Number 39
May 1996

This issue is being sent electronically to all AN subscribers who have given us their email addresses. This will save us hundreds of dollars, and a lot of time. If you have email and have not yet notified us, please send us an email! It is such an incredibly efficient way to communicate, especially between countries. We hope you will find the enclosed list of AN subscriber email addresses useful (see page 12). Also in this issue are two thesis abstracts, the abstracts from the Sept. 1995 annual meeting of the Zoological Society of Japan and the June 1995 meeting of the Italian Embryology Group. We now have our own HomePage on the Internet: http://nsm.fullerton.edu/~lamberts/ascidian where you can see all the issues of AN for the past six years, #31-#39.

Our 6 weeks in New Zealand over Christmas and January was very productive. It was gratifying to see that there were just as many ascidians with symbiotic eukaryotic algae in their tunics as during our last visit (see New Publications listing of our recent paper on this topic). We also spent some time gazing at the yellow eyed penguins. Charley worked further on brooding in Asterocarpa humilis. Gretchen continued work on biomineralization in Pyura pachydermatina and isolated several hundred mg of purified spicules that she brought to her colleagues in Israel in February when she gave an invited talk at the Bat-Sheva Seminar on Biomineralization in Honor of Heinz A. Lowenstam. Shigeko Ooishi came down to collect ascidicolous copepods from Aplidium and other ascidians and we had a great time in the field seeing the incredibly abundant ascidians and enteropneusts.
We are leaving Fullerton May 27 and from June 11-Aug. 20 we will be at the Station Biologique, BP 74, 29682 Roscoff Cedex, France; tel 33.98.29.23.05; fax 33.98.29.23.26. Our email addresses remain the same.

l. Congratulations to Dr. Hitoshi Michibata, who received the Inoue Science Prize (2,000,000 yen) for his work on vanadium accumulation. (contributed by M. Hoshi) This is in addition to the Zool. Soc. of Japan award we mentioned in AN38.

2. Internet Home Pages: There is now an ascidian home page in Italy, where you can also find an abridged version of Ascidian News #38 (December '95) as well as work in progress on a monumental task of compiling a year by year database of references on ascidians. http://biol.dgbm.unina.it:8080/ascidians/urochordate.html

The Japan home page is http://160.29.96.22/ascidians/Home.html (you must capitalize the word Home.)

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

1. O.Y. Chaga, Dept. Cytol. & Histol., St. Petersburg Univ., Russia. Blood cells of ascidian Styela (Goniocarpa) rustica.

I. Morphological and histological study. Twelve types of blood cells were identified in the blood of an ascidian, Styela rustica, by means of phase contrast microscopy, Nomarksy optics and histological analysis of differentially stained sections. They are haemocytoblasts (20%), giant cells (0.3%), hyaline amebocytes (30%), macrophages (2%), cells with eosinophilic grains (0.2%), cells with eosinophilic granules (2%), cells with colourless granules (2%), granulocytes (6%), vacuolated young intermediate and mature morula cells (about 40% all together). Four later cell types are presumably the consequent stages of morula cell differentiation pathway, macrophages - the final step of the hyaline amebocytes' maturation. The haemocytoblasts are mainly localised in connective tissue of endocarps, intestinal and subendostylar sinuses. These cells are quickly dividing, their mitotic index being about 2%, index of S phase about 10%. They give rise to all other cells, representing at least 6 independent cell lineages. Mature giant cells are located in the vessels of branchial sac, whereas mature cells with colourless granules, granulocytes and morula cells migrate into the tunic. All blood cells are ameboid at least in some degree, but only hyaline amebocytes, macrophages and morula cells are phagocytic. It seems that macrophages, cells with eosinophilic granules and cells with colourless granules are specific for members of family Styelidae, whereas other cell types, i.e. giant, hyaline, granulocytes and morula cells, are common for ascidians of all three families of the order Stolidobranchia. On the other hand, the signet ring and compartment cells so characteristic for other ascidian species are absent in the blood of S. rustica and presumably of other members of family Styelidae.

II. Cytochemical study. Blood cells of ascidian Styela rustica were studied by means of cytochemical methods. It has been shown that each cell type has its own cytochemical markers, which can be used for exact identification of the blood cells in other ascidian species. For example, haemocytoblasts, the proliferating stem cells, possess high amount of RNA in their cytoplasm, whereas the most ameboid and phagocytic cells - hyaline amebocytes - show the highest staining for filamentous actin. Granules of basophilic granulocytes are composed of proteins in complex with acid sulphated polysaccharides. Therefore these cells can be detected easily by PAS-alcian blue reaction or by toluidine blue staining. Specific granules of the cells with eosinophilic inclusions are composed of basic arginine-rich proteins without free amino groups, and granules of the cells with colourless inclusions (glycoprotein cells) contain neutral glycoproteins with high content of cystine and cystein. Four cell types - vacuolated, young, intermediate and mature morula cells - possess the same complex of unique cytochemical markers. They are the bound iron, cationic proteins with high content of free lysine (-amino groups, and enzyme 0-diphenoloxidase in inactive form. The fourth component of the granules of mature morula cells is represented by the o-diphenolic compounds. It has been shown that accumulation of o-diphenols starts at the late step of morula cell maturation, i.e. at the stage of intermediate morula cells. Therefore, during their differentiation the morula cells of S. rustica accumulate in their granules all the components of phenoloxidase system, which is used for sclerotization of tunic proteins as well as for realisation of different immune reactions.

III. Autoradiographic study on the metabolism of the blood cells.
Metabolism of the blood cells of ascidian Styela rustica was studied in autoradiographic experiments with labeled precursors of polysaccharide and protein synthesis. It has been shown tha t about 50-60% of haemocytoblasts incorporate glucose and different amino acids. These cells may correspond to the fraction of quickly dividing cells. In good agreement with the results of previous cytochemical studies, the cells with eosinophilic granules, containing arginine-rich basic proteins, incorporate only amino acids, whereas cells with colourless (glycoprotein) granules and basophilic granulocytes incorporate both amino acids and glucose.
Morula cells and their precursors show no uptake of labeled glucose or 14C-CO2 and do not participate in cellulose synthesis. On the other hand, intermediate and some mature morula cells demonstrate selective uptake of tyrosine, which is more than 50 times higher than incorporation of other amino acids. In continuous experiments the level of incorporation of amino acids by haemocytoblasts and glycoprotein cells does not show significant changes. On the contrary, the labelling of morula cells with tyrosine demonstrates a drastic decrease during the expriment. Thus, total radioactivity of morula cells in experiments with 7, 8-3H-tyrosine drops two times in 24 hours, and 10-15 times after 3-7 days. These data suggest that the morula cells use tyrosine not for protein synthesis, but mainly for the synthesis of o-diphenolic compounds. Therefore, the decrease of their radioactivity may be explained by the modification of tyrosine side chain during the process of o-diphenol production. Both cytochemical and autoradiographic data show clearly that the synthesis and accumulation of o-diphenolic compounds during morula cell differentiation in Styela rustica start at the stage of intermediate morula cells. The main part of the mature morula cell population can thus be considered as being composed of terminally differentiated and resting defensive cells.

1. Richards-Gross, Sarah E.E. 1993. Natural products chemistry of taxonomically diverse ascidians from southern Californian and Caribbean waters. Univ. Calif. San Diego M.S. thesis with Dr. W.H. Fenical.
The presence of secondary metabolites in marine invertebrates, especially those in tropical waters where competition and predation are more intense, has long been recognized. Recently, animals of the class Ascidiacea have proven especially rich chemically, possessing many novel compounds, often with unique antiviral or antitumor activity. Relatively little is known about the natural products chemistry of these animals and even less about the ecological and developmental implications of these metabolites. Ascidians from the tropical Caribbean waters have been investigated. Several species of colonial ascidians present in the mangrove and reef habitats were assayed for chemically based feeding deterrence. Four novel structures, the didemneimides A-D, composed of indole, maleimide, and imidazole moieties, have been isolated and identified. Ascidians from the temperate waters off La Jolla, California were similarly investigated. A broad screening program was undertaken to identify the chemical resources of this area. More than 20 species were collected at a variety of habitats and seasons.
Pharmacological activities as well as potential chemical defense mechanisms were tested. The colonial species Cystodytes lobatus was found to use chemical mediation to maintain a surface free of fouling organisms.
An extensive review of the chemical literature and the chemical ecology of ascidians through 1992 is included.

