Gretchen and Charles Lambert

12001 11th Ave. NW, Seattle, WA 98177

206-365-3734 or

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Number 56                                                                                                                               December 2004


We begin this issue with a worrisome, one might even say alarming, development. It has come to our attention that a new publication refers repeatedly to information in a recent issue of Ascidian News as if it were a publication, and even includes AN in the bibliography. Furthermore, some of the information is incorrect and not even what was in AN. We must remind you that, as stated on the first page of every issue, Ascidian News is not part of the scientific literature and should not be cited as such. Ascidian News has been a popular newsletter for over 30 years and this is the first time this has happened; we sincerely hope it will be the last. Newsletters are a wonderful venue for putting out new ideas and presenting new data prior to publication, but they will surely be stifled if information is inserted into published papers prior to official publication by the authors of that information without those authors’ knowledge and approval. Anyone wishing to include unpublished information in their papers of any kind from any source must contact the authors of that information and request permission; in the publication it must then be credited to that person and referred to as “personal communication”.

   During August we collected and identified ascidians on S. Padre Island, Texas for the Univ. of Texas Pan Am marine lab, and in Miami, Florida with the Smithsonian Invasions lab. In September we participated in an invasive species survey of southern England harbors along the English Channel, organized by Peter Dyrynda. We sampled 12 sites from Brighton to Plymouth. In Nov. we attended the 4th Intl. Symp. on the Molecular and Cell Biol. of Egg-and Embryo-Coats organized by Hitoshi Sawada in Ise-Shima, Japan. Ascidian abstracts from this meeting are contained below.

   Please save the dates of two exciting ascidian symposia in 2005, one in April and the other in July.  See below for more information, including websites for registration and abstract submission.  There are 6 thesis abstracts in this issue; we congratulate these and the many other young researchers on ascidians. At the end of this issue are listed 91 new publications on ascidians. Please send us reprints (preferably, for our permanent collection) or PDFs to assure inclusion of your papers in AN.


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




1. INTERNATIONAL INVASIVE SEA SQUIRT CONFERENCE April 21-22, 2005, Woods Hole Oceanographic Institution (WHOI), Woods Hole, Massachusetts.  Conference organizer/contact person: Mary Carman, Geology & Geophysics, WHOI, Woods Hole, MA 02543. Phone 508-289-2987; email   Abstract deadline 1 February 2005. Registration deadline April 1, 2005.  Conference proceedings will be published in J. Exp. Mar. Biol. and Ecol. (JEMBE).

   Invasive ascidians are impacting ecosystems, creating a nuisance for the aquaculture industry, and are a major component of fouling communities. The aim of this conference is to bring together marine biologists and other people concerned with invasive ascidians, to explore the biology, ecology, impacts, management options for control, and other relevant topics. The format of the two day conference will include invited plenary talks, contributed research presentations, posters, and discussions led by an expert panel. The conference target audience will be marine biologists, shellfishery scientists, representative of the shellfishery industry, members of local, state, and federal agencies concerned with coastal resources, and representatives from sponsoring organizations. Contributed posters will be displayed throughout the conference in Clark Laboratory, room 509. Poster abstracts will be included in the published proceedings.
2.  Mark your calendars! and send in your application for the Third International Urochordate meeting, July 9-13, 2005 at Univ. of California Santa Barbara. Organized by Bill Smith (UCSB) and Billie Swalla (Univ. of Washington Biol. Dept.) .  Go to the website for registration:

****Important: attendees from outside of the U.S.***  We really want to welcome all foreign visitors, but you may need to get a visa to attend the meetings. Check here to see if you are exempt:  Also, if you do not have a machine-readable passport you will need a visa. It is important to start the visa process early. Wait times for visas can be found here:


Preliminary Program

Saturday, July 9   Check-in with dinner as the first meal

Sunday July 10

Morning Session I: Urochordate genomes and genome evolution

Morning Session II: Genome-scale approaches and bioinformatics

Afternoon Session I: Oogenesis, gamete interactions and fertilization

Afternoon Session II: Ascidian development: germ layer formation and morphogenesis


Monday, July 11

Morning Session I: Ascidian development: neural development and function; specification and development of germ plasm

Morning Session II: Ecology; Natural products

Afternoon - Excursions

Evening: Round table discussion: ­ future collaborations, bioinformatic tools, NIH white papers for new species?


Tuesday, July 12

Morning Session I: Development and evolution of pelagic tunicates: Appendicularians and Thaliaceans

Morning Session II: Development and metamorphosis of ascidians

Afternoon Session I: Ascidian population biology and systematics

Afternoon Session II: Ascidian innate immunity


Wednesday, July 13

Morning Session I: Genetic networks in ascidian development

Morning Session II: Ascidian development and evolution





1. Special Issue of Canadian Journal of Zoology on the Protochordata will appear in 2005, with the following reviews:

Environmental factors affecting reproduction and development in ascidians and other protochordates.  W.R. Bates

The morphology, behavior, and biomechanics of swimming in ascidian larvae.  M.J. McHenry

Historical introduction, overview and reproductive biology of the protochordates.  Charles C. Lambert

Rejection patterns in botryllid ascidian immunity: The first tier of allorecognition.  Baruch Rinkevich

Amphioxus molecular biology: insights into vertebrate evolution and developmental mechanisms.  Sebastian M. Shimeld and Nicholas D Holland

Using ascidian embryos to study the evolution of developmental gene regulatory networks.  Robert Zeller, and A.C. Cone

Protochordate body plan and the evolutionary role of larvae: old controversies resolved? T.C. Lacalli

Ecology and natural history of the protochordates.  G. Lambert

The nervous system of amphioxus: Structure, development, and evolutionary significance.  H. Wicht, and T.C. Lacalli

