Ascidian News*
Charles and Gretchen Lambert
Dept. of Biol. Sci., Calif. State Univ., Fullerton, CA 92834-6850
tel. (714)278-3481 Fax (714)278-3426
e-mail: or

Number 43                                                                                                                          May 1998

    Charles is retiring at the end of this semester, after 28 years at Calif. State Univ. Fullerton.  On June 4 we are moving to Seattle, Washington. Our new address will be 12001 11th Ave. NW, Seattle, Wa 98177, telephone number 206-365-3734. We will continue to use our same email addresses temporarily. We will both be doing research in association with the Univ. of Washington. We will continue our studies on the introduction of non-indigenous ascidians to the U.S. Pacific Coast, but move our sampling from southern California to the Pacific Northwest. We also plan to do some travelling; we will be spending the month of October in Honolulu, with Charles doing research on ovulation in Herdmania momus and Gretchen working at the Bishop Museum identifying some of their ascidians.
  Gretchen will continue to produce Ascidian News twice a year on our web site. We have compiled this newsletter since 1975, and we hope you continue to find it useful.  Please keep in touch, and please continue to send us a copy of your new publications, which will assure that they are included in the Recent Publications section at the end of each AN. There are 60 new publications in this issue; if you have not yet sent us a copy of your latest papers, please send them to our new Seattle address.
 *Ascidian News is not part of the scientific literature and should not be cited as such.

                                                                    NEWS AND VIEWS
Dr. Paul Illg:   We are very sorry to report that our long term friend and author of books and numerous monographs on copepods that inhabit ascidian hosts passed away on May 10th after a long illness. His knowledge of invertebrate
biology was truly amazing. Paul was influential to both of our careers and we learned a great deal from him.  We greatly enjoyed talking to him on a daily basis when we were graduate students. He helped to generate our interest in systematics and we have counted upon him as a friend throughout our subsequent lives. He will be greatly missed by all who were fortunate enough to know him during his many years at the Friday Harbor Laboratories and in the Zoology department of the University of Washington.

                                                          WORK IN PROGRESS

Drs. V.K. Meenakshi, Dept. of Zool, APC Mahalaxmi college for Women, Tuticorin 628 002 and T.K. Renganathan, Dept. of Zool. VOC College, Tuticorin 628 008, India.
    We are working on the ascidians collected from the SE coast of India along the Gulf of Mannar from Tuticorin to Rameswaram.  A preliminary search has yielded many new records to Indian waters and a few species which are new to science.  Our papers on the new reports are under preparation. (We congratulate Dr. Meenakshi on receiving her Ph.D. in Dec. 1997; her thesis was "Biology of a few chosen ascidians" with a chapter on taxonomy which included 8 new species.)

                                                ABSTRACTS FROM RECENT MEETINGS

1.  Zool. Soc. of Japan 68th annual meeting, 1997. (Publ. in Zool. Sci. vol. 14. supplement, December 1997).
T. Irie1, T. Seki2, S. Kajiwara3 and M. Iwamoto4.   1Osaka Meijo Women's Coll., Osaka; 2Dept. Health Sci., Osaka Kyoiku Univ., Osaka; 3Dept. Biol., Fac. Educ., Iwate Univ., Iwate and 4Dept. Biophys. Engn., Fac. Engn. Sci., Osaka Univ. Osaka.
    Lipovitellin 1 (LV1), a yolk protein, was shown to be the retinal binding protein in Xenopus laevis eggs.  In the eggs of amphibian, teleost and urochordate, corresponding proteins to LV1 were found by SDS-PAGE.  For the confirmation of retinal binding to a specific yolk protein, rat antisera against LV1 of X. laevis, E1a of chum salmon (Oncorhynchus keta) and flatfish (Limanda yokohamae) eggs, and against retinal were obtained.  By western blotting analysis, antiserum against X. laevis stained only one band of LV1.  Using the anti-retinal antiserum, positive bands have not been observed, but retinyl product of X. laevis LV1 was found to be fluorescent on the blotted membrane. Among the egg proteins, two bands were stained with anti-O. keta E1a antiserum, while many protein bands of L. yokohamae were stained with anti-L. yokohamae E1a antiserum. Identification of the retinal binding protein with one or some stained bands is a problem for further study.  In the ascidian eggs, four bands corresponding to LV1 were observed, but none reacted with anti-retinal antiserum.

H. Izumi1, T. Marian2, K. Inaba1, Y. Oka1, M. Morisawa1.   1 Misaki Mar. Biol. Sta., Graduate Sch. of Sci., Univ. of Tokyo; 2 Positron Emission Tomograph Center, Univ. Med. Sch. of Debrecen, Hungary.
    Sperm-activating and -attracting factor (SAAF) released from egg at fertilization activates sperm motility in the ascidians Ciona savignyi or C. intestinalis through synthesis of cAMP and influx of Ca2+ (Yoshida et al. 1994).  We show here that the activation of  Ciona sperm motility was suppressed by extracellular K+. A K+ ionophore, valinomycin, substitutes the SAAF effect; it activated sperm motility in the absence of SAAF.  The effect of the ionophore is reduced by higher concentration of external K+ . On the other hand. measurement of membrane potential with a slow voltage-sensitive carbocyanine dye, DisC3(5) showed that both valinomysin and SAAF induce hyperpolarization of the sperm plasma membrane in the presence or absence of extracellular Ca2+ and the amplitude of the potential was dependent on external K+ concentration.  Valinomycin as well as SAAF caused an increase in intracellular cAMP.  A K+ channel blocker, MCD-peptide, suppressed the activation of sperm motility and  membrane hyperpolarization.   MCD-peptide also inhibited cAMP elevation  by SAAF but did not inhibit valinomycin-induced cAMP synthesis. These results suggest that hyperpolarization of the plasma membrane through an increase in K+ permeability causes the synthesis of cAMP, resulting in the activation of sperm motility. It is also suggested that Ca2+ influx is necessary for cAMP synthesis and sperm motility activation, but is unnecessary for the membrane potential changes.

M. Nomura, K. Inaba, M. Morisawa.  Misaki Mar. Biol. Station,  Graduate Sch.of Sci., Univ. of Tokyo.
     Sperm of the ascidian Ciona intestinalis initiates motility by unfertilized egg-derived factor, SAAF (Sperm Activating and Attracting Factor) which -raises intracellular cyclic-AMP (cAMP) level dependent on extracellular Ca2+ (1). On the other hand, demembranated sperm of C. intestinalis require cAMP to reactivate (2,3,4). In order to elucidate a relation between SAAF-dependent motility initiation and sperm protein phosphorylation, we used demembranated Ciona sperm with SAAF-dependent in vivo activation.  From experiment using demembranated sperm model, SAAF activated sperm model have already been prepared to motile with ATP alone, furthermore extracellular Ca2+ was required to initiate the motility in intact sperm prior to cAMP-dependent activation pathway. Intact sperm motility initiation in SAAF-dependent manner was inhibited by W-7 and ML-7. Remarkable sperm protein phosphorylation corresponding to SAAF-dependent sperm motility initiation, was seen in 21kDa and 26kDa proteins. These two proteins were phosphorylated in intact sperm which had been activated by SAAF. These protein phosphorylations may be regulated by CaM-like protein. Furthermore the 32P-labeled 21kDa protein was identified as one having a dynein light chain. We conclude that SAAF activates Ciona sperm via the dynein light chain, which is upregulated by Ca2+ efflux into sperm prior to regulation by  calcium binding protein. These support a previous report related to Ciona sperm activation by theophylline (4).   (1) Yoshida, M. et al. 1994. Develop. Growth & Differ. 36: 589-595. (2) Brokaw, CJ 1985. Ann. N. Y. Acad. Sci. 438, 132-141. (3) Dey, CS & Brokaw, CJ 1991. J. Cell Sci. 100, 815-824. (4) Chaudhry, PS et al.1994. Cell Motil. Cytoskeleton 32, 65-79.

M. Kamiimura, K. Hisata, S. Fujiwara, K. Kawamura and T. Yubisui.  Dept. of Biol., Fac.of Sci., Univ. of Kochi.
    It has been suggested that in the budding tunicate Polyandorocarpa misakiensis, retinoic acid (RA) is an endogenous regulator of bud development (Kawamura et al.).  In mammals, RA exerts a wide variety of activities via its nuclear receptor, retinoic acid recepter (RAR) that can form a heterodimer.  We have recently isolated almost the full length of RAR cDNA from Polyandrocarpa PmRAR (Hisata et al. 1995).  The aim of this study was to isolate Polyandrocarpa homologs of retinoid X receptor (PmRAR) that would be a partner for heterodimerization. In order to amplify cDNA fragments by PCR, degenerate primers were designed encoding conserved amino acid sequences of the known RXRs. The strands were extended by 3' RACE, and eventually a total of 2132 bp was sequenced. Deduced amino acid sequence was 80% identical with that of vertebrate RXRs.  Both DNA-binding domain and ligand binding domain were highly conserved RTPCR and Southern hybridization analyses showed that PmRXR was expressed in developing buds in the same developmental time course as PmRAR. Our results indicate that there is a good chance of PmRXR forming heterodimer together with PmRAR.