2. Kang, Heonjoong 1994. The natural products chemistry of marine ascidians: structural elucidation and molecular modeling studies of novel secondary metabolites. Univ. Calif. San Diego Ph.D. thesis with Dr. W.H Fenical.
Ascidians are well-known producers of amino acid-derived secondary metabolites. Recent chemical and biological investigations of taxonomically diverse ascidians have revealed a variety of pharmacologically potent natural products and the importance of these metabolites in the survival of physically vulnerable ascidians in predator-rich habitats. However, at issue are the real sources of novel secondary metabolites isolated from ascidians, which are related to symbiosis due to complex associations of the ascidians with microbial symbionts. Furthermore, rapidly developing resistance of infectious diseases and cancers toward traditional antibiotics and anti-cancer drugs needs unprecedented novel natural products. Despite great advance in separation science, marine natural products research in ascidians was relatively limited to nonpolar compounds. Furthermore, the aquatic environment implicates the significance of hydrophilic molecules in defensive adaptations and symbiosis. Therefore, the main objective of this research was to investigate biosynthetic diversity and limitation of ascidian secondary metabolism with emphasis of identification of hydrophilic bioactive molecules and unprecedented carbon skeletons. In this way, a broader understanding of ascidian secondary metabolism, and a chemical data base for studies of chemical ecology, could be established.
A variety of chromatographic methods, such as HPLC and high speed countercurrent chromatography, were used to isolate polar compounds. Extensive spectroscopic techniques, including a variety of two-dimensional NMR experiments and mass spectrometric methods as well as computer-molecular modeling, were utilized to elucidate their structures. This research resulted in the structural elucidation of 25 new secondary metabolites in 10 different structural classes, including 8 new carbon skeletons from marine sources. A high diversity of chemical structures along with various bioactivities, such as cytotoxicity against colon cancer cells, antimicrobial activity, and anti-inflammatory activity, were demonstrated. Several natural products showed cytotoxic activities directed against a drug-resistant human colon cell line, HCT 116. Some of these compounds are known to inhibit an enzyme which is frequently used as a chemotherapeutic target. In addition to them, some had antibacterial and anti-inflammatory activities. However, not every compound was bioactive. Perhaps some of them may explain the chemical defense mechanism of ascidians against predators.

Abstracts from the 66th annual meeting of the Zool. Soc. of Japan, Sept. 15-17, 1995, Tokyo.

MORPHOLOGY OF THE ROHDE CELLS IN THE SPINAL CORD OF THE AMPHIOXUS; A GOLGI STUDY
N. Iwahori, Dept. of Anat., Fac. of Med., Nagasaki Univ., Nagasaki.
The morphology of the Rohde cells (RC) located at the caudal level of the spinal cord in the amphioxus was studied with the use of the rapid Golgi method. The RC are distributed segmentally in the median region over the central canal of the spinal cord. The somata are nearly primodial , pyriform or flask-shaped with their long axis oriented transversally. Several thick dendrites arise from the somata, extend dorsally and dorsolaterally, and branch out numerous collaterals terminating in the dorsal and dorsolateral fiber columns. The axons arise from the apex of the somata travel ventrally through the lateral cord, and then turn rostromedially toward the lateral cord of the opposite side crossing the cord floor. In the lateral cord of the opposite side, the axons turn rostrally and form a lateral bundle of giant axons. At their proximal part, the axons emit a few collaterals terminating in the ventrolateral cord, while in the lateral bundle, the axons emit numerous collaterals of varying length and thickness distributing to the ventrolateral and lateral cords. At the their terminal, the axons branch out into numerous terminal collaterals.

COLONY SPECIFICITY IN APLIDIUM YAMAZII : SELF OR NONSELF RECOGNITION IN THE TUNIC.
T. Ishii1, E. Hirose2, and Y. Taneda3. 1Biol. Lab., Col. of Edu., Akita Univ.; 2Biol. Lab., Col. of Ag. / Vet. Med., Nihon Univ., Fujisawa, and 3Dept. of Biol., Fac. of Edu., Yokohama Natl. Univ.
Colony specificity has been well studied in botryllid ascidians that have developed interzooidal tunic vessels. In spite of the lacks of tunic vessels, Aplidium yamazii shows colony specificity. Since the fusion rate among the natural populations in A. yamaziiwas higher (101 fusion cases out of 115 combinations : 88%) than that of other colonial ascidians, allo-types may be small in number in this species. When two incompatible colonies were contacted with each other at their growing edges, some pieces of tissue masses were cut off at the contact region. Only one cell type in the tunic cells, phagocytic tunic cells, were contained in these tissue masses. Therefore, it is strongly suggested that these cells play an important role in allorecognition and rejection.

MINUTE PROTRUSIONS OF TUNIC CUTICLE IN APLOUSOBRANCH ASCIDIANS WITH SOME PHYLOGENETIC CONSIDERATIONS
Euichi Hirose1, Gretchen Lambert2 and Teruaki Nishikawa3. 1Col. Ag./Vet. Med., Nihon Univ., Fujisawa, 2Dept. Biol. Sci., Calif. State Univ., Fullerton, CA 92634 U.S.A., and 3Grad. Sch. Human Informatics, Nagoya Univ., Nagoya.
Fine structure of the tunic cuticle was studied in 28 species belonging to the suborder Aplousobranchia of the Class Ascidiacea to investigate their phylogenetic significance. The present results, together with previously published ones describe the cuticular structures in 36 species of aplousobranchs, covering all of the families and, where appropriate, subfamilies. Cuticular protrusions were found in all species of Polyclininae (fam. Polyclinidae) and Clavelininae (Polycitoridae). They were undetectable in Didemnidae (which lack subfamilies), Euherdmaninae (Polyclinidae), and in Polycitorinae and Holozoinae (both in the Polycitoridae), only except for Polycitor proliferus (Polycitorinae) and Distaplia dubia (Holozoinae) that have cuticle protrusions. This irregularity of P. proliferus is discussed with reference to its systematic position. Further, the inconsistency in this character-state distribution seen at the subfamily level in each of the two families suggests that we should reconsider the present taxonomy of the suborder.

INDUCTION OF DIFFERENTIATION IN CELL LINE FROM THE ASCIDIAN, POLYANDROCARPA MISAKIENSIS.
N. Shibata, S. Fujiwara, K. Kawamura and T. Yubisui. Dept. of Biol., Fac. of Sci., Kochi Univ.
During bud development of the ascidian, Polyandrocarpa misakiensis, most of new tissues are formed from the atrial epithelium. We have already established several cell lines from this epithelium. We examined how and to what extent these cell lines exhibit differentiation repertoire. Cells that grew exponentially looked undifferentiated, whereas long-term culture of confluent cells resulted in the expression of antigens specific for the atrial epithelium, gut and nerve cells. Aminopeptidase was purified from Polyandrocarpa colonies and applied to a clonal cell line. They were induced to proliferate and express the epithelium- and gut-specific antigens. Excess thymidine that blocked cell proliferation prevented the cells from expressing these antigens. These results showed the relationship between cell growth and differentiation. Next, we examined the control of cell differentiation by ECM components. The cells that were cultured in a dish coated with collagen type 4 expressed higher level of gut- specific antigen than those in a non-coated dish. These results suggests that both protease and ECM can control differentiation of the tunicate cell line.

MOLECULAR CLONING AND EXPRESSION OF A HOX GENE PHOX19 IN BUDDING ASCIDIANS.
S. Fujiwara, K. Kawamura and T. Yubisui. Dept. of Biol., Fac. of Sci., Kochi Univ.
We isolated cDNA fragments encoding a Hox homeodomain protein, termed Phox19, from the budding ascidian Polyandrocarpa misakiensis by 3'- and 5'-RACE methods. Amino acid sequence of the homeodomain deduced from the cDNA was closely similar to those of Antennapedia and Sex combs reduced. Quantitative RT-PCR analysis showed that Phox19 mRNA was expressed in both the adults and developing buds. The amount of the mRNA in the adults was much greater than that in the buds. Adults body was cut anteroposteriorly into five pieces, which were numbered 1 to 5 from anterior to posterior. RT-PCR was carried out using RNA extracted from each one-fifth fragment. The fragment No.4 contained Phox19 mRNA but the others did not. This fragment was at the position of atrial siphon and contained digestive tract and heart. The results suggest that the expression of Phox19 mRNA in the adult body is region- or tissue-specific.

EXPRESSION OF THE ALDEHYDE DEHYDROGENASES IN THE BUDDING TUNICATE, POLYANDROCARPA MISAKIENSIS.
N. Harafuji, S. Fujiwara, K. Kawamura and T. Yubisui. Dept. of Biol., Fac. of Sci., Kochi Univ.
Retinoic acid is an endogenous determinant of anteroposterior axis in the developing bud of the ascidian, Polyandrocarpa misakiensis. Since the activity of aldehyde dehydrogenase (ALDH) was detected specifically in the morphogenetic region of the bud, local activation of this enzyme is thought to trigger bud development. Previously, we isolated several clones of the fragments of Polyandrocarpa ALDH genes by PCR amplification. Northern blot analysis showed that two of these genes, termed ALDH-9 and ALDH-24, were expressed at high levels in both the adults and buds. Changes in the amount of mRNAs were not obvious during bud development. It appears that the expression of these genes are not regulated transcriptionally. Although the substrate specificity of products of these genes has not yet been determined, amino acid sequence deduced from ALDH-9 was closely similar to that of murine retinal dehydrogenase AHD-2.

