Gretchen and Charles Lambert

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Number 61                                                                                                                   November 2007


   There were several successful well-attended meetings this year devoted to our favorite organisms:  The 4th Intl. Tunicata Conference June 23-27, 2007, Villefranche-sur-Mer, France, organized by Christian Sardet; to see the program go to  (A few of the abstracts are included in this issue.) This was followed by a one-day meeting in Italy to honor Dr. Armando Sabbadin--  Fifty years of Botryllus schlosseri as a model organism in biology: results and perspectives, at the Univ. of Padova, June 29. Just held was the second Intl. Invasive Sea Squirt Conference Oct. 2-4, 2007, Prince Edward Island, Canada, organized by Mary Carman and others; the conference proceedings will be published next year in the journal Aquatic Invasions. For a complete program of the talks and posters go to

   We spent a few weeks of research and writing at the Friday Harbor Labs during June and July, then participated in a rapid assessment survey of New England from Maine to Cape Cod for invasive species. We gave a half day ascidian identification workshop for 80 people (!) at the recent Intl. Invasive Sea Squirt Conference; we could not have managed without the generous help of several knowledgeable colleagues. The conference organizers surprised us with a beautiful framed watercolor print with a plaque thanking us for our “Global Ascidian Assistance”!  We’ve been invited to lead a 5 day workshop on the taxonomy and biology of ascidians next August 4-8 in northern Ireland; more details will be sent by email when the preparations are further along.

  There are  131 new publications listed at the end of this issue.


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




   Dr. Xavier Turon, this year’s Paul Illg Distinguished Lecturer for the Friday Harbor Labs, gave two excellent lectures in early July and spent a week interacting with students and researchers. This was his and his wife’s first visit to the Labs but he promises it won’t be their last! He was very impressed with the facilities and location.


   I am still looking for ascidian recipes! Please send to Gretchen. I would like to include them in a future issue of AN.




1. Patrick Frank, Dept. of Chemistry, Stanford University, Stanford, CA 94305-5080.

    We have been using x-ray absorption spectroscopy (XAS) to look at the sulfur within Ascidia ceratodes. XAS permits examination of sulfur within living cells at ambient temperature, avoiding the artifacts of lysis. With our extensive library of sulfur spectra, we are able to interpret the sulfur XAS spectra of blood cells in terms of specific molecules. We are looking at whole blood, at Henze solution, at cell fractions, and at cell-free blood plasma. The results so far have been surprising, in that the blood cell sulfur chemistry is far richer than anticipated. The expected signatures of sulfate and sulfonic acid, and thiol and disulfide (similar to cysteine and cystine) were found in whole blood. However, although the latter dominate the reduced froms of sulfur, there was unexpected evidence for thiamine-like sulfur, among others. The oxidized forms of sulfur have also yielded the unexpected, including evidence for multiple sulfate esters. The majority sulfur species are not in trace amounts, but at relatively high (several mM) concentrations. In A. ceratodes at least, there is as large a metabolic commitment to sulfur as there is to vanadium.


2. Andy Davis, Institute for Conservation Biology, Sch. of Biol. Sciences, Univ. of  Wollongong, NSW 2522,  Australia.

   Xavier Turon (University of Barcelona) spent several months working in my lab at the University of Wollongong (Australia) in 2007.  We focused on two ascidian projects; (i) determinants of distribution of the zone-forming intertidal ascidian, Pyura praeputialis and (ii) the ecological significance of spicules in the large subtidal ascidian Herdmania grandis.  In the first project, we only observed field recruitment of P. praeputialis on turfing algae and adult conspecifics, although lab settlement experiments indicated that larvae were catholic in their settlement responses; settling on a large range of substrata.  Lab and field experiments confirmed that these were the only microhabitats on which recruits survived, with 100% mortality on exposed rock surfaces over surprisingly short time intervals.  It appears that the distribution and abundance of one ecosystem engineer is dependent on another ─ turfing algae.  The second project focused on the barbed calcareous spicules that dominate the mantle of the large solitary H. grandis.  They constitute up to 85% of the dry weight of the mantle wall, supporting the notion that they play a defensive role.   We sought to quantify the size and quantity of spicules in H. grandis from sites grazed by urchins vs ungrazed sites.  We detected little difference in the weight of spicules and are now quantifying their size. We maintained adult H. grandis in aquaria with a number of molluscan predators and they remained untouched while other pyurid species (which lacked spicules) were rapidly consumed.  It appears that the spicules do indeed play a defensive role.  Both of these projects are in early stages and would make excellent studies for postgraduate students. 


