Number 37
May 1995

This marks the 20th year that we have been doing Ascidian News. In 1975 we took over from Lu Eldredge, who with Don Abbott initiated the whole thing and put out the first 4 issues. We hope it has been useful all these years and worth the trouble! You will find another long list of new publications in this issue, a very valuable part of this newsletter in this age of shrinking library budgets. If you did not get an email note from us in April, it means we do not have your email address yet. If you have one, please take a minute right now and send us your email address, fax and phone #. We plan to include this information in the next issue. Hopefully we will soon be able to send AN via Internet, thereby saving a considerable amount of money now spent on duplicating and postage. Of course we will continue to send AN by postal mail for those of you without email. Our thanks to those who sent contributions for this issue. We will be at the Friday Harbor Labs, 620 Univ. Rd., Friday Harbor, WA 98250 from June 1-Aug. 25. Our email will remain the same. Please send us a contribution for the next AN; limit thesis abstracts to about 200 words. We greatly appreciate receiving a copy of all new publications.

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

1. Dr. Noriyuki Satoh has requested that if you have a copy of his book "Developmental Biology of Ascidians", please note the following errata: page 22, line 25: "(Fukumoto, 1983, 1984)" should read "(Cotelli et al., 1980)". Page 28, lines 12 & 13: "(Hoshi, 1984, 1985)" should read "(Fukumoto, 1983, 1988, 1990a)". Page 29, line 2: "(Usui, Takahashi, and Hoshi, 1987; Fukumoto, 1990a)" should read "(Fukumoto, 1990a)".

2. Bill Bates has moved his lab to Bamfield; his address is Dr. Wm. R. Bates, Bamfield Marine Station, Bamfield, British Columbia, Canada VOR 1B0 tel. 604-728-3301, fax 604-728-3452, email bbates@sfu.ca

3. From Euichi Hirose, Col. Agr./Vet. Med., Nihon Univ.

We thank Dr. Shigeki Fujiwara, Kochi Univ., for sending us the very interesting logos printed below. Japan Ascidian "Club": This is an informal meeting of ascidian biology, and held at the annual meeting of the Zool. Soc. of Japan. The name of the meeting is "Hoya no Seibutsugaku Danwa-kai" (Hoya=ascidian(s), no=of, Seibutugaku=biology, Danwa=informal talk, kai=meeting), shortened to Hoya-no-Kai. This meeting was established more than ten years ago. It does not require definite membership; anyone who attends the meeting are members that year. Hiroki Nishida and I organized it from 1990 to 1994; the 1995 meeting is organized by Shigeki Fujiwara (Kochi Univ.) and Taro Uyama (Hiroshima Univ.), and please communicate with Fujiwara-san about your plans. You can communicate with him at Dept. of Biol., Fac. of Sci., Kochi Univ., Akebono-cho, Kochi 780, Japan or by e-mail: tatataa@cc.kochi-u.ac.jp

We also have a branch meeting of the Kanto area of Japan (some prefectures around Tokyo) every spring, in which we alternate between the Misaki Marine Lab (Univ. Tokyo) or Manazuru Mar. Lab (Yokohama Natl. Univ.), and have a afternoon symposium and evening party. 1992: Manazuru, Y. Taneda & H. Koyama organizers; 1993: Misaki, M. Yoshida, T. Honneger & H. Nishida, organizers; 1994: Manazuru, Y. Taneda, T. Ishii & S. Takizawa, organizers; 1995: Misaki, Y. Sato, Ohtsuka, Yoshida, Terakado & Katsuyama, organizers.

1. Jon Havenhand: I've finished the initial phase of my work on the behaviour and dispersal patterns of Distaplia corolla larvae in Belize, and shall be writing that up in the near future. Although larvae are capable of exhibiting precise responses to light and dark in both the field and the laboratory, direct observations of over 60 larvae released naturally in a mangrove channel show that larval dispersal distance and settlement site are determined largely by chance events. These include transport of larvae by mysid shrimps, passive currents and random swimming patterns. Despite being close to potential settlement sites, many larvae settle on the soft substratum and perish.