MOLECULAR CLONING AND EXPRESSION OF A HOMOLOGUE OF RETINOIC ACID RECEPTORS IN THE BUDDING ASCIDIAN, POLYANDROCARPA MISAKIENSIS.
K. Hisata, S. Fujiwara, K. Kawamura and T. Yubisui. Dept. of Biol., Fac. of Sci., Kochi Univ.
Retinoic acid can induce the secondary anteroposterior axis of the developing bud in the colonial ascidian, Polyandrocarpa misakiensis. In addition, exogenous retinoic acid is quickly incorporated into mesenchymal cells, which acquire the organizer activity of morphallaxis, depending on de novo protein synthesis. In vertebrates, the effects of retinoic acid are mediated through regulation of gene expression by two subclasses of nuclear receptors called retinoic acid receptors (RARs) and retinoid X receptors (RXRs). As the first step to elucidate the mechanism by which retinoic acid regulates pattern formation in this animal, we isolated a cDNA fragment of a RAR homologue by PCR amplification from a Polyandrocarpa cDNA library. Amino acid sequence deduced from the fragment exhibited high similarity to vertebrate RARs. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis showed that in P. misakiensis the transcript of RAR homologue is expressed in the mesencymal cells of developing buds.

A STUDY ON RECOGNITION OF ORGANIC OR INORGANIC FOREIGN BODIES BY COELOMOCYTES OF A SOLITARY ASCIDIAN, HALOCYNTHIA RORETZI.
M. Mishima and M Dan-Sohkawa. Dept. of Biol., Osaka City Univ., Osaka.
Coelmocytes of Halocynthia roretzi play a self-defense role in vivo. We have developed an in vitro system for observing their behavior. Using this system, we observed their reactions against organic and inorganic foreign bodies. The coelomocytes vigorously attacked the organic foreign bodies, e. g. aggregates of starfish phagocytes, with a chemotactic reaction, while they neglected the inorganic ones, e. g. the latex beads (dia. 20 um). When the remnants of the sea water in which coelomocytes attacked either of these materials were given to the cells in culture through a microneedle, both of them induced a chemotactic reaction. However, only the sea water in which coelomocytes encountered the organic material showed a cytotoxic effect. These results suggest that coelomocytes recognize organic or inorganic nature of the foreign bodies.

GENETIC VARIABILITY OF MITOCHONDRIAL DNA AMONG REPRODUCTIVELY ISOLATED THREE TYPES IN Halocynthia roretzi.
T. Kakuda1, F. Takizawa1, A. Hino1, and T. Numakunai2. 1Dept. of Biol. Sci., Kanagawa Univ., Hiratsuka. 2 Marine Biol. Sta. , Fac. of Sci., Tohoku Univ., Asamushi.
Halocynthia roretzi is classified into three types on account of their reproductive behavior; spawning season and daily spawning time. Experimentally, the cross fertilization among these three types was succeeded and the resulting embryos gave rise to normal young adults, but F2 embryo has not been developed. There is a possibility that these three types of Halocynthia roretzi show an example of sympatric speciation. To estimate the genetic divergence of these types, 25 individuals of each type were examined by restriction-fragment pattern and by restriction site analysis of mtDNA. The degree of genetic differentiation was estimated by the proportion of shared restriction fragment and restriction site (Nei & Li '79). According to these estimates, the genetic divergence (d) within three types of Mutsu Bay was 4.4~6.2x10-4, and these standard deviations at each diverging point were over lapped among three types. It seems that the genetic differentiation among these types is not significant.

DISTRIBUTION AND SEASONAL VARIATION OF RETINAL IN THE ADULT OF THE ASCIDIAN, HALOCYNTHIA RORETZI.
T. Irie, S. Kajiwara and T. Seki. Osaka Meijo Women's Coll., Osaka, Dept. of Biol, Fac. of Educ., Iwate Univ., Morioka and Dept. of Health Sci., Osaka Kyoiku Univ., Osaka.
Retinoids in the several organs (blood cells, blood plasma, body wall muscle, endostyle, gill, gonad and hepatopancreas) of the ascidian, Halocynthia roretzi, were analyzed by high performance liquid chromatography. Most of the retinoid in every organ examined was retinal. The retinal was distributed mostly in the gonad and the body wall muscle (about 2:1) in summer. The body wall muscle of the ascidian is very thick, while the gonads are undeveloped in this season. On the other hand, the retinal was detected mainly in the gonad (more than 90 %), but only a little retinal in the body wall muscle in winter. The body wall muscle is thin, whereas the gonads are quite developed in this season, since the spawning season of the ascidian is winter. All other organs contained a small amount of retinal without any apparent seasonal variation. We have already reported that retinal is the major retinoid in the ascidian eggs, similarly to the eggs of several amphibians and teleosts. The results of the present experiments indicate that retinal is the major and the most important retinoid store not only in the eggs but also throughout the life cycle of the ascidian. The results also suggest that the retinal is accumulated in the body wall muscle prior to the spawning season and transported to the gonads accompanying the egg maturation.

DO HEMOCYTES OF HALOCYNTHIA HISPIDA EXERT ALLOGENEIC AND XENOGENEIC REACTION ?
S. Ohtake1, T. Abe1, F. Shishikura1, K. Tanaka1, M. Arai2 and J. Chiba2. 1Dept. of Biol., Nihon Univ. Sch. of Med., Tokyo and 2Dept. of Biol. Sci. Tech., Sci. Univ. of Tokyo, Chiba.
The allogeneic and xenogeneic responsibilities of H. hispida were studied by mixed-hemocytes aggregation in a U-plate (MHI-assay), electron microscopy and fluorescent viability test using calcein AM. We have reported the typical allogeneic cellular reactions of H. roretzi. Rapid cell aggregation, the degranulation of large granular amebocytes, cytolysis and the mutual phagocytosis (allogeneic phagocytosis) by small granular amebocytes (SG) were observed in the mixture of allogeneic hemocytes within the two min incubation. However, we could not observe these reactions in H. hispida by MHI-assay, electron microscopy and fluorescent viability test. When the hemocytes of H. hispida and H. roretzi were mixed each other, the rapid and drastic cell aggregation, cytolysis and phagocytosis by SG of each species were observed. Fluorescent viability test revealed that the hemocytes of H. hispida as well as that of H. roretzi responded to the hemocytes of another species. The hemocytes of H. hispida may lack any acute allogeneic responsibility although they have the xenogeneic responsibility to the hemocytes of H. roretzi.

IMMUNOLOGICAL ANALYSIS OF 58KDA PROTEINASE INHIBITOR FROM AN ASCIDIAN, HALOCYNTHIA RORETZI.
T. Abe1, F. Shishikura1, S. Ohtake1, K. Tanaka1, T. Iizuka2 and J. Chiba2. 1Dept. of Biol., Nihon Univ. Sch. of Med., Tokyo and 2Dept. of Biol. Sci. Tech., Sci. Univ. of Tokyo, Chiba.
A proteinase inhibitor from the hemolymph plasma of Halocynthia roretzi inhibits the activity of the coexisting plasma proteinase(s) and trypsin. The inhibitor was a single-chain protein with 58kDa and pI 9.2. This inhibitor seems to interact with trypsin to form a complex (approximately 80kDa) that was not dissociable under boiling in SDS. Its inhibition was accelerated by glycosaminoglycans. These finding and the partial amino acid sequence of the inhibitor suggest that the inhibitor is a member of the serpin superfamily. For further investigation on the inhibitor, we prepared antibodies against the purified inhibitor. We analyzed the inhibitor treated with N-glycosidase F and/or O-glycosidase by SDS-PAGE and immunoblot. The untreated inhibitor gave a single protein band of 58kDa on SDS-PAGE and immunoblot. After deglycosilation with N-glycosidase F, an immunoreactive band of 48.6kDa was detected. This result suggests that the inhibitor had some asparagine bound N-glycans by approximately 16% of molecular weight. However, O-glycosidase treatment did not affect the inhibitor in immunoreactivity and SDS-PAGE mobility. A band of the inhibitor-trypsin complex on PAGE was also immunoreactive.