Eutely, cell lineage and fate within the ascidian larval nervous system: determinacy or to be determined?   I.A. Meinertzhagen

Key characters uniting hemichordates and chordates:  Homologies or homoplasies?  E. Ruppert

The nervous system in adult tunicates: current research directions.  G.O. Mackie, and P. Burighel

Endocrinology of Protochordates.  N.M. Sherwood, B.A. Adams, and J.A. Tello

Morphological phylogeny of the hemichordates suggests an enteropneust-like protodeuterostome.  Christopher B. Cameron

Molecular phylogeny of the protochordates: Chordate evolution.  Billie J. Swalla


2. From Patrick Frank, Dept. of Chemistry, Stanford Univ. (

In work done with Bob and Elaine Carlson, and Keith Hodgson, we have been following the fate of vanadyl ion [V(IV)] taken up in vitro by whole blood cells of Ascidia ceratodes. We used EPR and x-ray absorption spectroscopies to track the vanadium in the intact cells.  It appears that vanadyl ion is actively partitioned among at least three quite dissimilar intracellular environments. One of them seems to be a monomeric complex that is either a protein site or an organic sequestering agent. It seems most likely that all three environments are within the signet ring cell. There also seem to be shifts in the environment of trivalent vanadium following uptake of [V(IV)] consistent with increased vacuolar acidity. Hemocytic vanadium is clearly under very active control, with blood cells rapidly accommodating opportunistic vanadyl ion.


3. From Gretchen Lambert: The nonindigenous Didemnum that is abundant on both coasts of the U.S., in New England on the Atlantic side and in northern California on the Pacific side, has now appeared in several locations in the Pacific Northwest region of the U.S., in Puget Sound. In addition, new surveys by the U.S. Geological Survey, using remote cameras, show that it now covers a much larger area of the Georges Bank than previously documented: 50-90% cover over a 40 square mile region. The following website contains many photos and updated distribution locations: 


4. Update on ANISEED (Ascidian Network for In Situ Expression and Embryological Data).

    Communicated by Olivier Tassy, Vanessa Fox, David Salgado, Kaz Makabe*, and Patrick Lemaire

IBDM, Campus de Luminy, Case 907, F-13288 Marseille France; *Dept. Integrated Arts & Sci., Tokushima Univ., Tokushima, Japan.  (

    The Aniseed system is a community resource for ascidian developmental studies. It is composed of a relational SQL database hosting anatomical, embryological, molecular and expression data and which was publicly released at the beginning of 2004 ( It is developed and kept in Marseille, and hosts data from several collaborating labs, foremost of them those of the Lemaire group and of the group of Kaz Makabe who communicated the Magest data (see below). In the past few months, we have been working on updating the content of the database, rendering the system generic (that is that it can be used for other model organisms), and developing new query tools.

   The database now includes all ESTs and cDNAs from Ciona intestinalis deposited into Genbank (691,000 sequences), 80% of which are put in relation to a JGI gene model. It also includes 60540 ESTs from Halocynthia roretzi (Magest Data communicated by K. Makabe). Clustering of these latter ESTs is underway, so as to generate tentative consensus sequences which will be submitted to the entry pipeline for functional annotation. This will allow identification and comparison of orthologous genes in both species. Expression data includes 500 in situ expression patterns from Ciona as well as 5963 expression patterns from Halocynthia. New query tools to be made available within the next few weeks include: Differential Digital Display, search for neighboring cells in the embryo, search for progenitors or progeny of a given cell, and a tool to combine tools sequentially. This will allow, for example, to search for genes with a given GO function expressed in the neighbors of a given cell. Watch the Aniseed site for further developments!




1. A comparative study of self-fertilization in the life histories of three ascidian species with contrasting dispersal patterns. Aimee L. Phillippi, Univ. of Maine. Ph.D. thesis; advisor Dr. Philip O. Yund.

   A survey of the published data on self-fertilization in animals reveals that self-fertility is widely distributed among invertebrate species.  However, sufficient data to determine the complete role of self-fertilization is lacking for many self-compatible species.  For the more extensively studied species, patterns were identified that suggest an association between dispersal distance and self-fertilization.  Although most discussion of self-fertilization concerns the detrimental effects (i.e., inbreeding depression) it can have for individuals and populations, theory and data suggest it can provide positive consequences under some circumstances.  For example, self-fertilization provides reproductive success when other mates are unavailable or it can maintain gene combinations that have already proven successful.  Individuals of species with limited dispersal potential which cannot move to locate mates and where progeny exist in the same environment as parents, may be more likely to benefit from self-fertilization than others.  To investigate the potential for a correlation between dispersal and self-fertility, the ability to self-fertilize, the likelihood of self-fertilizing under natural conditions, and the consequences of self-fertilization were examined in three ascidian species with contrasting dispersal patterns.  Ascidians are sessile as adults, making dispersal the result of gamete and larval movement, or occasional events of rafting.  Molgula provisionalis, a direct developer exhibiting the most limited dispersal potential, readily self-fertilized even in water of moderate flow velocity and did not suffer detectable inbreeding depression.  The results suggest that M. provisionalis utilizes self-fertilization as a regular component of its reproductive strategy.  Although Botryllus schlosseri has limited larval dispersal, it may realize greater gene flow distances from long-distance fertilizations by long-lived sperm.  Colonies of this species exhibited high levels of self-fertilization when self-sperm were available; however, the life history of B. schlosseri effectively prevents self-fertilization under natural conditions.  Self-fertilized progeny of B. schlosseri suffered severe inbreeding depression, limiting the role self-fertilization could have in its reproductive biology.  Ciona intestinalis, the most dispersive species, exhibited intra-specific variation in self-fertility with approximately 16% of individuals being self-compatible.  Self-compatible individuals were unable to self-fertilize in limited water flow (~ 1 cm s-1), and self-fertilized C. intestinalis progeny exhibited reduced fitness at all life stages examined.  Because intra-specific variability exists for self-fertility in C. intestinalis, potential genetic and environmental influences on the trait of self-fertility were also investigated.  Results from these three study species are discussed and compared to dispersal and self-fertility data from other species. 


