Number 44                                                                                                                        December 1998

    After retiring at the end of spring semester, after 28 years at Calif. State Univ. Fullerton, we moved to Seattle, Washington in June. In September we joined a group of about 14 invertebrate specialists on an 8 day expedition to survey a large number of marinas in Puget Sound, the San Juan Islands, and north to the Canadian border, to document  recent introductions of non-indigenous marine invertebrates to the U.S. Pacific Northwest coast.  There are a large number of new records for this state; see the WORK IN PROGRESS section for details of the ascidian results and information on how to receive a copy of the full report. Gretchen  received a small grant from the Bishop Museum in Honolulu to identify their ascidian collection, so we spent the month of November in Honolulu. Charles investigated aspects of fertilization in Phallusia nigra at the Univ. of Hawaii Kewalo Marine Biol. Lab. We put on three ascidian workshops this year, the first in May at CSUFullerton, the second in October at the  Paulsbo marine station near Seattle, and the third at the Kewalo lab in Honolulu in November. All were focussed on helping people identify the local species, introduced as well as indigenous, in each of these localities, with many live and preserved specimens to examine. Gretchen was also invited to the Univ. of Guam to help them identify their local ascidians, with another modest grant to defray expenses, so in February we will spend several weeks there and Charles will continue his research on ascidian fertilization.
    We are pleased to report that there are 84 citations in the New Publications section. Keep in touch, and send us an article for Work in Progress, abstracts from recent meetings attended, or your students' thesis abstracts on ascidian topics.  Please send us a copy of your new publications to our Seattle address. This will assure that they are included in the Recent Publications section at the end of each AN.  We use our ascidian reprints every day and they are very valuable to us.
 
 *Ascidian News is not part of the scientific literature and should not be cited as such.

1. Sea squirt die off - Korea: Request for information:
Albert Kwang-Sik Choi, Dept. of Aquaculture, Cheju National University, 1 Ara 1 Dong, Cheju City, Cheju, 690-756 Korea  (skchoi@cheju.cheju.ac.kr) & Jeffrey Shields (jeff@vims.edu)  http://shellfish.cheju.ac.kr
    The sea squirt Halocynthia roretzi is commercially cultured in the southern coast of Korea. Due to an unidentified disease, large numbers of sea squirts in the farming areas died this summer and the industry is now in bad shape. Water temperature during last summer was abnormally high due to the El Nino. The dying sea squirts usually exhibited thinning of the skin and the meat become quite soft and dissolving. We do not know the agents involved in this disease and if anybody has any information on Sea Squirt disease, please help us.

2. While searching for new references for this newsletter, we found an extremely useful website maintained by the University of Washington library in Seattle. http://www.lib.washington.edu/   While not all databases are accessible to the public, many are, such as CarlUncover, in which you can search for papers using keywords as well as author or journal.

3. Our colleague Alan Kohn recently brought to our attention a long-forgotten poem which we reproduce here from The Poetical Works of Andrew Lang (1923) by Mrs. Lang. vol. 3, pp. 133-135.

1. The following is taken, with minor changes, from a recently prepared document, Report of the Puget Sound Expedition September 8-16, 1998: A Rapid Assessment Survey of Non-indigenous Species in the Shallow Waters of Puget Sound. Washington State Dept. of Natural Resources, Olympia, WA and U.S. Fish and Wildlife Service, Lacey, WA. For a copy of the complete report please contact Helen Berry at the Washington State DNR.  helen.berry@wadnr.gov

