ASCIDIAN NEWS*

 

Gretchen Lambert 

12001 11th Ave. NW, Seattle, WA 98177

206-365-3734   gretchen.lambert00@gmail.com

home page:  http://depts.washington.edu/ascidian/

 

Number 73                                                                                                                 June 2014

 

I  taught an Ascidian Taxonomy Workshop with Noa Shenkar March 17-21 at the Inter-University Institute in Eilat, Red Sea coast of Israel, followed by a few days working on some of the samples in Tel Aviv University’s museum collection. I greatly enjoyed learning about the interesting projects Noa’s students are working on, snorkeling in the Red Sea, and sightseeing around Tel Aviv and southern Israel, and I am very appreciative that my trip was supported by the Israel Taxonomy Initiative.

   I am now looking forward to teaching a two week workshop with Rosana Rocha June 17-July 2 at the Smithsonian Tropical Research Institute (STRI) at Bocas del Toro, Panama. This will be the 4th ascidian workshop at Bocas; the first was in 2006. It is very gratifying to see that many of today’s biologists working on ascidians have taken our workshops over the years. In July Susanna López-Legentil (Univ. of N. Carolina) and I will conduct an ascidian survey of many marinas and some aquaculture sites along the North Carolina coast, a region that has not been surveyed for ascidians for many decades. We will report our findings at the next Invasive Sea Squirt meeting (see below).

   There are 79 new publications listed at the end of this newsletter.

        

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

 

NEWS AND VIEWS

 

1. From Mary Carman: The next Intl. Invasive Sea Squirt Conference (IISSC5) will be held at Woods Hole, Massachusetts Oct 29-31, 2014. Please save the date! Submit abstracts to: mcarman@whoi.edu. Registration at https://www.whoi.edu/main/sea-squirt-conference-v .

 

2. Congratulations to Mari Carmen Pineda, whose Ph.D. thesis entitled “A global wanderer: Biology, phylogeography and resilience of the introduced ascidian Styela plicata”, defended September 25, 2012 and advised by Xavier Turon and Susanna Lopez-Legentil, was recently awarded a Doctoral Extraordinary award by the University of Barcelona. (http://www.ub.edu/irbio/ENnoticia.php?id=222)

 

3. Charles Lambert’s last publication has finally appeared! Lambert, C. C. 2014. Obtaining gametes and embryos of ascidians. In: Carroll, D. J. and Stricker, S. A. (eds.), Developmental Biology of the Sea Urchin and Other Marine Invertebrates. Humana Press, pp. 27-33. Charley finished the final revisions for this chapter before he died 3 years ago, and was very pleased with it. He was a whiz at getting gametes to fertilize and develop, so if you are having methodological problems obtaining gametes or raising embryos from either solitary or colonial species, this chapter will be very useful.

 

4. From Noa Shenkar (Zoology Dept., Tel Aviv University, Israel):  In March 2014, an Ascidian Taxonomy workshop was held for the first time at the Inter-University Institute in Eilat, Israel, taught by myself and Gretchen Lambert. 16 participants successfully learned general aspects of ascidian biology and ecology, and how to identify the local species. My new laboratory at Tel-Aviv University is continuing to study non-indigenous ascidians in the Mediterranean and biodiversity of ascidians in tropical regions.

    The WoRMS Ascidiacea World Database is being updated and revised constantly. http://www.marinespecies.org/  Please e-mail PDF files of new species publications to noa.shenkar@gmail.com. Any comments and corrections to the database are greatly appreciated.

 

5. The 8th Intl. Tunicata meeting will be held on July 13-17, 2015 in Aomori city, Japan. Aomori city is located at the northern end of the Japan main island, being surrounded by well-known scenic places such as the Shirakami Mountains World Heritage Site and Lake Towada. July in Aomori is a pleasant season as the temperature averages approximately 22o C. The organizers are Kazuo Inaba, Director, Shimoda Marine Research Center, University of Tsukuba, and Gaku Kumano, Asamushi Research Center for Marine Biology, Tohoku University, Asamushi.  kumano@m.tohoku.ac.jp

 

6. It is with great sadness that we report the death of Dr. Ivan Goodbody on April 16, 2014, at his home near Aberdeen, Scotland. Ivan worked for many decades on ascidian physiology and taxonomy and produced many important publications, at least 36 of which are either specifically on ascidians or including ascidians. His lengthy 1974 review of the physiology of ascidians still stands as the standard reference, as are his numerous taxonomic papers of Caribbean ascidians. Ivan and his wife Charlotte worked closely together for many years. They produced a CD of color photos of many Caribbean ascidians in 2006.

   I am very grateful to Charlotte and her family for contributing the following biography and tribute:

 

   Ivan Goodbody was born and raised in Dublin, Ireland where he developed an interest in Natural History at an early age.  His interest in Ascidians began during his undergraduate studies at Trinity College Dublin, thanks to a challenging exam question on the life cycle of Botrylloides.  He received a First Class Honours degree in Zoology and spent a year at Oxford University studying Ornithology (a lifelong passion).  He then moved to the University of Aberdeen, Scotland where he completed a Ph.D. on the Biochemistry and Physiology of Nitrogen Excretion in Ascidians.  In Aberdeen he met and married Charlotte Fraser, with whom he celebrated 60 years of marriage in September, 2013. 

   In 1955 Ivan and Charlotte moved to Jamaica, where he took up an appointment of Lecturer in Zoology at the University College of the West Indies (later University of the West Indies).  In 1962 he was appointed Senior Lecturer and became Professor and Head of department in 1964.

   When he moved to Jamaica Ivan’s research involved the taxonomy and the ecology of ascidians. In his early research in Jamaica he demonstrated the phenomenon of continuous breeding in certain ascidians and also discovered mass mortality in tropical marine invertebrates when exposed to fresh water runoff from heavy tropical rains.   His scientific publications drew attention to the diversity of ascidians in the Caribbean, putting him in touch with ascidian researchers elsewhere in the world, notably Patricia Mather in Brisbane, Robin Millar at Oban in Scotland and Donald Abbott in Monterey.  Furthermore, his many publications on tropical marine invertebrates and the publication of his Ph.D. thesis on the metabolism of nitrogen in ascidians drew attention to his research leading to an invitation from Sir Frederick Russell to contribute a review on ascidian physiology to Advances in Marine Biology.  This review has stood the test of time and continues to be widely cited.

   His research on ascidians was further enhanced when he was invited to undertake a study of ascidians in Belize in Central America, as part of a major tropical marine research program set up by the United States National Museum of Natural History.  The programme's field research laboratory was based in the central sector of the Meso-American Barrier Reef at Carrie Bow Cay, Belize.  In the fifteen years during which he participated in this program, he identified 70species from the Pelican Cays and 40 from Twin Cays and documented his findings in 8 scientific papers.  He regarded this work on ascidians as the culmination of his career as an ascidiologist.

   While ascidian biology remained his primary research interest throughout his scientific career, Ivan put great energy and drive into building applied research and educational programs at UWI.  Along with other colleagues in the Zoology Department, he was instrumental in the founding of the Port Royal marine lab in the 1950s which continues to operate as a key research and teaching facility in marine biology.  Under his leadership, research programs into pollution in Kingston Harbour, aquaculture and fisheries all flourished at UWI.  He successfully promoted and managed multi-disciplinary research, believing such collaborations as key to addressing complex environmental questions.   Throughout his career he was a tireless advocate for conservation and habitat preservation.  He served on many national committees and received numerous awards in Jamaica in recognition of his many and diverse contributions to research, conservation and education.  

   After living a in Jamaica for 52 years, Ivan and Charlotte returned to Aberdeen where Ivan became Honorary Research Fellow in the School of Biological Sciences at the University of Aberdeen.  His passion for ascidian biology never waned, and he continued writing up his research from his new home base. 