2. Community structure and genetic variability of colonial ascidians of the rocky intertidal zone. Gustavo Muniz Dias, M.S. thesis, Depto. de Zoologia, sala 70, UNICAMP, Brazil. (

   The diversity, seasonality, spatial distribution and microhabitat occupation by colonial ascidians on a rocky intertidal zone were studied at Praia da Baleia and Praia Grande, both in the São Sebastião district, on the northern coast of São Paulo State. Samples were collected, monthly from January 2001 to January 2003 by raffling 1m2 quadrats. Thirty-three species of colonial ascidians were identified, with the family Didemnidae being the most represented (19 species, of which two were previously unknown). Praia Grande had a greater richness and diversity than Praia da Baleia, probably because of differences in the hydrodynamic processes at the two beaches. Richness and dominance varied among the three areas of the intertidal zone (low, middle and high), with species zonation occurring only at Praia Grande. Variation in these parameters during the year showed that this taxocoene was more diversified and richer during the warmest months. The undersurface of rocks was the microhabitat most used by these organisms, probably because of the photophobic behavior of the larvae. The genetic variability of the colonial ascidian Symplegma rubra found on the coasts of the States of São Paulo and Rio de Janeiro was assessed by allozyme analysis. Extensive genetic variability and a moderate structure (FST=0.051) were observed among populations separated by up to 363 km. These results differed from those for colonial ascidians in temperate areas. The maintenance of gene flow observed here reinforces the importance of alternative ways, such as rafting, for dispersal over great distances, particularly since colonial ascidians have lecithotrophic larvae that spend only a short time in plankton. Variations in the allele frequencies for the locus Pgi suggested that were selective pressures related to differences in seawater temperature among the studied areas. This finding reinforced the importance of temperature in determining the distribution of colonial ascidians.


3. Characterization of cortical polarity from maturation to the 8 cell stage in oocytes and embryos of three ascidian species (Ciona intestinalis, Halocynthia roretzi and Phallusia mammillata). François Prodon, Univ. of Nice-Sophia Antipolis (UNSA, France) Ph.D. thesis.  ( until 1/1/05; then

   The ascidian egg cortex is highly polarized along the animal-vegetal (a-v) axis at the end of oogenesis, and along the Dorso-Ventral (D-V) axis and Antero-Posterior (A-P) axis between fertilization and first cleavage. Mature ascidian oocytes display (a-v) gradients of 1) a mitochondria-rich subcortical domain (called myoplasm), 2) a network of cortical Endoplasmic Reticulum (cER), and several cortical maternal mRNAs called postplasmic/PEM RNAs. We show that these domains and mRNAs acquire their polarized distribution during oocyte maturation. After fertilization the oocyte cortex undergoes 2 major phases of reorganization. The cortical (cER) and subcortical (myoplasm) domains are first concentrated in the vegetal contraction pole (future dorsal pole) during an acto-myosin dependant cortical contraction(first major phase of reorganization). The myoplasm, cER/mRNA domains are then translocated posteriorly by a microtubule-dependant movement of the sperm aster with respect to the cortex (second major phase of reorganization). The domains are distributed equally between blastomeres during the first cleavage. At the 2-4 cell stage, the myoplasm, cER and postplasmic/PEM RNAs accumulate in posterior blastomeres. At the 8 cell stage, cER and postplasmic/PEM RNAs are concentrated in a cortical macroscopic structure called Centrosome Attracting Body (CAB) located in the vegetal posterior-most blastomeres (B4.1). The CAB is involved in the formation of three successive unequal cleavages and in mRNA segregation in small posterior blastomeres. We have characterized for the first time the  evolution and dynamics of this cortical polarity using cortex isolation and characterization in oocytes, zygotes and early embryos (8 cell stage). We observe that two postplasmic/PEM RNAs, PEM1 and macho1 respectively involved in axes formation and primary muscle cell formation, are anchored to the surface of the polarized network of cortical rough ER. After fertilization these cortical RNAs are concentrated in the vegetal cortex with the cER (forming a cER/mRNA domain). The

cER/mRNA domain moves posteriorly before the first cleavage and compacts into the CAB at the 8 cell stage.  We discuss how the cytoskeleton relocates the cER/mRNA domain and how the CAB may form from the translocation and compaction of polarized cER/mRNA domain already present in the oocyte. We also discuss how the segregation of postplasmic/PEM RNAs into specific blastomeres directs development and differentiation of the posterior region of the embryo and particular primary muscle cell formation.


4. Ion currents involved in oocyte maturation, fertilization and early development in Ciona intestinalis (ascidians).  Annunziata Cuomo, Stazione Zoologica “Anton Dohrn” Naples, and Univ. of Calabria, Italy. Ph.D. thesis; advisor Dr. Elisabetta Tosti.