PRODUCTION OF MONOCLONAL ANTIBODIES THAT BLOCK CYTOTOXIC REACTION BY HEMOCYTES OF THE ASCIDIAN HALOCYNTHIA RORETZI
1M. Arai, 2S-I. Ohtake, 1Y. Ohga, 1T. Iizuka, 1H. Ohba, 2K. Tanaka, and 1J. Chiba. 1Dept. of Biol. Sci. and Technol., Sci. Univ. of Tokyo, Noda, Chiba 278 and 2Dept. of Biol., Nihon Univ. Sch. of Med., Tokyo 173.
Hemocytes from H. roretzi undergo rapid lysis in vitro when those derived from different individuals come in contact with each other. This allogeneic response appears to be a specific cytotoxic reaction of the ascidian hemocytes, which may represents the evolutionary precursors of the vertebrate immune system, although cellular mechanism of the cytotoxic reaction and recognition molecules that participate in the response remain to be elucidated. In order to investigate cellular mechanism of the cytotoxic reaction and find out the molecules that take part in self- and non-self recognition in the ascidian, we prepared monoclonal antibodies (mAbs) against membrane fractions of hemocytes from H. roretzi and selected mAbs that block the cytotoxic reaction. The cytotoxic reaction by the allogeneic hemocytes was quantitatively measured by the newly developed fluorometric assay using Calcein AM. Six mAbs that inhibit the cytotoxic reaction were prepared. They were divided into two groups according to the antigens recognized by them and termed as mAbs CRB1.1, CRB1.2, and CRB1.3 and CRB2.1, CRB2.2, and CRB2.3

TWO TYPES OF HEMOCYTES THAT MAY PLAY CENTRAL ROLES AND RELEASE CYTOTOXIC FACTOR(S) THAT INVOLVE(S) IN CYTOTOXIC REACTION IN THE ASCIDIAN HALOCYNTHIA RORETZI
T. Iizuka1, S-I. Ohtake2, R. Midorikawa1, M. Arai1, H. Ohba1, K. Tanaka2, and J. Chiba1. 1Dept. of Biol. Sci. and Technol., Sci. Univ. of Tokyo, Noda, Chiba 278 and 2Dept. of Biol., Nihon Univ. Sch. of Med., Tokyo.
Most of all types of H. roretzi hemocytes undergo rapid lysis in vitro when those derived from different individuals come in contact with each other (Fuke, 1980). One of us has distinguished five types of cells; vacuolated cells (VC), small granular amoebocytes (SGA), large granular amoebocytes (LGA), lymphoid cells, and dense granule cells in the hemocytes with respect to their morphology by light and electron microscopy. Eight sub-types; VC1, 2, 3, 4, 5, 6, 7, and 8 have been also set apart in VC (Ohtake, et al., Zool. Sci. 11, 681-691, 1994). We prepared two kinds of monoclonal antibodies (mAbs) that reacted with membrane fractions of the ascidian hemocytes and blocked cytotoxic reaction by allogeneic hemocytes. We report here that both of the blocking mAbs with different specificity were strongly reactive with the cell surface of VC (especially VC5) and LGA but hardly with that of SGA by immunofluorescence assay. VC (especially VC5) and LGA may play central roles in specific recognition of the allogeneic hemocytes in the ascidian. After incubation of hemocytes from different individuals in artificial sea water containing Ca2+ and Mg2+ for 30 min at 23C, nonspecific cytotoxic factor (s) was detected in supernatants of reaction mixtures. The cytotoxic factor, named ACF, had low molecular weight and was resistant to the heat treatment. Incubation of hemocytes from unrelated individuals with ACF resulted in the same cytotoxic reaction as that caused by allogeneic hemocytes, in which numerous cell death and discharge of cytoplasmic contents were observed. ACF may nonspecifically enhance the cytotoxic reaction in the ascidian.

ANALYSIS OF CELL SURFACE ANTIGEN(S) RELATED TO CYTOTOXIC REACTION BY HEMOCYTES OF THE ASCIDIAN HALOCYNTHIA RORETZI
M. Suzuki1, T. Iizuka1, H. Ohba1, T. Abe2, F. Shishikura2, K. Tanaka2, and J. Chiba1. 1Dept. of Biol. Sci. and Technol., Sci. Univ. of Tokyo, Noda, Chiba 278; and 2Dept. of Biol., Nihon Univ. Sch. of Med., Tokyo.
Hemocytes from H. roretzi undergo rapid lysis in vitro when those derived from different individuals come in contact with each other (Fuke, 1980). This allogeneic response appears to be a specific cytotoxic response of the ascidian hemocytes, which may represent the evolutionary precursors of the vertebrate immune system. If such evolutionary homology does exist it is likely to find out an ''ancestor'' gene of the immunoglobulin super gene family such as major histocompatibility complex of higher vertebrates in the species. In order to find out the molecules that take part in self- and non-self recognition in the ascidian, we prepared monoclonal antibodies (mAbs) against membrane fractions of hemocytes from H. roretzi and selected mAbs that block the cytotoxic reaction. Two groups of mAbs, CRB1 and CRB2, were found to react with different antigens in membrane fractions of hemocytes by the Western blot analysis. Purification of the antigen molecule(s) and biochemical analysis of the antigens are now in progress.

FURTHER STUDIES OF THE COLONY SPECIFICITY IN THE COLONIAL ASCIDIAN Botryllus scalaris.
Maki Shirae and Yasunori Saito. Shimoda Marine Research Center, Univ. of Tsukuba, Shimoda.
Colony specificity in botryllid ascidians is manifested by fusion and nonfusion between two colonies. The processes of fusion are essentially same among those botryllids, but the processes of rejection are different from each other. In Botryllus scalaris, the rejection reaction occurs after the establishment of vascular connection between two nonfusible colonies, though in other species the sign of rejection appears before connection of vascular vessels. To get more detail information on this unique rejection reaction of B. scalaris, we performed histological and histochemical examination by using light and electron microscopy. In this species, neither infiltration and degranulation of blood cells in early stage of rejection reaction, nor blackening of the infiltrated cells in later stage was observed although those phenomena were common in other botryllids. The major reactions were phagocytosis and encapsulation by blood cells, which were restricted only inside the connected blood vessels. Blood cells of B.scalaris showed lower activity of phenoloxidase than those of B.primigenus that might cause blackening of blood cells in the reaction area. In B.scalaris, the allorecognition and rejection reaction might be caused by cellular factors rather than by humoral factors restrictively within vessels.

HRSNA, AN ASCIDIAN HOMOLOGUE OF THE SNAIL GENE, IS EXPRESSED IN THE PARAXIAL MESODERM.
S. Wada and H. Saiga. Dept. Biol., Fac. Sci., Tokyo Metropol. Univ.
Morphogenesis in the development of ascidian embryos shows some striking similarities to that of vertebrate embryos. It is, therefore, possible that a molecular basis for the morphogenesis may be conserved between them. To learn extent of the conservation, we isolated and examined expression of Hrsna of the ascidian, Halocynthia roretzi. This is an ascidian homologue of vertebrate snail gene that has been known to encode a transcription factor expressed mainly in the paraxial mesoderm and the somites in vertebrate embryos. We found that Hrsna is expressed in the muscles that are the paraxial mesoderm locating bilaterally in the tail of ascidian embryos. This suggests that a part of mechanism that directs morphogenesis or determination of the paraxial mesoderm may be conserved between ascidian and vertebrate embryos.

NEUROSECRETORY CELLS SCATTERED ALONG THE DORSAL STRAND IN ASCIDIANS. 2. PRORACTIN-POSITIVE CELLS
K. Terakado1 and M. Ogawa2. 1Dept. of Regul. Biol. and 2 Dept. of Molec. Biol., Fac. of Sci., Saitama Univ., Urawa.
By means of the immunocytochemical method, proractin (PRL)-like immunoreactivity was demonstrated in many secretory cells scattered along the dorsal strand and in numerous neurons of cerebral ganglion of the ascidian Halocynthia roretzi. Neural complex of adult specimens were fixed with Bouin's fluid without acetic acid. Sections were treated with one of the following primary antiserum: Anti-bullfrog PRL, anti-salmon PRL and anti-human PRL. The specificity of the antibody to bullfrog PRL, which reacted positively to the neural complex, was tested by preabsorption with bullfrog PRL. Many granule-containing (secretory)cells scattered along the dorsal strand and neumerous neurons in the cerebral ganglion were positively stained only when the antibody to bull frog PRL was used. the immunoreactivity was lost by preabsorption with bullfrog PRL. The presence of PRL-positive cells along the dorsal strand (and in the cerebral ganglion) may have some evolutionary significance to vertebrate pituitary.