   Ivan’s career and contributions to the University of the West Indies (UWI) are captured in this excellent tribute from his former colleagues in Jamaica:  

http://myspot.mona.uwi.edu/marcom/newsroom/entry/5671

 

WORK IN PROGRESS

 

1.  Introduced ascidians in South and Northern Chile. Xavier Turon. Centre for Advanced Studies of Blanes (CEAB), Spanish Research Council.  xturon@ceab.csic.es

     I had the opportunity to spend a couple of months in 2013 in Chile, in the framework of a biodiversity project led by Prof. Ivan Cañete (Univ. of Magallanes, Punta Arenas), with the participation of Prof. Javier Sellanes (Universidad Católica del Norte, Coquimbo). Our goal was to assess ascidian diversity in these two areas, with particular emphasis on introduced ascidians. Punta Arenas is a hub for shipping activities through the Magallanes Strait, and the Coquimbo region hosts important aquaculture facilities. We surveyed ports and artificial structures, scuba dove (an unforgettable experience in the freezing waters of Patagonia) and took video footages from the surface. I was very glad to coincide with Rosana Rocha (Univ. Federale do Paraná) in Punta Arenas and share with her the morphological study. The samples are now being genetically barcoded by Susanna López-Legentil (Univ. of N. Carolina). The results so far point to a relatively poor ascidian fauna (ca. 25 species), but clearly differentiated between the two regions surveyed. No species was found at both latitudes. In addition, while there was a high prevalence of introduced ascidians in the Coquimbo region, no invasive ascidian was found in the Punta Arenas region, which was indeed a surprising result given our exhaustive sampling of ports and artificial structures.

 

2. From Patrick Frank, Dept. of Chemistry, Stanford Univ. : pfrank@slac.stanford.edu

    We have extended our XAS studies of sulfur in the blood cells of Ascidia ceratodes to the Bodega Bay population. Free sulfate dominates total sulfur (~50%) but alkyl sulfate esters are again richly present. Biological thiol - disulfide fractions are similar to those recently reported for the A. ceratodes population in Monterey Bay, and are likewise consistent with the participation of thiol in the reduction of blood cell vanadium. A suite of other sulfur secondary metabolites included functional groups similar to benzothiophene and thianthrene. Blood plasma also, surprisingly, included a complex mixture of sulfur compounds. The study includes single animals, which display much wider variations in signet-ring vacuolar pH than the previously examined aggregated samples. I'll be reporting these results in more detail at a symposium talk for Ken Kustin at the August ACS meeting in San Francisco.

 

3. From Benedikte Vercaemer, Dawn Sephton and Claudio DiBacco Fisheries & Oceans Canada, Dartmouth, NS, Canada. Benedikte.Vercaemer@dfo-mpo.gc.ca;  Dawn.Sephton@dfo-mpo.gc.ca; Claudio.DiBacco@dfo-mpo.gc.ca

     A suspicious marine tunicate (Didemnidae) specimen collected in October 2013 by a recreational diver off the coast of Parrsboro, north of Minas Basin, Nova Scotia (NS) was brought to the Bedford Institute of Oceanography (BIO) for identification. The tunicate was sampled from a depth of about 10 m, distributed over an area of about 100 sq. ft. (i.e., the size of a room) and attached to small rocks representative of hard substrate in the area. Taxonomic (BIO) and molecular identification (Dr. Sarah Clark, Dalhousie University) confirmed the sample as the invasive tunicate Didemnum vexillum, which represents the first detection of this species in Eastern Canada. Annual rapid assessment surveys conducted in Passamaquoddy Bay, New Brunswick and areas near Eastport, Maine since 2009 as well as southwestern NS (2013) did not detect this species. Didemnum species have never been detected along the Bay of Fundy or in nearshore coastal habitats of Nova Scotia despite DFO biofouling monitoring initiated in 2006.

     There is considerable suitable habitat for D. vexillum throughout the Maritimes and given its known establishment in Eastport, Maine since 2003, it is likely that other regions of the Bay of Fundy are also affected. The DFO Aquatic Invasive Species group (BIO) completed a rapid assessment survey in early April 2014 to confirm the presence of D. vexillum in the Minas Basin, upper Bay of Fundy, and to collect samples for taxonomic confirmation. Molecular screening of 41 tissue samples collected from 23 stations sampled (10-30 m depth) in the Minas Basin and Minas Channel were confirmed to be D. vexillum. DFO recently initiated a bay-wide assessment to delineate and map the distribution of D. vexillum throughout the Bay of Fundy as well as regions of the southern Scotian Shelf, including Georges Bank, German Bank and Browns Bank.

 

4. Investigations into the reproductive biology of non-native species of ascidians in UK waters. Anaëlle Lemasson, Master’s project. Supervisors: John D.D. Bishop (MBA); Andy Foggo (Plymouth University).  analem@mba.ac.uk

Asterocarpa humilis, the compass ascidian, and Corella eumyota, the orange-tipped ascidian, are two non-native species recently identified on UK shores. They are solitary brooders originating from the southern hemisphere. Since their first record in the Northern Hemisphere, in France during the 2000s, they have rapidly spread through the coasts of France and the UK. As part of my Master’s thesis, undertaken with Plymouth University (UK) and the Marine Biological Association, I am studying various aspects of their reproductive biology in UK waters. The project holds three sub-studies: the brooding seasonality of A. humilis, the seasonal variation in the fecundity potential of A. humilis and C. eumyota, and larval dispersal and settlement preferences of A. humilis, C. eumyota and seven other species of ascidian commonly found on UK shores.

   A. humilis broods larvae throughout the year in UK waters, with a possible peak in winter. Early results for the seasonal variation in fecundity indicate the ability of both species to release thousands of larvae at a time, with an apparent decrease in fecundity from February to April for both species. Future work will also compare their reproductive output with ones of typical solitary broadcast spawners in order to determine whether their brooding strategy is costly in terms of fecundity. The larval dispersal and settlement experiment, not completed yet, will provide crucial information regarding the species’ dispersal potentials and settlement substrate choices, which will lead to a better understanding the type of environment they are likely to colonize in the field.

 

5. From Rosana Rocha, Univ. Federal do Parana, Curitiba, Brasil. rmrocha@ufpr.br

   I have two new doctoral candidates working on ascidians, and a summary of their research projects follows:

Rodolfo Corrêa de Barros: Population genetics and phylogeography of Styela canopus Savigny, 1816.

   Styela canopus is a small and solitary ascidian with high external morphological variability. It’s a fouling animal found on artificial substratum in harbors and disturbed environments. Styela canopus is registered in several places around the world and in Brazil it is found from Ceara to Santa Catarina. The main goal of this project is to understand the geographical distribution of S. canopus through the study of genetic relationships among populations and investigate patterns of dispersal and invasion. I will use COI and ITS1 molecular markers. So, I will need help from researchers to collect samples of S. canopus from distinct populations around the world. If you can help, please contact me: rodolfo@ufpr.br. Since early detection is an important tool in controlling bioinvasions, this project will also aim to establish protocols for molecular identification of S. canopus, S. plicata and C. intestinalis in samples of seawater.

 

Livia de Moura Oliveira: Morphology and systematics of the genera Didemnum Savigny, 1816 and Polysyncraton Nott, 1892.