3.  Charles Lambert, University of Washington Friday Harbor Laboratories, Friday Harbor WA.

Germinal vesicle breakdown in ascidian oocytes.

   Oocytes of stolidobranch ascidians undergo “spontaneous” germinal vesicle breakdown (GVBD) after passing from the ovary into pH 8.2 sea water. They can be prevented from GVBD by pH 4 sea water. Germinal vesicle breakdown can then be stimulated by increasing intracellular cAMP levels by inhibiting its breakdown. This can be accomplished by blocking phosphodiesterases with methyl xanthines or stimulating its synthesis with forskolin. Oocytes of the solitary ascidian Boltenia villosa undergo GVBD in response to these drugs. However the nearly universal cAMP receptor, protein kinase A (PKA) does not appear to be a target for cAMP as the PKA inhibitor H-89 has no effect upon GVBD.  In addition, the PKA independent epac agonist 8CPT-2Me-cAMP stimulates GVBD under acidic conditions. GVBD is partially inhibited in calcium free sea water. The intracellular calcium chelator BAPTA-AM blocks GVBD at 10 µM. GVBD is also inhibited when the calcium induced calcium release channels are blocked by tetracaine or ruthenium red. Protein phosphorylation is important in ascidian GVBD as oocytes inhibited from GVBD by BAPTA, ruthenium red or tetracaine undergo GVBD in response to dimethylbenzanthacene, a protein kinase stimulator.


4. V. K. Meenakshi, Dept. of Zoology, A.P.C. Mahalaxmi College For Women, Tuticorin, India. 

Screening of a few chosen ascidians of Tuticorin coast for antimicrobial activity, sponsored by the University Grants Commission, Hyderabad was completed with some interesting findings.

   Ascidians were collected from 25 stations along the Tuticorin sea coast. More than 100 samples of ascidians belonging to twenty genera in ten families were identified. Nine species of ascidians were chosen for antibacterial studies. Crude extracts of the chosen ascidians were prepared with methanol, methylene chloride and hexane separately.

  Ten species of pathogens – Enterobacter aerogenes, Streptococcus pneumoniae, Streptococcus pyogenes, Bacillus subtilis, Shigella flexneri, Klebsiella pneumoniae, Psedomonas putida, Staphylococcus aureus, Nocardia corynebacteroid, Actinomyces humiferus – infecting the respiratory, gastrointestinal, urinary tract and wounds were obtained from the Microbial Type Culture Collection (MTCC), Chandigarh.  Nine species of pathogens – Streptococcus viridans, Corynebacterium diphtheriae, Salmonella typhi, Vibrio cholerae, Shigella sp., Enterococcus faecalis, Staphylococcus epidermidis, Nocardia sp., Actinomyces sp., were isolated from samples collected from infected patients.

  Antibacterial activity was studied by disc diffusion method.  Preliminary screening indicated the presence of antibacterial activity in all the ascidians studied. Of the three solvents used for extraction, methylene chloride extracts showed maximum activity followed by methanol and hexane.  Methanol and methylene chloride extracts of Aplidium indicum were active against all pathogens including hospital isolates. Streptococcus pneumoniae, Corynebacterium diphtheriae, Vibrio cholerae, Salmonella typhi, Psedomonas putida, Enterococcus faecalis showed sensitivity to methylene chloride extracts of all the ascidians studied.