2. Wm. E. Robinson,Univ. of Mass.-Boston and K. Kustin and L. Linebaugh, Brandeis Univ.:

We have been collaborating with Drs. Pat Frank and Keith Hodgson of Stanford Univ., to study vanadium oxidation states and complexation in ascidian blood cells using X-ray Absorption Spectroscopy (XAS, EXAFS). In Ascidia ceratodes, for example, over 90% of this vanadium is present as vanadium(III) ion. XAS and EXAFS analyses on whole blood cell pellets (a mixture of up to 9 different blood cell types) indicate that the majority of the vanadium that is present in vivo may not be complexed to a chelator such as tunichrome. Instead, it appears that the vanadium(III) exists in an environment coordinated by water molecules, similar to that shown for a vanadium(III)-sulfuric acid solution. Since it is known that at least three types of A. ceratodes blood cells may concentrate vanadium (signet ring, compartment and morula), the question remains open as to whether vanadium(III) may be stored in different forms in different blood cells. To address this question, we prepared four separate pools of A. ceratodes blood cells. Differential blood cell counts revealed a vastly different makeup of blood cells (e.g. morula cells ranged from 36 to 52 %; signet rings from 5 to 12 %; compartment from 17 to 30 %). Nevertheless, the vanadium K-edge XAS spectra were virtually identical for all four samples. Following XAS and EXAFS analyses, the samples were digested and metal contents (vanadium, iron and chromium) were analysed by AA. As with the differential blood cell counts, vanadium content of the samples also showed a wide range of values (from 35 to 305 (g).

Using the differential blood cell counts and the results of the metal analyses, we were able to calculate the most probable vanadium concentrations in the morula, signet ring, compartment and the "remaining" cells using a non-linear regression model. Applying estimates of the density of total blood cells in our samples, our preliminary calculations indicate that the vanadium content of a morula cell is 8.7 X 10-8, a signet ring is 1.9 X 10-7 and a compartment cell is 9.0 X 10-8 micromole/cell. The finding that the signet ring cell has over 2X more vanadium than the morula cell agrees nicely with the previously published results of Oltz et al. (1989). The combination of our vanadium-modeling work, the differential cell counts, the variation in vanadium content in our samples and the XAS/EXAFS results indicate that vanadium is present in a similar internal environment in all of the various blood cells in which it is found. These results lend credence to the hypothesis that all of the vanadium-containing blood cells are ontogenetically related. We are currently finishing experiments to test this hypothesis, in which we have been following the development of the vacuolated blood cells following a one-time labeling of the cells with the thymidine analog bromodeoxyuridine. We are also conducting XAS/EXAFS analyses of A. nigra blood cell samples on which we have made differential blood cell counts.

3. from George Mackie: I am interested in the fate of the motor nerves innervating the branchial stigmata and mantle muscles after brain removal in Chelyosoma productum. This species can tolerate brain removal and survive for many months without regenerating a new one. So far, it looks as if the motor nerves do not degenerate at all, despite the fact that removal of the brain removes their cell bodies. We are using cholinesterase histochemistry and immunofluorescence microscopy with anti-tubulin antisera to follow the peripheral nerves before and after debraining. In the course of this study, we have found we can visualize the sensory nerves in the mantle quite well. Their cell bodies show up nicely with antitubulin. With Don Deibel, I have been trying to locate GnRH-immunoreactive neurons in Oikopleura, so far without success. We expected to find a system comparable to Fedele's "visceral nervous system", which I recently described in Ciona. (see current issue of J.M.B.A., Feb 1995)