   Didemnum Sluiter, 1816 is the most diverse genus in the Family Didemnidae with about 230 species. Polysyncraton Nott, 1892 is the second largest genus in the family with 97 known species, and both genera have similar zooids. This study intends to test the monophyly of these two genera and to clarify the phylogenetic relationship between them, using morphology and molecular data, including secondary structure models. Many samples will come from the Brazilian coast, where we already know that there are many new species to be described. But I would also like to include species from different geographical origins to have a better representation of both genera. If you can help, please contact me: lihmoura.oliveira@yahoo.com.br

 

6. Guidelines for the nomenclature of genetic elements in tunicate genomes.

A. Stolfi, Y. Sasakura, D. Chalopin, Y. Satou, L. Christiaen, C. Dantec, T. Endo, M. Naville, H. Nishida, B. J. Swalla, J.-N. Volff, A. Voskoboynik, D. Dauga, and P. Lemaire. patrick.lemaire@crbm.cnrs.fr  [A manuscript recently submitted for a special issue of Genesis on tunicates. I thank all the authors for permission to include it in AN. And a reminder to all readers of AN: as stated on the first page of every newsletter, Ascidian News is not part of the scientific literature and should not be cited as such.]

    Tunicates are invertebrate members of the chordate phylum, and are considered to be the sister group of vertebrates. Tunicates are composed of ascidians, thaliaceans and appendicularians. With the advent of inexpensive high throughput sequencing, the number of sequenced tunicate genomes is expected to rise sharply within the coming years. To facilitate comparative genomics within the tunicates, and between tunicates and vertebrates, standardized rules for the nomenclature of tunicate genetic elements need to be established. Here we propose a set of nomenclature rules, consensual within the community, for predicted genes, pseudogenes, transcripts, operons, transcriptional cis-regulatory regions, transposable elements, and transgenic constructs. In addition, the document proposes guidelines for naming transgenic and mutant lines.

 

7. Chemical and pharmacological activities of a simple ascidian. C.Stella Packiam, Assistant Professor of  Chemistry under the guidance of  Dr. R. Jothibai Margret, Dept. of Chemistry, Pope’s College, Sawyerpuram, Tirunelveli and coguidance of Dr. V.K. Meenakshi, Dept. of Zoology, A.P.C.Mahalaxmi College for Women, Tuticorin, Tamilnadu, India.

     GC-MS analysis, IR Spectral studies and spectrophotometric studies are in progress in a simple ascidian Ascidia sydneiensis. Ascidians have tremendous potential in pharmaceutical and biomedical field. Ascidia sydneiensis is commonly called as crevice ascidian. It has been subjected to Infrared (IR) spectral study which indicates the presence of aromaticity, hydroxyl and carbonyl group in the ethanolic extract. GC-MS studies revealed the presence of ten chemical constituents -Tetradecanoic acid, Bis-(2-methylpropyl)ester of 1,2-benzenedicarboxylic acid, n-Hexadecanoic acid, 9-Hexyl-heptadecane, Diisooctyl ester of 1,2- benzenedicarboxylic acid, 3-ethyl-5-(2-ethylbutyl)-octadecane, Squalene, Cholest-5-en-3-ol(3∝)-carbonochloridate, Cholesterol, 2-[[2-[(2-ethylcyclopropyl)methyl]cyclopropyl]methyl]-methylester of cyclopropaneoctanoic acid.

     Infra red spectrum for ethanolic extract of Ascidia sydneiensis (Figure-1) shows broad band at 3416.97 cm-1 which is due to the presence of moisture or hydroxyl groups in the compound and that at 2930.39 cm-1 is characteristic for C-H stretching vibration indicating aliphatic chain. The band at 2350.80 cm-1 for O-H stretching gives evidence for the presence of carboxylic acid and the band at 1620.06 cm-1 for C=O stretching vibration indicates carbonyl group. The strong bands above 3000 cm-1 shows evidence for aromatic rings. Estimation of the total carbohydrates, proteins, lipids, phenols and flavonoids from Ascidia sydneiensis were analysed by Spectrophotometer. The results showed that the specimen was rich in lipids (25.33%) followed by flavonoids (19.01%), proteins (10.61%), phenols (10.09%) and carbohydrates (5.58%). Ascidia sydneiensis possess higher percentage of lipid and lower carbohydrate. Following observations suggest that this commonly available ascidian has active biochemical potential possessing antioxidant, anti-inflammatory and antimicrobial properties for curing various ailments which can lead to the isolation of new and novel compounds.

 

8. Chemical Investigation and anticancer activities of selected tunicates. S. Sankaravadivu, Assistant Professor of  Chemistry, under the guidance of  Dr. R. Jothibai Margret, Dep.t of Chemistry, Pope’s College, Sawyerpuram, Tirunelveli and coguidance of Dr. V.K. Meenakshi, Dept. of Zoology, A.P.C.Mahalaxmi College for Women, Tuticorin, Tamilnadu, India.

      GC-MS analysis, IR Spectral studies and spectrophotometric studies are in progress in a colonial ascidian Ecteinascidia venui. GC-MS analysis of the ethanolic extract of the animal revealed the presence of twelve components. The peak with retention 10.38 corresponds to 1-(2-Ethyl-3-cyclohexenyl)ethanol, 14.26 to (E,E)-methyl ester of 9,12-Octadecadienoic acid, 14.34 to (E)-methyl ester of 9-Dodecenoic acid, 16.96 to 9-Octadecenal, 18.71 to (Z)6,(Z)9-Pentadecadien-1-ol, 19.39 to (Z,Z)-9,12-Octadecadien-1-ol, 19.46 to 13-methyl-Oxacyclotetradecane-2,11-dione, 19.73 to Eicosane, 21.13 to Tetradecyloxirane, 22.14 to (R)-(-)-14-Methyl-8-hexadecyn-1-ol, 22.52 to Nonadecane and  23.90 to 1-Iodo-2-methylundecane. These chemical components have not been reported elsewhere from ascidians specially Ecteinascidia venui and hence can be considered as a first report. Infrared spectrum for ethanolic extract of Ecteinascidia venui indicated a broad band at 3409.83 cm-1 due to the presence of moisture or hydroxyl group in the compound. The band at 1632.09 cm-1 is characteristic of carbonyl group and that at 1412.10 cm-1 shows the presence of C-H bending. Presence of peaks above 3000 cm-1 indicates aromaticity and C-H stretching. Analysis of carbohydrates, proteins, lipids, phenols and flavonoids were determined by Spectrophotometry. A maximum of 45.82% flavonoids, 20.68% proteins, 9.77% lipids, 9.21% phenols and 4.57% carbohydrates were observed in Ecteinascidia venui. These studies clearly indicate that Ecteinascidia venui is rich in many biochemical compounds.

 

ABSTRACTS FROM RECENT MEETINGS

 

1. Benthic Ecology Meeting, Jacksonville Florida, March 19-22, 2014

 

Unraveling the diversity of the ascidian microbiome. López-Legentil, S.1; Pineda, M.C.2; Webster, N.2; Turon, X.3; Erwin, P.M.1  1Department of Biology & Marine Biology, and Center for Marine Science. 5600 Marvin K. Moss Ln, Wilmington NC 28409, USA. lopezlegentils@uncw.edu; 2Australian Institute of Marine Science, Townsville Mail Centre, Qld 4810, Australia; 3Center for Advanced Studies of Blanes (CEAB-CSIC), Accés Cala S. Francesc 14, 17300 Blanes, Girona, Spain.

     Symbiotic microbes are known to play a critical role in host metabolism and in nutrient cycles; however, the ascidian microbiome remains largely unexplored. Here, we provide the most comprehensive characterization to date of the ascidian microbiome by investigating the diversity, structure and specificity of microbial symbionts in 42 Great Barrier Reef ascidians using 16S rRNA gene tag pyrosequencing. Results revealed high bacterial biodiversity (3,217 OTU0.03) and the widespread occurrence of ammonia-oxidizing Archaea. The ascidian microbiota was clearly differentiated from free-living seawater microbial communities and included symbiont lineages shared with other invertebrate hosts as well as unique, ascidian-specific phylotypes. Several rare seawater microbes were markedly enriched in the ascidian microbiota, suggesting that the rare biosphere of seawater may act as a conduit for horizontal symbiont transfer among hosts. However, most OTUs (71.2%) were rare and specific to a single host while core communities were comprised of only 7 OTUs, indicating an overall high degree of host-specificity. We hypothesize that the complex ascidian microbiota revealed herein is maintained by a dynamic tunic microenvironment, offering periodic windows of optimal conditions for different metabolic pathways. For example, ample chemical substrate (ammonia-rich host waste) and physical habitat (high oxygen, low irradiance) that support nitrification processes.