K. Kurita, K. Kawamura and T. Yubisui, Dept. Biol., Fac. Sci., Kochi Univ.
 NDADH-cytochrome b5 reductase is considered to play an important role in the development of ascidians, based on the studies of the mammalian enzymes.  Therefore, we studied the properties of NDADH-cytochrome b5 reductase of  S. plicata.  In this study, the enzymes of  S. plicata were partially purified by an affinity chromatography on a 5'-AMP-S column, and we investigated some kinetic properties.  Optimal pH of the enzyme was determined to be 6.2, and apparent Km values for NADH and cytochrome b5 were determined to be 1.4, and 3.6 uM, respectively.  An apparent Vmax value was 3.0 umol/min/mg.  An immunoblot study with an antibody against human enzyme showed a distinct band at around 67kD, which is about double the size of mammalian enzymes.

M.Fujimura and K.Takamura, Div. of Biotech., Fac. of Engineering, Fukuyama Univ., Fukuyama.
   We isolated a DEAD-box gene from Ciona ovary cDNA library by PCR method with degenerate primers and RACE method. The protein deduced from this cDNA sequences showed good homology to vasa homolog of other animals. The mRNA for this cDNA was about 2.8 kb by nothern blot analysis, and was detected through embryogenesis by RT-PCR. In situ hybridization showed that this mRNA existed in the inner region of eggs and was segregated into most posterior region during the second cleavage (2 to 4 cell stages). These results suggest that the segregation mechanism of this mRNA is different from that of ooplasm which occurs immediately after fertilization. As a result, this mRNA was inherited by most posterior blastomeres through succeeding cleavages and, finally, was detected in a part of the caudal endodermal strand at the early tailbud stage.

 K.Takamura and S. Kawano, Div. of Biotech., Fac. of Engineering, Fukuyama Univ., Fukuyama.
  We isolated two types of cDNA for an ascidian netrin homolog by PCR methods. Each cDNA was designated CINETA and CINETB, respectively. Their sequences were determined and compared with known netrin homologs of C.elegans, D.melanogaster and G. gallus. CINETA had all conserved domeins, while CINETB lacked last laminin-like domein. RT-PCR showed that both mRNAs for these cDNAs were detected from the middle gastrula stage and decreased from the late tailbud stage.  In situ hybridization with RNA probes for CINETA showed that this mRNA expressed in the primary notochord lineage blastomeres (A-line) at the gastrula stage and in the notochord at the early tailbud stage. Additional expression was detected in the brain lineage at the neurula stage and in the ventral region of brain at the early tailbud stage. These results show that the expression pattern of netrin in ascidians is different from that in other vertebrates whose netrin expresses in the ventral spinal cord.

Y. Ohtsuka1, Y. Okamura2 and T. Obinata1.  1Dept. of Biol., Fac. of Sci., Chiba Univ., Yayoi-cho, Chiba; 2Natl. Inst. of Biosci. & Human Technology Agency of Industrial Science and Technology M.I.T.I.,Tsukuba .
   Previously, we found that gelsolin, an actin filament severing and capping protein, is present abundantly in epidermal sensory neurons of the ascidian larva. In this study, we examined how vegetal blastomeres are involved in sensory neuron differentiation during ascidian larval development by using gelsolin as a molecular maker. When ascidian embryos at the 8-cell stage were manipulated to remove vegetal cells, either pairs of A4.1 cells or pairs of B4.1 cells, and then cultured until reaching the late tailbud stage, gelsolin-positive cells were detectable by anti-gelsolin antibody (AS23) in each case. The anterior half (a4.2+A4.1) and posterior half (b4.2+B4.1) embryos cleaved at the 8-cell stage produced gelsolin positive cells, whereas isolated (b4.2+A4.1), vegetal half (A4.1+B4.1) and animal half (a4.2+b4.2) embryos did not produce the neurons. These results indicate that signals from A4.1 and B4.1 are necessary for gelsolin expression in a-line and b-line blastomeres, respectively. At the16-cell stage, all of progenies derived from the A4.1 and B4.1 showed the ability to induce the neurons in animal blastomer when cultured in combination with them.

T. Kawakami, Y. Ohtsuka, T. Endo & T. Obinata. Dept. of Biol., Fac. of Sci., Chiba Univ., Yayoi-cho, Chiba.
   Troponin T (TnT) is a component of troponin, a Ca2+-dependent regulator for striated muscle contraction in a variety of vertebrates and invertebrates.  TnT is not detectable in vertebrate smooth muscle, but we previously demonstrated that TnT exists in adult body wall smooth muscle of ascidian, Halocynthia roretzi, as well as in the larval striated muscle, and in addition, that the embryonic TnT (eTnT) and adult TnT (aTnT) are present in this animal, which are encoded by distinct genes (Endo et al.1996. J. Biol. Chem. 271: 27855-27862).  In the present study, we examined the expression of the two types of TnT during ascidian embryogenesis and metamorphosis using specific cDNA probes and an anti-TnT monoclonal antibody (NT302).  The results show that mRNA for eTnT is detectable in the nuclei of several blastomeres at the 110-cell stage or a little earlier, while TnT protein is detectable only after the neurula stage.  The switching from eTnT to aTnT occurs about 7 days after tail absorption (onset of metamorphosis). Co-expression of eTnT and aTnT was not observed at any stage, suggesting that expression of the two TnT isoforms is precisely regulated.

H. J. Yuasa and T. Takagi, Biol. Inst., Graduate Sch. of Sci., Tohoku Univ., Sendai.
   Previously we identified two distinct cDNAs of troponin C (TnC) isoforms from the ascidian, Halocynthia roretzi.  These two TnCs (larval TnC and adult TnC) were encoded by a single gene, and expressed through alternative selection of a third exon (1).  This time, we determined cDNA sequences of three TnI isoformes from H. roretzi.  One of the three isoforms (adult TnI) was composed of 173 amino acids, and expressed in body-wall smooth muscle and heart muscle.  The other two isoforms (larval TnIƒ¿ and ƒÀ) were isolated from a cDNA library of larvae.  Both larval TnIs are composed of only 142 amino acids, and are the smallest of all known TnIs.  They are ca. 30 amino acid residues shorter at the C-termini than other TnIs.  The position of the intron of these TnIs C-terminal regions was also determined.  When compared with vertebrate TnI genes, the intron of the ascidian adult TnI gene is located at 6 nucleotides downstream, and the introns of the two larval TnIs are positioned at 9 nucleotides upstream.  These results suggest that H. roretzi TnI is encoded by at least three genes (2).  (1) Yuasa et al. 1997. J. Biochem. 121, 671-676. (2) Yuasa et al. 1997. J. Biochem. 122, 374-380.

R. Toyoda1, S. Sato1,T. Numakunai2, T. Gojobori3, K. Ikeo3 and H.Yamamoto1.
1Biol. Inst. Tohoku Univ., Sendai; 2Mar. Biol. Stn. Tohoku Univ., Asamushi, 3Natl. Inst. Genet. Mishima.
    Tadpole larvae of the Japanese ascidian Halocynthia roretzi deve1op two sensory pigment cells in the brain, the  otolith and ocellus. These melanin -containing cells specificaIly express tyrosinase activity and play an important role in detection of gravity and light. Genes encoding tyrosinase, a key enzyme in melanin biosynthesis, have been isolated from various vetebrate species. The 5' flanking sequences of the vertebrate genes have several conserved sequences, but their function as cis-elements remains to be solved. To carry out a phylogenetic study of the regulation of tyrosinase gene expression, we cloned a putative tyrosinase gene and a gene encoding tyrosinase c-related protein TRP from Halocynthia roretzi. To localize the sequences regulating expression of these genes, each 5' flanking sequence was ligated to a lacZ reporter gene and used for microinjection into fertilized ascidian eggs. 1.8 kb of the upstream sequence of the ascidian tyrosinase gene drives expression of the reporter gene in pigment precursor cells at the tailbud stage. On the other hand, 573 bp of the ascidian TRP gene were found to be sufficient to express in pigment precursor cells.

Y. Sasakura, M. Ogasawara, K.W. Makabe, Dept. of Zool., Grad. Sch. of Sci., Kyoto Univ., Kyoto.
     To understand the roles of maternal mRNAs in early development, we have started a mass screening of cDNAs of the fertilized egg of  Halocynthia roretzi. Here we report the cloning and characterization of  a member of the Wnt family. This Wnt-related gene shows the highest homology with Wnt-5, thus we named it HrWnt-5. The maternal transcripts of HrWnt-5 are distributed at the cortical region of the unfertilized egg. After secondary ooplasmic segregation, the transcripts are strongly localized at the posterior region of the egg.  This localization pattern continues to the tailbud stage, and at that stage maternal transcripts are seen only in the endodermal strand. Zygotic transcripts are detected at the 64-cell stage  in various regions of the embryo. At  the tailbud stage, zygotic transcripts are seen only in the notochord cells.

 S. Yoshida and N. Satoh, Dept. of Zool., Graduate School of Science, Kyoto Univ., Sakyo-ku, Kyoto.
     Maternal factors are implicated in the establishment of the animal body plan.  Ascidian embryogenesis is regarded as the typical "mosaic" type, and recent studies have provided convincing evidence that the posterior-vegetal cytoplasm of the fertilized egg is associated with the determinant and the establishment of the antero-posterior axis of the embryo.  We report here an analysis for the role of a novel maternal gene, posterior end mark (pem).  The transcript is initially concentrated in the posterior-vegetal cytoplasm of the fertilized egg, and later marks the posterior end of developing ascidian embryos.  The predicted pem protein showed no significant homology to known proteins.  Overexpression of this gene by microinjection of synthesized pem mRNA into fertilized eggs resulted in development of tadpole larvae lacking the anterior-most adhesive organ, dorsal brain and sensory pigment-cells.  Lineage tracing analysis revealed that the anterior dorsal neuronal cells were translocated posteriorly into the tail region, and this translocation could be rescued by LiCl treatment. These data suggest that pem plays a role in patterning of the ascidian embryo.