4. Cloney, R. A. and L. J. Hansson. 1995. Ascidian larvae: The role of test cells in preventing hydrophobicity. Acta Zool. in press, Oct. Abstract: Ascidian test cells co-differentiate on the surface of each ovarian oocyte beneath the vitelline coat. They become vacuolated and later occupy the perivitelline compartment of each egg and embryo. In some species their vacuoles contain submicroscopic granules or filaments called "ornaments" and acidic glycosaminoglycans. These test cells deposit their products on the surface of the larval tunic in late embryogenesis. In these species the test cells are lost at hatching. In other species the test cell vacuoles contain acidic glycosaminoglycans, but no ornaments. In these species the test cells attach to the larval tunic and probably secrete acidic glycosaminoglycans. We deprived the embryos of seven species of ascidians of their test cells and vitelline coats during mid-embryogenesis. After completing their development the larvae of both kinds of species were hydrophobic. They easily become trapped on the surface of sea water in cultures. Normal larvae (controls), bearing test cell secretions, are hydrophilic and never become trapped. We infer that negatively charged secretions of the test cells make normal larvae hydrophilic. Some molgulids with direct development have no test cells, no fins and no swimming larva. We reason that the test cells of these species may have been lost during evolution because they no longer had an important role in preventing hydrophobicity.

5. From Sjaak Lemmens: Following further experimentation on clearance rates, we have now prepared a manuscript titled: "The clearance rates of four ascidians from Marmion Lagoon, Western Australia." Authors: J.W.T.J. Lemmens, D. Kirkpatrick & P. Thompson. This MS is now ready to be presented to external reviewers.

6. From Peter Holland: Since my last progress report in AN, my research group has moved from Oxford University to Reading University, UK. Our research continues to focus on developmental control genes in protochordates, and the genetic evolution of developmental mechanisms in chordates. Over the last year, we have focussed principally on homeobox genes in amphioxus (see Nature 370: 563-566), although we also cloned a partial fragment of a Hox gene from a larvacean (mentioned in Development 1994 Suppl., 125-133). Work in progress in our lab includes cloning and embryonic expression analysis of Msx, Otx, Emx, Cdx and Xlox homeobox genes in amphioxus (Nic Williams, Anna Sharman, and collaboration with Jordi Garcia-Fernandez, now at the Univ. of Barcelona), zinc-finger genes in amphioxus (Nic Williams) and gene linkage analysis in amphioxus (Simon Patton). A collaboration with Hubert Ortner and Bernhard Hermmann (Frieburg, Germany) on Brachyury genes in amphioxus has also just been completed. Hopefully, these molecular level investigations will eventually combine with similar work from ascidians and other chordates, to give a fuller picture of how genes, development and morphology interact in chordate evolution. This is already proving to be a fruitful approach for understanding the evolution of the chordate neural tube (from comparison of homeobox gene expression patterns between taxa) and the evolution and homology of the notochord (from Brachyury genes).

7. Charles and Gretchen Lambert: We continue to work on the problems of introduced ascidians in southern California harbors, biomineralization and egg activation. Our manuscript on brooding in Corella is in press in Canad. J. of Zool., and we have two other manuscripts submitted: one on algae in the tunics of New Zealand ascidians (with R. Waaland) and the other on activation of ascidian eggs (A. McDougall, C. Sardet and C.C. Lambert).

1. Noriko Akita, Dept. of Life Sci., Tokyo Inst. of Technol., Yokohama 226, Japan. Ph.D. thesis with Dr. M. Hoshi. Allo-recognition in the ascidian, Halocynthia roretzi.