  The crude extracts were fractioned using column chromatography.  Forty fractions were collected.  Methanol fraction 17 and methylene chloride fractions 1, 3, 5, 13, 23 of Aplidium indicum showed activity to 80 and 70 % of the pathogens respectively.  Methylene chloride fractions 30, 40 of Ascidia sydneiensis and 18, 19, 27 of Microcosmus exasperatus showed activity to 100, 90 % of the pathogens respectively.  A comparison of the antibacterial sensitivity of ascidian extracts with selected antibiotics to the pathogens showed that the methylene chloride extracts of Microcosmus exasperatus were more sensitive with a maximum zone of inhibition of 23mm. Thus the current studies revealed the presence of potent antibacterial compound from ascidians of Tuticorin coast.




1. 2nd International Symposium on Environment, Athens, Greece, Aug. 2-5, 2007.


 Potential estuarine water quality improvement via marine invertebrate bioremediation. Draughon, L.1, Scarpa, J.2, Keating, P. 1 and Hartmann, J. X. 1   1Florida Atlantic University, Boca Raton, Florida; 2Harbor Branch Oceanog. Inst., 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.


2. The evolution of the animals: a Linnaean tercentenary celebration. The Royal Society, London, 18-19 June 2007.


Phylogeny of living and fossil Deuterostomia. Billie J. Swalla1 & Andrew Smith2.  1Dept. of Biol., Univ. of Washington, Seattle, WA.

   Deuterostomia is a monophyletic group of animals that include echinoderms, hemichordates, xenoturbellids, cephalochordates and vertebrates. Molecular clock estimates suggest that the deuterostomes diverged before the Cambrian, so all major groups and perhaps some surviving stem groups, should be present in the Cambrian fauna.  Genomics has allowed insight into the phylogenetic relationships of the chordates and their invertebrate relatives, and comparison of the shared genetic pathways in related embryos. Molecular data has shown that echinoderms, hemichordates and xenoturbellids form a monophyletic group of animals with very divergent adult body plans. Fossil evidence suggests that echinoderms slowly evolved radial symmetry from stem groups showing bilateral symmetry. Hemichordates evolved coloniality, resulting in highly divergent adult body plans within the phylum. Tunicates, lancelets and vertebrates have traditionally been considered a monophyletic group, the chordates, which share five morphological characters: a notochord, a dorsal neural tube, an endostyle, a muscular, post-anal tail and pharyngeal gill slits. However, genomic DNA sequences and mitochondrial DNA sequences come to contrasting conclusions about whether tunicates or cephalochordates are the sister group of the vertebrates. Morphological characters shared by vertebrates, hemichordates and stem group echinoderms are presumably primitive features of the latest common ancestor of all deuterostomes.  These include the pharyngeal gill slits, an endostyle and a post-anal tail. They were also thought to share a notochord homolog, the stomochord, and a dorsal neural tube in the neck region, although the evidence for this is much less secure.  The chordate ancestor for years has been considered to be a filter-feeding, tunicate-like animal with a tiny chordate tadpole larva.  However, recent evidence suggests that the chordate ancestor was more likely a benthic worm, with a mouth and pharyngeal gill slits, supported by cartilaginous gill bars.  


 3.  4th Intl. Tunicata Conference, Villefranche-sur-Mer, France, June 23-27, 2007.


a. A cis-regulatory atlas of the ascidian Ciona intestinalis. Jamie E. Kugler1,#, David N. Keys2, Janice Imai1, Izumi Oda-Ishii1,, Byung-in Lee2, Paul M. Richardson2 and Anna Di Gregorio1,*  1Dept. of Cell and Dev.Biol., Weill Medical College of Cornell Univ., New York, NY 10021.  2U.S. Dept. of Energy Joint Genome Institute, Walnut Creek, CA.