 M. Ogasawara & N. Satoh, Dept. of Zool., Grad. Sch. of  Sci., Kyoto Univ., Kyoto.
     The endostyle is an organ key to an understanding of the molecular mechanism underlying the origin and evolution of chordates.   We reported previously an isolation of two cDNA clones of the Halocynthia roretzi endostyle-specific genes, HrEnds1 and HrEnds2. In this study, we isolated and examined novel endostyle-specific genes of Ciona intestinalis, which belongs to another order of ascidians.  Transcripts of the CiEnds1 and CiEnds3 were detected in the protein secreting element of zone 6, and CiEnds2 transcripts were detected in other protein secreting element of zone 2 under our in situ hybridization.  These genes may serve as probes for further analysis of molecular mechanisms involved in the formation and evolution of the endostyle.

Ciona savignyi EMBRYOS.
  S. Chiba1,2 , Y. Satou2, T. Nishikata1 and N. Satoh2.  1Dept. of Biol., Konan Univ., Kobe;  2Dept. of Zool., Grad. Sch. of Sci., Kyoto Univ., Kyoto.
    We prefer Ciona eggs and embryos as an experimental system for future studies, in particular with respect to those with genetic approaches, because their spawning season is basically all year round and because their generation time is about 3 months. But, at present, we have isolated few tissue-specific genes in Ciona species that are useful items for the marker. Therefore we tried to isolate cDNA clones for tissue-specific genes from a library of tailbud-embryo mRNAs subtracted with fertilized-egg mRNAs of Ciona savignyi. Thus far we were able to isolate several cDNA clones for tissue-specific genes. We characterized two cDNA clones for muscle-specific genes and two  clones for epidermis-specific genes. All of the genes are useful for the marker. One of the muscle-specific genes is identified to be a muscle actin gene of Ciona savignyi.

S. Hori, T. Saitoh,& H. Nishida.  Dept. of Life Sci., Tokyo Inst. of Tech., Yokohama.
  Ascidian embryogenesis involves the various cell-cell interactions to specify the particular cell fate. To know the molecular basis of such cell interactions, we isolated the full-length cDNA of the ascidian homologue of the Drosophila neurogenic gene Notch (HrNotch).  HrNotch transcripts encode a protein of 2352 amino acids and share the principal features of the Notch gene family; extracellular EGF-like repeats, three Notch/Lin-12 repeats, six intracellular ankyrin repeats and RAM domain. Yet ascidian Notch contains only 33 EGF-repeats in the putative extramembrane domain and specifically lacks the three EGF-like repeats. We have previously reported the zygotic expression of HrNotch in sensory pigment cell precursors, which suggests its roles in the determinative events in the sensory pigment cell equivalence group. In this report, the zygotic expression of HrNotch is also detected in the nerve cord lineage (b-line cells) and the presumptive larval adhesive organ (a-line cells).

M. R. Wada, Y. Ohtani, Y. Shibata, K. J. Tanaka, & T. Nishikata, Fac. of Sci., Konan Univ., Kobe.
    The localization of maternal mRNAs and their segregation to different embryonic cells has been proposed to mediate cell fate determination during early development.  Recently, Y-box protein family which is known as the nucleic acid-binding protein has been suggested to have an ability to repress the  translation of mRNAs.  In this paper, we isolated three cDNA clones for Y-box protein mRNAs (CiYB1, CiYB2 and CiYB3) from eggs and embryos of the ascidian, Ciona intestinalis.  The genomic sequence analysis of CiYB gene revealed that the three mRNAs were produced by the alternative splicing. CiYB1 and CiYB2 were expressed in the very early period of oogenesis and suggested to be recruited into maternal ribonucleoprotein particles (mRNPs). CiYB1, CiYB2 and CiYB3 expressed zygotically in tissue restricted manners. Using gel-mobility shift assays, we found that CiYB1 protein have an RNA-binding ability. This could suggest that the ascidian Y-box proteins play an important role for the storage and/or repression of the translation of the maternal informations.

T. Hirano & H. Nishida, Dept. of Life Sci., Tokyo Inst. of Technology, Yokohama.
     To study the origin of adult tissues of the ascidian Halocynthia roretzi, we traced the cell fate by intracellular injection of horseradis peroxidase (HRP) into identical blastomeres at 110-cell stage.  Descendants of the injected cells were histochemically detected at juvenile stage.  In the previous meeting, we reported the results of the cell lineage analysis of mesodermal cells of ascidian larvae and suggested that the cell lineages during metamorphosis were also invariant.   In this meeting, we present the results about endodermal cells of larvae.  Endodermal cells of the larvae were indicated to give rise to adult endodermal tissues, that is endostyles, peribranchial epithelium, and digestive organs and so on.  Furthermore these results suggested that the boundaries of adult-endoderm precursors do not correspond to the clonal boundaries of larval endodermal cells.

M. Matsumoto and M. Hoshi, Dept. of Life Science, Tokyo Inst.of Tech., Kanagawa.
         Genetic variety of rRNA gene (rDNA) repeated units was studied in Ciona savignyi and Ciona intstinalis. We previously reported that the internal spacer region between 18S rDNA and 5.8S rDNA had a wide variety in length. For further investigation, we analyzed animals from three bays where both  species live alongside (Onagawa, Honmoku and Qingdao).  Contrary to the previous data, all animals so far studied gave very similar PCR products of  the spacer region regardless of species and of geography, at least within these three bays.

A. Yamada and H. Nishida, Dept. of Life Sci., Tokyo Inst. of Technology, Yokohama.
     Cell number during ascidian embryogenesis were counted every hour. Number of nuclei of the squashed embryos and larvae were counted on photographs.  Cell number of a whole larva was about 2700 just after hatching. Next, animal and vegetal egg fragments were made by bisecting unfertilized eggs to alter the egg volume and the nucleocytoplasmic ratio. Egg fragments were fertilized and cell number of the resultant larvae were estimated. The results suggest that three kinds of mechanisms are  involved in control of cell division number during embryogenesis: (1) Mechanism that depends on the nucleocytoplasmic ratio.  (2) Mechanism that depends on the absolute volume of cytoplasm. (3) Mechanism that is not affected by changes of both factors.

H. Sawada, J. Fujino, Y. Takahashi, & H. Yokosawa,  Dept. Biochem., Fac.Pharm. Sci., Hokkaido Univ., Sapporo.
   We have previously reported that the sperm 20S proteasome and 930-kDa prot easome are involved in the sperm binding to and in the sperm penetration through the vitelline coat of the eggs of the ascidian, Halocynthia roretzi, respectively. In the present study, we investigated the mode of action of the proteasomes in fertilization.  MG115 (an inhibitor for proteasome and cathepsin B/L) inhibited the fertilization of intact eggs, while E-64-d (an inhibitor for cathepsin B/L and calpain) showed no inhibition. This potent inhibitory ability of MG115 against fertilization appears to be diminished by using the eggs treated with acidic seawater (pH 3.0), which elicits the release of 70-kDa component of the vitelline coat.  In addition, MG115 inhibited the sperm binding to the vitelline coat of the glycerinated eggs. The anti-proteasome antibody also inhibited the fertilization. These results suggest that the proteasome is involved in the sperm binding to and penetrating through the vitelline coat, and that the 70-kDa component of the vitelline coat may be a candidate substrate for the proteasome. It was also revealed that the proteasome functions extracellularly during fertilization.

Y. Aburatani & H. Yokosawa, Dept. of Biochem., Fac. of Pharm. Sci., Hokkaido Univ., Sapporo.
   The ubiquitin-dependent proteolysis is required for the progression of cell cycle. We previously reported that the proteasome is activated in response to intracellular calcium increase during activation of ascidian and Xenopus eggs. To define factors functioning in calcium-induced proteasome activation, we investigated effects of   three protein kinase inhibitors and an anti-microtubule compound on proteasome activation in ascidian eggs, which was triggered by calcium ionophore. Protein kinase inhibitors (olomoucine, curcumin, and erbstatin analog) and an inhibitor of microtubule polymerization (nocodazole) inhibited the proteasome activation, which is due to the assembly of the 26S proteasome from the 20S proteasome.  This result indicates that  protein kinases play important roles in the calcium-induced proteasome activation and that the presence of intact microtubules is indispensable for this event.

H. Michibata, J. Wuchiyama, Y. Nose, T. Uyama. Mukaishima Mar. Biol. Lab., Fac. Sci. and Lab. Marine Molec. Biol., Graduate Sch. Sci., Hiroshima Univ., Hiroshima.
   Recently we reported that a vanadium-associated protein (VAP) was extracted from the blood cells of an ascidian, Ascidia sydneiensis samea.  VAP was estimated to associate with vanadium at an approximate ratio of 1 mol : 16 mols.  In the present experiment, we raised a monoclonal antibody, designated F8DH, against VAP in blood cells of a vanadium-rich ascidian, A. sydneiensis samea.  Immunoblot analysis showed that F8DH recognized 2 related peptides of 15 kDa and 16 kDa of VAP.  Using F8DH, VAP was shown to be in the cytoplasm of vanadocytes and compartment cells, both of which cells were reported to contain vanadium.  F8DH also stained the vanadocytes distributed in the connective tissues around the alimentary canal, suggesting that vanadocytes in the connective tissue contained VAP. Furthermore, blood cells of 3 different species of ascidian having high levels of vanadium, A. sydneiensis samea, A. ahodori, and Ciona intestinalis, showed reactivity of F8DH but little reactivity was observed in 2 species having less vanadium, Halocynthia roretzi and Pyura michaelseni, suggesting that VAP recognized by F8DH is a common protein in vanadium-rich ascidians.