Two types of allo-recognition phenomena are known in the ascidian, Halocynthia roretzi; self-sterility in gametes and contact reaction in hemocytes. Contact reaction is an experimental and morphological expression of allogeneic recognition, namely hemocytes from two individuals undergo an exocytotic burst if they are mixed in vitro. We have found that contact reaction is accompanied by a burst of cyanide-insensitive oxygen consumption that, unlike exocytotic events of mammalian phagocytosis, is Ca2+ -dependent and does not culminate in the production of superoxide anions (O2-) or H2O2. Instead, this burst was caused by a surge of phenoloxidase from the hemocytes as known for the self-defense systems in insects and crustaceans. The activity of phenoloxidase released from hemocytes corresponds well to the degree of contact reaction. This finding enabled us to quantify the contact reaction simply by measuring the activity of phenoloxidase released from hemocytes. In an attempt to find a molecule(s) th at is responsible for both self-sterility and contact reaction, the activity of phenoloxidase released from hemocytes were assayed after mixing them with sperm, eggs, follicle-free eggs or follicle cells. Hemocytes released phenoloxidase when they were mixed with non-self, but not with self follicle cells. However, the activity was detected when they were mixed with sperm or follicle-free eggs regardless of their origin. Out of many monoclonal antibodies against hemocytes, two antibodies suppressed the contact reaction to an appreciable extent. One antibody that suppressed 80-90 % of the phenoloxidase surge from hemocytes upon contact reaction recognized 50 and 25 kDa proteins in the hemocytes and follicle cells. It is therefore suggested that hemocytes and follicle cells share a common molecule(s) that appears to be involved in the allo-recognition in Halocynthia roretzi.

2. Noritaka Hirohashi, Dept. of Life Sci., Tokyo Inst. of Technol., Yokohama 226, Japan. Ph.D. thesis with Dr. M. Hoshi. Role of Acidic O-glycans In Sperm-Vitelline Coat Interactions In The Ascidian, Halocynthia roretzi.

A series of sperm-egg interactions is required for fertilization. Sperm binding to the vitelline coat (VC), an extracellular matrix surrounding the oocytes, is an essential step for successful fertilization in ascidians. This thesis is aimed to identify the molecules that are involved in sperm-VC binding. It was revealed by histochemical staining of VCs with FITC-labeled lectins that several oligosaccharides exist on the outer surface of the VC. Furthermore, these oligosaccharides located in the regions where the sperm bind. From these data, it was proposed that the interaction(s) between the oligosaccharide(s) on VC and the oligosaccharide binding molecule(s) on sperm mediates sperm-VC binding. Acidic and neutral oligosaccharides were obtained from VC after hydorazinolysis. Chemical analysis showed the majority of acidic oligosaccharides had galactose at the reducing termini, sulfate and phosphate groups, suggesting that most of the acidic oligosaccharides were O-linked. It was showed that not the neutral but the acidic oligosaccharides from the VC inhibited sperm-VC binding. Moreover, the acidic oligosaccharides digested with B-L-fucosidase or mild-methanolysis (0.05N HCl/MeOH 37o C, 4h), lost their activity to inhibit sperm-VC binding. These results suggest that terminal fucose and sulfate groups in the acidic oligosaccharides play an important role for sperm-VC binding. Also, in ascidians, several glycosidases in spermatozoa are involved in sperm-egg interactions. In Halocynthia roretzi, several glycosidase activities were detected in spermatozoa. Deoxyfuconojirimycin (DFJ), a potent inhibitor against B-L-fucosidase, inhibited B-L-fucosidase activity in living spermatozoa in normal sea water. DFJ inhibited sperm-VC binding in a dose-dependent manner, suggesting that sperm B-L-fucosidase is involved in the sperm binding to VC. From our data, we conclude that sperm-VC binding is achieved in part by the interaction between sperm B-L-fucosidase and acidic oligosaccharides on VC.

3. Kathryn H. Wiltshire (1994): BSc (Hons) thesis under Dr. Jon Havenhand, Flinders Univ., Adelaide, Australia. Being prepared for publication (with Jon- contact him for more detail). Separating the physiological and mechanical effects of water temperature on the filter feeding characteristics of the solitary ascidian Herdmania momus (Savigny).