    The aim of this study has been to characterize the presence and space-time distribution of plasma membrane ion currents from the oocyte at the GV stage up to 8 cell stage embryo in the marine invertebrate Ciona intestinalis. This Ph.D. thesis shows that the plasma membrane of Ciona intestinalis oocyte undergoes a profound electrical modification through meiosis completion and subsequent development. The work describes in detail an oscillatory pattern of calcium and sodium current activities and steady state conductance. The results obtained are in agreement with previous findings in different ascidian species demonstrating a major role for ion currents during fertilization and development. In this thesis, using the electrophysiological technique “whole cell voltage clamp” an electrophysiological characterization of plasma membrane in oocytes at the GV stage has been reported for the first time. In more detail, electrical and pharmacological studies showed a prevalence of L type Ca2+ currents in the immature stage that decreased thorough meiosis, strongly suggesting that these currents are involved in meiosis resumption following germinal vesicle breakdown. When oocytes reached MI (metaphase I) stage, Ca2+ currents decreased and disappeared. Accordingly, L type Ca2+ channels play a role in meiosis progression in molluscs, pleurodeles and mammals. In the mature stage of C. intestinalis oocytes, Na+ currents appeared and their amplitudes remained high up to the zygote stage, suggesting a functional role for Na+ during fertilization. This work shows, for the first time, that fertilization current is mainly driven by Na+ currents and that inhibition of Na+ entry is associated with abnormal embryo development. This result supports the hypothesis that fertilization current plays a key role in the subsequent embryo development. Using calcium imaging it was shown that the calcium stores are abundant in MI with respect to GV; Ca2+ currents in GV were devoted to fill the stores subsequently emptied at MI phase to sustain calcium wave and contraction at fertilization.  Stages from 2 to 4 cells represented a quiescent moment electrophysiologically, suggesting that the first cleavages did not rely on ion current activities. The 8 cell stage was critical because a resumption of ion activities was observed. Particularly, Na+ current amplitudes were higher than Ca2+. However, in these embryos, no significant differences between blastomeres were observed, excluding, in such a way, the presence of a distribution gradient of currents. The only exception was blastomere B4.1.

    These data demonstrate that ion currents cannot be considered good markers for cell lineage in C. intestinalis, at least up to the 8 cell stage. Finally, the pattern of total conductance suggested a major role for plasma membrane permeability of and a minor function for specific currents in the cell line segregation event. Overall, the findings described in this thesis provide new information and insight into mechanism and dynamics of meiosis and embryo development.


5. Life history variation in a colonial hermaphroditic ascidian, Botryllus schlosseri.  E. Nicole Kroutter, Master's thesis, Univ. of New Orleans Dept. Biol. Sci.; advisor J. Stewart-Savage.

        Botryllus schlosseri, a subtidal hermaphroditic ascidian, has a cosmopolitan distribution in temperate waters. Variations in non-reproductive and reproductive life history characteristics provide a valuable opportunity for evaluating the effects of genetics and environment in these populations. Analysis of these life history characteristics was performed on 100 oozooids reared from two locations (DM, CI) ten km apart within the Damariscotta River estuary, Maine, USA. Micro-scale genetic variation between the two cohorts was found in the timing of the onset of female reproduction with a significant interaction between collection site and the asexual cycle of development. Other life history characteristics, growth rate, mortality, and the timing of male reproduction onset, while influenced by temporal components, did not significantly differ between the two cohorts. These results are discussed together with life history data from three other geographic locations: Woods Hole, Mass., USA, the Mediterranean Sea, and Monterey Bay, Calif., to evaluate the macro-scale genetic differentiation and evolution of this species.


6. Reproductive Strategies of Didemnum rodriguesi (Aplousobranchia, Didemnidae). Nicole Ritzmann, Universidade Federal do Paraná, Dept. de Zool., Master Program in Zoology; Thesis advisor Rosana Moreira da Rocha.

Reproductive strategies are very important for understanding population dynamics, geographic distribution and survival. Understanding how resources are allocated for growth, maintenance and reproduction is also important, since organisms should optimize their allocation efforts to maximize fitness.  This study examines reproductive strategies of D. rodriguesi at Arvoredo Island, southern Brazil. Specifically, we compare investment in sexual and asexual reproduction between winter and summer. Phenotypic plasticity was compared between two locations with lighted and shaded treatments. We predicted that variability in investment in the two types of reproduction shows that reproductive traits are not genetically controlled, but rather are environmentally adaptable. Estimates of fragmentation, fusion, colony size, growth rates, mortality and recruitment were based on digital photographs. Photographs were taken in spring 2003 and summer and winter 2004. In winter and summer samples, photographs were taken at intervals of 7 to 10 days to accompany relatively rapid changes in the colonies. Monthly samples were also collected from July 2003 until May 2004 to estimate the number of eggs and larvae, colony size and zooid density. Comparison between sexual and asexual reproduction showed seasonal changes in sexual and asexual reproduction. Sexual reproduction was most important during warmer water temperatures (from November to May, the southern summer). Asexual reproduction, associated with greater growth rates and larger colony sizes, was more important during the winter. Colony fragmentation, also a type of asexual reproduction, was constant over seasons and treatments. Colony fusions were also constant over seasons. Fusion occurred more often than fragmentation, and since colony size and the “per-capita” number of eggs or larvae are correlated, larger size is favored to increase reproductive output. Mortality is constant over seasons.  In the experimental manipulation of light, mortality, recruitment and number of brooded eggs were greater in the natural light treatment compared to the shaded treatment. The number of larvae was constant over light treatments. Growth rates and colony size were greater in the shaded treatments, while fusions and divisions were similar among treatments.  Thus, we show that the reproductive strategies of D. rodriguesi vary seasonally, but also include phenotypic plasticity with respect to light conditions.




A. 4th Intl. Symposium on the Molecular and Cell Biology of Egg- and Embryo-Coats (MCBEEC), Nov. 8-13, 2004, MielPearl Ise-Shima, Japan.