T. Uyama1, H. Takahashi2, N. Satoh2, and H. Michibata1.
1 Mukaishima Marine Biol. Lab., Fac. Sci. and Lab. Marine Molec. Biol., Graduate Sch. Sci., Hiroshima Univ., Hiroshima and 2Dept. Zool., Graduate Sch.Sci., Kyoto Univ., Kyoto
   To characterize a 45 kDa-antigen recognized by the monoclonal antibody S4D5, specific to vanadocytes in vanadium-rich ascidian Ascidia sydneiensis samea, the gene encoding the antigen was screened using S4D5 monoclonal antibody as a probe from a cDNA library prepared from the blood cells.  Three positive clones were isolated.  The longest insert was found to cover other two clones by nucleotide sequencing.  A full length cDNA designated sd45 for the newly cloned molecule was subcloned into the expression vector according to the sequencing strategy of the cDNA.  Consequently, sd45 encompassed 92-bp of the 5' untranslated region, a 1542-bp open reading frame, and 449 bp of the 3' untranslated sequence.  A search of the SwissProt sequence data base for similarities with sd45 detected a match with 6-PGDH both in levels of nucleotide and amino acid sequences.  The open reading frame encoded a protein of 483 amino acids with 74.6% identity and 87.1% similarity to 6-PGDH of sheep.  A 45 kDa-antigen recognized by the monoclonal antibody S4D5 is, therefore, shown to be 6-PGDH which is an enzyme of the pentose phosphate pathway.

 T. Kinoshita, T. Uyama, and H. Michibata, Mukaishima Marine Biol. Lab., Fac. Sci. and Lab. Marine Molec. Biol., Graduate Sch. Sci., Hiroshima Univ., Hiroshima.
  A 45 kDa-antigen recognized by the monoclonal antibody S4D5, specific to vanadocytes in vanadium-rich ascidian Ascidia sydneiensis samea, was found to be 6-phosphogluconate dehydrogenase (6-PGDH) which is an enzyme of the pentose phosphate pathway, based on cDNA isolation of RNA samples from blood cells of the ascidian.  Western blot analysis confirmed an abundance of 6-PGDH protein in the vanadocytes and localization of 6-PGDH in soluble extract in the blood cells.  Soluble extract further exhibited a correspondingly high level of 6-PGDH enzymatic activity. Km for the substrate and Vmax were calculated to be 62.5 micromol/l and 129 nmol/min at pH 8.9.  Almost all vanadium ions are reduced to the +3 oxidation state via the +4 oxidation state in vanadocytes, although vanadium ions are dissolved in the +5 oxidation state in sea water.  Some reducing agents must, therefore, participate in the accumulation process. These observations suggest that NADPH produced in the pentose phosphate pathway conjugates reduction of vanadium from the +5 oxidation state through the + 4 oxidation state in vanadocytes of ascidians.

K. Yamamoto, T. Uyama, and H. Michibata. Mukaishima Marine Biol. Lab., Fac. Sci. and Lab. Marine Molec. Biol., Graduate Sch. Sci., Hiroshima Univ., Hiroshima.
   Ascidians are sessile marine animals known to accumulate high levels of vanadium selectively in vanadium-containing blood cells (vanadocytes).  Almost all the vanadium accumulated in the vacuoles of vanadocytes is reduced to the +3 oxidation state via the +4 oxidation state, although vanadium is dissolved in the +5 oxidation state in sea water.  Some of the reducing agents that participate in the reduction have been found.  By chemical study, vanadium in the +5 oxidation state was reported to be reduced to the +4 oxidation state in the presence of NADPH.  The present study revealed the existence of glucose-6-phosphodehydrogenase (G6PDH), the first enzyme to produce NADPH in the pentose phosphate pathway, in vanadocytes of a vanadium-rich ascidian.  Western blot analysis with anti-G6PDH antibody demonstrated that a positive band of 58 kDa exhibited in soluble proteins.  The soluble proteins were confirmed to exhibit a high level of G6PDH enzymatic activity.  Km for the substrate and Vmax were 78.6 micromol/l and 220 nmol/min, respectively, at pH 8.0.  Taken together, it is suggested that G6PDH conjugates the reduction of vanadium from the +5 through to the +4 oxidation state in vanadocytes of ascidians.

S. Ohtake, T. Abe, F. Shishikura, and K. Tanaka, Dept. of Biol., Nihon Univ. Sch. of Med., Tokyo.
   In order to clarify the nature of the tunic cells of H. roretzi, we examined by transmission electron microscopy their fine structure after hatching out, and compared their morphological characteristics to those of the hemocytes in juvenile individuals. The cells were observed first in tunic immediately after the beginning of tail absorption distributed at the frontal part of the tadpole larva. The cells were characterized by their electron-dense cytoplasm and large nucleus. The cytoplasm wa packed with ribosome-like granules and well-developed endoplasmic reticulum. Mitochondria were relatively rich and Golgi were found occasionally. The number of cells, here called larval tunic cells, increased in tunic, and granules and vacuoles progressively appeared in their cytoplasm during metamorphosis.  14 days after tail absorption, we could distinguish in the tunic of juveniles two types of tunic cells.  One was probably the final stage of the larval tunic cell and the other, characterized by numerous dense granules, corresponded to the dense-granular cell in adult tunic.  The larval tunic cells probably stay in the tunic from metamorphosis to juvenile stage.  However, the fine structure of these cells differs from hemocytes of the juveniles.

E. Hirose1 and K. Chiba2.  1Dpt. Chem. Biol. & Mar. Sci., Univ. of the Ryukyus, Nishihara, and 2Dpt. Biol., Ochanomizu Univ., Tokyo.
    In the colonial ascidian Clavelina miniata, physical stimulation induces strong luminescence of yellow-green in the tunic.  The light source is a particular type of tunic cells (free cells in the tunic), and we call them tunic luminocyte.  Under blue light excitation, tunic luminocytes showed a weak yellow-green autofluorescence that is similar in color to the luminescence.  It is possible that this fluorescent molecule is concerned with the luminescence system in the cell.  Staining with acridine orange or LysoSensor (Molecular Probes Inc.) demonstrated the presence of acidic vacuoles in the tunic luminocytes.  The pair images of Nomarski DIC suggested that the acidic vacuoles seem to correspond to the clear vacuoles that are characteristic to the luminocytes.  Luminescence occurs when water is added to the freeze dried zooids.  The amount of luminescence is much smaller in acidic condition, e.g., pH 3, than that in neutral condition.  This might suggest that the acidic vacuole contributes to keeping the luminescent system silent within the cell.

T. Ishii1, Y. Saito2 and Y. Taneda3.  1Biol. lab., Coll. of Edu., Akita Univ., Akita; 2Shimoda Mar. Res. Center, Univ. of Tsukuba, Shimoda, Shizuoka; 3Dept. of Biol., Fac. of Edu., Yokohama National Univ., Yokohama.
        In the compound ascidian P. misakiensis, there are two different strains of body color pattern, white-spot and spotless. In this species, situs inversus or reversed zooids are easily induced experimentally from the normal zooids. We created chimeric zooids between body fragments with reversed white-spot and ones with normal spotless. Then we analyzed the visceral pattern of asexual offsprings from the chimeric zooids. The blastozooids with normal white-spot and reversed spotless appeared from the fused area of the chimeric zooids. These blastozooids were new combination of body color pattern and visceral pattern. A bud consists of two cell layers, epidermal layer and peribranchial wall. We already had revealed that the epidermal layer is the carrier of color pattern. Therefore, our results suggest that the information of visceral pattern exists in the peribranchial wall.

Y.Taneda, M. Ochmai, and Y. Arimoto.   Dept. of Biol., Fac. of Edu., Yokohama Natl. Univ., Yokohama.
    In botryllid ascidians a unique budding called vascular budding is well known.  In Botryllus primigenus it occurs in intact colony.  However in Botryllus schlosseri it  does not occur in intact colony, but it occurs when all zooids and buds are removed from the co1ony.  Why does it occur in B. schlosseri only when all zooids are removed from the colony?  First of all, we investigated the condition under which vascular budding could occur.  Not only in the case without any zooids and any buds but also in that with some zooids and no buds, vascular budding occurred.  In the latter case vascular budding occurred on the process of physiological regression of functional zooids.  Next we investigated the ability of vascular budding of the colony.  Colony with one functional zooid (oozooid or first blastozooid) did not have an ability of vascular budding,  while that with two or more functional zooids produced vascular buds.  Ability of vascular budding may be acquired between the colony with functional first blastozooid and that with functional second blastozooids.