This thesis investigates the physiological and mechanical effects of water temperature on filter feeding characteristics of the solitary ascidian Herdmania momus. The observed variation in pumping rate with temperature has been traditionally attributed to physiological effects of temperature. However, water viscosity more than doubles over the range 30 to 0¡C. The Reynolds number of filtering elements is much less than one, indicating that viscous forces dominate over inertia. This study aims to separate the physiological and mechanical (viscous) effects of water temperature on pumping rate, and to determine the effects of viscosity on pump pressure and retention efficiency. Herdmania momus is a ciliary-mucoid filter feeder. In this type of organism viscous forces operate in the propulsion of water by cilia, in pressure drops through the mucous net, and in frictional losses through the siphons, as well as in particle capture by the mucous net. In these experiments viscosity was manipulated by the addition of Ficoll to enable the mechanical and physiological effects of temperature to be separated. The methodology of RiisgŒrd (various papers in Mar. Ecol. Prog. Ser. 1979-1993) was used to assess the impact of temperature and viscosity on pumping rate and pump pressure. Pumping rate showed a positive trend with water temperature in unmanipulated treatments, however, the overall effect of temperature on pumping rate was found not to be significant. Hence, the changes in pumping rate with temperature at constant viscosity, and with viscosity at constant temperature were also not significant. No evidence was found that viscous effects on pumping rate were greater at lower temperatures. Maximum pumping pressure of the ascidian pump was found to vary with temperature in the constant viscosity trials. Maximum pump pressures from 12¡C to 21¡C were not significantly different, but maximum pump pressure at 24¡C was significantly higher than at 12¡C or 15¡C. This suggested a thermo-neutral response over the range 12¡C to 21¡C, a temperature range which may reflect the normal environmental conditions of this species.

Retention efficiency of particles in the size range 3-10 µm was found to be independent of both temperature and viscosity and near 100% for all trials, suggesting that particles in this size range were removed by sieving, in which case viscosity should not affect retention. The pressure loss through the mucous net was found to be the major component contributing to pressure loss through the ascidian pump system, with kinetic losses at the exhalant siphon being the only other important factor. A sample size of approximately 30 would be needed for the observed changes in pumping rates with either temperature or viscosity to be significant (p < 0.05), and a sample size of around 50 needed for the observed change in maximum pump pressure to be significant at the 5% level.

For complete abstracts of the ascidian papers presented at the following recent meetings please request copy of AN37.

Southern Calif. Acad. of Sciences annual meeting, Calif. State Univ. Fullerton, May 5-6, 1995

Intl. Conf. on Environmental & Biol. Aspects of Main-Group Organometals, Bordeaux, France. Sept. 6-9, 1994.

8th Intl. Conf., Intl. Soc. of differentiation, Hiroshima, Japan. Oct. 22-26, 1994.

3rd Intl. Temperate Reef Symp.

Zool. Soc. of Japan annual meeting, Nagoya, Japan. Oct. 5-8, 1994. Published in Zool. Sci. 11, suppl., Dec. 1994.

Sensory Ecology & Physiol. of Zooplankton Symp., Honolulu, HI. Jan. 9-12, 1995.

Abraham, R.T. et al. 1994. Cellular effects of olomoucine, an inhibitor of cyclin-dependent protein kinases. Biomed. J. 1:32-44.

Andersen, V. & J. Sardou 1994. Pyrosoma atlanticum (Tunicata, Thaliacea): diel migration and vertical distribution as a function of colony size. J. Plankton Res. 16:337-349.

Attaway, D.H. & O. Zaborsky, eds. 1994. Marine biotechnology. Plenum Press, NY, 500 pp.

Ballarin, L., F. Cima & A. Sabbadin 1994. Phenoloxidase in the colonial ascidian Botryllus schlosseri (Urochordata: Ascidiacea). Anim. Biol. 3:41-48.

Bates, W.R. 1993. Evolutionary modifications of morphogenetic mechanisms and alternate life history strategies in ascidians. Micros. Res. & Tech. 26:285-300.

Bates, W.R. 1994. Ecological consequences of altering the timing mechanism for metamorphosis in anural ascidians. Amer. Zool. 34:333-342.