1. Sperm Chemotaxis of Ciona by SAAF: Ca2+-dependent cell signaling, and identification and synthesis of the chemoattractant. M. Yoshida1, H. Tsuchikawa2, T. Oishi2, M. Murata2, and M. Morisawa1. 1Misaki Mar. Biol. Sta., Grad. Sch. Sci., Univ. of Tokyo, Miura 238-0225, and 2Dept. of Chem., Grad. Sch. of Sci., Osaka Univ., Toyonaka 560-0043, Japan.

   Sperm of the ascidian, Ciona intestinalis, were immotile or move slightly when they were suspended in seawater, and sperm are intensely activated near the egg, and then the activated sperm showed chemotactic behavior toward the egg, since the ascidian egg releases sperm-activating and -attracting factors around the egg (1).  The chemotactic behavior of the ascidian sperm requires extracelluar Ca2+.  The chemotactic behavior of the activated sperm was analyzed using the linear equation chemotaxis index (LECI), a parameter that is based on a linear equation of time vs. the distance between the micropipette tip and the sperm head.  The modulators of store-operated Ca2+ channel inhibited sperm chemotactic behavior of ascidian sperm, but any blocker for voltage-dependent Ca2+ channel did not, while the sperm activation operated by voltage-dependent Ca2+ channel.  These blockers of store-operated Ca2+ channel also inhibited asymmetrical flagellar form and turning movement of sperm that is the typical sign of sperm chemotaxis.  These results suggest that increase in [Ca2+]i through the store-operated Ca2+ channel causes asymmetrical flagellar movement to establish the sperm chemotaxis (2). Recently, we purified the factor, and named it as Sperm-Activating and Attracting Factor (SAAF).  We elucidated the chemical structure of SAAF using NMR and ESI/TOF-MS, and proposed it as a novel sulfated steroid (3).  Based upon the proposed structure, two epimers were synthesized from chenodeoxycholic acid in 16 steps, and comparison between synthetic and natural compounds led to the unambiguous structure determination of SAAF to be (3R,4R,7R,25S)-3,4,7,26-tetrahydroxycholestane-3,26-disulfate (4).  Sperm-activating and -attracting activities are existed in both synthetic SAAF and its epimer, (25R)-SAAF.  They activated and attracted the Ciona sperm at 3.7 - 10 nM.  Furthermore, the sperm-activating and attracting activities of the desulfated SAAF analogs, 3-desulfate SAAF, 26-desulfate SAAF, and 3,26-bisdesulfate SAAF were reduced.  Therefore, the sulfate groups on 3 and 26 portions of SAAF are indispensable for SAAF activities.1. M. Yoshida, K. Inaba, M. Morisawa, Dev. Biol. 157, 497-506 (1993).  2. M. Yoshida, M. Ishikawa, H. Izumi, R. De Santis, M. Morisawa, PNAS 100, 149-154 (2003). 3. M. Yoshida, M. Murata, K. Inaba, M. Morisawa, PNAS 99, 14831-14836 (2002). 4. T. Oishi, H. Tsuchikawa, M. Murata, M. Yoshida, M. Morisawa, Tetrahedron 60, 6971-6980 (2004).


2. Genomic and proteomic approaches to the molecular mechanism of sperm activation induced by egg-derived substance in Ciona intestinalis. K. Inaba, Shimoda Mar. Res. Center, Univ. Tsukuba, Japan.

   Prior to fertilization, spermatozoa undergo dynamic physiological changes, including motility initiation, activation and chemotaxis to the egg. In Ciona, a sulfated steroid, called SAAF (sperm-activating and attracting factor) is released from the egg to activate sperm motility and to attract sperm to the egg (1). Analysis of whole sperm proteins before and after the activation by two-dimensional gel electrophoresis reveled 12 proteins that change the quantity or the isoelectric points. To identify these proteins, we have developed a database (MSCITS) and a search program (PerMS) for the analysis by peptide mass fingerprinting (2), based on the information from extensive cDNA (EST) analysis (3) or genome project (4) in the ascidian Ciona intestinalis. The results showed that these proteins include an intermediate chain of an inner arm dynein IC116, a radial spoke protein LRR37 (5) and a novel axonemal protein. They also included a regulatory subunit of cAMP-dependent protein kinase, which is known to be associated with the axoneme. These proteins appear to be involved in the activation of the motile machinery, the axoneme. Other proteins are thought to be the components of plasma membrane or intracellular matrix of sperm and suggested to be involved in the signal transduction in the downstream of SAAF reception. Immunoprecipitation or a series of column chromatography resulted in the isolation of protein complexes containing these proteins. Following peptide mass fingerprinting has shown another components that are associated with or localized in the complex of these proteins. These lines of study should shed light on the molecular architecture or the function of protein complexes making up a molecular signal network for sperm activation.   (1) Yoshida et al., 2002. PNAS 99, 14831-14836. (2) Hozumi et al., 2004, Biochem. Biophys. Res. Commun. 319, 1241-1246. (3). Inaba et al., 2002, Mol. Reprod. Develop. 62, 431-445. (4) Dehal et al., 2002, Science 298, 2157-2167. (5) Padma et al., 2003, Mol. Biol. Cell, 14, 774-785.


