are necessary to understand the differential labeling of sperm heads by CTB and filipin, and to understand the origin of lipid raft-like structures in ascidian sperm cells.




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