T.Okada 1,  K.Nakajo 2,  R.Okagaki 3,  L.Chen 1, Y.Okamura 1,2,4.
1Lab of Cell Biochem., NIBH.,Tsukuba, 2Univ.of Tokyo.,Tokyo, 3Kasumigaura Hospital, Tsuchiura, 4 PRESTO, Japan Science and Technology Corp., "Intelligence and Synthesis".
    Recently  we cloned a cDNA putatively coding for voltage-gated Ca2+ channel, TuCa1, from young tadpole larvae of the ascidian, Halocynthia roretzi. The amino acid sequence of TuCa1 was 50% identical to that of the rabbit skeletal muscle Ca2+ channel alpha1-subunit. Here we studied expression of TuCa1 by whole mount in situ hybridization. TuCa1 mRNA was expressed in neurons, some epidermal cells, and all of muscle cells. We also compared development of calcium channel currents in cleavage-arrested neuronal and muscular blastomeres with the  two-electrode voltage clamp method. Kinetic properties were similar between Ba2+  currents through Ca2+ channels from neuronal and muscular blastomeres.  These results indicate that the single Ca2+ channel ƒ1-subunit gene encodes Ca2+ currents in ascidian neurons and muscle cells.This is compatible with the idea that the EC-coupling of ascidian striated muscle cell is similar to vertebrate "cardiac" type rather than to "skeletal" type.
K. Kyozuka1, R. Deguchi2, T. Mohri2 and S. Miyazaki2.
1Asamushi Mar. Biol. Stn., Tohoku Univ., Aomori and 2Natl. Inst. for Phys. Sci., Okazaki.
   We examined effects of sperm extract (SE) of Ciona savignyi, a supernatant of the centrifuged sperm homogenates, on changes in intracellular free Ca2+ concentration ([Ca2+]i) and the associated meiotic process in the oocyte.  Microinjection of SE into the unfertilized oocyte caused two sets of [Ca2+]i spikes, mimicking the [Ca2+]i pattern at normal fertilization. When SE was microinjected into the cortical region of the oocyte, the [Ca2+]i rise started from the nearest cortex and propagated across the oocyte.  A similar Ca2+ wave from the cortex was detected even when SE was injected into the central region.  SE injection also triggered meiosis resumption from metaphase I, resulting in emission of both first and second polar bodies.  SE was heat-unstable and was speculated to contain a molecule(s) between 30 kDa and 100 kDa.  These results support the ''sperm content model'' that sperm introduce soluble substances inside the egg following gamete fusion to activate the egg.

K. Terakado, Dept. Regul. Biol., Fac. of Sci., Saitama Univ., Urawa.
   Using antiserum against to human or salmon GnRH, we stained immunohistochemically the neural complex and its neighboring regions by streptavidin-biotin-peroxidase complex (ABC) method in adults and juveniles of the ascidian Halocynthia roretzi.  Adult neural complex were also examined with immunoelectron microscopy.  There were numerous GnRH immunoreactive neurons in the cerebral ganglion and along the dorsal strand.  In addition, many immunoreactive axons ran in the connective tissue matrix to pharyngeal epithelium, epithelium of dorsal tubercle, ciliated epithelium of gill baskets, etc. These axons originated from distributed in the connective tissue matrix.  GnRH neurons along the dorsal strand seemed not to extend to these regions and remained within the blood sinus.  In small juveniles (about 2mm in length), there were already a number of GnRH immunoreactive neurons arranged longitudinally in the cerebral ganglion and some cell bodies arranged in lines, presumably along the dorsal strand.  However, juveniles reared for 30 days in refrigerator were GnRH negative in whole body.  Immunoelectron microscopy revealed that GnRH in localized in the 100-250nm granules in the GnRH neurons associated with the dorsal strand.  These results suggest that GnRH functions as a neurotransmitter or a neuromodulator as well as a hormone.

G. Kawahara1, K. Terakado2, K. Yamamoto1, S. Kikuyama1
1Dept. Biol., Schl. Education, Waseda Univ., Tokyo, and 2Dept. Regul. Biol., Fac. of Sci., Saitama Univ., Urawa.
    The neural complex (dorsal strand, cerebral ganglion and neural gland) of  Halocynthia roretzi was examined using light and electron microscopy.  Cells scattered along the dorsal strand and in the cerebral ganglion contained electron-dense secretory granules with diameters of 100-250nm.  Immunoelectron-microscopic studies with an antiserum against bullfrog prolactin (PRL) revealed that the secretory granules of some of the granulated cells contained a PRL-like immunoreactive substance.  Gel chromatography and western blot analysis revealed that the PRL-like substance is a protein with an apparent molecular mass of 75kDa.  The cells of the  neural gland and the dorsal strand neither contained the dense secretory granules nor showed PRL immunoreactivity.  These findings raise the possibility that the granulated cells along the dorsal strand and those in the cerebral ganglion are phylogenetic progenitors of vertebrate adenohypophyseal cells.

T.Iwasa, T.Tajika, K.Kanehara, M.Ohkuma, M.Tsuda. Dept. of Life Sci., Fac. of Sci., Himeji Inst. of Technol., Hyogo.
     The small number of neural cells in the head and the well-characterized cell lineage based on the stereotyped cell cleavage pattern make the ascidian larva a simple system for examining neural differentiation and neural signal transduction. We have isolated a novel G protein subunit, Gn, from the larvae of  Halocynthia roretzi .  Gn has the consensus sequence of N-terminal myristylation, which is characteristic for various Gi class a subunits.  Gn has a unique amino acid sequence in its C-terminal and lacks a cystein residue for ADP-ribosylation by pertussis toxin. These data suggest that Gn defines a novel subclass within the Gi family of a subunits.  This is also supported by a phylogenetic tree of Ga subunits made by the NJ-method.  In situ hybridization and northern analysis of the Gn message revealed that a small amount of the message is maternal, evenly distributed within the blastomere, and the expression of the message is first detected at the 64-cell stage in eight blastomeres containing neural lineage cells.  At the early tailbud stage, it is expressed in several cells of brain and mesenchyme. Four more cDNA clones were isolated from H. roretzi larvae.  They were Gs, Gi, Gq and one similar to Gq.  The class of the last one is not clear yet.

M. Ohkuma and M. Tsuda.  Dept. of Life Sci., Fac. of Sci., Himeii lnst. of Tech., Hyogo.
    Light signals can shift the phase of the circadian oscillator of the biological clock for most animals.  In the avian pinealocyte, it was shown that the retinal protein pinopsin is a photoreceptor that resets the phase of the circadian pacemaker controlling the rhythmic production of melatonin D.  Spawning of ascidians has been reported to be triggered by exposure to light following a period of darkness.  It was suggested that the cerebral ganglion is a possible photoreceptor that is related to gamete release in the adult ascidian.  ln order to determine the localization of retinal protein in the cerebral ganglion of ascidians, the fluorescence of retinal protein were used for histochemical studies.  As in many other ascidian species, the thickest nerves arise as a pair from the anterior and posterior end of the ganglion.  Ye11ow- green fluorescence due to retinal protein appeared on both sides of the crossing point of the branch, indicating the presence of retinal protein.

F. Nakaya1, Y. Saito2 and T. Motokawa1.  1Basic Biol., Fac. of Biosci. & Biotechnol., Tokyo Inst. of Technol.; 2Shimoda Mar. Res. Center, Univ. of Tsukuba.
     The allometric relationship between metabolic rate and body size is well established in organisms with individuality. In colonial organisms, however, there is little research on the relationship between metabolic rate and body size. We measured the metabolic rate of the colonial ascidian Botryllus schlosseri to investigate the relationship between metabolic rate and colony size. A small colony was put on a slide glass which was set in a container submerged in the open sea water. The colony spread on a slide glass as it grew to be a sheet. The zooid size did not change with colony growth. We measured oxygen consumption of these reared colonies of various sizes with 30-800 zooids by oxygen electrodes. The allometric relationship obtained is:
  VO2 = 0.0643 W 0.674  (VO2: oxygen consumption rate, ml/h; W: wet weight, g; 20)
A clear allometric relationship exists between the metabolic rate and colony size of this colonial ascidian.

T. Kakuda, Dept. of Biol. Sci., Kanagawa Univ., Hiratsuka.
    Genetic differences among two morphological forms of the ascidian Halocynthia hispida - igaboja and ritteri forms, sympatric at Shimokita Peninsula - were investigated by restriction fragment analysis of mitochondrial DNA (mtDNA) and allozyme analysis.  Based on restriction patterns of eight restriction enzymes, there were no shared restriction patterns between two forms.  The nucleotide divergence d which is estimated by the proportion of shared restriction fragments (Nei & Li '79) were 4.246x10^-2.  In allozyme analysis, a remarkably different electrophoretic pattern was observed in SOD, between the two forms.  These differences are far larger within reproductive isolated three types of H. roretzi, and equal to the rate between different species, H. roretzi and H. aurantium.  In addition, by some morphological characters, H. hispida were definable to the two forms.  At the end of September, the igaboja form was observed spawning, but ritteri form did not.  These results suggest that the two forms of H. hispida may represent separate species.

S. Minowa1, C.Nishijima1,C. Haraguchi1, M. Hashimoto1,H. Fujisawal and T. A. Nomaguchi2.
1Fac. of Educ., Saitama Univ., Urawa and 2Dept. of Cell Biol., Tokyo Metropolitan lnst. of Gerontol., Tokyo.
    Embryonic thermotolerance of the ascidian Ciona savignyi was examined with special reference to acclimatization to seasonal changes in seawater temperature. This ascidian spawns throughout the year. Its life span is completed within six months and depends on the cumulative environmental temperature. The optimal temperature range for development from early cleavage stage to metamorphosis in embryos produced by individuals raised in warmer seasons differed significantly from that of individuals raised in colder seasons.  Within the common optimal temperature range, developmental times at any given temperature were the same for both groups of embryos. The thermal acclimation of ascidian embryos is discussed in comparison with that of echinoid embryos.