    The ascidian C. intestinalis offers an unique combination of experimental advantages for the fast identification and characterization of cis-regulatory elements, including a rapid embryonic development and the availability of both biomolecular resources and a straightforward protocol for transient transgenesis.

    We have carried out a survey of ~16 Megabases (~10%) of the C. intestinalis genome, which has led to the identification of over 200 novel enhancer elements, directing expression in one or more of the main tissues that compose the larval body. All these enhancers have been mapped to the corresponding sequence scaffolds, their neighboring genes have been identified and the expression of these genes has been either retrieved from the existing EST expression database ( or, in the case of selected representative examples, analyzed by whole-mount in situ hybridization to validate the enhancers’ activity. These data have been organized in the form of a searchable atlas which will be made available to the community in the near future.


b. Blastogenetic cycle and haemocytes in the colonial ascidian Botryllus schlosseri: a matter of life and death. Loriano Ballarin, Adams Menin, *Giuseppe Basso, *Elena Fortunato, Francesca Cima.  Dept. of Biol. and *Dept. of Pediatry, University of Padova, Italy.

   A recurrent blastogenetic cycle characterizes the colonies of the ascidian Botryllus schlosseri. It starts when a new zooid generation opens its siphons and ends with take-over, when adult zooids cease filtering, are progressively resorbed and replaced by a new generation of buds, reaching functional maturity. During the generation change, massive apoptosis occurs in the colony, mainly in the tissues of old zooids.  In the present study, we investigated the behaviour of haemocytes during the colonial blastogenetic cycle, in terms of occurrence of cell death and expression of molecules involved in the induction of apoptosis. Results indicate that, during take-over: i) caspase-3 activity in haemocyte lysates increases; ii) about 20-30% of haemocytes express phosphatidylserine on the outer leaflet of their plasma membrane, show DNA fragmentation, are immunopositive for caspases-3 and –8, and express the death receptor Fas on their surface; iii) FasL is massively expressed by immunocytes; iv) there is an increase in the fraction of cells expressing molecules recognized by the anti-Bax antibody, and a parallel decrease in the number of cells immunopositive to anti-Bcl-2. On the whole, these data are consistent with the involvement of both the extrinsic, death-receptor-mediated, and the intrinsic, stress-related pathway in the induction of haemocyte apoptosis. Effete cells and corpses are quickly ingested by phagocytes and replaced by a new generation of cells which appears in the circulation during the generation change.


c. Stem cell lineages in colonial botryllid ascidians Federico D. Brown, Stefano Tiozzo, Billie J. Swalla, and Anthony De Tomaso.

   One of the first decisions a cell has to make during lineage specification is whether it shall contribute to the germline or to somatic lineages. To test the hypothesis that germline stem cells belong to a different lineage of cells from other somatic stem cells, we investigate the expression and function of vasa and sox2 that are known to be involved in the specification and maintenance of germline and somatic lineage progenitor cells respectively. Within the stolidobranch ascidians, coloniality has evolved once and this transition was likely accompanied by the evolution of an ability to bud by the retention of multipotent stem cells in adults. In the solitary stolidobranch ascidian Boltenia villosa, maternally provided vasa is segregated into the germline early in development and distinguishes this lineage from other somatic lineages typical of other solitary ascidians, including Ciona intestinalis. Here we report the occurrence and function of vasa in the colonial botryllid ascidians Botrylloides violaceus and Botryllus schlosseri. In reproductive colonies, vasa is expressed in germ cells of the gonads as well as in a subset of blood cells throughout the colony. In contrast, the somatic precursor gene sox2, is found to be expressed in a different subset of cells in colonies of Botryllus schlosseri. We propose that these two different expression patterns of progenitor stem cell markers may represent two distinct populations of precursor stem cells for somatic and germline lineages respectively. We are currently exploring the effect of these two genes during the asexual blastogenic cycles of Botryllus using siRNA to help us elucidate the ultimate function of these putative lineages of stem cells.