Ascidians in Puget Sound and the San Juan Islands, September 1998. Gretchen Lambert, Charles Lambert and Claudia Mills.
    Ascidians are prominent members of the fouling community and are often present in very high densities on marina floats and boat hulls.  We found that, after mussels and barnacles, they comprised perhaps the most important group of dock inhabitants in Puget Sound. Boat harbors were surveyed from a northern extreme of Blaine at the Canadian border to a southernmost point near Olympia on the Washington mainland, as well as sites on Fidalgo and Whidbey Islands, and San Juan,Orcas and Lopez Islands in the San Juan Islands.  Sampling was accomplished by reaching under floating docks to remove adherent ascidians and by raising ropes, hoses and wires to examine attached organisms.  Salinity and temperature were measured at each collection site.
    Summarizing our findings from north to south, the harbor at Blaine contained the native species Distaplia occidentalis and Corella inflata along with the introduced Botrylloides violaceus and Styela clava.  This is the first report of S. clava in American waters north of San Francisco Bay in the NE Pacific, although we observed a population on Vancouver Island  several years ago that is still flourishing. Bellingham marina with reduced salinity had only a few colonies of Botrylloides violaceous.  The Edmonds marina contained numerous colonies of Botrylloides violaceus as well as the natives Distaplia occidentalis, Corella inflata, Styela gibbsii and Diplosoma listerianum. Similar ascidians were encountered in the Elliott Bay Marina.  At the Des Moines Marina several native species were common along with the non-natives Botryllus schlosseri and Botrylloides violaceus, but the floats were dominated by huge numbers of Ciona savignyi, a species introduced into California in 1985 but never before noted north of San Francisco. In Tacoma we found numerous Botryllus schlosseri and a few native S. gibbsii and Distaplia occidentalis.  At the Shelton Yacht Club both B. violaceus and B. schlosseri were very numerous as was a third introduced ascidian Molgula manhattensis, together covering nearly all submerged objects. At the Brownsville Yacht Club we found a number of native species along with B. violaceus and a few Ciona savignyi.  Port Townsend marina had all the previously mentioned native species and also the native Perophora annectens and Ascidia callosa.  In addition B. violaceus was present in large numbers.
     Ascidians are thus an important component of nearly every float-fouling community in Puget Sound. Non-indigenous species comprise a substantial part of this ascidian load, in places forming the dominant biota.  At the Des Moines Marina (south of the Seattle-Tacoma airport), Ciona savignyi occupied an estimated 90% of the concrete float surface area under covered (shaded) docks, although it was much less abundant in uncovered areas and limited to float bottoms. At many sites, the non-indigenous B. violaceus accounted for at least 10-20% of the float cover, often overgrowing other organisms.
    We further examined 7 boat harbors on San Juan Island and 6 on Orcas Island and found only the native species in most harbors but B. violaceus was in Roche Harbor, Mitchell Bay and Westcott Bay on San Juan Island, in most harbors on Orcas Island, and at Fisherman's Bay on Lopez Island.  Ciona savignyi, Molgula manhattensis and Styela clava seem to be absent from the San Juan Archipelago at this time.
    There is thus an indication of a north-south gradient in the number of introduced ascidian species on the U.S. Pacific coast, with 5 recorded so far for Puget Sound, 8 in San Francisco Bay and 14 in southern California [see Lambert and Lambert 1998, Mar. Biol. 130:675-688].

2. Sulfur allocation and vanadium-sulfate interactions in whole blood cells from the tunicate Ascidia ceratodes, investigated using X-ray absorption spectroscopy. P. Frank, B. Hedman, and K.O. Hodgson. Dept. Chem, Stanford Univ., Stanford, CA 94305.  In press,  Inorganic Chemistry (1998). frank@SSRL01.slac.stanford.edu
    Sulfur K-edge x-ray absorption spectroscopy (XAS) has been used to investigate the distribution of sulfur types in two whole blood cell samples, in selected subcellular blood fractions, and in cell-free plasma from the tunicate Ascidia ceratodes. Whole blood cells are rich in sulfate, aliphatic sulfonate, and low-valent sulfur. The sulfur K-edge XAS spectrum of washed blood cell membranes revealed traces of sulfate and low-valent sulfur, but no sulfate ester or sulfonate.  Sulfonate is thus exclusively cytosolic. Cell-free blood plasma contains primarily sulfate sulfur. Gaussian fitting and sulfate quantitation yielded average sulfur type concentrations for two whole blood cell samples, each representing dozens of individuals from separate collections taken per annum, yielded average sulfur type concentrations for the two populations (first year, second year): [sulfate], 110 mM, 150 mM; [sulfonate], 99 mM, 70 mM, and; [low-valent sulfur], 41 mM, 220 mM. On titration of sulfate with acid or V(III) in aqueous solution the strong 2482.4 eV 1s-->(valence t2) sulfur K-edge XAS transition of tetrahedral SO4(2-) splits into 1s-->a1 and 1s-->e transitions because HSO4(-) and VSO4(+) are of C3v symmetry. Gaussian fits and appropriate comparisons allow the following assignments: (compound/complex, 1s-->a1(eV), 1s-->e (eV)): myo-inositol hexasulfate, 2480.8, 2482.8; HSO4(-), 2481.4, 2482.7; VSO4(+), 2481.2, 2482.9.  The energy separating the a1 and e states of complexed sulfate appears to be solvation dependent. From these studies an explicit inorganic spectrochemical model is derived for biological V(III) and sulfate. The average endogenous equilomer concentrations of sulfate complexed with V(III) and/or acid within the two blood cell samples are calculated from the model. The results provide a natural explanation for the observed biological broadening of A. ceratodes blood cell sulfur K-edge XAS spectra.