 

2. XV Meeting of the Italian Association of Developmental and Comparative Immunobiology (IADCI), Univ. of Ferrara, Ferrara, February 12-14, 2014.

 

a. The production of amyloid requires cross-talk between immunocytes in the compound ascidian Botryllus schlosseri. L. Ballarin1, R. Girardello2, A. Grimaldi2, N. Franchi1, M. De Eguileor2 1Dept .of Biology, Univ. of Padua, Padua, Italy; 2Dept. of Biotechnology and Life Sci., Univ. of Insubria, Varese, Italy ballarin@bio.unipd.it

Two main immunocyte types are present in the hemolymph of the compound ascidian Botryllus schlosseri, i.e., phagocytes and cytotoxic morula cells (MCs). Previous studies have demonstrated that MCs work as sentinel cells able to sense foreign molecules and, as a consequence, release cytokines which activate phagocytes leading to the synthesis and release of rhamnose-binding lectin. The latter, in turn, acts as a chemotactic factor for phagocytes and opsonizes foreign particles stimulating their clearance. In addition, upon the contact with foreign molecules, MCs can degranulate and release the content of their vacuoles, mainly phenoloxidase (PO) and its polyphenol substrata. In a recent investigation on Botryllus cytotoxic cells, we found abundance of amyloid inside MC vacuoles which likely, once released, act as a scaffold to prevent the diffusion of PO and cytotoxicity to the whole organism. In addition, the study of the molecular cascade leading to the synthesis of amyloid, is revealing a non-classical pathway in which both the phagocytes and MC are involved.

 

b. Phagocytosis-induced apoptosis in the compound ascidian Botryllus schlosseri.  F. Schiavon, N. Franchi, L. Ballarin, Dept. of Biology, Univ. of Padua, Padua, Italy.

     Colonies of the ascidian Botryllus schlosseri contain three blastogenetic generations: functional zooids, their buds and budlets on buds. Generation change or take-over (TO) occur cyclically and assure the recurrent renewal of the colony. During these events, lasting 24 - 36 h, diffuse apoptosis occurs in zooid tissues, as indicated by morphological, biochemical and molecular investigations. Tissues are rapidly infiltrated by circulating phagocytes, selectively recruited by dying cells, which recognize and greedily ingest them. Using hemocytes as selected cell population for investigation, we studied the transcription rate of three recently characterized genes involved in apoptosis, bsbax, bsaif1 and bsparp1, during the TO as compared to colony developmental phases far from it. In addition, we observed that the massive ingestion leads phagocytes themselves to undergo apoptosis, probably as a consequence of the oxidative stress related to the sustained respiratory burst, as suggested by biochemical analysis. Therefore, a large fraction of circulating phagocytes needs to be replaced by new, young hemocytes, entering the circulation at the end of the generation change.

c. New evidences of conserved pathways in complement system dynamics from the colonial ascidian Botryllus schlosseri. N. Franchi, L. Ballarin, Dept. of Biology, Univ. of Padua, Padua, Italy.

     In recent years, it has been widely demonstrated that complement, although often depicted as a ‘first line of defense’, is more than just a defender against microbial intruders and acts as a tightly integrated surveillance system. It is not only important against microorganisms, but also for the clearance of apoptotic cells and corpses. Botryllus schlosseri belongs, as vertebrates, to the phylum Chordata and, for this phylogenetic trait, it is unanimously considered a reliable model organism for the studies of the evolution of the immune system. Moreover it is also characterized by a peculiar life cycle with a cyclical, massive apoptosis. This two key features render B. schlosseri a good research tool for the study of the evolution of the complement system. Here we report the first results on the expression of BsC3 and BsFactorB, both components of the alternative pathway (AP) of complement activation, which form the AP C3 convertase.

     Since studies on mammalian models have shown that 80 % of the observed complement response is derived from AP convertase-mediated C3 amplification, even if initially induced by the classical pathway (CP), studies of complement activation in an organism that lack the adaptive immunity, as B. schlosseri, could lead to a better comprehension of the AP cascade and the behavior of C3 convertase not only in invertebrates, but also in vertebrates, mammals included. As in mammals, BsC3 is highly transcribed at basal level and over-expressed after incubation with non self (zymosan) while BsFactorB always shows limited expression. In the presence of compstatin, a 13-residue cyclic peptide able to inhibit the activation of C3 by C3 convertases, the percentage of phagocytosing hemocytes collapses. In the presence of both zymosan and compstatin, the transcription of BsC3 by hemocytes increases with respect to cells exposed only to zymosan: this suggests the presence of a conserved molecular machinery able to control and modulate B. schlosseri as well as the mammalian complement.

 

d. Evidences for antimicrobial peptides in the colonial ascidian Botryllus schlosseri. F. Schiavon, N. Franchi, L. Ballarin. Dept. of Biology, Univ. of Padua, Padua, Italy

     Invertebrates have acquired many mechanisms of defence in order to overwhelm the risk of pathogen attack. In particular, the presence of various types of antimicrobial peptides (AMP) guarantees an efficient response, making them able to kill both gram positive and negative bacteria, fungi and viruses. In ascidians, AMPs have been isolated and described in different species, such as in Styela clava (Clavanins and Styelins) and in Ciona intestinalis (the recent Ci-MAM). Previous studies have revealed molecules, including phenoloxidase and rhamnose-binding lectins, with an antimicrobial effect also in Botryllus schlosseri. Here, we report that the growth of some bacteria strains is highly inhibited by extracts of haemocytes, showing alterations in their surface. Moreover, molecular analyses allowed us to identify a Botryllus sequence similar to Styelins that is abundantly transcribed in phagocytes.

 

e. Characterization of proxiredoxins’ coding genes in Ciona intestinalis. R. Benevenia1, D. Ferro2, L. Ballarin1, G. Santovito1. 1Dept. of Biology, Univ. of Padua, Padua, Italy; 2Institute for Evolution and Biodiversity, Westfälische Wilhelms-Universität, Münster, Germany.

Ascidians represent an interesting model from an evolutionary and ecotoxicology point of view, because of their large distribution in temperate sea and their phylogenetic position of invertebrate chordates. Immune responses imply an increase in oxygen consumption with a consequent risk of oxidative stress. With the aim to study the components of the antioxidant defense system in the solitary ascidian Ciona intestinalis, we characterized gene sequences encoding peroxiredoxins (Prxs), non-selenium peroxidases that are able to reduce hydrogen peroxide, organic hydroperoxides and peroxynitrite. Thus they represent a class of important antioxidant enzymes, that protect cells against oxidative stress. In the GeneBank database five Prx sequences are present, Prx2, 3, 4, 6a and 6b. The multi-alignment analysis, conducted with orthologous sequences of vertebrates and invertebrates, showed that in Ciona’s Prxs the amino acids essential for their enzymatic activity are highly conserved, namely the catalytic tetrad consisting of proline, threonine, cysteine and arginine. Preliminary phylogenetic reconstruction indicates that Prx3 and 4 emerge as sister group of Prxs of the respective vertebrates groups (or isoforms), while Prx2, 6a and 6b have an uncertain position. A partial confirmation of phylogenetic results was obtained with the analysis of homology modeling, according to which Prx3 and 4 present a structure similar to that of two vertebrate proteins, Bos taurus Prx3 and Larimichthys crocea Prx4, respectively, while Prx2 and 6 six have a three dimensional structure similar to that of two invertebrate proteins, Ancylostoma ceylanicum Prx1 and Arenicola marina Prx6, respectively. The transcription of all these genes, measured by qRT-PCR, resulted variable in different organs (intestine, ovary, pharynx, stomach). In particular, the ovary is the organ that expresses the highest level of messenger for all Prxs. Preliminary data were also collected about the possible circadian expression of Prx.