3. Glycosidase functions in sperm-egg coat interaction in ascidians: a reconsideration and a new approach.  T.G. Honegger and R. Koyanagi, Dept. Zool., Univ. Zurich, Switzerland

   Ascidian eggs are surrounded by an egg investment consisting of the acellular vitelline coat (VC) with follicle cells (FCs) on its outer and test cells on its inner side. This complex egg coat is the site of species-specific gamete recognition and in self-sterile species the site of self-nonself discrimination. It was first proposed by Hoshi and collaborators (Hoshi et al. 1983, 1985; Hoshi 1984, 1986) that in ascidians binding of the sperm to the egg coat could be mediated by an enzyme-substrate complex established between a sperm surface glycosidase and glycoside moieties on the egg coat. Since glycosidases usually have a low activity at neutral and alkaline conditions, a stable lectin-like enzyme-substrate binding would be established in sea water. Fertilization-induced alterations of the sperm ligands on the egg coat would result in a block to polyspermy. In fact, eggs of ascidians were reported to release glycosidase within seconds after fertilization (Lambert 1986,1989). The enzyme was suggested to modify VC sperm ligands thus preventing supernumerary sperm from binding and setting up a major block to polyspermy. Although abundant evidence for an essential role of gamete associated glycosidases have been presented, several controversial issues exist and the currently favored model needs  to be approved. One issue concerns the location and nature of the binding sites for the sperm glycosidase which usually have been traced by binding of appropriate FITC-lectins. These experiments showed a strong labeling of the VC but not of the FCs (Honegger 1986; Lambert 1989) suggesting the VC as primary sperm binding site. However, we found recently that in Phallusia mammillata and Ascidia mentula FCs also possess glycoside residues corresponding to the predominating sperm-bound glycosidase. Furthermore, we showed that a number of sperm surface proteins bind to the follicle cell surface but not to the VC and that in P. mammillata FCs are indispensable for fertilization. These findings attribute a key role in sperm binding and possibly sperm activation to the follicle cells. In both processes, glycosidase-mediated binding is an essential but most probably not the only receptor-ligand interaction. A second issue concerns the localization and the target of the egg glycosidase suggested to mediate a block to polyspermy. In P. mammillata, initially N-Acetyl-b-D-hexosaminidase had been suggested to be released from the egg cell proper but in recent years the FCs were favored as its source (Lambert et al. 1997). We have shown by in situ hybridization that in P. mammillata b-hexosaminidase is also expressed in the test cells. However, the three egg b-hexosaminidases are not readily distinguishable and so far approaches to purify and/or characterize egg glycosidases produced diverging results (Matsuura et a. 1995; Eisenhut 2001). Thus, it remains to be elucidated which of the egg b-hexosaminidases participate in the block to polyspermy, what mechanisms triggers the release and what the target of the egg glycosidases is. To approach these questions, we constructed  a GFP-fusion protein from P. mammillata b-hexosaminidase and traced its binding to unfertilized and fertilized eggs by light and electron microscopy. The observed binding to the FCs will be discussed in context with the binding properties of b-hexosaminidase and the involvement of other candidate molecules in sperm-egg coat interactions in P. mammillata.


4. Multiple Functions of Ascidian Follicle Cells: The Cellular Swiss Army Knife.  C.C. Lambert, Univ. of Washington Friday Harbor Labs, Friday Harbor, WA

   The ascidian egg is surrounded by test cells within an acellular vitelline coat (VC = chorion) and a single layer of follicle cells on the outer surface of the VC.  These cells have diverse morphologies and functions in different ascidian species. They contribute to egg maturation in Halocynthia roretzi2 and Boltenia villosa (unpublished) and synthesize proteins and RNA in Styela clava3 . They also have many other functions during fertilization and development.  In several species including H. roretzi4,5 and B. villosa6 , they are required for fertilization. They also block self-fertilization in H. roretzi4 and Ciona intestinalis7 eggs.  In others, they function in egg localization, by synthesizing and sequestering ammonia for flotation in Corella inflata8 or by release of adhesive secretions which anchor the eggs in the atrium in Corella eumyota9 or substrate in Molgula pacifica10.  Enlarged follicle cells are present in many other ascidians with floating eggs including Corella parallelogramma, C. japonica, C. willmeriana and Ascidiella aspersa. At fertilization, the follicle cells release glycosidases in response to sperm, which function in the early block to polyspermy by blocking sperm receptors on the VC in Phallusia mammillata11 , Ascidia ceratodes and A. columbiana (unpublished).  The interaction between sperm cells and follicle cells is not species-specific. This can lead to interspecific sperm competition: sperm from Ascidia sydneiensis and Phallusia julinea interfere with the fertilization of Phallusia nigra eggs12.  Thus ascidian follicle cells are highly diverse and among the most active of cell coats. 1. Burighel P. & Cloney R.A. 1997. In Microscopic Anatomy of Invertebrates. pp. 221-347. 2. Sakairi & Shirai 1991. Dev. Growth Differ. 33:155-162. 3. Jeffery 1980. J. Exp. Zool. 212:279-289. 4. Fuke 1983. Roux's Arch. Dev. Biol. 192:347-352.  5. Hoshi et al. 1981. Dev. Biol. 86:117-121. 6. Hice & Moody 1988. Dev. Biol. 127:408-420. 7. Marino et al. 1999. PNAS 96:9633-9636. 8. Lambert & Lambert 1978. Science 200:64-65. 9. Lambert et al. 1995. Can. J. Zool. 73:1666-1671. 10. Young et al. 1988. Biol. Bull. 174:39-46. 11. Lambert et al. 1997. Mol. Reprod. Dev. 48:137-143. 12. Lambert 2000. Biol. Bull. 198:22-25.


5. Self/nonself-recognizable sperm receptor on the vitelline coat is degraded by the sperm ubiquitin-proteasome system during ascidian fertilization.  H. Sawada1,2*, S. Ban1,2, Y. Harada1, and H. Yokosawa2. 1Sugashima Mar. Biol. Lab., Grad. Sch. Sci., Nagoya Univ., Japan; 2Dept.Biochem., Grad. Sch. Pharmaceut. Sci., Hokkaido Univ., Sapporo, Japan.