Bates, W.R. 1995. Direct development in the ascidian Molgula retortiformis (Verrill, 1871). Biol. Bull. 188:16-22.

Belogortseva, N.I., R.G. Ovodova, S.V. Moroz, N.A. Odintsova, A.V. Yermak & Y.S. Ovodov 1994. Isolation and characterization of a structurally unusual lectin from ascidian Didemnum ternatum (DTL). Bioorganicheskaya Khimiya 20:975-983. (in Russian; English abstract)

Biseswar, R. & T. Maslamoney 1994. An ultrastructural study of spermatogenesis and the mature spermatozoon of the ascidian Pyura stolonifera. S. Afric. J. Zool. 29:93-98.

Boden, C. & G. Pattenden 1994. Total synthesis of lissoclinamide 5, a cytotoxic cyclic peptide from the tunicate Lissoclinum patella. Tetrahedron Lett. 35:8271-8274.

Britten, R.J., T.J. McCormack, T.L. Mears & E.H. Davidson 1995. Gypsy/Ty3-class retrotransposons integrated in the DNA of herring, tunicate, and echinoderms. J. Molec. Evol. 40:13-24.

Brunetti, R. 1994. Ascidians of the northern Adriatic Sea (Aplousobranchia). Boll. Zool. 61:89-96.

Carlton, J.T. & J.B. Geller 1993. Ecological roulette: the global transport of nonindigenous marine organisms. Science 261:78-82.

Cavey, M.J. 1994. Spermatogenesis in the ovotestes of the solitary ascidian Boltenia villosa. pp. 64-76 in Reproduction and Development of Marine Invertebrates, ed. by Wilson, W.H.J., S.A. Stricker & G.L. Shinn.

Chang, W. & R.J. Lauzon 1995. Isolation of biologically functional RNA during programmed death of a colonial ascidian. Biol. Bull. 188:23-31.

Cloney, R.A. 1994. Test cell secretions and their functions in ascidian development. pp. 77-95 in Reproduction and Development of Marine Invertebrates, ed. by Wilson, W.H.J., S.A. Stricker & G.L. Shinn. Baltimore: Johns Hopkins Univ. Press. No reprints were made available by the publisher.

Cloney, R.A. 1995. Origin and differentiation of the inner follicular cells during oogenesis in Molgula pacifica (Urochordata), an ascidian without test cells. Acta Zool. 76:89-104.

Cohen, S. 1990. Outcrossing in field populations of two species of self-fertile ascidians. J. Exp. Mar. Biol. Ecol. 140:147-158.

Compagnone, R.S., D.J. Faulkner, B.K. Carte, G. Chan, A. Freyer, M.E. Hemling, et al. 1994. Pentathiepins and trithianes from two Lissoclinum species and a Eudistoma sp.: inhibitors of protein kinase C. Tetrahedron 50:12785-12792.

Conradi, M., P.J. LopezGonzalez & J.C. GarciaGomez 1994. Botryllophilus conicus n. sp. (Copepoda: Ascidicolidae) associated with a compound ascidian from the Strait of Gibraltar. Syst. Parasitol. 29:97-104.

Crispino, A., A. DeGiulio, S. DeRosa, S. DeStefano, A. Milone & N. Zavodnik 1994. A sulfated normonoterpenoid from the ascidian Polycitor adriaticus. J. Nat. Prod. 57:1575-1577.

Daponte, M.C. & G.B. Esnal 1994. Differences in embryological development in two closely related species: Ihlea racovitzai and Ihlea magalhanica (Tunicata, Thaliacea). Polar Biol. 14:455-458.

Epel, D. 1994. Developmental Biology of Ascidians: a review of the book by N. Satoh. Science 266:1086.

Fielding, P.J., K.A. Weerts & A.T. Forbes 1994. Macroinvertebrate communities associated with intertidal and subtidal beds of Pyura stolonifera (Heller) (Tunicata, Ascidiacea) on the natal coast. S. Afric. J. Zool. 29:46-53.