PHYSIOLOGICAL COLOR CHANGE IN THE COLONIAL ASCIDIAN, POLYCITOR PROLIFERUS.
Y. Taneda. and K.Matsuyama. Dept. of Biol., Fac. of Educ., Yokohama Natl. Univ.
It is well known that there are two color types, bluish white and brownish orange, in the colonial ascidian, Polycitor proliferus. Field survays revealed that bluish white colonies were always seen at sun-filled areas and brownish orange ones were at shade areas. Color type of the ascidian may depend on the light conditions in their habitats. To clarify the participation of light in color change of the animal, we carried out the exchange of their habitats between two different color types. When brownish orange colonies were transfered to sun-filled area, they varied to bluish white ones after several days. Histological research revealed that difference between two color types depend upon the white pigment cells distributed beneath the tunic surface. The pigment cells were more abundant in bluish white colonies than brownish orange ones. The pigment cells were observed as dark brown under a light microscope, while as white under dark contrast. Two types of the pigment cells were observed under a light microscope. These cells were similar to punctate and reticulate states in chromatophore. Long-term (propagation of the pigment cells) and short-term (morphological change in the pigment cells) are indicated under light condition in this species.

APPEARANCE OF ACTIVITIES INDUCING THE OOCYTE MATURATION AND THE EXPANSION OF PERI-VITTELLINE SPACE BY TREATMENT WITH TRYPSIN OF INHIBITORY FRACTIONS FROM THE OVARY EXTRACT OF THE ASCIDIAN, HALOCYNTHIA RORETZI.
1T. Numakunai, and 2H. Yokosawa. 1Mar.Biol. Sta., Tohoku Univ., Asamushi; 2Dept. of Biochem., Fac. of Pharmaceut. Sci., Hokkaido Univ., Sapporo.
When the extract of ovarian egg mass of the spawning animals is subjected to gel filtration on Sephadex G-75 column (the gel filtration), two peaks of activities inducing the oocyte maturation and the expansion of peri-vitelline space (self-sterility) are detected. The first peak shows a trypsin-like enzyme activity. In the second peak the inhibitory activity against the oocyte maturation is observed. A similar inhibitory activity is detected in the crude extract of ovarian egg mass. The inhibitory activity was lost after dialisis using a membrane (molecular weight cut-off, 1000), while the activity was retained in the tube using another membrane (MW cut-off, 500). When fraction showing the inhibitory activity was treated with trypsin and the trypsin was removed by ultrafiltration through a centricut filter (MW cu-off, 1000), the inhibitory activity in the filtrate disappeared and the activities inducing the oocyte maturation and the expansion of perivitelline space appeared in the filtrate.

CHARACTERIZATION OF ENDODERM-SPECIFIC ALKALINE PHOSPHATASE IN LARVAE OF THE ASCIDIAN Halocynthia roretzi .
G.Kumano, H.Nishida. Dept. of Life Sci., Tokyo Inst. of Technol., Yokohama.
Endoderm-specific alkaline phosphatase is present in larvae of the ascidian Halocynthia roretzi by NBT/BCIP histochemical staining. We characterized alkaline phosphatases from Halocynthia larvae. First, two bands were detected by zymographic technique after SDS-PAGE of membrane preparation. This suggests the existence of two types of alkaline phosphatase in the larvae. These two enzymes were separated into the adsorbed and the non-adsorbed fractions by Cibacron Blue-3GA affinity chromatography. Alkaline Phosphatase in the adsorbed fraction is likely to be endoderm-specific one by two reasons. One is the coincidence of sensitivity against alkaline phosphatase inhibitors between the adsorbed fraction and larval histochemical staining. The other is the difference of activities between these fractions when BCIP was used as substrate. Now we are trying to purify endoderm-specific alkaline phosphatase from the adsorbed fraction.

THE CYTOPLASMIC DETERMINANTS ARE PRESENT IN GRADIENT IN THE UNFERTILIZED EGGS OF THE ASCIDIAN HALOCYNTHIA RORETZI
A. Yamada and H. Nishida. Dept. of Life Sc., Tokyo Inst. of Technol., Yokohama.
Ascidian embryogenesis has been regarded as a typical example of mosaic development. But when unfertilized eggs were cut into two equal pieces and then fertilized, both fragments developed into normal larvae. To investigate the localization of cytoplasmic determinants in unfertilized eggs of the ascidian Halocynthia roretzi, we carried out cytoplasmic-transfer experiments by fusing blastomeres and egg fragments from various regions of unfertilized eggs. We transplanted cytoplasmic fragments of five different position along the animal-vegetal axis. The results suggested that endoderm and muscle determinants are present in gradient with maximum activity at the vegetal pole, and epidermis determinant exists with maximum activity at the animal pole.

ACID MUCOPOLYSACCHARIDE IN THE BRANCHIAL BASKET OF AN ASCIDIAN, ASCIDIA SYDNEIENSIS SAMEA.
J. Hirata Dept. of Biol., Tsurumi Univ. Sch. of Dental Medicine, Yokohama.
Acid mucopolysaccharide is present on the surface of the branchial basket of the ascidian and may be involved in predation and uptake of food molecules from seawater. In this paper, histochemical localization of the acid muco-polysaccharide in the branchial basket was studied using Alcian blue (pH1.0) and electron microscope. A large number of vesicles containing acid mucopolysaccharide were observed in the cells of papilla. The dorsal lamina was also stained with Alcian blue. On the other hand, neither cells in stigma nor the endostyle were stained with the dye.These results suggest that acid mucopolysaccharideis supplied from papilla and dorsal lamina.

CHARACTERIZATION OF PHENOLOXIDASE ISOLATED FROM ASCIDIAN HEMOCYTES
S. Hata, K. Azumi, and H. Yokosawa. Dept. of Biochem., Fac. of Pharmaceutical Sci., Hokkaido Univ., Sapporo.
We have previously reported that a phenoloxidase (PO)-like activity toward 4-methylcatechol is detected in hemocytes of the solitary ascidian, Halocynthia roretzi, and that PO was released from the hemocytes during the allogenic reaction (contact reaction) but not by treatment of PMA or calcium ionophore. We purified PO from H. roretzi hemocyte extract using a procedure including SP-Sephadex ion-exchange chromatography and Sephadex G-100 gel filtration under the acidic conditions (pH 4.5). The isolated PO was stable only around pH 4, whereas it was unstable at neutral pH. The PO activity was inhibited by 1-phenyl-2-thiourea and diethyldithiocarbamic acid, but not by metal chelaters, such as EDTA and o-phenanthroline. It was demonstrated that PO was released from H. roretzi hemocytes by addition of sheep erythrocytes (SRBC) or yeasts, as well as during "contact reaction": PO-like activity was undetectable in the extract of SRBC or yeasts. However, latex beads had little ability to release this enzyme. Thus, we proposed that PO is released from H. roretzi hemocytes when hemocytes contact foreign substances, and that the enzyme may play a role in host defense of H. roretzi.

ENHANCEMENT OF PHAGOCYTIC ACTIVITY OF ASCIDIAN HEMOCYTES BY SUPEROXIDE DISMUTASE ISOLATED FROM ASCIDIAN PLASMA
G. Ishikawa, K. Azumi, and H. Yokosawa. Dept. of Biochem., Fac. of Pharmaceutical Sci., Hokkaido Univ., Sapporo.
We have previously reported that phagocytosis by hemocytes of Halocynthia roretzi is promoted by addition of H. roretziplasma, and is inhibited by diethyldithiocarbamate, an inhibitor of superoxide dismutase (SOD). It was also we found that a SOD activity was detected in H. roretzi plasma. To clarify a role of H. roretzi plasma SOD in the phagocytosis by H. roretzi hemocytes, we first purified the SOD from ammonium precipitates of H. roretzi plasma using a procedure including DEAE ion-exchange and heparin-agarose chromatographies, and Superose 12 gel filtration. Its molecular weight was estimated to be 17K by SDS-PAGE, and 30K by gel filtration. Since the SOD activity was susceptible to KCN, H. roretzi plasma SOD is thought to belong to the Cu-Zn SOD family as H. roretzi cytosol SOD. The purified plasma SOD was found to enhance phagocytosis of sheep erythrocytes by H. roretzi hemocytes. Thus, SOD in H. roretzi plasma may function as an "opsonin" in the phagocytosis by H. roretzi hemocytes.

INVOLVEMENT OF TYROSINE KINASE IN AN EARLY EVENT OF FERTILIZATION OF THE ASCIDIAN, CIONA SAVIGNYI
K. Ueki and H. Yokosawa. Dept. of Biochem., Fac. of Pharm. Sci., Hokkaido Univ., Sapporo.
Fertilization induces egg activation and initiation of cell division. It has been proposed that a tyrosine kinase plays an essential role in a process leading to egg activation. But, however, definitive evidence for its involvement has not been presented. In the ascidian egg, insemination triggers the first ooplasmic segregation, the first observable structural change (surface contraction) of egg which occurs several minutes after insemination. To define the involvement of tyrosine kinase in an early event of ascidian fertilization, we investigated the effects of several tyrosine kinase inhibitors on the contraction and cleavage of naked eggs of the ascidian, Ciona savignyi, and found that erbstatin analog inhibited the contraction and cleavage of naked eggs when added before insemination, but not when added several minutes after insemination. These results strongly suggest that a tyrosine kinase functions in the early stage of fertilization, leading to the ooplasmic segregation. Analyses of tyrosine kinases and tyrosine phosphorylation of proteins, susceptible to erbstatin analog, are now in progress.