d. The making of a urochordate without retinoic acid. Cristian Cañestro1, Ricard Albalat2, Roser Gonzàlez2 and John Postlethwait1.  (1) Institute of Neurosci., Univ. of Oregon, Eugene, OR, (2) Dep. Genetica, Facultat de Biologia, Universitat de Barcelona (Spain).  and 
    Developmental signaling by retinoic acid (RA) is thought to be an innovation essential for the origin of the chordate body plan. The larvacean Oikopleura dioica maintains a chordate body plan throughout life, and yet its genome appears to lack genes for RA synthesis, degradation, and reception (Aldh1a1, Cyp26 and Rar). This situation suggests the hypothesis that the RA-machinery was secondarily lost during larvacean evolution, and predicts that Oikopleura development has become independent of RA-signaling. This prediction raises the problem that the anterior-posterior organization of a chordate body plan can be developed without the classical morphogenetic role of RA. To address this problem, we performed pharmacological treatments (all-trans-RA, 9-cis-RA and the retinaldehyde dehydrogenase inhibitor DEAB) and analyzed expression of developmental molecular markers (Hox1, Otxa-c, Pax2/5/8a-b, Rxr1, Rxr2, Aldh2 and endogenous beta-galactosidase) to investigate whether RA acts in anterior-posterior axial patterning of the CNS, epidermis and endoderm in Oikopleura embryos. Results revealed that RA does not cause homeotic posteriorization in Oikopleura as it does in vertebrates and cephalochordates, and showed that a chordate can develop the phylotypic body plan in the absence of the classical morphogenetic role of RA. A comparison of Oikopleura and ascidian evidence suggests that the lack of RA-induced homeotic posteriorization is a shared derived feature of urochordates. Our findings raise the possible existence of alternative physiological pathways sensitive to RA but not mediated by Rar. We discuss possible relationships between genomic events (e.g. rupture of the Hox-cluster) and the altered roles of RA in the temporal collinear regulation of Hox genes during development in the context of a new model of chordate phylogeny in which urochordates are the sister group of vertebrates.


4. 53rd Meeting of the Italiano Embriologists (GEI), Giardini Naxos (Sicily) June 6-9, 2007.


Blood cell renewal during the colonial generation change in the ascidian Botryllus schlosseri. L. Ballarin, F. Schiavon, A. Menin, F. Cima. Dipto. di Biol., Università di Padova, Italy.

   Colonies of the ascidian Botryllus schlosseri are characterized by the succession of blastogenetic cycles starting when a new zooid generation open its siphons and ending with the take-over, when adult zooids cease filtering and are progressively resorbed and replaced by a new generation of buds reaching functional maturity. During the generation change, massive apoptosis occurs in tissues of adult zooids with infiltration of professional circulating phagocytes. In the present study, we focused our attention on the behavior of hemocytes during the colonial blastogenetic cycle. In the course of the cycle we observed changes in the distribution of circulating hemocytes. The fraction of cells with a diameter ≥ 7 µm is significantly higher at the take-over than in mid-cycle stages. During the take-over, all the main circulating hemocyte types change their frequency. In particular, we observed a significant decrease in the quantity of hyaline amoebocytes, which represent the active phagocytes, paralleled by an increase in the frequency of macrophage-like cells, representing phagocytes having resorbed their cytoplasmic projection, upon the ingestion of foreign or senescent cells, acquiring a globular shape. As regards cell death, 20-30% of circulating hemocytes undergo apoptosis during the take-over, as indicated by the expression of phosphatidylserine on the outer leaflet of plasma membrane and their positivity to TUNEL and COMETA assay for DNA fragmentation. This cell loss is counterbalanced by the appearance of new, young circulating cells during the resorption, as demonstrated by both hystochemical and cytofluorimetric analysis. These cells result immunopositive to anti-FasL antibodies and will express membrane positivity to anti-FasL in the following blastogenetic cycle. Therefore, there is a short time interval in which a wave of new cell enter the circulation, corresponding to the known period of a new stem cell-mediated organogenesis leading to the appearance and the development of a new bud generation.