K. Kuobkawa1 and M. Nozaki2. 1Ocean Research Inst., Univ. of Tokyo, Tokyo,  2 Marine Biol. St., Niigata Univ., Niigata.
      Amphioxus were collected in the coast of the Atsumi peninsula, Aichi prefecture during  the summer and early autumn of 1995, 1996 and 1997.  They were fixed in a 10% formaldehyde solution except for individuals collected in September, 1995. The latter individuals were fixed in a Bouin-Hollande sublimate fixative solution for immunohistochemistry.  When an anti-human LH antibody was used, a strong immunoreactivity was detected in the apical area of Hatschek's pit, but no immunoreactions were detected against antibodies of Salmon GTH I and II, and sturgeon GTH. The positive reaction was observed in all individuals examined which possessed different stages of the gonad, matured, regressed and developing.  Interestingly, small spots showing a strong immunoreactivity were found in the abdominal part of the neural tube extending to Hatschek's pit in individuals possessing the developing gonad.  This suggests that the immunopositive substance to anti-human LH antigen exists not only in Hatschek's pit but also in the neural tube, although the relation between these two regions is still to be elucidated. Judging from the gonadal condition, this species breeds once a year between July and August. In September, gonads are completely regressed and only early stage germ cells were observed . Mature gonads were usually observed in individuals with longer body length than 30 mm.  This species seems to become sexually mature within a short time, about 6 weeks after metamorphosis and settlement.
S. Kimura1, Y. Kawamura1, A. Watanabe1, K. Kubokawa2, and K. Maruyama1.  1Dept. of Biol., Fac. of Sci., Chiba Univ., Chiba,  and 2Ocean Res. Inst., Univ. of Tokyo.
     Connectin/Titin is a 3000 kDa elastic protein that positions the myosin filament at the center of a sarcomere in vertebrate striated muscle myofibrils.  It has been shown that connectin is present in all the vertebrate skeletal muscles examined from mammals to fishes including lamprey (Cyclostomata). In the present study, it was demonstrated that the 3000 kDa band of Amphioxus striated muscle in an SDS gel electrophoresis reacted with polyclonal antibodies to chicken breast muscle connectin (Pc1200).  Although other kinds of antibodies to connectin did not give positive reactions, it is regarded that a connectin-like 3000kDa protein is present in protochordate skeletal muscle. In addition, it is observed that a 750 kDa band of Amphioxus striated muscle crossreacted with polyclonal and monoclonal antibodies to skeletal muscle nebulin. This information is of particular interest because nebulin is present in invertebrate skeletal muscle as well as in vertebrate skeletal muscle.

2.  Italian Embryol. Group 43rd annual meeting, Grottammare, Italy June 8-11, 1997. Publ. in Anim. Biol. 6(3).

L. Ballarin, F. Cima and A. Sabbadin.  Dpto. di Biol., Univ. di Padova, Padova, Italy.
    Even if ascidian hemocytes are the subject of a vast scientific literature, controversial aspects still exist mainly regarding their differentiation pathways and their biological role. For instance phagocytosis raises interesting questions about the analogies, homologies of this process with its counterpart in vertebrates. We focussed our attention to phagocytosis in
the colonial ascidian B. schlosseri, both in vitro and in vivo. In vitro investigations led us to identify the phagocytes (hyaline amoebocytes and macrophage-like cells) and at least two receptors involved in the recognition and adhesion of foreign/non-self particles to the phagocyte surface. One is the mannose receptor, required to bind yeast in the absence of any opsonizing molecule and playing a similar role in mammals. The other is the antigen CD36 involved in the recognition of apoptotic cells by mammalian macrophages and probably required for the recognition of apoptotic cells of degenerating zooids ingested during the takeover phase of the colonial life cycle. The study of phagocytosis in vivo took advantage of the peculiar life cycle of Botryllus colonies, which is the sum of several blastogenetic cycles: at 19° C old zooids are weekly resorbed and replaced by their palleal buds reaching maturity and opening the siphons. During the takeover we observed a significant increase in the frequency of circulating macrophage-like cells containing digested material inside their vacuoles and a parallel decrease in the frequency of hyaline amoebocytes. This fact supports our hypothesis of a single phagocyte-type in Botryllus, with two functional stages: the hyaline amoebocyte, moving actively and ingesting foreign particles, which changes its morphology to the macrophage-like one upon ingestion. These changes were coupled with a cytoskeletal reorganization, both in the actin and tubulin components. The takeover phase is characterized by a massive infiltration of phagocytes inside the tissues of the old zooid (not into the bud tissues) and by an intense phagocytosis: we observed, analogously to that reported for the same process in mammals, a significant increase in the concentration of reactive oxygen metabolites, such as peroxides, and of hydrolytic lysosomal enzymes, such as acid phosphatase.  We also observed a significant increase in the frequency of circulating macrophage-like cells containing apoptotic cells inside their vacuoles, as revealed with the TUNEL reaction, thus confirming the morphological data indicating the occurrence of apoptosis associated with zooid regression. At the beginning labeling was limited to the nucleus of the ingested cells whereas, as regression went on, it diffused inside the vacuoles, due to the release of small DNA fragments from the nuclear envelope. We are now interested in defining the morphological changes on the surface of senescent cells responsible for their recognition by phagocytes.

C. Sotgia, U. Fascio, S. Groppelli,* M. De Vincentiis and F. De Bernardi.
Dept. of Biol., Univ.of Milan; *Dept. of Genetic, General & Molec. Biol., Univ. of Naples.
    Ascidian embryos showed malformations similar to those described in many vertebrate embryos. Previous researches on amphibian embryos (Xenopus laevis) have shown that treatment with RA at early gastrula stages affects the development of head structures leading to dose-dependent progressive truncation of the antero-posterior axis, preventing the formation of forebrain, cement gland and hatching glands (Durston et al., 1989. Nature 340:140-144). The results of RA treatment are fully described in two species of solitary ascidian: Phallusia mammillata (De Bernardi et al.1994. Anim. Biol. 3:75-81) and Halocynthia roretzi (Katsuyama et al.1995. Development 121:3197-3205). In both species malformations were detectable in the larvae, the anterior part of which were truncated, and adhesive papillae failed to develop; the posterior part of the larvae was not affected.  Phallusia embryos at the two blastomere stage treated with 10-7 M RA developed larvae that were unable to hatch. On the contrary, Halocynthia embryos treated with higher RA concentration developed larvae that normally hatched. We tried to understand the reason for the different effect of RA on the two species and we performed RA experiments on Ciona intestinalis, a solitary ascidian belonging to a different family. C. intestinalis embryos were treated at early cleavage stage with 10-6 M RA for at least four hours in the dark. We obtained hatched swimming larvae with rather normal tail. but the cephalenteron was small and rounded. Papillae were absent and papillary nerves were damaged. The brain vesicle was reduced, lacking sensory organs whose pigment was extruded. The mesenchymal cells beneath the papillae were absent and the other mesenchymal cells were kept close to the epithelium of the pharynx. The malformations obtained confirmed that the effect of RA is always the same in ascidian embryos and it is comparable to that in vertebrate embryos. We used a polyclonal antibody raised against Ciona intestinalis hatching enzyme to perform whole mount immunofluorescence experiments. By confocal microscopy bright fluorescence was localized in the peripheral secreting cells of the adhesive papillae and in the cells of the epidermis of the tip of the tail of newly hatched C. intestinalis larvae. The same antibody recognized hatching enzyme in Phallusia mammillata newly hatched larvae, in which only the peripheral secreting cells of the adhesive papillae react. Since RA causes truncation of the anterior region of the larvae, papillae do not develop, and consequently Phallusia larvae cannot hatch. Ciona intestinalis larvae lacking the papillae after RA treatment do hatch by hatching enzyme secreted by the tail epidermal cells, which are not affected by RA treatment.

U. Fascio, S. Groppelli,**A. D’Aniello, *M. De Vincentiis, *S. Scippa and F. De Bernardi.
Dept. of Biol., Univ. of Milan;*Dept. of Genetic, Gen. & Molec. Biol., Univ. of Naples; **Dept. Biochem. & Molec. Biol., Staz. Zoologica “Anton Dohrn”,Naples.
    A polyclonal antibody raised against the hatching enzyme of Ciona intestinalis (D’Aniello et al., 1997 Biochim. Biophys. Acta, 1339:155-163) was used on larvae of different ages in whole mount immunofluorescence experiments to localize  the cells secreting the enzyme. After staining with FITC-conjugated second antibody, the larvae were observed by confocal microscopy. The larvae just before hatching (9-10 hours after fertilization) showed the presence of the enzyme in the peripheral cells of the adhesive papillae. These cells secrete and produce the hatching enzyme which digests the egg chorion. The newly hatched larvae showed fluorescence both on the adhesive papillae and in the epidermal cells of the tip of the tail. Higher magnification confocal images of the papillae revealed bright fluorescence both in peripheral cells of the papillae and in the cavity between the tunic and the apex of the papillae (hyaline cap). The secreting cells were still fluorescent. The swimming larvae maintain the fluorescence in the peripheral cells and in the hyaline cap for some hours until the beginning of metamorphosis, when the fluorescence of the tip of the tail disappears. We also used the same antibody on larvae of another ascidian, Phallusia mammillata. The peripheral cells of the papillae of the newly hatched larvae were fluorescent, and a bright fluorescence was also present between the two layers of the tunic above the papillae and the anterior part of the cephalenteron. We never observed fluorescence in the cells of the tail epidermis. When the larvae start the tail resorption, just before settlement and the beginning of metamorphosis, some vesicles with a positive fluorescent reaction appeared at the tip of the adhesive papillae. These tunic vesicles, already observed by SEM, soon break and some microvilli protrude from the central sensorial cells (De Bernardi et al., 1996 Anim. Biol. 5:83). We hypothesize that at this stage the hatching enzyme, or a modification of it, could help the next protrusion of the microvilli interacting with protein components of the tunic.

