Number 47                                                                                                                                                      June 2000

In December we worked at the Friday Harbor Labs where Gretchen prepared Guam didemnid spicules for SEM and Charley worked on sperm proteases in Ciona savignyi.  January-February were spent at the University of Guam laboratories in Mangilao where Gretchen continued her work on the endemic ascidians of Guam with Gustav Paulay.  She now has about 115 species for her monograph on the ascidians of Guam.  Charley worked on germinal vesicle breakdown in 2 stolidobranch ascidians.  During this time we also worked at the Coral Reef Research Foundation in Palau for two weeks at the invitation of Patrick and Lori Colin; Gretchen identified many of the harbor ascidians for them and Charley continued with maturation studies.  In May we returned to the Friday Harbor labs.  Gretchen continued her work on the monographs of ascidians from Guam and Alaska. This summer we will take part in two surveys of harbor faunas; the first in Massachusetts and Rhode Island and the second in southern California.  The southern California survey will give us the opportunity to see which of the 11 species of introduced ascidians we reported in our 1998 paper have persisted to the present and to see if there have been further introductions.

It is only a few weeks now until The First Intl. Symposium on the Biology of Ascidians will be held in Sapporo, Japan, from June 26-30. Dr. Hitoshi Sawada, with the help of several colleagues, has worked very hard to put together an excellent program, and we are looking forward to seeing many of you there. The symposium proceedings will be published by Springer-Verlag Tokyo. The latest information, including the full schedule of talks, can be found at the website http://www.hokudai.ac.jp/pharma/seika/ISOBA.htm

As always, this issue of AN includes citations of many new publications covering every aspect of ascidian biology. If you wish to correspond with some of the authors, a list of email addresses of ascidian researchers can be found in AN #45. Please send us a copy of YOUR new publications. We rely on your contributions, including abstracts from recent meetings, work in progress, and your students’ thesis abstracts. If you find this newsletter useful, please let us know!

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

Dr. Hiroshi Watanabe retired in March from the presidency of Tsukuba Womens University after a long and very distinguished career.

Dr. Motonori Hoshi moved from Tokyo Inst. of Technology on April 1 and has a new position at the Center for Life Science and Technology, Graduate School of Science and Technology, Keio University, Yokohama.

Dr. Joel Weintraub, Dept. of Biol. Sci. at Calif. State Univ. Fullerton recently brought to our attention this old song about Amphioxus by Philip H. Pope 1910, sung to the tune of "It’s a Long Long Way from Tipperary." He writes that "it came from a songbook from BBB (probably the Beta Beta Beta Songbook). This was an early (1910s) biology club/organization that fostered the love of biology. I know that parts of the Amphioxus Song found their way into the Woods Hole Songbook of the late 1910s..... so the song is earlier than that..... and could date to the turn of the century."

A fish-like thing appeared among the annelids one day
It hadn't any parapods or setae to display
It hadn't any eyes or jaws or a ventral nervous chord
But it had a lot of gill slits and it had a notochord!

Chorus:
It's a long way from Amphioxus
It's a long way to us
It's a long way from Amphioxus
To the meanest human cuss
Good-bye fins and gill slits
Welcome lungs and hair
It's a long, long way from Amphioxus
But we all came from there. (Note..I added the all for better match to the song)

It wasn't much to look at and it scarce knew how to swim
And Nereis was very sure it didn't spring from him
The Molluscs wouldn't own it and the arthropods got sore
So the poor thing had to burrow in the sand along the shore.

It wriggled in the sand before a crab could nip its tail
It said, "Gill slits and myotomes are all of no avail
I've grown some metapleural folds, and sport an oral hood,
But all these fine new characters don't do me any good"

It sulked a while down in the sand without a bit of pep
Then it stiffened up its notochord and said, "I'll beat em yet,
I've got more possibilities within my slender frame
Than all these proud invertebrates that treat me with such shame"

"My Notochord shall grow into a chain of vertebrae;
As fins my metapleural folds shall agitate the sea;
This tiny dorsal nervous tube shall form a mighty brain
And the vertebrates shall dominate the animal domain."