 

3. The 9th Intl. Vanadium Symposium will be held at Padova, Italy, from June 29- July 3. The topics will cover coordination chemistry, speciation, biological chemistry, toxicology, enzymology, catalysis, therapeutic applications, batteries, novel V-containing materials, and environmental/occupational exposure to V agents. Dr. Tatsuya Ueki will give an invited presentation on the accumulation of vanadium in ascidians. http://www.chimica.unipd.it/V9/ .

 

THESIS ABSTRACTS

 

1. Studies on the antiproliferative activity of a chosen ascidian of Tuticorin coast. Ph.D. thesis by M. Paripooranaselvi, Assistant Professor, Dept. of Zoology, A.P.C. Mahalaxmi College for Women, Tuticorin, under the guidance of Dr. V.K. Meenakshi. Submitted to Manonmaniam Sundaranar University, Tirunelveli, Tamil Nadu, India. mparipooranaselvi@gmail.com

Phallusia nigra are common persistent biofoulants found mostly attached to cement blocks, rocks, pilings and oyster cages in Tuticorin harbour area. The thesis consists of two parts. Part I Biofouling deals with an assessment of the species composition, distribution and seasonal variation in the occurrence of ascidian biofoulers at three different stations - Tuticorin Green Gate Barge, Harbor installations and Tuticorin Green Gate intertidal rocky shores. 50 species of ascidians belonging to 20 genera coming under 9 families were identified in all the three stations together and the species composition was rich in station 3 with the total number of 44 species. Part II deals with the in vitro and in vivo antiproliferative and immunomodulatory studies of the ethanolic extract of simple ascidian Phallusia nigra against DLA, EAC, S-180, HLCA-549 and MDA-MB-231 bearing mice. Acute oral toxicity studies using the ethanolic extract of P. nigra to Swiss albino mice administered with 2000 mg/kg body weight did not show any mortality. 100% cytotoxicity to the above mentioned cells were observed at a concentration of 0.60, 0.40, 0.60, 0.80 and 0.60 mg/ml respectively. Administration of the extract increased median survival time, % life span, non viable cells, hemoglobin, RBC, lymphocyte, eosinophil, bone marrow cellularity, β-Esterase positive cells, antibody titer and the plaque forming cells in spleen. A reduction in the body weight, relative organ weight, solid tumor volume, packed cell volume, viable cells, WBC, neutrophils, serum Gamma Glutamyl Transpeptidase (GGT), cellular Glutathione (GSH) and restoration of NO levels were observed on treatment with the extract in DLA, EAC, S-180, HLCA-549 and MDA-MB-231 tumor bearing mice. The activity was comparatively greater than that observed for the standard drug - Vincristin in all the parameters tested. Quantitative hemolysis of sheep red blood cells, lymphocyte proliferation, NK cytotoxic activity and phagocytosis rate increased on treatment with the extract indicating activation of immune function.

 

2. Chemical screening and pharmacological evaluation of ascidians, Ph.D. thesis by S. Gomathy, Assistant Professor, Dept. of Zoology, A.P.C. Mahalaxmi College for Women, Tuticorin under the guidance of Dr. V.K. Meenakshi. submitted to Manonmaniam sundaranar University, Tirunelveli, Tamil Nadu, India. gomathyapcm@gmail.com

     Ascidians, an important group of marine, sedentary organisms found distributed along the Indian coast throughout the year has been screened for their chemical constituents and pharmacological properties. The thesis consists of five chapters. Chapter 1 deals with the study of the distribution of ascidians at 3 different stations Kanyakumari, Thoothukudi and Mandapam along the Southeast coast of India during January to December 2011. 53 species of ascidians belonging to 22 genera coming under 9 families were identified in all the three stations together. Chapter 2 deals with Chemical screening and pharmacognostic studies of Microcosmus exasperatus. The specimen of Microcosmus exasperatus was analyzed macroscopically, microscopically and extracted using different solvents such as petroleum ether, benzene, chloroform, ethanol, methanol, methylene chloride and water. Alkaloids, terpenoids, steroids, coumarins, tannins, saponins, flavonoids, quinones and anthraquinones were observed. Ethanolic and Methanolic extract of Microcosmus exasperatus has been subjected to GC – MS analysis which showed the presence of twenty and nine compounds respectively. HPTLC studies revealed the presence of phenolic compounds such as Gallic acid, Ferulic acid and Caffeic acid. Rutin, Isoquercitrin and Quercetin were some of the flavonoids identified. Fat soluble vitamins D3, A, K and water soluble vitamins riboflavin, thiamine were noticed in high concentration. In Chapter 3, studies on hepatoprotective activity of Microcosmus exasperatus has been performed in Carbon tetrachloride induced liver damaged Wistar albino rats which indicated an increase in body weight, protein, albumin, globulin, total conjugated, unconjugated  bilirubin, GGT, GPX, GR, SOD, CAT, GSH and decrease in the level of LPO, SGPT, SGOT, ALP in serum. Antidiabetic studies in alloxan induced diabetic rats in Chapter 4 recorded an increase in insulin level, protein, albumin, globulin, SOD, CAT, GPX, GSH, GR and a decrease in blood glucose, urea, creatinine, HbAlc, SGPT, SGOT, ALP, TC, TG, LDL – C, VLDL, HDL, PL and LPO activities in serum. Antifertility activity of the ethanolic extract of Microcosmus exasperatus in male albino rats in Chapter 5 noted a significant decrease in the weight of body, testis and the accessory sex organs. A dose related reduction in sperm count of testis, epididymis, motility and increase in the level of abnormal sperms were observed. The serum biochemical parameters and liver marker enzymes did not show any significant variation.

 

3. Studies on a few aspects of ascidians from the  Gulf  of Mannar.  Ph.D. thesis by S. Senthamarai, Research Scholar, PG and Research Dept. of Zoology, V.O. Chidambaram College, Tuticorin. Submitted to Manonmaniam Sundaranar University, Tirunelveli, Tamil Nadu, India under the guidance of Dr. V.K. Meenakshi.

       Marine biodiversity of the Gulf of Mannar is poorly known. Knowledge of the ascidian biodiversity of the ocean makes it one of the greatest resources for drug discovery. The thesis consists of two parts. Part 1 deals with the biodiversity of Gulf of Mannar ascidians, which includes the section on Taxonomy giving detailed descriptions of the newly recorded species. The biological resources study of ascidians of Gulf of Mannar could give as many as 26 species of ascidians as new records to Indian waters belonging to 3 families and 9 genera. Of the 26 species, one species - Aplidium digitalis is new to science. The total number of species of ascidians from Gulf of Mannar water has now gone up to 99.  All the 26 species which are new to India have been dealt in detail with photographs and camera Lucida diagrams. Part 2 deals with the biomedical applications of a selected ascidian from the Gulf of Mannar and contains two sections. In the first section the screening of antibacterial activity of the simple ascidian Microcosmus exasperatus against MTCC and clinical isolates of human pathogens affecting the various tracts has been carried out. The present study reveals that the crude Methylene Chloride extract was more active, exhibiting a broad spectrum antibacterial activity than the selected commercially available antibiotics. Maximum zone of inhibition (23 mm) was observed against Streptococcus pneumoniae (655) of respiratory tract and Actinomyces sp. of wound pathogen. The second section focuses the in vitro and in vivo antitumor and immunomodulatory studies of the ethanolic extract of simple ascidian Microcosmus exasperatus against DLA bearing mice. Acute oral toxicity studies using the ethanolic extract of M. exasperatus to Swiss albino mice administered with 2000 mg/kg body weight did not show any mortality. 100% cytotoxicity to DLA cells was observed at a concentration of 0.80 mg/ml. Administration of the extract of M. exasperatus increased median survival time, % of life span, non viable cells, heamoglobin, RBC, lymphocyte, eosinophil, bone marrow cellularity, β-esterase positive cells, antibody titer and the plaque forming cells in spleen. A reduction in the body weight, relative organ weight, solid tumor volume, packed cell volume, viable cells, WBC, neutrophils, serum Gamma Glutamyl Transpeptidase (GGT), cellular Glutathione (GSH) and restoration of NO levels in DLA tumor bearing mice. The activity was comparatively greater than that observed for the standard drug (Vincristin) in all the parameters tested.