L. Manni, G. Zaniolo & P. Burighel.  Dpto. di Biol., Univ. di Padova, Italy.
    The brain of adult ascidians is composed of an oval cerebral ganglion lying within the connective tissue of the mantle between the two siphons. Some nerves, directed to the periphery, emerge from its ends. Knowledge of the peripheral nervous system (PNS) and, in particular, of its development in ascidians, is incomplete. Detailed information on branchial basket innervation was recently obtained (Arket et al.1989, Cell Tiss. Res. 257:285-294) by staining nerves for acetylcholinesterase (AChase), which is not restricted to synaptic sites but occurs throughout the neurons. Using the same method, we studied the complete organization of the cholinergic PNS and its development in relation to forming organs in blastozooids of the colonial ascidian Botrylllus schlosseri. B. schlosseri forms colonies of  numerous zooids, each possessing its own nervous system. Colonies cultured in the laboratory were followed daily under the stereomicroscope, and buds and adults were dissected and processed at appropriate stages of development to reveal AChase activity. Whole mount preparations and thick sections of embedded specimens were observed.
    Achase activity appears in buds at the stage at which all organs are present in rudimentary form, before the heart begins to beat. In these young buds, 4 or 5 bundles of neurites emerge from each side of the developing ganglion, running in the mantle toward the latero-ventral surface of the bud. During development, the number of nerve roots increases to 10-15 in both sides of the ganglion, in addition to some anterior, posterior and ventral nerves. Nerves split repeatedly into thinner branches penetrating bud organs. In particular, nerves from the anterior end of the ganglion innervate the most anterior region of the animal (especially the oral siphon) and give rise to two main parallel nerves, directed ventrally to the endostyle. Nerves from the posterior end run in the dorsal-posterior mantle to the developing atrial siphon, and a bundle of neurites (the visceral nerve) passes from the ventral surface of the ganglion to the viscera, running along the roof of the branchia. The viscera are also innervated by branches of nerves coming from the mantle and passing through the trabeculae, and from the endostyle region. Nerves display numerous swellings along their length, thus looking like a string of pearls, sometimes very large and regularly arranged.  Nerve development usually parallels that of the organs they accompany. In the adult, local nerve plexa may be recognized innervating branchia, gut (stomach, intestine and pyloric gland), heart and the two siphons. Activity-dependent selection of neurites probably occurs during development, so that adults possess fewer nerves than buds. In fact, only two pairs of anterior and posterior nerves, one visceral nerve, and 4 or 5 minor lateral nerves lead  from the brain in adults.

3. RETINOIDS ’97  European Retinoid Research Group, Nice (France) Sept. 28- Oct. 1, 1997.

F. De Bernardi, S. Groppelli and C. Sotgia.  Dept. of Biol., Univ. of Milan, Milan, Italy.
    Embryos of Phallusia mamillata and Ciona intestinalis were treated at two-cell or at four-cell stage with different concentrations of all-trans retinoic acid (RA) for two hours, rinsed with sea water and allowed to develop at 20°C to the swimming larva stage. The RA-treated embryos give rise to hatching or non-hatching larvae that show dose-dependent reduction of anterior adhesive papillae, alterations of the brain vesicle and of sensory organs and extrusion of pigment. Pharynx structures appeared to be unaffected, but the button cells of P. mammillata larvae appeared unable to move and remained close to the endodermal cells. The larvae, still inside the chorion, retracted the tail and carried out metamorphosis quite regularly, but without settlement. The Ciona intestinalis larvae hatched but failed to develop adhesive papillae. The effect of RA on both ascidian species was to truncate the anterior structures, as it does in amphibian and in other vertebrate embryos, probably acting on migrating cells.

4. Gordon Conf. on natural products research, Ventura, Calif. Feb. 22-27, 1998.

M.S.G. Pavao1,2, K. Rebeca1, L.C.F. da Silva1, P.A.S. Mourao1, N.S. Colwell2 & D.M. Tollefsen2.
1Dept. Biochquim., Inst. Ciencias Biomedicas, Univ. Fed. Rio de Janeiro, C.P. 68041, Rio de Janeiro, RJ 21941-540, Brazil; 2Div. of Hematol., Depts. Internal Med, Biochem & Mol. Biophys., Washington Univ. Sch. Med., St. Louis, MO.
    We previously isolated an oversulfated dermatan sulfate-like glycosaminoglycan from the body of the ascidian Ascidia nigra, composed of disaccharide units of [IdoAc(2S04) - GalNAc (6S04)] (J. Biol. Chem. 270:31027-31036, 1995). Different from mammalian dermatan sulfate, the ascidian glycosaminoglycan had no HCII-mediated thrombin inhibitory activity because of the absence of sulfation on carbon 4 of the galactosamine residues. We now describe a new oversulfated dermatan sulfate with a high HCII activity, isolated from the body of the ascidian Halocynthia pyriformis. The structure was determined by analysis with specific glycosidases and sulfatases, and by 1H Nuclear Magnetic Resonance.  The polymer is composed of repeating disaccharide units of [IdoAc(2S04) - GalNAc (4S04)]. It has a very high HCII-mediated thrombin inhibitory activity, 13 times more potent than that of the mammalian counterpart, estimated by a chromogenic substrate assay.  The activity of the ascidian glycosaminoglycan is specific for HCII, and experiments using native or mutated recombinant HCII indicate that it binds to the dermatan sulfate binding site on the inhibitor. We also describe the occurrence of a heparin-dependent thrombin inhibitor in the ascidian hemolymph, which inhibited thrombin activity in the presence of 1ug of heparin.  The inhibitor forms a 97,000 MW covalent complex with human thrombin.  No inhibition or complex is observed in the presence of mammalian dermatan sulfate.