1. From Kazuhiro W. Makabe kwmakabe@ascidian.zool.kyoto-u.ac.jp We are making a database of Halocynthia maternal mRNAs: MAGEST: MAboya (the ascidian, Halocynthia roretzi) Gene Expression Patterns and Sequence Tags. http://www.genome.ad.jp/magest

This database supplies data of DNA sequences and expression patterns of ESTs (Expressed Sequence Tags) from maternal mRNAs of the ascidian egg. We constructed an arrayed cDNA library from uncleaved fertilized eggs of Halocynthia roretzi (Urochordata, Ascidiacea). Both termini of many Cdna clones were sequenced, and whole-mount in situ hybridization to the staged embryos was carried out to obtain information about localization and/or expression sites of the clones.
    In the phylum Chordata, a genome duplication event took place twice in the process of vertebrate evolution. Ascidian, which is lower Chordata, however, has a non-duplicated genome that can be regarded as a basic set of chordate-type genome. This suggests that ascidian is a good model system to investigate functions of genomes of chordates, espacially when considering that functional analysis of genes is easily accessible by gene introduction to the ascidian eggs.
    Fertilized eggs cleave many times to give rise to multicellular organisms. Within embryos, embryonic blastomeres develop into various types of tissues such as epidermis, muscles and nervous systems. In the processes of early embryogenesis, maternal factors stored in the egg cytoplasms are known to play various significant roles. But the functions of the maternal factors in chordate eggs remain elusive compared with those of fruitfly and nematode. Since the last century, ascidian egg has been well known as a mosaic egg in which many blastomeres in the early embryo differentiate autonomously. Recent works have revealed that there exist cytoplasmic determinants that direct formation of epidermis, muscle and endoderm as well as cytoplasmic factors involved in axis specification of the embryo and in gastrulation. Thus, various processes of ascidian embryogenesis are mediated by maternal factors in the egg.
    In this project, we aim at all-inclusive and systematic description of maternal transcripts stored in fertilized eggs of the Japanese ascidian, Halocynthia roretzi; cDNA sequences from ca. 10,000 different genes and their expression patterns during embryogenesis. This will provide an intellectual estate which would allow us to investigate the maternal factors involved in the early developmental events, and consequently, to study the comprehensive maternal genetic information, which cannot be achieved by specific investigations on some particular phenonena of embryogenesis. Finally this will also enable us to understand molecular mechanisms of establishment of embryonic body plans of chordates and to understand evolution from invertebrates to vertebrates in future.

2. Maturation of ascidian oocytes   Charles Lambert.
   In Guam I examined maturation of two stolidobranch ascidians Herdmania momus (Pyuridae) and Cnemidocarpa irene (Styelidae). Both had stores of ovulated prophase oocytes, unlike the situation in Halocynthia roretzi (Sakairi, K., and H. Shirai 1991. Dev. Growth Differ. 33: 155-162) in which the ovary has numerous unovulated ovarian follicles.  Confirming the findings in H.roretzi, the oocytes of Herdmania undergo maturation in a few minutes in normal sea water but fail to do so in low pH SW.  Oocytes of Cnemidocarpa remain in prophase even after overnight incubation in SW.  The ionophore A23187 stimulates resumption of meiosis in both species and work is in progress investigating roles for phosphorylation and dephosphorylation in the maturation of ascidian oocytes.