5. SECOTOX Conference and the Intl. Conf. on Environmental Management, Engineering, Planning and Economics, Skiathos (Greece), June 24-28, 2007.


  Immunotoxicity in ascidians of antifouling compounds alternative to organotins: the case of Diuron and TCMS pyridine. F. Cima, L. Ballarin. Dipto. di Biol., Univ. di Padova, Italy.

   Since ascidians are benthic filter-feeding organisms living in the water-sediment interface, they rapidly bioaccumulate great amounts of xenobiotics and can be employed as useful biosensors in marine pollution monitoring of a wide range of environmental contaminants. In particular, compound ascidians has been reported to be very sensitive to organotin compounds (OTCs), a class of biocides which present in the coastal ecosystems mainly due to the release of antifouling paints from boats and harbour structures. As previously shown, one of the most important toxic effects of these compounds is represented by an immunosuppressant activity both in vertebrates (teleosts and mammals) and filter-feeding marine invertebrates (bivalves and ascidians). After OTC ban by many world's countries due to their severe impact to coastal ecosystems, alternative biocides have been massively introduced in formulations of antifouling paints. Diuron (3-(3,4-dichlorophenyl)-1,1-dimetylurea) and TCMS pyridine (DENSIL100; 2,3,5,6-tetrachloro-4-(metylsulphonyl)pyridine) are two of these new generation biocides, previously used as herbicide and leather tanning additive, respectively. However, up to now, no data are available concerning their potential target organisms, long-term toxic effects on biocenoses, mechanisms of action, bioaccumulation and environmental fate. In the present study, short-term haemocyte cultures of the colonial ascidian Botryllus schlosseri were exposed to various concentrations of xenobiotics for evaluating the toxic effects by means of a series of bioindexes. Similarly to OTCs, at sublethal concentrations (from 25 to 500 µM for Diuron and from 5 to 20 µM for TCMS pyridine) both the pollutants negatively affected the phagocyte morphology and induced apoptosis, but did not inhibit the oxidative phosphorylation and Ca2+ homeostasis. Differently from OTCs, Diuron was able to increase the lysosomal hydrolytic activities of phagocytes, and TCMS pyridine caused glutathione oxidation and inhibited the phenoloxidase activity of the cytotoxic cells. Although these compounds showed a higher LC50 than their environmental concentrations, our assays do open many questions on the long-term effects related to bioaccumulation. On the other hand, as observed for OTCs, the settlement of new toxicity indexes will furnish crucial information about the pesticide impact on the coastal ecosystems and contribute to the choice of antifouling compounds with less dangerousness for the environment. This work was supported by CoRiLa.


6. Exploratory Workshop: High-throughput sequencing applied to marine organisms, a European perspective - Roscoff (France), April 15-17, 2007.


The application of genomic approaches to colonial ascidian B. schlosseri.  Nicola Franchi, Lorìano Ballarin, Fabio Gasparìni, Università degli Studi di Padova, Italy.

   The colonial ascidian Botryllus schlosseri is emerging as a model species for studies of immunity, developmental and evolutionary biology and there is a feasibility to have and maintain Botryllus lab culture in Padova.  We are a team that studies the development and immunity of B. schlosseri, we are principally morphologists and we are acquiring know-how in genomics and molecular biology Thanks to a MGE course at Berlin MPI we have prepared a full-length cDNA library from B. schlosseri colonies, (asexual reproduction); a 384 well plate sequencing result gives to us information about a developed and tested ability to obtain it. Now we are planning to obtain same result with embryos and larvae (sexual reproduction).  At the moment our main research is focussed on the characterization of a rhamnose binding lectin which pays a key role in immune responses of marine invertebrate; up to now we have identified three full transcripts which result high homologous to known genes for rhamnose-binding lectins. The putative amino acidic sequences contain the tryptic peptide sequences we got In a previous biochemical analysis. Now we Intend to produce some probes for ISH to study the expression of these proteins during embryonic development and immune response and to identify the cells responsible of their secretion.