As-T mRNA PROMOTES NOTOCHORD DIFFERENTIATION WITHOUT INDUCTION
H. Yasuo, N. Satoh Dept. of Zool., Grad. Sch. of Sci. Kyoto Univ., Kyoto.
An ascidian homolog of Brachyury(T), As-T, starts its expression at the 64-cell stage in the primordial notochord blastomeres. It has been shown that inductive interactions at the 32-cell stage are required for both the determination of notochord cells and the expression of As-T. In present study, we injected in vitro synthesized mRNA of As-T into the fertilized eggs and then notochord lineage blastomeres were isolated just before inductive signals were elicited on these blastomeres. The resultant partial embryos expressed notochordal features (the vacuolation and the expression of a material in notochordal sheath). On the other hand, control embryos injected with lacZ mRNA didn't express these characters. These results suggest that the expression of As-T in the notochord lineage blastomeres is sufficient to promote the expression of some differentiation markers of the notochord.

EXPRESSION OF REL GENE IN THE ASCIDIAN, Halocynthia roretzi
M. Shimada 1, K. Azumi 1, H. Yokosawa 1, T. Miya 2 and N. Satoh 2. 1Dept. of Biochem., Fac. of Pharm. Sci., Hokkaido Univ., Sapporo, and 2Dept. of Zool., Fac. of Sci., Kyoto Univ., Kyoto.
Members of the Rel/NF-kB family are found in mammals, Xenopus and Drosophila, and have a conserved region of the Rel homology domain (RHD). It has been proposed that they mediate an immune response and participate in establishing the dorsal-ventral polarity. To define expression of rel genes in the ascidian, H. roretzi, we first obtained the PCR fragment that was amplyfied from ascidian genomic DNA and was cross-hybridized with the RDH domain of mouse c-rel cDNA. Next, using this fragment, we examined the expression of rel mRNAs were detected in H. roretzi blood cels, and fertilized eggs and embryos at various developmental stages of H. roretzi. Cloning of full-length cDNAs of H. roretzi rel gene is now in progress.

ISOLATION OF BRAIN PIGMENT-CELL-SPECIFIC GENES FROM TWO CLOSELY RELATED CONGENERIC SPECIES OF ASCIDIAN, WHICH TAKE DIFFERENT DEVELOPMENTAL MODES
K. F. Tagawa1, W. R. Jeffery 2, N. Satoh 1. 1Dept. of Zool., Grad. Sch. of Sci. Kyoto Univ.; 2Dept. of Zool. and Bodega Marine Lab., UC Davis., P.O. Box 247, Bodega Bay, California 94923.
Two closely related congeneric ascidians, Molgula oculata and Molgula occulta, living in the sand flats near Roscoff, France, take different developmental modes. The former exhibits urodele development, while the latter exhibits anural development. The larva of the urodele species has differentiated muscle, notochord, and a pigment cell ( otolith ), the larva of anural species does not have these cells. We report here an isolation of pigment-cell-specific genes in order to investigate how these closely related species of ascidian have evolved to take different developmental modes.

TIMING OF GENE EXPRESSION IN EMBRYOS WITH REDUCED CYTOPLASM VOLUME OF Halocynthia roretzi
K. Ishida and N. Satoh. Dept. of Zool., Grad. Sch. of Sci. Kyoto Univ., Kyoto.
We are interested in how a gene initiates its zygotic expression at the appropriate developmental stage. Using Halocynthia roretzi embryos, we examined the timings of the onset of expression of muscle actin gene, ascidian Brachyury homolog (As-T) and an epidermis-specific gene in half embryos by in situ hybridization. Half embryos were obtained by cutting an unfertilized egg at equatorial plane. They developed nomally after insemination. Transcriptional activation of these genes, however, occurred at the same timing as those in the normal embryo.

LOCALIZATION OF ACIDIC ORGANELLES IN THE ASCIDIAN, ASCIDIA SYDNEIENSIS SAMEA
M. Hayashi 1, J. Asai-Hirata 2, H. Yamada 1, T. Uyama 1, Y. Moriyama 1 and H. Michibata 1. 1 Marine Biol. Lab., Fac. of Sci., Hiroshima Univ.; 2 Dept. of Biol., Dental School of Tsurumi Univ.
Central vacuolar systems of eucaryotic cells such as lysosomes and endosomes are acidic, which are established by vacuolar-type proton ATPase (V-ATPase). The acidic pH is important for various cellular functions such as proteolysis and concentration of metabolites. In this study, we investigated the localization of acidic organelles in ascidians,one of the marine invertebrate evolutionally very close to vertebrates, using vital staining with acridine orange and immunostaining with anti-V-ATPase antibody. A large number of acidic organelles were identified in branchial sac. The acidic pH was increased by the addition of proton conductors and bafilomycin A1, an inhibitor of V-ATPase, while neither vanadate (P-type ATPase inhibitor) nor azide (F-type inhibitor) did not affect the internal pH. Furthermore, antibody against V-ATPase subunit A recognized the acidic organelles. Similar acidic organelles are present in branchial sac from other marine invertebrates. These results suggested that acidic organelles containing V-ATPase play an important role(s) in the branchial sac in vertebrates.

THE VANADOCYTES SPECIFIC MONOCLONAL ANTIBODY RECOGNIZED A 70kDa PROTEIN OF THE FAN WORM, PSEUDOPOTAMILLA OCCELATA
T. Uyama, Y. Moriyama and H. Michibata. Mukaishima Marine Biol. Laboratory, Fac. of Sci., Hiroshima Univ.
The vanadocytes, one type of blood cells, contains high levels of vanadium in ascidians, up to 1000,000 to 10,000,000 times the levels in seawater. We previously prepared S4D5 monoclonal antibody specific to the vanadocytes of Ascidia sydneiensis samea. During development of the ascidian, the expression of the S4D5 antigen was coincided with the accumulation of vanadium. In the present study, we examined the property of the S4D5 antigen and the possible presence of the S4D5 antigen in the bipinnate radiole of the fan worm, Pseudopotamilla occelata, which is newly discovered as a vanadium accumulator. The S4D5 antigen was found to be a soluble protein with apparent molecular mass of 45kDa and was localized in the cytoplasm in the vanadocytes of the ascidian. In P. occelata, the S4D5 antibody specifically reacted with protein of about 70kDa.

PURIFICATION OF VANADIUM-BINDING PROTEIN FROM ASCIDIA SYDNEIENSIS SAMEA
T. Kanda, J. Wuchiyama, T. Uyama, Y. Moriyama and H. Michibata. Mukaishima Mar. Biol. Lab., Fac. of Sci., Hiroshima Univ.
Ascidians, marine animals living in sea water, have been known to accumulate high levels of vanadium in their blood cells. To reveal the mechanism on accumulation of vanadium uptake in vanadium-rich ascidians, In this study, we isolated vanadium-binding protein from blood cell membrane of Ascidia sydneiensis samea. Apparent molecular weight of the protein was caluculated to be 15kDa on SDS-PAGE. The protein bound vanadium about 0.6mol/mol. We will show some properties of the protein.

LOCALIZATION OF A VANADIUM-BINDING PROTEIN EXTRACTED FROM BLOOD CELL MEMBRANES OF THE ASCIDIAN, ASCIDIA SYDNEIENSIS SAMEA
J. Wuchiyama, T. Kanda, Y. Moriyama and H. Michibata. Mukaishima Marine Biological Laboratory, Faculty of Science, Hiroshima University, Hiroshima.
Ascidians are known to accumulate vanadium in signet ring cells (vanadocytes), one type of blood cell, up to 350mM. We have attempted to solve the mechanism how to accumulate vanadium in the vanadocytes from sea water. Recently we succeeded to isolate a vanadium-binding protein from the blood cell membranes of Ascidia sydneiensis samea, one of vanadium-rich ascidians. In this report, we studied the localization of vanadium-binding protein using monoclonal and polyclonal antibodies recognized signet ring cells among eleven types of blood cells, suggesting that vanadium-binding protein was expressed in the vanadocytes.