Fu, X., M.B. Hossain, D. vanderHelm & F.J. Schmitz 1994. Longithorone A: unprecedented dimeric prenylated quinone from the tunicate Aplidium longithorax. J. Amer. Chem. Soc. 116:12125-12126.

Fukuzawa, S., S. Matsunaga & N. Fusetani 1994. Ritterazine A, a highly cytotoxic dimeric steroidal alkaloid, from the tunicate Ritterella tokioka. J. Org. Chem. 59:6164-6166.

Fukuzawa, S., S. Matsunaga & N. Fusetani 1995. Isolation and structure elucidation of ritterazines B and C, highly cytotoxic dimeric steroidal alkaloids, from the tunicate Ritterella tokioka. J. Org. Chem. 60:608-614.

Goodbody, I. 1993. The ascidian fauna of a Jamaican lagoon - 30 years of change. Revista de Biologia Tropical 41:35-38.

Goodbody, I. 1994. The tropical western Atlantic Perophoridae (Ascidiacea): I. The genus Perophora. Bull. Mar. Sci. 55:176-192.

Goudeau, H., Y. Depresle, A. Rosa & M. Goudeau 1994. Evidence by a voltage clamp study of an electrically mediated block to polyspermy in the egg of the ascidian Phallusia mammillata. Dev. Biol. 166:489-501.

Hikosaka, A., T. Kusakabe & N. Satoh 1994. Short upstream sequences associated with the muscle-specific expression of an actin gene in ascidian embryos. Dev. Biol. 166:763-769.

Hirose, E., T. Ishii, Y. Saito & Y. Taneda 1994. Seven types of tunic cells in the colonial ascidian Aplidium yamazii (Polyclinidae, Aplousobranchia): morphology, classification, and possible functions. Zool. Sci. 11:737-743.

Hirose, E., Y. Saito & H. Watanabe 1994. Surgical fusion between incompatible colonies of the compound ascidian, Botrylloides fuscus. Develop. and Comp. Immunol. 18:287-294.

Kanamori, K. & H. Michibata 1994. Raman spectroscopic study of the vanadium and sulphate in blood cell homogenates of the ascidian, Ascidia gemmata. J. Mar. Biol. Ass. U. K. 74:279-286.

Kawahara, H. & H. Yokosawa 1994. Intracellular calcium mobilization regulates the activity of 26 S proteasome during the metaphase-anaphase transition in the ascidian meiotic cell cycle. Dev. Biol. 166:623-633.

Kawamura, K. & S. Fujiwara 1995. Establishment of cell lines from multipotent epithelial sheet in the budding tunicate, Polyandrocarpa misakiensis. Cell Structure and Function 20:97-106.

Kawamura, K., K. Hara & S. Fujiwara 1993. Developmental role of endogenous retinoids in the determination of morphallactic field in budding tunicates. Development 117:835-845.

Lambert, C.C. and G. Lambert 1995. Developmental Biology of Ascidians--a review of the book by N. Satoh. Quart. Rev. of Biol. 70:79.

Lindquist, N. & W. Fenical 1989. Ascidiatrienolides A-C, novel lactonized eicosanoids from the colonial marine ascidian Didemnum candidum. Tetrahedron Lett. 30:2735-2738.

Lindquist, N. & W. Fenical 1990. Polyandrocarpamides A-D, novel metabolites from the marine ascidian Polyandrocarpa sp. Tetrahedron Lett. 31:2521-2524.

Lindquist, N., W. Fenical, L. Parkanyi & J. Clardy 1991. Polyclinal, a new sulfated polyhydroxy benzaldehyde from the marine ascidian Polyclinum planum. Experientia 47:503-506.

Mancuso, V. 1994. Folliculum formation in the ascidian Phallusia mammillata ovary. Anim. Biol. 3:3-14.