 

4. Phenotypic plasticity and adaptation potential to salinity in early life stages of the tunicate Ciona intestinalis sp. B. Elin Renborg, PhD thesis. Awarded April 29, 2014 from University of Gothenburg, Tjarno Marine Biological Laboratory; Jon Havenhand supervisor.

See Renborg, E., Johannesson, K. and Havenhand, J. 2014. See: Renborg, E., Johannesson, K. and Havenhand, J. 2014. Variable salinity tolerance in ascidian larvae is primarily a plastic response to the parental environment. Evol. Ecol. 28: 561-572. Abstract:

     Both phenotypic plasticity and local genetic adaptation may contribute to a species’ ability to inhabit different environmental conditions. While phenotypic plasticity is usually considered costly, local adaptation takes generations to respond to environmental change and may be constrained by strong gene flow. The majority of marine species have complex life-cycles with pelagic stages that might be expected to promote gene flow and plastic responses, and yet several notable examples of local adaptation have been found in species with broadcast larvae. In the ascidian, Ciona intestinalis (Linnaeus, 1767),—a common marine species with broadcast spawning and a short larval stage—previous studies have found marked differences in salinity tolerance of early life-history stages among populations from different salinity regimes. We used common-garden experiments to test whether observed differences in salinity tolerance could be explained by phenotypic plasticity. Adult ascidians from two low salinity populations [2–5 m depth, ~25 practical salinity units (PSU)], and two full salinity populations (25–27 m depth, ~31 PSU) were acclimated for 2–4 weeks at both 25 and 31 PSU. Gametes were fertilized at the acclimation salinities, and the newly formed embryos were transferred to 10 different salinities (21–39 PSU) and cultured to metamorphosis. Adult acclimation salinity had an overriding and significant effect on larval metamorphic success: tolerance norms for larvae almost fully matched the acclimation salinity of the parents, independent of parental origin (deep or shallow). However we also detected minor population differences that could be attributed to either local adaptation or persistent environmental effects. We conclude that differences in salinity tolerance of C. intestinalis larvae from different populations are driven primarily by transgenerational phenotypic plasticity, a strategy that seems particularly favourable for an organism living in coastal waters where salinity is less readily predicted than in the open oceans.

 

5. Ecology and genetics of invasive ascidians in the Western Mediterranean. Víctor Ordóñez. Ph.D. thesis 2013, Univ. of Barcelona, Catalonia, Spain. Advisors: Marta Pascual, Xavier Turon.  xturon@ceab.csic.es

     Biological invasions are of special concern to marine biologists. The Mediterranean is one of the seas’ most threatened from this point of view. The main goal of this PhD thesis is to study ecological and genetic aspects of the biology of four invasive ascidians in the western Mediterranean. In the first chapter, we delve into the post-border processes of the ascidian Microcosmus squamiger, by analysing its population genetic structure. This ascidian has a relatively high capacity of post-border dispersion, which allows gene flow between populations on a small scale. Moreover there are two genetic clusters related to the two original sources of the introduced worldwide populations. These genetic pools are mixed at the population level but not at the individual level. In the second chapter, we evaluate biological interactions in early-life history stages of Microcosmus squamiger and Styela plicata, among themselves and with mussels. No interactions between the two ascidians were found in fertilization or larval settlement and metamorphosis processes. In contrast, larvae of both ascidians were consumed by juvenile mussels, and the ascidians also presented shifts in larval behavior in the presence of the mussels. This highlights the importance of the resident community for the establishment of introduced species. In the third chapter, we study the life cycle and population genetic structure of the invasive Didemnum vexillum, which is reported for the first time in western Mediterranean. This ascidian showed a marked seasonal growth and reproductive cycle, negatively influenced by warm summer temperatures. Additionally, introduced populations in the Mediterranean showed low diversity, suggesting a bottleneck process in the introduction. Finally, in the fourth chapter, we study the life cycle of Clavelina oblonga. This ascidian was in fact introduced in the Mediterranean for more than 80 years now, but it was erroneously described as a new species, C. phlegraea, purportedly native to the Mediterranean. Their reproductive and growth cycle was markedly seasonal, favored by warm summer temperatures, and hibernating in colder months.

 

6. Species invasion in the marine fouling communities of British Columbia: factors that influence invasion dynamics and how they may affect Botrylloides violaceus. Jocelyn Nelson, M.S. thesis, Univ. of British Columbia, Vancouver, BC, Canada. Advisors Drs. Christopher D. G. Harley and Thomas W. Therriault.  jocelyn.nelson@gmail.com

     Species invasion has been recognized as a major threat to biodiversity. Knowledge of the factors that limit the establishment and spread of non-indigenous species (NIS), such as biotic resistance and unfavourable environmental conditions, are important to their effective management. To test the biotic resistance and environmental favourability hypotheses in the fouling communities of British Columbia (BC), 22 locations were compared using settlement tiles in a large-scale survey. Biotic resistance is believed to be stronger in more diverse communities, therefore NIS richness and abundance were compared to native species richness and environmental conditions to investigate their importance using generalized and linear mixed models. Invader taxonomic group may influence biotic resistance, and environmental tolerances vary by species, therefore factors that affected Botrylloides violaceus presence and abundance were investigated as a case study. The biotic resistance hypothesis was not supported for NIS richness or NIS abundance, but could not be fully discounted due to a trend toward a negative slope between native species richness and B. violaceus presence and abundance, and the absence of predator data. Environmental variables affected NIS: salinity had a positive influence on NIS richness, NIS abundance, and B. violaceus presence, and temperature had a positive effect on B. violaceus presence and abundance. Salinity had a positive impact on native species richness as well, supporting the environmental favourability hypothesis. This suggests that knowledge of relevant environmental conditions is more important for the management of invasive species than the species richness of vulnerable communities.

     Environmental conditions are not static, so species invasion must be considered in the context of climate change. To understand how climate change may influence species invasion, B. violaceus presence and abundance in BC were compared to a range of abiotic conditions. This comparison informed a GAMLSS model that used linear trends from historical shore station data to project potential abundance in BC forward 50 years. Overall, the abundance of B. violaceus in BC was projected to increase. A larger increase in B. violaceus abundance was forecast for locations where conditions increased into the range favourable for growth. If temperature and salinity become more favourable for B. violaceus, as projected, climate change could intensify the invasion.

    The full text can be found online at http://circle.ubc.ca/handle/2429/46606.

 

NEW PUBLICATIONS

 

Aldred, N. and Clare, A. S. 2014. Mini-review: Impact and dynamics of surface fouling by solitary and compound ascidians. Biofouling 30: 259-270.

Ankisetty, S., Khan, S. I., Avula, B., Gochfeld, D., Khan, I. A. and Slattery, M. 2014. Chlorinated didemnins from the tunicate Trididemnum solidum. Mar. Drugs 11: 4478-4486.

Arkhipkin, A. and Laptikhovsky, V. 2013. From gelatinous to muscle food chain: rock cod Patagonotothen ramsayi recycles coelenterate and tunicate resources on the Patagonian Shelf. J. Fish Biol. 83: 1210-1220.