                                                                        NEW PUBLICATIONS
    Abbott, D.P., A.T. Newberry & K.M. Morris  1997.  Reef and Shore Fauna of Hawaii. 6B: Ascidians (Urochordata). G. Lambert, ed. .  Bishop Mus. Press, Honolulu, 64 pp.
    Ayre, D.J., A.R. Davis, M. Billingham, T. Llorens & C. Styan 1997.  Genetic evidence for contrasting patterns of dispersal in solitary and colonial ascidians.  Mar. Biol. 130:51-62.
    Bruno, J.G., S.B. Collard & A.R.J. Andrews 1997.  Further characterization of tunicate and tunichrome electrochemiluminescence.  J. Biolumin. & Chemilumin. 12:155-164.
    Burighel, P., N.J. Lane, G. Zaniolo & L. Manni 1998.  Neurogenic role of the neural gland in the development of the ascidian, Botryllus schlosseri (Tunicata, Urochordata).  J. Comp. Neurol. 394:230-241.
    Cáceres, C.E. 1997.  Dormancy in invertebrates.  Invert. Biol. 116:371-383.
    Cammarata, M., V. Arizza, N. Parrinello, G. Candore & C. Caruso 1997.  Phenoloxidase-dependent cytotoxic mechanism in ascidian (Styela plicata) hemocytes active against erythrocytes and K562 tumor cells.  Europ. J. Cell Biol. 74:302-.
    Caracciolo, A., I. Gesualdo, M. Branno, F. Aniello, R. Di Lauro & A. Palumbo 1997.  Specific cellular localization of tyrosinase mRNA during Ciona intestinalis larval development.  Develop. Growth Differ. 39:437-444.
    Cima, F., L. Ballarin, G. Bressa, A. Sabbadin & P. Burighel 1997.  Triphenyltin pesticides in sea water as immunotoxins for tunicates.  Mar. Chem. 58:267-274.
    Coles, S.L., R.C. DeFelice, L.G. Eldredge & J.T. Carlton 1997.  Biodiversity of Marine Communities in Pearl Harbor, Oahu, Hawaii with Observations on Introduced Exotic Species. Bishop Museum Press, Honolulu, 76 pp. + appendices.
    Coniglio, L., A. Morale, C. Angelini & C. Falugi 1998.  Cholinergic activation of settlement in Ciona intestinalis metamorphosing larvae.  J. Exp. Zool. 280:314.
    Dalby, J.E. 1997.  Reproductive and electrophoretic evidence for genetic maintenance of dimorphism in the ascidian Pyura stolonifera near Melbourne, Australia.  Ophelia 47:227.
    Erives, A., J.C. Corbo & M. Levine 1998.  Lineage-specific regulation of the Ciona snail gene in the embryonic mesoderm and neuroectoderm.  Dev. Biol. 194:213-225.
    Ford, P.W. & B.S. Davidson 1997.  Plakinidine D, a new pyrroloacridine alkaloid from the ascidian Didemnum rubeum.  J. Nat. Prod. 60:1051-1053.
    Freyer, A.J., A.D. Patil, L. Killmer, N. Troupe, M. Mentzer, B. Carte, et al. 1997.  Three new pseudodistomins, piperidine alkaloids from the ascidian Pseudodistoma megalarva.  J. Nat. Prod. 60:986-990.
    Gabriele, M., V. Putrone & R. Brunetti 1997.  Morphometrics and energetic value of Adriatic ascidians.  Cah. Biol. Mar. 38:169-174.
     Galinis, D.L., T.C. McKee, L.K. Pannell, J.H. Cardellina & M.R. Boyd 1997.  Lobatamides A and B, novel cytotoxic macrolides from the tunicate Aplidium lobatum.  J. Org. Chem. 62:8968-8969.
    Gianguzza, M. & G. Dolcemascolo 1997.  Adhesive organs in the swimming larvae of Ascidia malaca: an ultrastructural and cytochemical study.  Anim. Biol. 6:53-60.
    Grumetto, L., M. Wilding, M.L. DeSimone, E. Tosti, A. Galione & B. Dale 1997.  Nitric oxide gates fertilization channels in ascidian oocytes through nicotinamide nucleotide metabolism.  Biochem. Biophys. Res. Commun. 239:723-728.
    Hatcher, A.M. 1997.  Comparison of biomass and percentage cover of sessile epibiota on the Poole Bay Artificial Reef.  Ophelia 47:55-62.
    Hibino, T., T. Nishikata & H. Nishida 1998.  Centrosome-attracting body: a novel structure closely related to unequal cleavages in the ascidian embryo.  Develop. Growth Differ. 40:85-96.
    Hirano, T. & H. Nishida 1997.  Developmental fates of larval tissues after metamorphosis in ascidian Halocynthia roretzi. I. Origin of mesodermal tissues of the juvenile.  Dev. Biol. 192:199-210.
    Hogarth, A.N., P.S.P. Thong, D.J.W. Lane & F. Watt 1998.  A quantitative, comparative study of element variations found within the range of blood cells from the tropical ascidian Phallusia philippinensis, using the nuclear microscope.  Acta Zool. 79:75.
    Hori, S., T. Saitoh, M. Matsumoto, K.W. Makabe & H. Nishida 1997.  Notch homologue from Halocynthia roretzi is preferentially expressed in the central nervous system during ascidian embryogenesis.  Dev. Genes & Evol. 207:371-380.
    Kondow, A., S. Yokobori, T. Ueda & K. Watanabe 1998.  Ascidian mitochondrial tRNA(Met) possessing unique structural characteristics.  Nucleos. & Nucleot. 17:531-540.
    Kusakabe, R., T. Kusakabe, N. Satoh, N.D. Holland & L.Z. Holland 1997.  Differential gene expression and intracellular mRNA localization of amphioxus actin isoforms throughout development: implications for conserved mechanisms of chordate development.  Dev. Genes & Evol. 207:203-215.
    Kusakabe, T. 1997.  Ascidian actin genes: developmental regulation of gene expression and molecular evolution.  Zool. Sci. 14:707-718.
    Lacalli, T.C. 1994.  Apical organs, epithelial domains, and the origin of the chordate central nervous system.  Amer. Zool. 34:533-541.
    Lacalli, T.C. 1996.  Landmarks and subdomains in the larval brain of Branchiostoma: vertebrate homologs and invertebrate antecedents.  Israel J. Zool. 42:S131-S146.
    Lacalli, T.C. 1997.  The nature and origin of deuterostomes: some unresolved issues.  Invert. Biol. 116:363-370.
    Lambert, C.C. & G. Lambert 1998.  Non-indigenous ascidians in southern California harbors and marinas.  Mar. Biol. 130:675-688.
    MacLean, D.W., T.H. Meedel & K.E.M. Hastings 1997.  Tissue-specific alternative splicing of ascidian troponin I isoforms--redesign of a protein isoform-generating mechanism during chordate evolution.  J. Biol. Chem. 272:32115-32120.
     Marino, R., M.R. Pinto, F. Cotelli, C.L. Lamia & R. De Santis 1998.  The hsp70 protein is involved in the acquisition of gamete self-sterility in the ascidian Ciona intestinalis.  Development 125:899-907.
    Matsushita, M., Y. Endo, M. Nonaka & T. Fujita 1998.  Complement-related serine proteases in tunicates and vertebrates.  Current Opinion in Immunology 10:29-35.
    Miller, R.L. & J. Cosson 1997.  Timing of sperm shedding and release of aggregates in the salp Thalia democratica (Urochordata; Thaliacea).  Mar. Biol. 129:607-614.
    Miya, T., K. Morita, A. Suzuki, N. Ueno & N. Satoh 1997.  Functional analysis of an ascidian homologue of vertebrate Bmp-2/Bmp-4 suggests its role in the inhibition of neural fate specification.  Development 124:5149-5159.
    Monniot, C. 1997.  Ascidies phlebobranches du canal du Mozambique.  Zoosystema 19:557-571.
    Nakatani, Y. & H. Nishida 1997.  Ras is an essential component for notochord formation during ascidian embryogenesis.  Mechanisms of Development 68:81-90.
    Nishikawa, T. & M. Nishida 1997.  Problems in lancelet systematics.  pp. 241-246 in Advances in Comparative Endocrinology. Proc. 13th Intl. Cong. Comp. Endocrinol,  ed. vol. 1, ed. by Kawashima, S. & S. Kikuyama.
    Olguin-Uribe, G., E. Abou-Mansour, A. Boulander, H. Debard, C. Francisco & G. Combaut 1997.
6-bromoindole-3-carbaldehyde, from an Acinetobacter sp. bacterium associated with the ascidian Stomozoa murrayi.  J. Chem. Ecol. 23:2507-2522.
    Pietra, F.  1990.  A Secret World: Natural Products of Marine Life. Birkhauser Verlag, Basel, Switzerland, 279 pp.
    Plubrukarn, A. & B.S. Davidson 1998.  Arnoamines A and B, new cytotoxic pentacyclic pyridoacridine alkaloids from the ascidian Cystodytes sp.  J. Org. Chem. 63:1657-1659.
    Pond, D.W. 1998.  Lipid composition of the pelagic tunicate Dolioletta gegenbauri (Tunicata, Thaliacea).  J. Plankton Res. 20:169.
    Riisgård, H.U. & P.S. Larsen 1995.  Filter-feeding in marine macro-invertebrates: pump characteristics, modelling and energy cost.  Biol. Rev. 70:67-106.
    Satou, Y. & N. Satoh 1997.  posterior end mark 2(pem-2), pem-4, pem-5, and pem-6; Maternal genes with localized mRNA in the ascidian embryo.  Dev. Biol. 192:467-481.
    Shimauchi, Y., H. Yasuo & N. Satoh 1997.  Autonomy of ascidian fork head/HNF-3 gene expression.  Mechanisms of Development 69:143-154.
    Shishikura, F., T. Abe, S.I. Ohtake & K. Tanaka 1997.  Purification and characterization of a 39,000-Da serine proteinase from the hemolymph of a solitary ascidian, Halocynthia roretzi.  Comp. Biochem. Physiol. B. 118:131-142.
    Stewart-Savage, J. & P.O. Yund 1997.  Temporal pattern of sperm release from the colonial ascidian, Botryllus schlosseri.  J. Exp. Zool. 279:620-625.
    Stock, D.W., J.M. Quattro, G.S. Whitt & D.A. Powers 1997.  Lactate dehydrogenase (LDH) gene duplication during chordate evolution: the cDNA sequence of the LDH of the tunicate Styela plicata.  Molec. Biol. & Evol. 14:1273-1284.
    Swalla, B.J. 1993.  Mechanisms of gastrulation and tail formation in ascidians.  Micros. Res. & Tech. 26:274-284.
    Takamura, K. 1998.  Nervous network in larvae of the ascidian Ciona intestinalis.  Dev. Genes & Evol. 208:1-8.
    Taylor, S.W., B. Kammerer, G.J. Nicholson, K. Pusecker, T. Walk, E. Bayer, et al. 1997.  Morulin Pm: a modified polypeptide containing TOPA and 6-bromotryptophan from the morula cells of the ascidian, Phallusia mammillata.  Arch. Biochem. Biophys. 348:378-390.
    Tsukamoto, S., H. Kato, H. Hirota & N. Fusetani 1998.  Antifouling and metamorphosis-promoting compounds from the marine sponges Pseudoceratina purpurea and Agelas mauritiana [induce metamorphosis in 2 ascidian spp.].  pp. 399-412 in Sponge Sciences - Multidisciplinary Perspectives.  Proc. of the 1996 Intl. Conf. on Sponge Science,  ed. vol., ed. by Watanabe, Y. & N. Fusetani.
    Vorontsova, M.N., L.P. Nezlin & I.A. Meinertzhagen 1997.  Nervous system of the larva of the ascidian Molgula citrina (Alder and Hancock, 1848).  Acta Zool. 78:177-186.
    Wada, H., H. Saiga, N. Satoh & P.W.H. Holland 1998.  Tripartite organization of the ancestral chordate brain and the antiquity of placodes: insights from ascidian Pax-2/5/8, Hox and Otx genes.  Development 125:1113-1122.
    Wang, G.Y.S., B.M. Borgeson & P. Crews 1997.  Pitholides A-D, polyketides from a marine tunicate-derived culture of Pithodes sp.  Tetrahed. Lett. 38:8449-8452.
    Welsch, U. 1984.  Chapter 44: Urochordata.  pp. 800-816 in Biology of the Integument,  ed. vol. 1. Invertebrates, ed. by Bereiter-Hahn, J., A.G. Matoltsy & K.S. Richards.
    Wilding, M. & B. Dale 1997.  Sperm factor: what is it and what does it do?  Molec. Human Repro. 3:269-273.
    Young, C.M., M.A. Sewell, P.A. Tyler & A. Metaxas 1997.  Biogeographic and bathymetric ranges of Atlantic deep-sea echinoderms and ascidians: the role of larval dispersal.  Biodiver. & Conserv. 6:1507-1522.
    Zaniolo, G., L. Manni, R. Brunetti & P. Burighel 1998.  Brood pouch differentiation in Botrylloides violaceus, a viviparous ascidian (Tunicata).  Invert. Repro. & Develop. 33:11-24.