7. Second Intl. Invasive Sea Squirt Conference, Prince Edward Island, Canada, Oct. 2-4, 2007.


a. Monitoring the distribution of indigenous and non-indigenous ascidians and macroinvertebrates in harbours around Newfoundland. Callahan, A.G.1, Deibel, D.1, McKenzie, C.H.2 & Sargent P.1 1Ocean Sciences Centre, Memorial University, St. John's, Newfoundland Labrador, Canada A1C 5S7;  2Northwest Atlantic Fisheries Centre, Department of Fisheries and Oceans Canada, St. John's, Newfoundland Labrador, Canada A1C 5X1 

    Invasive, non-indigenous ascidians have been a significant biofouling problem for the aquaculture industry in Nova Scotia and Prince Edward Island since the mid-1990s.  Given the high level of vessel traffic between Newfoundland and the Maritime Provinces, it may be a matter of when rather than if one or more of these species invade Newfoundland harbours. My thesis work is part of the first assessment of non-indigenous invertebrates in Newfoundland harbours. Field work has been conducted in the fall of 2006 and summer of 2007 in four ports (Port-aux-Basques, Corner Brook, Botwood and Argentia) to assess the abundance and biodiversity of macroinvertebrates on wharf pilings, including indigenous and non-indigenous ascidians.  All four ports are visited regularly by a variety of ships sailing from locations in the southern Gulf of St. Lawrence.  We are also exploring the use of sequence analysis of the cytochrome oxidase I gene (COI) of mtDNA to confirm taxonomic identity and to develop species-specific primers for early detection of larvae and juveniles of indigenous and non-indigenous ascidians. Quadrat samples, visual surveys and photographic records were taken at each harbour and when encountered ascidians were fixed for genetic analysis.

    At this time, I am processing my field samples and am in the early stages of my genetic work.  Early results have shown the presence of indigenous Halocynthia pyriformis, Ascidia prunum, Molgula citrina, Boltenia echinata, Didemnum albidum, Molgula spp. and the non-indigenous Botryllus schlosseri in high densities on the hull of vessels in three ports in Placentia Bay (Argentia, Long Harbour and Arnold’s Cove). I plan to present quantitative quadrat data from my samples, which are dominated by mussels. I am in the final stages of sequencing a portion of COI from the indigenous Halocynthia pyriformis, and will present data from this and the other species. As a result of the discovery of B. schlosseri, we are conducting a more intensive survey of several small harbours in Placentia Bay during 2007 to determine the spatial distribution of this potentially invasive species.


b. Adventures of a sea squirt sleuth: the remarkable story of Didemnum sp. A, a global ascidian invader. Lambert, Gretchen1 and Stefaniak, Lauren2.  1Univ. of Washington Friday Harbor Laboratories, Friday Harbor, WA 98250  2Dept. of Marine Sci., Univ. of Connecticut, 1080 Shennecossett Rd, Groton, CT 06340.

    The route to the identity of Didemnum sp. A has been long and tortuous. The magnitude of its worldwide invasions has taken a number of years to be comprehended. During that time, it was identified as various species depending on its location—D. carnulentum in California, D. lutarium or D. vestum in New England, D. lahillei or D. helgolandicum in France and the Netherlands, D. vexillum in New Zealand, D. pardum in Japan. This talk summarizes a chronology of the steps in the development of our awareness and understanding of this species, lists the known invaded sites and the approximate minimum length of time it has been known in each area and will (hopefully!) unveil its true identity and origin, based on both comparative morphology and genetics.