Brine, O., Hunt, L. and Costello, M. J. 2013. Marine biofouling on recreational boats on swing moorings and berths. Manag. Biolog. Invasions 4: 327–341.

Bruce, A. J. 2014. Periclimenaeus colemani sp. nov. (Crustacea; Palaemonidae; Pontoniinae) from Heron island, Queensland. Zootaxa 3774: 258-264.

Bruce, A. J. 2014. Periclimenaeus denticulodigitus sp nov (Crustacea: Decapoda: Palaemonidae: Pontoniinae), from Heron Island, Queensland, Australia. Zootaxa 3753: 71-78.

Bullard, S. G., Davis, C. V. and Shumway, S. E. 2013. Seasonal patterns of ascidian settlement at an aquaculture facility in the Damariscotta River, Maine. J. Shellfish Res. 32: 255–264.

Carman, M. R., Grunden, D. W. and Ewart, D. 2014. Coldwater reattachment of colonial tunicate Didemnum vexillum fragments to natural (eelgrass) and artificial (plastic) substrates in New England. Aquatic Invasions 9: 105-110.

Crean, A. J., Dwyer, J. M. and Marshall, D. J. 2013. Adaptive paternal effects? Experimental evidence that the paternal environment affects offspring performance. Ecology 94: 2575-2582.

Daponte, M. C., Palmieri, M. A., Casareto, B. E. and Esnal, G. B. 2013. Reproduction and population structure of tlhe salp Iasis zonaria (Pallas, 1774) in the southwestern Atlantic Ocean  (34°30' to 39°30'S)  during three successive winters (1991-2001). J. Plankton Res. 35: 813-830.

de Barros, C. M., Emrich, L. C., Mello Ade, A., da Fonseca, R. N. and Allodi, S. 2014. Regulation of nitric-oxide production in hemocytes of the ascidian Phallusia nigra. Nitric Oxide: Biology and Chemistry 38: 26-36.

Delibaltov, D. L., Ghosh, P., Rodoplu, V., Veeman, M., Smith, W. and Manjunath, B. S. 2014. A linear program formulation for the segmentation of Ciona membrane volumes. Med. Image Comput. Comput. Assist. Interv. 16: 444-451.

Dishaw, L. J., Flores-Torres, J., Lax, S., Gemayel, K., Leigh, B., Melillo, D., Mueller, M. G., Natale, L., Zucchetti, I., De Santis, R., Pinto, M. R., Litman, G. W. and Gilbert, J. A. 2014. The gut of geographically disparate Ciona intestinalis harbors a core microbiota. PLoS ONE 9: 1-8.

Erwin, P., Pineda, M. C., Webster, N., Turon, X. and López-Legentil, S. 2014. Down under the tunic: Bacterial biodiversity hotspots and widespread ammonia-oxidizing Archaea in Great Barrier Reef ascidians. ISME J. 8: 575–588.

Fletcher, L. M., Forrest, B. M., Atalah, J. and Bell, J. J. 2013. Reproductive seasonality of the invasive ascidian Didemnum vexillum in New Zealand and implications for shellfish aquaculture. Aquaculture Environment Interactions 3: 197–211.

Fletcher, L. M., Forrest, B. M. and Bell, J. J. 2013. Impacts of the invasive ascidian Didemnum vexillum on green-lipped mussel Perna canaliculus aquaculture in New Zealand. Aquaculture Environment Interactions 4: 17-30.

Forrest, B. M., Fletcher, L. M., Atalah, J., Piola, R. F. and Hopkins, G. A. 2013. Predation limits spread of Didemnum vexillum into natural habitats from refuges on anthropogenic structures. PLoS One 8: e82229.

Forrest, B. M. and Hopkins, G. A. 2013. Population control to mitigate the spread of marine pests: insights from management of the Asian kelp Undaria pinnatifida and colonial ascidian Didemnum vexillum. Management of Biol. Invasions 4: 317-326.

Franchi, N., Piccinni, E., Ferro, D., Basso, G., Spolaore, B., Santovito, G. and Ballarin, L. 2014. Characterization and transcription studies of a phytochelatin synthasegene from the solitary tunicate Ciona intestinalis exposed to cadmium. Aquatic Toxicol. 152: 47–56.

Frank, P., Hedman, B. and Hodgson, K. O. 2014. XAS spectroscopy, sulfur, and the brew within blood cells from Ascidia ceratodes. J. Inorg. Biochem. 131: 99–108.

Freestone, A. L., Osman, R. W., Ruiz, G. M. and Torchin, M. E. 2011. Stronger predation in the tropics shapes species richness patterns in marine communities. Ecology 92: 983-993.

Frey, M. A., Simard, N., Robichaud, D. D., Martin, J. L. and Therriault, T. W. 2014. Fouling around: vessel sea-chests as a vector for the introduction and spread of aquatic invasive species. Manag. Biolog. Invasions 5: 1-10.

Gewing, M.-T., Rothman, S. B. S., Nagar, L. R. and Shenkar, N. 2014. Early stages of establishment of the non-indigenous ascidian Herdmania momus (Savigny, 1816) in shallow and deep water environments on natural substrates in the Mediterranean Sea. BioInvasions Records 3: 1-5.

Griggio, F., Voskoboynik, A., Iannelli, F., Justy, F., Tilak, M.-K., Turon, X., Pesole, G., Douzery, E. J. P., Mastrototaro, F. and Gissi, C. 2014. Ascidian mitogenomics: comparison of evolutionary rates in closely related taxa provides evidence of ongoing speciation events. Genome Biol. & Evol. 6: 591–605.

Hirose, E. 2014. Photosymbiotic ascidians (Ascidiacea: Didemnidae) from Minami-Daitojima Island. Biol. Mag. Okinawa 52: 31-38.

Hirose, E. 2014. Ascidian photosymbiosis: diversity of cyanobacterial transmission during embryogenesis. Genesis 52: 1-11.

Hirose, E., Nozawa, Y. and Hirose, M. 2014. First record of a photosymbiotic ascidian Lissoclinum midui (Ascidiacea: Didemnidae) from Lyudao (Green Island), Taiwan. Mar. Biodiversity Rec. 7: 1-5.

Holland, L. Z. 2014. Genomics, evolution and development of amphioxus and tunicates: The goldilocks principle. J. Exp. Zool. B Mol. Dev. Evol. 16: 10844-10853.

Ikeda, T., Matsuoka, T. and Satou, Y. 2013. A time delay gene circuit is required for palp formation in the ascidian embryo. Development 140: 4703-4708.

Imperatore, C., D'Aniello, F., Aiello, A., Fiorucci, S., D'Amore, C., Sepe, V. and Menna, M. 2014. Phallusiasterols A and B: two new sulfated sterols from the Mediterranean tunicate Phallusia fumigata and their effects as modulators of the PXR receptor. Mar. Drugs 12: 2066-2078.

Ishii, H., Shirai, T., Makino, C. and Nishikata, T. 2014. Mitochondrial inhibitor sodium azide inhibits the reorganization of mitochondria-rich cytoplasm and the establishment of the anteroposterior axis in ascidian embryo. Dev. Growth & Differ. 56: 175-188.

Khandelwal, M. and Windle, A. H. 2014. Small angle X-ray study of cellulose macromolecules produced by tunicates and bacteria. Int. J. Biol. Macromol. 68: 215-217.

Kim, Y. O., Park, S., Nam, B. H., Jung, Y. T., Kim, D. G. and Yoon, J. H. 2014. Ruegeria meonggei sp. nov., an alphaproteobacterium isolated from ascidian Halocynthia roretzi. Antonie Van Leeuwenhoek 105: 551-558.

Kumano, G., Negoro, N. and Nishida, H. 2014. Transcription factor Tbx6 plays a central role in fate determination between mesenchyme and muscle in embryos of the ascidian, Halocynthia roretzi. Dev. Growth & Differ. 56: 310-322.