   We previously reported that two sperm trypsin-like proteases, acrosin (1) and spermosin (2), and the sperm proteasome (3) play key roles during fertilization of eggs from the ascidian Halocynthia roretzi (4, 5).  Purified preparations of the two trypsin-like proteases exhibited no proteolytic activity toward the vitelline coat, but the sperm 26S-like proteasome lysed the vitelline coat protein.  A major component of the vitelline coat, HrVC70, was found to be degraded by the ubiquitin-proteasome system contained in sperm exudate, a supernatant fraction of the reacted sperm (6).  Immunocytochemistry using a monoclonal antibody (FK2) specific to ubiquitinated proteins revealed that vitelline coats were ubiquitinated after sperm-egg interaction (6).  In addition, it was also discovered by Western blotting that HrVC70 in the vitelline coat was specifically ubiquitinated during fertilization.  Here, we show that HrVC70, which consists of 12 EGF-like repeats, functions not only as a sperm receptor (6) but also as a candidate self/nonself-recognition molecule in the fertilization of H. roretzi eggs (7), a hermaphroditic animal with strict self-sterility.  Fertilization was strongly inhibited by pretreatment of sperm with HrVC70 from a different individual, but not from the same individual, and the number of nonself sperm bound to HrVC70-agarose was significantly higher than that of self-sperm.  A sequence analysis of HrVC70 disclosed that several amino acid residues in a restricted region are substituted at an individual level.  Furthermore, genomic DNA analysis revealed that the EGF-like domains correspond to the exons, and each intron is highly conserved among even- and odd-numbered introns.  It was also found that diversity in cDNA sequences is derived from genomic DNA polymorphism, probably elicited by crossing over and specific nucleotide substitutions. These results suggest that HrVC70 is a candidate allorecognition molecule in gamete interaction and that this molecule is degraded by the sperm ubiquitin-proteasome system during fertilization.  We recently found that a VC70-homologue in Halocynthia aurantium (HaVC80) consists of 13 EGF-like repeats and that this molecule seems to play a key role in allorecognition in the fertilization of this species.  (1) Kodama, E., et al. 2001. J. Biol. Chem. 276, 24594-24600. (2) Kodama, E., et al. (2002. Eur. J. Biochem. 269, 657-663. (3) Sawada, H., et al. 2002. Mol. Reprod. Dev. 62, 271-276. (4) Sawada, H. 2002. Zool. Sci. 19, 139-151. (5) Sakai, N., Sawada, M.T., & Sawada, H. 2004. Int. J. Biochem. Cell Biol. 36, 776-784. (6) Sawada, H., et al. 2002. Proc. Natl. Acad. Sci. USA 99, 1223-1128. (7) Sawada, H., et al. 2004. PNAS 101, 15615–15620.

6. Lineage analysis of the germline in ascidian embryogenesis.  M. Shirae-Kurabayashi1, T. Nishikata2, K. Takamura3, C. Nakamoto1 and A. Nakamura1. 1RIKEN Ctr. Dev. Biol., 2Dept. Biol., Konan Unv., 3Dept. Marine Biotech., Fukuyama Unv., Japan.

   In animal embryos, there are two modes of germ cell formation: 1) germ cells are predetermined by maternally inherited germ plasm; and 2) germ cell formation is induced by a signal or signals from somatic tissues. In spite of those differences in germ cell formation among species, products of germ cell-specific genes are often highly conserved. In an ascidian Ciona intestinalis, germ cells are thought to be determined by germ plasm. However, it also appears that germ cells are capable of being regenerated after metamorphosis. As the first step to understand the mechanism of germ cell formation in ascidians, we have conducted a detailed description in the process for germline formation during embryogenesis. For this purpose, we have identified two conserved germline-specific genes, vasa homolog (CiVH) and tudor homolog (CiTud3) from C. intestinalis. In cleavage stage embryos, CiVH mRNA and CiVH protein were localized in a specialized cortical structure in a pair of posterior-most blastomeres, called the centrosome-attracting body (CAB). It has been shown that many maternal factors including determinants for somatic cell differentiation are localized in the CAB. The CAB becomes partitioned into the B7.6 blastomeres in the late cleavage stage. The B7.6 cells had been proposed to remain transcriptionally repressed and mitotically inactive until larval stage. However, we found that, B7.6 cells became positive for phospho-histone H3, a mitosis marker, at gastrulaion, and subsequently underwent asymmetric cell division to produce two morphologically distinct daughter cells. In the larger daughter cells, CiVH formed aggregates around the nucleus like “nuage”, conserved germline-specific structures among animal species. In the smaller daughter cells, CiVH was detected in the entire cytoplasm without aggregation. The distributions of CiTud3 mRNA and CiTud3 protein were the same as that of CiVH during embryogenesis. In contrast, other CAB components, which seem to be involved in somatic cell differentiation, were segregated into the smaller daughter cells. Furthermore, we found that the CiVH promoter became active only in the larger cells in the tailbud embryo. These results suggest that segregation of the maternal factors in the CAB by asymmetric cell division causes the initiation of zygotic expression of germline-specific genes, leading to the differentiation into functional germ cells.

7. Localization and functions of glycosidases in fertilization: Clues from molecular and experimental approaches in the ascidian, Phallusia mammillata. R. Koyanagi and T. G. Honegger, Zool. Inst., Univ. Zürich,  Switzerland. 