Marikawa, Y., S. Yoshida & N. Satoh 1995. Muscle determinants in the ascidian egg are inactivated by UV irradiation and the inactivation is partially rescued by injection of maternal mRNAs. Roux's Arch. Dev. Biol. 204:180-186.

Martoja, R., P. Gouzerh & F. Monniot 1994. Cytochemical studies of vanadium, tunichromes and related substances in ascidians: possible biological significance. Oceanogr. Mar. Biol. Ann. Rev. 32:531-556.

McDonald, L.A., G.S. Eldredge, L.R. Barrows & C.M. Ireland 1994. Inhibition of topoisomerase II catalytic activity by pyridoacridine alkaloids from a Cystodytes sp. ascidian: a mechanism for the apparent intercalator-induced inhibition of topoisomerase II. J. Med. Chem. 37:3819-3827.

McDougall, A. & C. Sardet 1995. Function and characteristics of repetitive calcium waves associated with meiosis. Current Biol. 5:318-328.

Miller, R.L. 1994. Mechanisms for enhancing fertilization success in two species of free-spawning invertebrates with internal fertilization. pp. 106-117 in Reproduction and Development of Marine Invertebrates, ed. vol., ed. by Wilson, W.H.J., S.A. Stricker & G.L. Shinn.

Mourao, P.A.S. & A.M.S. Assreuy 1995. Trehalose as a possible precursor of the sulfated L-galactan in the ascidian tunic. J. Biol. Chem. 270:3132-3140.

Muller, W.E.G., Z. Pancer & B. Rinkevich 1994. Molecular cloning and localization of a novel serine protease from the colonial tunicate Botryllus schlosseri. Molec. Mar. Biol. & Biotech. 3:70-77.

Nishida, H. 1994. Localization of determinants for formation of the anterior-posterior axis in eggs of the ascidian Halocynthia roretzi. Development 120:3093-3104.

Nontratip, A., S. Wada & H. Yamanaka 1991. Post-mortem glycolysis and ATP degradation in the muscle of ascidian Halocynthia roretzi. Nippon Suisan Gakkaishi 57:761-766.

Nontratip, A., S. Wada & H. Yamanaka 1992. Changes in polyamines and amino acids in ascidian Halocynthia roretzi muscle during storage. Nippon Suisan Gakkaishi 58:315-322.

Nontratip, a & H. Yamanaka 1993. Activities of glycogen degrading enzymes in ascidian muscle. Nippon Suisan Gakkaishi 59:1265.

Nontratip, A. & H. Yamanaka 1993. A simple method for confirmation of inosine monophosphate in ascidian Halocynthia roretzi muscle. Nippon Suisan Gakkaishi 59:1635-1636.

Nontratip, A. & H. Yamanaka 1994. Seasonal variations of activities of glycogen-degrading enzymes in ascidian muscle. Fisheries Sci. 60:77-82.

Ohtake, S.-I., T. Abe, F. Shishikura & K. Tanaka 1994. The phagocytes in hemolymph of Halocynthia roretzi and their phagocytic activity. Zool. Sci. 11:681-691.

Oldewage, W.H. 1994. Description of Doropygus pyurus n. sp. (Copepoda, Notodelphyidae) from Pyura stolonifera (Ascidiacea) in South Africa. S. Afric. J. Zool. 29:212-216.

Olsson, R. 1993. Reissner's fiber mechanisms: some common denominators. pp. 33-39 in The Subcommissural Organ, ed. vol., ed. by Oksche, A., E.M. Rodriguez & P. Fernandez-Liebrez.

Olsson, R., R. Yulis & E.M. Rodriguez 1994. The infundibular organ of the lancelet (Branchiostoma lanceolatum, Acrania): an immunocytochemical study. Cell Tiss. Res. 277:107-114.

Peddie, C.M. & V.J. Smith 1994. Mechanism of cytotoxic activity by hemocytes of the solitary ascidian, Ciona intestinalis. J. Exp. Zool. 270:335-342.

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