Kumaran, N. S., Bragadeeswaran, S. and Meenakshi, V. K. 2014. Mitochondrial cytochrome oxidase I (COI) DNA sequencing of the ascidians Didemnum granulatum (JQ013198) and D. psammathodes (JN624758). Mitochondrial DNA 25: 131-134.

Kuwajima, M., Kumano, G. and Nishida, H. 2014. Regulation of the number of cell division rounds by tissue-specific transcription factors and Cdk inhibitor during ascidian embryogenesis. PLoS One 9: e90188.

Kwan, J. C. and Schmidt, E. W. 2014. Bacterial endosymbiosis in a chordate host: long-term co-evolution and conservation of secondary metabolism. PLoS One 8: e80822.

Kwan, J. C., Tianero, M. D., Donia, M. S., Wyche, T. P., Bugni, T. S. and Schmidt, E. W. 2014. Host control of symbiont natural product chemistry in cryptic populations of the tunicate Lissoclinum patella. PLoS One 9: e95850.

Lambert , C. C. 2014. Obtaining gametes and embryos of ascidians. In: Carroll, D. J. and Stricker, S. A. (ed.), Developmental Biology of the Sea Urchin and Other Marine Invertebrates. Humana Press, pp. 27-33.

Lee, C. M., Kafle, K., Park, Y. B. and Kim, S. H. 2014. Probing crystal structure and mesoscale assembly of cellulose microfibrils in plant cell walls, tunicate tests, and bacterial films using vibrational Sum Frequency Generation (SFG) spectroscopy. Phys. Chem. Chem. Phys. 16: 10844-10853.

Liberti, A., Melillo, D., Zucchetti, I., Natale, L., Dishaw, L. J., Litman, G. W., De Santis, R. and Pinto, M. R. 2014. Expression of Ciona intestinalis variable region-containing chitin-binding proteins during development of the gastrointestinal tract and their role in host-microbe interactions. PLoS ONE 9: e94984.

Manni, L., Gasparini, F., Hotta, K., Ishizuka, K. J. and Ricci, L. 2014. Ontology for the asexual development and anatomy of the colonial chordate Botryllus schlosseri. PLoS ONE 9: 1-15.

Matozzo, V., Franchi, N. and Ballarin, L. 2014. In vitro effects of the nonsteroidal anti-inflammatory drug, ibuprofen, on the immune parameters of the colonial ascidian Botryllus schlosseri. Toxicol. in Vitro 28: 778–783.

McCann, L. D., Holzer, K. K., Davidson, I. C., Ashton, G. V., Chapman, M. D. and Ruiz, G. M. 2013. Promoting invasive species control and eradication in the sea: options for managing the tunicate invader Didemnum vexillum in Sitka, Alaska. Mar. Pollution Bull. 77: 165-171.

McDougall, A., Lee, K. W. and Dumollard, R. 2014. Microinjection and 4D fluorescence imaging in the eggs and embryos of the ascidian Phallusia mammillata. Methods Mol. Biol. 1128: 175-185.

McLaughlin, J., Bourque, D., LeBlanc, A. R. and Fortin, G. 2013. Effect of suspended inorganic matter on fertilization success, embryonic development, larval settlement, and juvenile survival of the vase tunicate Ciona intestinalis (Linnaeus, 1767). Aquatic Invasions 8: 375-388.

Mecho, A., Aguzzi, J., Company, J. B., Canals, M., Lastras, G. and Turon, X. 2014. First in situ observations of the deep-sea carnivorous ascidian Dicopia antirrhinum Monniot C.,1972 in the Western Mediterranean Sea. Deep Sea Res. 1 83: 51–56.

Menna, M. 2014. Important classes of bioactive alkaloids from marine ascidians: structures, isolation and bioactivity. Curr. Topics in Med. Chem. 14: 207-223.

Murcia, C., Coello, L., Fernandez, R., Martin, M. J., Reyes, F., Francesch, A., Munt, S. and Cuevas, C. 2014. Tanjungides A and B: new antitumoral bromoindole derived compounds from Diazona cf formosa. isolation and total synthesis. Mar. Drugs 12: 1116-1130.

Nishikawa, T., Oohara, I., Saitoh, K., Shigenobu, Y., Hasegawa, N., Kanamori, M., Baba, K., Turon, X. and Bishop, J. D. D. 2014. Molecular and morphological discrimination between an invasive ascidian, Ascidiella aspersa, and Its congener A. scabra (Urochordata: Ascidiacea). Zool. Sci. 31: 180–185.

Oliveira, L. M., Gamba, G. A. and Rocha, R. M. 2014. Eudistoma (Ascidiacea: Polycitoridae) from tropical Brazil. Zoologia 31: 195-208.

Ooishi, S. 2014. Three species of Enterocola van Beneden (Copepoda: Cyclopoida: Ascidicolidae) living in compound ascidians from Madagascar, with a revision of Enterocola setiferus Hansen. Proc. Biol. Soc. Wash. 126: 343-368.

Oonuma, K., Hirose, D., Takatori, N. and Saiga, H. 2014. Continuous expression of Otx in the anterior neural lineage is supported by different transcriptional regulatory mechanisms during the development of Halocynthia roretzi. Dev. Growth & Differ. 56: 189-198.

Paetzold, S. C., Giberson, D. J., Hill, J., Davidson, J. D. P. and Davidson, J. 2012. Effect of colonial tunicate presence on Ciona intestinalis recruitment within a mussel farming environment. Management of Biol. Invasions 3: 15-23.

Park, K. H., Zeon, S. R., Lee, J. G., Choi, S. H., Shin, Y. K. and Park, K. I. 2014. In vitro and in vivo efficacy of drugs against the protozoan parasite Azumiobodo hoyamushi that causes soft tunic syndrome in the edible ascidian Halocynthia roretzi (Drasche). J. Fish Diseases 37: 309-317.

Patanasatienkul, T., Revie, C. W., Davidson, J. and Sanchez, J. 2014. Mathematical model describing the population dynamics of Ciona intestinalis, a biofouling tunicate on mussel farms in Prince Edward Island, Canada. Manag. Biolog. Invasions 5: 39–54.

Pezzotti, M. R., Locascio, A., Racioppi, C., Fucci, L. and Branno, M. 2014. Auto and cross regulatory elements control Onecut expression in the ascidian nervous system. Dev. Biol. 390: 273-287.

Reeves, W., Thayer, R. and Veeman, M. 2014. Anterior-posterior regionalized gene expression in the Ciona notochord. Dev. Dyn. 243: 612-620.

Reinhardt, J. F., Gallagher, K. L., Stefaniak, L. M., Nolan, R., Shaw, M. T. and Whitlatch, R. B. 2012. Material properties of Didemnum vexillum and prediction of tendril fragmentation. Mar. Biol. 159: 2875-2884.

Reinhardt, J. F. and Hudson, D. M. 2012. A review of the life history, invasion process, and potential management of Clavelina lepadiformis Müller, 1776: a recent invasion of the northwest Atlantic. Management of Biol. Invasions 3: 1-13.

Renborg, E., Johannesson, K. and Havenhand, J. 2014. Variable salinity tolerance in ascidian larvae is primarily a plastic response to the parental environment. Evol. Ecol. 28: 561-572.

Rinkevich, B. and Fidler, A. E. 2014. Initiating laboratory culturing of the invasive ascidian Didemnum vexillum. Manag. Biolog. Invasions 5: 55-62.

Rius, M., Clusella-Trullas, S., McQuaid, C. D., Navarro, R. A., Griffiths, C. L., Matthee, C. A., von der Heyden, S. and Turon, X. 2014. Range expansions across ecoregions: interactions of climate change, physiology and genetic diversity. Global Ecol. Biogeogr. 23: 76–88.

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