INCREASE IN CELL POPULATION OF VANADOCYTES IN THE ASCIDIAN, ASCIDIA SYDNEIENSIS SAMEA, BY ADMINISTRATION OF NH4CL
Mitsuko Hayashi, Yasuhiro Nose, Makoto Nakamura, Taro Uyama, Yoshinori Moriyama and Hitoshi Michibata. Mukaishima Marine Biological Laboratory, Faculty of Science, Hiroshima University, Hiroshima.
Some ascidians are known to accumulate high concentration of vanadium (up to 350mM) in their blood cells called vanadocytes. Vanadocyte contains a single and large vacuole which is maintained to be acidic internally by vacuolar H+-ATPase (V-ATPase). It seems to be possible that the proton gradient established by V-ATPase is energetically linked to the accumulation of the vanadium. Administration of ammonium chloride (50mM) or bafilomycin A1 (4uM), a specific inhibitor of V-ATPase, into adult specimen of Ascidia sydneiensis samea caused neutralization of vacuoler pH of the vanadocyte. Under similar conditions, it was found that vanadocyte increased in their population more than 2 fold without affecting number of other kinds of blood cells. Content of vanadium also increased slightly. We will discribe some features of the specific increase of vanadocyte and discuss significance of the phenomenon.

***The Following is the abstract of the lecture of the winner of the Japan Zoological Society Prize, 1995

THE MECHANISM OF ACCUMULATION OF METALS, ESPECIALLY HIGH LEVELS OF VANADIUM, BY ASCIDIANS
Hitoshi Michibata. Mukaishima Mar. Biol. Laboratory, Fac. of Sci. and Laboratory of Mar. Molecular Biol., Graduate School of Sci., Hiroshima Univ., Mukaishima-cho, Hiroshima 722, Japan.
High levels of vanadium were found by mere chance in the blood cells of an ascidian, known as sea squirt, by the German chemist M. Henze in 1911. Subsequently, many investigators including analytical chemists, physiologists, bioinorganic chemists and chemists of natural product, joined in attempts to characterize the metal in living organisms partly because of the initial interest in its very presence in such organisms and partly because of the primary interest in its involvement in carrying oxygen as the third metal following iron and copper. However, studies on the mechanism of accumulation and physiological significance of vanadium have made very slow progress, since the problem needed to interdisciplinary investigations, to be elucidated. First, we reexamined the vanadium content of several tissues from 20 ascidian species, employing the extremely sensitive method of neutron-activation analysis for the quantification of this metal. Consequently, the highest concentration of 350 mM vanadium was found in the blood cells of Ascidia gemmata belonging to the suborder Phlebobranchia, which concentration corresponds 10,000,000 times higher than that in sea water. Ascidian blood cells have been classified morphologically as being about 10 types. Morula cells, one type of vacuolated cell, have been thought to be the so-called vanadocytes that might be involved in the accumulation of vanadium. Our combination techniques of cell fractionation and neutron-activation analysis revealed that signet ring cells, another type of vacuolated cell, were the true vanadocytes rather than the morula cells. Furthermore, the oxidation state of vanadium in the blood cells has been a subject of controversy. Our ESR (electron spin resonance) measurements of the oxidation state in the fractionated blood cells revealed that the vanadocytes contained vanadium, 97.6% of which was in the +3 oxidation state with the rest being in the +4 oxidation state. After Henze's initial discovery of 1 N acidity in ascidian blood cells, low pH values have been reported by several investigators and the mechanism of accumulation and oxidation state of vanadium have been suggested to be associated with a very low pH within the cells. However, K. Kustin's group recently claimed that the intracellular pH was neutral on the basis of measurements made by their new technique. We considered that one of the reasons for this extreme controversy was that the measurements of pH were made using the entire population of blood cells, that is to say, without the separation by fractionation of the subpopulations of blood cells. Applying ESR spectrometry to the measurement of pH values of fractionated subpopulations, we could confirm that the vanadocytes in A. gemmata had a low pH value of 2.4 while these cells contained the highest level of vanadium. These facts suggest the possibility that proton ions concentrated by a H+-ATPase are energetically linked to the accumulation of vanadium. We made, therefore, immunological search for H+-ATPase in the vacuolar membrane of signet ring cells as the first step. It was consequently revealed that antibody raised against the vacuolar-type H+-ATPase reacted with the vacuolar membranes of signet ring cells and addition of bafilomycin A1, a specific inhibitor of vacuolar-type H+-ATPase, actually inhibited the uptake of protons by the vacuoles of signet ring cells, with resultant neutralization of the contents of the vacuoles. Hereupon, there have been certain complications involved in the identification of signet ring cells, the so-called vanadocytes, partly because of difficulties associated with morphological discrimination between several types of blood cells and partly because of our inadequate knowledge as to the cell-lineages of blood cell. We postulated that monoclonal antibodies would be powerful tools for resolving these issues. The antibody that we generated reacted with the signet ring cells not only of A. sydneiensis samea used as antigen but also with those of the other species and recognized a protein of about 45 kDa. This antibody was also a useful tool to determine the time at which the accumulation of vanadium commenced during embryogenesis. Using neutron activation analysis and an immunofluorescence method, we found that amounts of vanadium per individual increased dramatically two weeks after fertilization, and by two months the amount accumulated in larvae was about 600,000 times greater than that in the unfertilized eggs of A. gemmata. A vanadocyte-specific antigen, recognized by a monoclonal antibody specific to the signet ring cells, first appeared in the body wall coinciding with the significant accumulation of vanadium. Characterization of vanadium-binding proteins extracted from the blood cells of vanadium-rich ascidians is in progress, which must have a key how ascidians selectively and highly accumulate vanadium from sea water. The unusual phenomenon whereby some ascidians accumulate vanadium to levels more than ten million times higher than that in sea water has attracted researchers in various fields. Ascidians with this unusual physiological characteristic may help up to clarify not only how ascidians but also how other living organisms accumulate transition metal, as well as the physiological roles that these metals play in living organisms.

Abstracts from the 41st annual meeting of the Italian Embryology Group, June 5-7, 1995, Modena. Published in Animal Biology 4, 1995.

106. Development of siphons in the blastozooid of Botryllus schlosseri (Tunicata).
P. BURIGHEL, M. CELEBRE and G.B. MARTINUCCI Dipartimento di Bio1., Universita' di Padova, Padova. Italy.
Oral and atrial siphons are obvious structures of the ascidian body, which are often important also for taxonomy. However, despite their importance, detailed studies on siphon organization are scarce. We followed the development of oral and atrial siphons in the blastozooid of B. schlosseri. In this species, the colony is composed of numerous small zooids embedded in a common tunic and arranged to form a number of starshaped systems. In these, each zooid opens its oral siphon individually, while the atrial siphon converges with those of other zooids to form the common cloacal siphon at the centre of the system. B. schlosseri grow in series of blastogenic generations. Buds arise from the mantle of the parental zooid and develop, synchronizing with all the buds of the same generation. At maturity, they replace the regressing parental generation, as new adults. B. schlosseri was reared in the laboratory in tanks with sea-water, adhering pieces of glass. Blastozooid development was followed in vivo under a light microscope (LM) and staged according to Sabbadin, 1960 [Arch. Ocean. Limno1., 12: 97-107) Fragments of colonies were selected at opportune stages and processed for LM and electron microscopy, according to the conventional procedure, after fixation in 1.5% glutaraldehyde buffered with 0.2 M Na cacodylate plus 1.6% NaCl. In the early buds, at the two-vesicle stage, the epidermis surrounds the internal vesicle, which originates from the atrial epithelium. This vesicle then forms the rudiments of a branchial chamber, flanked laterally and dorsally by the atrial chamber. Oral and atrial siphon rudiments may precociously be recognized as thickened areas of branchial and atrial epithelia, each covered by a disc of thick epidermis. Nervous fibers extend beween the two epithelia and migrating cells begin to differentiate as myocytes. At stage 8, when organogenesis is particularly intense, rudiments of siphons evaginate slightly, while branchial and atrial epithelia produce a layer of tunic matrix and cuticle facing the internal cavity. The cuticle is provided with numerous small papillate protrusions, looking like those of adults. Later, the tunic is invaded by cells coming from the blood, after crossing the epithelia. The myocytes differentiate to form pluricellular smooth muscle around and along the siphons. Before the epidermis contacts the underlying adjacent epithelium, primary sensory cells differentiate and the crown of tentacles begins to evaginate. Sensory cells are represented by single cells penetrating the tunic matrix, having long cell extensions presumably directed to the cerebral ganglion. Before fissuration occurs, the tunic continues to grow and bores out the lumen of the siphons. Only in advanced stages do the inner epithelia evaginate to contact the epidermis and fissuration occurs. It is noteworthy that the lumen of the differentiating siphons is occupied by tunic secreted by epithelia lining internal cavities; these epithelia are both of atrial origin, being derived from the inner vesicle of the bud.