3.  Phenoloxidase and cytotoxicity in the compound ascidian Botryllus schlosseri. L. Ballarin, F. Cima & A. Sabbadin, Dipartimento di Biologia, Univ. di Padova. In press, Dev. Comp. Immunol., 22, 1998. ballarin@civ.bio.unipd.it
     The vacuoles of morula cells (MC) of the colonial ascidian Botryllus schlosseri contain phenoloxidase (PO). As the release of their vacuolar content at the border of incompatible contacting colonies is associated with the formation of necrotic masses which characterize the rejection reaction, the role of PO in Botryllus cytotoxicity was investigated.  When hemocytes are incubated with blood plasma from incompatible (heterologous) colonies, MC degranulate and, after 60 min, the cytotoxicity index becomes significantly greater than that observed in controls incubated with autologous plasma. The rise in cell mortality is completely inhibited by the addition of PO inhibitors sodium benzoate, tropolone and phenylthiourea, and serine protease inhibitors phenylmethylsulfonyl fluoride, benzamidine, N-tosyl-L-phenylalanine chloromethyl ketone and N-tosyl-L-lysine chloromethyl ketone. The addition of either reducing agents L-cysteine and ascorbic acid or reactive oxygen species scavenger enzymes superoxide dismutase and catalase has a similar effect. Significant inhibition of cytotoxicity is also observed with the quinone scavenger 3-methyl-2-benzothiazolinone hydrazone. In the presence of sodium benzoate and phenylthiourea, there is a significant reduction in the number, size and color intensity of necrotic masses along the contact border of incompatible colonies. A significant increase in superoxide anion production, completely inhibited by Na-benzoate, is observed when hemocytes are incubated with heterologous blood plasma. These results indicate that: i) PO is the enzyme responsible for the cytotoxicity observed in both hemocyte cultures and rejection reactions; ii) PO is present inside MC vacuoles as a proenzyme which is activated, upon release, by humoral proteases; iii) cytotoxicity appears to be mainly due to oxidative stress generated by PO during oxidation of polyphenols to quinones without the involvement of other oxidases such as NADPH oxidase and peroxidase.
 

1. Intl. Conference on Environmental and Biological Aspects of Main group Organometals. Helnaes, Denmark, June 28 - July 1, 1998.

TBT-INDUCED APOPTOSIS IN TUNICATE HEMOCYTES. F. Cima1, P. Fonti2 and L. Ballarin1
1Dept. of Biology, University of Padova, Via U. Bassi 58/B, 35131 Padova     ballarin@civ.bio.unipd.it
2Centro Studi Ambientali, Via al Torrente 22, 47037 Rimini - Italy
     Early events in apoptosis include chromatin condensation followed by DNA fragmentation as well as translocation of phosphatidylserine (PS) in the plasma membrane. Organotin compounds increase intracellular Ca2+ levels and cause apoptosis in mammalian cells. In previous reports we demonstrated the immunotoxicity of TBT in the ascidian Botryllus schlosseri, a filter-feeding marine invertebrate, as it inhibits phagocytosis and respiratory burst. The effect is mediated by an extensive cytosolic calcium increase resulting in volume loss and morphological changes related to cytoskeletal alterations. In order to investigate whether TBT can also induce apoptosis in ascidian hemocytes we exposed Botryllus blood cells to 10 µM TBT - the dose which significantly changes cytoskeletal structure and does not cause mortality - for 1, 2, 4 h. After 1 h we observed a significant collapse and cleavage of nuclear chromatin following a post-treatment with acridine orange or Pfitzner’s safranin, also revealed with TUNEL reaction which is specific to oligonucleosome-sized DNA fragments. Moreover, cytoplasmic blebbing occurred together with surface alterations triggered by PS translocation from the inner side of the plasma membrane to the outer side as shown with annexin-fluorescein method. Hemocyte mortality, detected with trypan blue, took place only after 2 h. We suggest that all these apoptotic events are strictly related with a TBT-triggered cytosolic calcium increase resulting in inhibition of calmodulin and Ca2+-dependent enzyme activities, e.g. Ca2+-ATPase and endonucleases.

2. 8th Intl. Congress on Invertebrate Reproduction & Development, Amsterdam, 10-14 August 1998.