   A number of observations suggested that primary sperm-egg binding in ascidians is mediated by a glycosidase on the sperm and a carbohydrate chain on the egg coat. Furthermore, to prevent polyspermy, the egg binding sites become unavailable after fertilization due to substances released from the egg including glycosidase activities. To confirm this model, we constructed a GFP-fusion protein from one of the glycosidases, Phallusia mammillata b-hexosaminidase (GFP-Hex), and observed its behaviour with regard to fertilization. GFP-Hex bound to the follicle cells (FC) of unfertilized P. mammillata eggs, but not to those on fertilized eggs. The binding was also eliminated in the presence of a specific inhibitor of b-hexosaminidase. Eggs were not fertilizable in GFP-Hex seawater, but recovered their normal state after washing in fresh seawater. These results clearly denote the FCs as a primary target for the P. mammillata b-hexosaminidase and are the first direct proof that egg glycosidase alone is sufficient to establish a block against polyspermy.


8. Genetic studies on the mechanism of self-sterility in the ascidian Ciona intestinalis.  Y. Harada, K. Kobayashi, Y. Takagaki and H. Sawada.  Sugashima Mar. Biol. Lab., Grad. Sch. Sci., Nagoya Univ., Sugashima, Japan.

   Ciona intestinalis is a hermaphroditic species, the egg-coat of which can discriminate between self- and nonself-derived sperm to prevent self-fertilization.  This allorecognition process is known to be genetically controlled, but the mode of inheritance and molecular nature of the gene(s) involved in the process are not known.  We previously identified a promising candidate for the allorecognition molecule, HrVC70, from another ascidian species, Halocynthia roretzi (1).  It is reported that HrVC70 molecules accumulate into the egg-coat during oocyte maturation, which is a process of acquisition of self-sterility.  Mature oocytes lose the self-sterility by an acidic seawater treatment, which is capable of eluting the HrVC70 molecules from the egg-coat.  HrVC70 consists of 12 EGF-like repeats, which are highly polymorphic between individuals.  In addition, it is also known that HrVC70 precursor HrVC120 contains a ZP-motif at the C-terminus.  On the basis of these features, we searched the genome database of C. intestinalis for the homolog of HrVC70, and we found three genes that are abundantly expressed in the gonad and contain both ZP-motif and EGF-like repeats.  In C. intestinalis, artificially self-fertilized F1 siblings show highly frequent mutual infertility (2).  We have embarked on a study to isolate the gene(s) that cause the infertility in such an incompatible combination.  In order to address this issue, we investigated whether mutual infertility is closely related to the genetic polymorphism in the above-mentioned putative HrVC70 homologs.  The results implied that at least one of three genes may play a role in allorecognition during fertilization of C. intestinalis. (1) Sawada, H. et al. 2004. PNAS 101, 15615–15620.  (2) Murabe, N. and Hoshi, M. 2002. Zool. Sci. 19:527-538.


9. Cloning and Structural Analysis of Polymorphic Vitelline-coat Protein HaVC80 from the Ascidian, Halocynthia aurantium: Implication in Self-sterility and Speciation.  S. Ban1,2, Y. Harada1, H. Yokosawa2, and H. Sawada1,2.  1Sugashima Mar. Biol. Lab., Grad. Sch. Sci., Nagoya Univ.,  Sugashima, Japan; 2Dept. Biochem., Grad. Sch. Pharmaceut. Sci., Hokkaido Univ., Sapporo. Japan.

   We previously reported that a 70-kDa sperm receptor on the vitelline-coat, HrVC70, consisting of 12 EGF-like repeats is a candidate self/nonself-recognition molecule during fertilization of the ascidian, Halocynthia roretzi.  Here, we report the cDNA and genomic DNA cloning of HaVC80, an HrVC70 homologue, from Halocynthia aurantium, which is an animal classified into the same genus with H. roretzi.  We found that HaVC80 is attached to the vitelline coat during oocyte maturation, a process of the acquisition of self-sterility, and is detached from the vitelline coat by acid treatment, which allows self-fertilization.  This suggests that HaVC80 is involved in self/nonself recognition during fertilization.  A cDNA clone of HaVC80 precursor HaVC130 was isolated from an H. aurantium gonad cDNA library.  HaVC130 contained a signal sequence, 14 EGF-like repeats, a ZP domain, and a transmembrane domain.  The structure of HaVC130 was similar but not identical to those of HrVC70 precursor HrVC120: HaVC80 region contained 13 EGF-like repeats, which was one-repeat longer than HrVC70.  Analysis by RT-PCR and TA cloning of HaVC80 mRNAs from 6 individuals revealed that HaVC80 is a highly polymorphic protein at an individual level.  The results of genomic DNA cloning also suggested that HrVC80 is evolutionarily generated by gene duplication in the 8th EGF domain of HrVC70.  Possible participation of HaVC80 in self-sterility and speciation will be discussed.


B. 75th meeting of the Zoological Society of Japan, Sept. 10-12, Kobe, Japan.


The ultrastructure of sensory cells associated with a tentacular tunic in the atrial tentacles of the ascidian Polyandrocarpa misakiensis.  H. Koyama, Coll. Nurs., Yokohama City Univ., Yokohama, Japan.

    I studied the ultrastructure of possible sensory hair cells within the epithelium surrounding the tentacular tunic, which penetrates into the atrial tentacles of Polyandrocarpa misakiensis.  The bottle?shaped sensory cells usually exist at the bottom of the tentacular tunic as a single unit, and they are supported by unspecialized epithelial cells of the descending or ascending epithelium of the siphons.  The sensory cells bear apical microvilli and a cilium.  There is no accumulation of  matrix around the cilium, as there is in the sensory cells associated with branchial tentacles.  The cytoplasm of the sensory cells is enriched by rough endoplasmic reticulum, mitochondria, lipid droplets, and dense bodies. There are many thin cell processes in the test matrix of the tentacles. Although we have not identified a basal process with the ultrastructural features of an axon, the morphology of these cells suggests that they might be primary sensory neurons, like many peripheral sensory cells of other protochordates.




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