1. Mating system dynamics in a free-spawning colonial ascidian. Sheri L. Johnson, School of Marine Sciences, University of Maine. Ph.D. Supervisor: Dr. Philip O. Yund

   Successful fertilization in marine organisms that release sperm into the seawater was traditionally thought to be limited by the rapid dilution of gametes.  More recent work has documented that egg brooders that possess mechanisms to concentrate sperm out of the water appear to be much less sperm limited than egg broadcasters.  The ability of an organism to overcome dilution effects and successfully fertilize distant mates can have important consequences for both male and female reproductive success.  The research presented in this dissertation demonstrates how the free-spawning colonial ascidian, Botryllus schlosseri, appears to be remarkably efficient at acquiring dilute, long-lived sperm from the water column.  In part because of this enhanced sperm longevity, distant fertilizations have been recorded in the field.   Past investigations of the selective pressures shaping the allocation of resources to sperm production have focused on the influence of sperm competition and local mate competition, but what we now know about sperm dispersal distances suggest that fertilization distance may be important as well.  A study exploring the influence of fertilization distance on the relationship between sperm production and resulting male reproductive success demonstrates that fertilization distance may be one of the selective pressures driving the allocation of resources to sperm production in B. schlosseri.  Although numerous studies have demonstrated the existence of multiple paternity in marine free-spawners, few studies have characterized the frequency of multiple paternity in natural populations or explored possible causes of this variation.  A study exploring variation in multiple paternity in B. schlosseri broods from natural populations demonstrates that multiple paternity is common, but highly variable, with a few broods displaying unequal contributions.  Indirect benefits from increasing the genetic diversity of broods are a possible explanation for the high level of multiple paternity in this species.  A study exploring the relationship between multiple paternity and subsequent fusion-rejection interactions in B. schlosseri demonstrates how fertilization processes can subsequently affect interactions among later life history stages.  In combination, these studies suggest that brooding free-spawners, with efficient sperm transmission, have very different mating dynamics than some broadcast spawning species.

Yund, P. O., Murdock, K. and Johnson, S. L. 2007. Spatial distribution of ascidian sperm: two-dimensional patterns and short vs. time-integrated assays. Mar. Ecol. Prog. Ser. 341: 103–109. 

Johnson, S. L. and Yund, P. O. 2007. Variation in multiple paternity in natural populations of a free-spawning marine invertebrate. Molec. Ecol. 16: 3253-3262.


2. The taxonomy and molecular phylogeny of solitary ascidians genus Polycarpa (Stolidobranchia: Styelidae) in Taiwan.  Tsai-Ming Lu. Institute of Oceanography, National Taiwan University. Master’s thesis; advisor Dr. Chang-Feng Dai.

   The species of Polycarpa (Stolidobranchia: Styelidae) are solitary ascidians that generally occur on the surface of coral reefs or rocks in tropical and subtropical shallow waters. They are important members in marine sessile communities because their leathery tunic provides habitats for other organisms, such as bivalves, polychaetes, echinoderms and other ascidians. There are about 40 species of Polycarpa worldwide, but only one species is previously recorded in Taiwan. The objectives of this study are to explore Polycarpa species diversity in Taiwan and to infer their molecular phylogeny. In total, 102 individuals of 5 Polycarpa species were collected from northern and southern Taiwan in 2005 and 2006. Among them, 4 Polycarpa species are newly recorded in Taiwan. Partial mitochondrial cytochrome C oxidase subunit I (COI) and the nuclear ribosomal internal transcribed spacer (ITS) DNA sequences were amplified and sequenced. These sequences were applied to phylogenetic reconstruction using neighbor joining, maximum parsimony, maximum likelihood, and Bayesian methods. The topologies of phylogenetic trees reconstructed by four methods are similar. Based on the phylogeny of Polycarpa, there are two major findings: (1) brooding modes of reproduction in the Polycarpa species have evolved independently; (2) there may be a cryptic species within the Polycarpa rima complex.




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