ASCIDIANS HAVE A RELAXIN HORMONE AND AN IGF-I RELATED PEPTIDE. D. Georges (Grenoble, France), M. Reinecke (Zürich, Switzerland).  DGeorges@europost.org
    Ascidians have been considered, for a long time, as very simple invertebrates. The first experiments demonstrated the role of the nervous complex (nervous ganglion and neural gland) in the regulation of spawning, during the tidal cycle. The main regulation is the same as in vertebrates with a negative feed-back control by the ovary, at the time of spawning (Georges,Gen.comp.Endocrinol.32, 454-473, 1977).  Immunofluorescence techniques allowed then to show that vertebrate-like peptides could be found in the nervous system, as well as in the digestive tract or the ovary, peptides related to ACTH, LHRH, methionine-enkephalin, serotonin, NGF, insulin, IGF-I and relaxin. Relaxin, a hormone from the insulin family, is known in vertebrates, specially in mammals as a hormone widening the birth canal. It has been localized only in the follicle cells of mature oocytes of various ascidians (Georges et al., Gen.comp.Endocrinol.79, 429-438, 1990).The amino acid sequence of relaxin studied in most classes of vertebrates shows a low level of homologies even between mammals themselves.
   For the first time, relaxin has been sequenced in an ascidian. Surprisingly, the nucleotide sequence of relaxin in Ciona intestinalis is about 98 % similar to that of porcine relaxin and is the same as whale relaxin. The danger of a contamination was eliminated as parallel experiments were running by C. Schwabe, in USA, who confirms the results with the amino-acid sequence. Moreover, control experiments using intestine tissue performed in the same time were negative. As results were the same in genomic and cDNA, it appears that : 1) there is no intron in the connecting peptide of the ascidian prorelaxin, 2) the exchanges between pig and ascidian relaxin concern a change in the first or second base in the codon.  IGF-I occurs in the endocrine cells of the digestive tract and in neurons of C. intestinalis (Reinecke et al., Histochemistry, 99, 277-285, 1993). Using gel chromatography and RIA, IGF-I immunoreactivity was detected in two peaks with apparent molecular weights of approximately 16 kDa and 3 kDa both in the gastrointestinal tract and in ovary. Results obtained by immunofluorescence techniques (antibodies raised in mammals against insulin, IGF-I and relaxin)
demonstrate that an IGF-I related peptide coexists 1) with an insulin-related peptide in the digestive tract and 2) with relaxin in the ovary, but there, only in the medium-sized and mature follicle cells. IGF-I exists alone in young oocytes.
   In another ascidian, Chelyosoma productum, McRory & Sherwood (DNA & Cell Biology, 16, 939-949, 1997) have sequenced two insulin and IGF genes : the preproinsulin has 87 % identity with the preproIGF, and the mature form of ascidian insulin shows 64% identity with the human insulin. All these results suggest that an ancestral insulin/IGF/relaxin
molecule precedes the ascidians.

3. 19th Conference of European Comparative Endocrinologists, Nimegen, 1-5 September 1998. 
Ascidians have a relaxin hormone. Danielle Georges (Grenoble, France). [similar to the above abstract]

4. 33rd EMBS meeting, Wilhelmshaven, Germany, September 1998.

ENERGY BUDGET OF TWO SPECIES OF BENTHIC ANTARCTIC SUSPENSION FEEDERS.  Jens Kowalke, Alfred Wegener Institute for Polar and Marine Research, Bremerhaven, Germany
    Although suspension feeders constitute the majority of many Antarctic benthic communities, little information about their biology or ecology is known. Complete energy budgets concerning growth, reproduction, egestion or respiration are still lacking but are essential for understanding ecosystems and interpreting communities over time and space. In this work energy budgets during austral summer of both the abundant ascidian Cnemidocarpa verrucosa  (Lesson) and the lamellibranch Laternula elliptica (King & Broderip) were established. Based on measured data of energy contents in the water column, filtration and egestion rates combined with respiration data taken from the literature, the energy available for growth and reproduction was calculated. The field work was carried out in Potter Cove, an inlet at King George Island, South Shetlands. This subsytem is characterized by a low annual primary production in the water column and enormous terrestrial sediment input due to the inflow of glacial meltwater during summer and autumn months as well as frequent disturbances through stranding icebergs and growlers. These physical effects  are the main structuring forces affecting the soft bottom communities found in the cove, which are, in contrast to less disturbed areas, dominated by solitary ascidians and bivalves. The summer months are characterized by a low water column primary production, resuspension being the major process of generating particles for suspension feeding animals. The main nutritional components are benthic diatoms, protists and bacteria. The overall energy budget is lower than described for species of
temperate and tropical zones, but the net growth efficiency, which is the energy disposable for growth and reproduction expressed in percentage of the energy assimilated, is at the upper margin of ranges measured for species of temperate regions. The values are 81% for C. verrucosa  and 79% for L. elliptica respectively (dry wt). Field data of L. elliptica for somatic and gonad production show a net growth efficiency of 42% on an annual basis. These findings reflect the
important gonad production of this species during the summer months, as L. elliptica spawns in the Antarctic autumn. During the extended winter period most of the ingested energy is, in contrast, used for respiration, thus reducing the yearly net growth efficiency to 42%. As field data for C. verrucosa are are not yet available, a validation of the calculated production values remains open.
 