109. Differentiation of blood cell lines in the colonial ascidian Botryllus schlosseri.
F. CIMA, L. BALLARIN and A. SABBADIN Dipartimento di Biologia, Universita' di Padova, Padova. Italy.
Ascidian blood contains a wide variety of morphologically different hemocytes which, in spite of numerous studies, are still matter of debate due to the persisting doubts and uncertainties about their differentiation pathways, mutual relationships and functions. Our interest in the role of blood cells in defence strategies of the colonial ascidian 8otryllus schlosseri has led us to study and characterize two differentiation lines of hemocytes involved in two defence reactions, namely phagocytosis which preserves the organism from invading microorganisms and histoincompatibility which prevents the fusion of genetically remote colonies. Three hemocyte types take part in phagocytosis, i.e. hyaline amoebocytes, macrophage-like and signet-ring cells: all of them share a common enzymatic pattern rich in hydrolytic enzymes. Hyaline amoebocytes can rapidly attach and ingest test particles; phagosomes inside macrophage-like and signet-ring cells appear after 30 min of incubation with test particles. These data suggest that they may represent different functional aspects of a single cell-type. This hypothesis is supported by the study of hemocyte frequencies in the weekly life cycle of Botryllus: during the resorption of the old blastogenetic generation, characterized by intense phagocytic activity, a significant decrease in the number of hyaline amoebocytes occurs together with a significant increase of macrophage-like cells. Histoincompatibility in Botryllus manifests itself as rejection between contacting colonies and is characterized by the appearance of dark-brown necrotic masses at the border line. In this reaction the so called morula cells (MC) play an important role, as they accumulate at the apices of contacting ampullae, cross the ampullar epithelium and migrate into the tunic where they finally release the content of their vacuoles and degenerate. MC have granular amoebocytes as their precursors and contain polyphenolic substance and phenoloxidase (PO) inside their vacuoles: in our hypothesis P0, by transforming polyphenol substrata into quinones, is the enzyme responsible of the localized cytotoxicity during the rejection reaction. We have demonstrated that MC degranulate and release the vacuolar content when incubated with incompatible blood plasma [BP) and an increase in cell mortality is observed when hemocytes are incubated for 60 min. with incompatible BP.

113. Expression of the (-smooth muscle actin in protochordates and lower craniates.
I. DI ROSA, A. FAG0T TI, F. SIMONCELLI, S. DELTO5T0, R. CARDELLICCHIO, F. PANARA and R. PASCOLINI
Istituto di Anatomia Comparata, Universita'di Perugia, Perugia. Italy.
Higher vertebrates express six major actin isoforms: two cytoplasmic actins (( and (), two smooth muscle actins [( and (), and two sarcomeric actins, (-cardiac and (-skeletal. Although there is a high homology in the aminoacid sequence of vertebrate actin isoforms, (-smooth muscle (SM) actin is selectively recognized by anti(SM-1, a monoclonal antibody directed against its amino terminal decapeptide. Incubation with synthetic-peptides related to the NH2-terminal decapeptide of (SM actin Ac-EEEDSTALVC have done that Ac-EEED is the epitope for anti-(SM-1 and that this sequence is important for regulating in vitro and in vivo polymerization of this actin isoform. In endothermic vertebrates aSM actin is used as a marker of origin and differentiation of smooth muscle tissues and is expressed in fibroblastic cells during remodelling and repair phenomena. Blocking anti-(SM-1 by the epitope peptide has recently indicated that this epitope sequence is also expressed in lower tetrapod smooth muscle cells and myofibroblasts. These results led us to further investigate the anti-(SM-1 immunoreactivity in protochordates and lower craniates. An urochordate (ascidian Ascidia ceradotes), a cephalochordate [amphioxus Branchiostoma floridae), a jawless fish (lamprey Lethenteron zanandreai), a cartilaginous fish (shark Mustelus mustelus) and two bony fishes (the primitive actinopterigian Calamoichthys calabaricus and the African lungfish Protocterus annectens) have been screened using anti-(SM-1. The results indicate that (SM actin is expressed in: 1) ascidian fibroblastic cells; 2) amphioxus transverse muscles; 3) lamprey astrocytes; 4) jawed craniate smooth muscle cells. The data obtained suggest that (SM actin appeared early in chordate evolution. Supported by MURST [40% and 60%) CNR (grant N. 94.02752.CT04)

121. Isolation and characterization of the hatching enzyme in Ciona intestinalis.
S. SClPPA*, A. D'ANIELL0, M.J. DENUCÉ* and M. DE VINCENTIIS* *Dpt. di Genetica, Bio1.Gen. e Mo1ec., Fac. di Sci., Univ. di Napo1i, Napoli. Ita1y  Stazione Zoo1. di Napoli, Ita1y* Dept. Zool., Univ. Cattolica di Nijmegen, Nijmegen, The Netherlands.
In ascidians hatching is regulated by a proteolytic enzyme (Berrill, 1929. Phy1. Trans. Roy. Soc. London, 8218: 37-78). In Ciona intestinalis it shows proteolytic and glycolytic activity [Caggegi et al., 1974. Rend. Accad. Naz. Lincei, 56 s. 8: 803-807), and would be a serine-protease [Denuce', 1975. Arch. Intern. Phisio1. Biochim., 83: 958-959). In Ha1ocynthia roretzi, the hatching enzyme is a trypsin-like proteinase (Hoshi and Numakunai, 1981. Acta Embryol. Morphol. Exper. n.s., 2: 163-169). The present note reports on the isolation purification and characterization of the hatching enzyme in Ciona intestinalis. The enzyme was purified by column chromatography on Bio-Gel P- 10 and low-pressure liquid chromatography (FPLC) using Superose- 12 . The purified enzyme has a molecular weight of 17,000 and consists of 131 aminoacid residue/molecule. The most abundant aminoacid residues are aspartic acid/aspargine, glutamic acid/glutamine and alanine. The enzyme exhibits maximum activity at pH 8.5. Leupeptin and EDTA inhibit enzyme activity. The purified enzyme hydrolyses the chorion of unfertilized eggs of C. intestinalis.

122. The role of inner follicular cells in embryo-parent interactions in ascidians.
G. ZANI0L0, M. DELLA BARBERA, L. MANNI and P. BURIGHEL Dept. di Biol., Universita' di Padova, Padova. Ita1y
Ascidian eggs are typically surrounded by three cellular envelopes, outer follicle cells (OFC), inner follicle cells (lFG) and test cells [TC), and by the acellular vitelline coat (VC), separating IFC from TC and oolemma. The differentiation and probably the role of each envelope show variations among species, particularly owing to the different modes of reproduction, respectively in oviparous solitary forms and in ovoviviparous or viviparous colonial forms. Commonly, OFC are discharged at ovulation, while IFC remain to surround the oocyte and, later, the embryo. IFC may be highly specialized: in solitary species such as Ciona intestinalis, they have large vacuoles which allow the embryo to float; in other cases, especially in anural species, adhesive material is secreted to attach the larva to the substratum. In several colonial species, IFC envelop the embryo, which develops in a brood pouch in the parents body (Botrylloides leachi, Botrylloides viviparous) or hangs in the atrial chamber by means of a cup-like placenta (Botryllus schlosseri). In the latter, IFC participate with atrial and oviducal epithelia to form the placenta and secrete a kind of glue for attachment to other placental epithelia. We studied recently two ovoviviparous colonial species, Clavelina lepadiformis and Aplidium pallidum, whose embryos apparently develop free inside the atrial chamber, enveloped by IFC. The latter form a continuous layer and arc extremely polymorphic cells with their basal lamina intermingled with the fibers of the VC layer. IFC have numerous, well-developed Golgi fields which bud vesicles of various sizes with a homogeneous dense content. A peculiar aspect is shown by the IFC of contiguous embryos when they contact each other. In the contact zones, the apical regions of the adhering IFC seem to fuse with one another, so that only a single layer remains to separate embryos. Each of these cells appear to possess on opposite sides a basal lamina, resting on the VC fibers of different embryos. Thus, they possess a peculiar polarity, because the basal region seem to be present on opposite poles. Moreover, the IFC appear to be involved in the secretion of Golgi vesicles, producing adhesive material that helps to bind together the embryos at the bottom of the atrial chamber. This probably prevents sea-water from fowing through the zooid and driving the embryos out of their parents before they have completely developed. This capacity for fusion is not shown by the IFC contacting the atrial chamber epithelial cells. However, in this case a fuzzy coat is observed in the contact zones which, together with the adhesive secretion, helps to keep the embryos together inside the parent.

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Larsson, P.T., U. Westermark & T. Iversen 1995. Determination of the cellulose 1a allomorph content in a tunicate cellulose by CP/MAS C-13-NMR spectroscopy. Carbohydr. Res. 278:339-344.

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Littler, D.M. & D.S. Littler 1995. A colonial tunicate smothers corals and coralline algae on the Great Astrolabe Reef, Fiji. Coral Reefs 14:148-161.

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