1. ENERGY BUDGETS OF BENTHIC SUSPENSION FEEDING ANIMALS OF POTTER COVE (KING GEORGE ISLAND, ANTARCTICA). Reports on Polar Research 286, 1998; 147pp. Dr. Jens Kowalke, Alfred Wegener Institute for Polar and Marine Research, Bremerhaven, Germany.   jkowalke@awi-bremerhaven.de
    The objective of this work was the first comprehensive calculation of the energy budgets of dominating groups of suspension feeders in Potter Cove, Antarctica. The field work was carried out in the Dallmann Laboratory, King George
Island, during Antarctic summer field campaigns. 27 species of sponges were recorded in the area, 13 of which were newly described for the South Shetland Islands and the adjacent Antarctic peninsula. The 2 dominant species, Mycale acerata and Isodictya kerguelensis were choosen for further ecological studies. Of the 15 ascidian species already known, Ascidia challengeri, Cnemidocarpa verrucosa, Corella eumyota and Molgula pedunculata were investigated, as well as the bivalve Laternula elliptica. A quantification of the amount of energy present in the particulate fraction in the water column at 30m depth shows seasonal variation throughout the year, but no extreme limitation of energy supply during the winter months. This is due to resuspension of particulate matter from the seafloor, induced by currents and, during no-ice conditions, by wave action. Bacteria and protista seem to play an important role in the nutrition of these suspension feeders, especially in winter, when photosynthesis plays a minor part. It is inferred that at least in the coastal maritime Antarctic habitats, energy is provided sufficiently throughout the year. The pumping rates of all Antarctic species, reflecting reduced metabolism at low temperatures and higher viscosity of the seawater, ranged below those of animals of temperate zones. The bottom sediments receive a considerable amount of fecal material produced by the ascidians and bivalves and thus support a variety of coprophag deposit feeders. The energy budgets were calculated using respiration and production data from the literature. All species showed a surplus energy uptake which could be related to an underestimation of the biodeposition rates. The results do not display a very harsh environment for the animals in general but reveal different adaptations among the investigated taxa to the special physical conditions of the Potter Cove, which make the ascidians and L. elliptica dominant in contrast to other Antarctic sites, where sponges dominate the communities.

2.  Eudistoma carolinense VAN NAME, 1945 (TUNICATA: ASCIDIACEA) AS A BIOLOGICAL SUBSTRATE, AND ITS ASSOCIATED MACROFAUNA. Tatiane Regina Moreno Master's thesis. Advisor: Dr. Rosana Moreira da Rocha. Dept. de Zool., Univ.Federal do Parana, Centro Politicnico, Cx.Postal 19020, 1531-990 Curitiba/ PR, Brasil.  rmrocha@bio.ufpr.br
    Eudistoma carolinense is a colonial ascidian with vertical growth. The colony consists of groups of digitiform expansions arising from an expanded incrusting base, which forms a belt in the low intertidal. E. carolinense is a live substrate and its type of growth and localization permit settlement of many invertebrates. We studied the structural features of the colonies:  belt width, colony  thickness, density of expansions, and the volume and grain size of accumulated sediment, during one year. Fifteen samples were collected in February, (Summer), May (Fall), August (Winter) and November (Spring) of 1996. The abundance of the associated fauna was 2021 in Summer, 3077 in Fall, 2710 in Winter and 1872 in Spring. The following taxonomic groups were recorded: Porifera, Cnidaria, Plathyhelminthes, Nemertinea, Nematoda, Polychaeta, Sipuncula, Mollusca, Crustacea, Pycnogonida, Bryozoa, Echinodermata, and Chordata and 107 species were identified. From these, Nicolea venustula, Typosyllis hyalina, Pseudonereis gallapagensis, T. variegata, Elasmopus pectenicrus, Sphenia antillensis and Molgula phytophila were considered characteristic species, because these had the greatest general Biological Index Values. The volume of sediment, total number of individuals and associated species were all positively correlated with the ascidian volume and density of expansions. Colony thickness appears to be related to a decrease in diversity. A comparison of various biological substrates with different architectures with patterns in the associated fauna composition, abundance and diversity did not reveal any evident relationships. Other important substrate characteristics should be considered, such as: location of the substrate on the shore, presence of sediment or other inert materials, chemical composition of the surface and the possibility that E. carolinense eliminates alellopathic substances in the water.