1. Canadian Soc. of Zoologists meetings, May 2-6, 2000, St. Andrews, New Brunswick, Canada

Embryogenesis in the ascidian Ciona intestinalis lacks cell death. Cole, A.G.*, T. Koropatnick, & I.A. Meinertzhagen, Life Sciences Centre, Dalhousie University, Halifax, NS B3H 4J1.
    Programmed cell death [apoptosis] is a feature of development in most organisms, prominent in the development and reorganization of functional nervous systems. The nervous system of the ascidian tadpole larva is suggested to develop in the absence of cell death. To investigate this possibility, embryos were fixed at 30 minute intervals from 12 h after fertilization till hatching, and stained with TUNEL to visualize apoptotic cells. Additionally, 2 h- and 12 h-old larvae were analyzed; metamorphosing larvae provided positive controls. TUNEL staining occurred in various tissues in 12 h- and metamorphosing larvae, but embryonic stages lacked staining, as did 2-hr larvae. Thus, embryogenesis of the ascidian larva is novel in lacking apoptosis.

CELL-LINEAGE OF THE LARVAL CNS AFTER NEURULATION IN THE ASCIDIAN Ciona intestinalis. Cole, A.G.* & I.A. Meinertzhagen, Life Sciences Centre, Dalhousie University, Halifax, NS B3H 4J1.
    The ascidian CNS is composed of fewer than 400 cells and develops from invariant cleavage patterns in the absence of cell death or extensive cell migration, suggesting that the mitotic history of each cell should be accountable. Using a nucleic acid stain, 3-D confocal image stacks from a series of fixed, staged embryos have been created in order to catalogue the mitotic history and relative positions of CNS cell nuclei. Cells of the CNS undergo 10 to 14 cell divisions from the fertilized egg, at which point mitotic activity ceases. Cell-lineage analyses have been completed for cells which constitue the caudal nerve cord and visceral ganglion. Resultant cell maps will form the basis for further experimental studies.

2. Soc. for Integrative and Comparative Biology (SICB): January 4-8, 2000, Atlanta, GA, USA.

ASCIDIAN LARVAL NEUROGENESIS; A 4-D ANALYSIS OF CELL LINEAGE IN Ciona intestinalis. Alison G. Cole and Ian A. Meinertzhagen, Life Sciences Centre, Dalhousie University, Halifax Nova Scotia, Canada B3H 4JI.
    Ascidian larvae constitute the simplest invertebrate group exhibiting chordate characteristics. One chordate feature of particular interest is the development of the central nervous system [CNS] in the tadpole larva. Ascidian development is largely determinate; pre-localized cytoplasmic factors specify differentiation early in embryonic development of the larva, thus the pattern of mitotic descent becomes important in the specification of embryonic cell fate. To investigate the extent of variation in late cleavage patterns within the developing CNS, we have created a consecutive series of detailed embryonic cell maps of the larval CNS from the ascidian, Ciona intestinalis. Utilizing confocal microscopy, 3-D images of cell nuclei in wholemount preparations have been generated, and used to reconstruct the relative positions of each cell in the CNS throughout development. The entire lineage of cell lines forming the visceral ganglion and the caudal nerve cord has been worked out, showing little variation in cleavage patterns. The anterior rows of the neural plate incorporate into the neurohypophysis [NH], however, the boundary between the NH and the sensory vesicle remains unclear. The development of these 3-D cell maps should facilitate further analysis of ascidian embryogenesis, particularly with respect to the localization of identified gene products to particular cell lines. Supported by NSERC grant OGP0000065 (to I.A.M.) and Dalhousie Graduate Scholarship (to A.G.C.).

1. Cell-lineage of the larval CNS in the ascidian Ciona intestinalis: Neurula stage through to hatched larva. Alison G. Cole M.Sc. thesis, Biology Dept., Dalhousie University. Dr. Ian Meinertzhagen, thesis advisor.
    Ascidians, or tunicates, constitute the simplest invertebrate group exhibiting chordate features. Chordate features such as the notochord and hollow dorsal nerve cord are found within the tadpole larva, whereas gill slits are a feature of the adult ascidian. Ascidian larval development is characterized by invariant cleavage patterns which produce a small number of cells, about 2600 which include: epidermis, mesenchyme, notochord, endoderm, neurons and neural related cells. The central nervous system [CNS] forms from a flat plate of cells which rolls into a tube on the dorsal side of the embryo, reminiscent of vertebrate neurogenesis. The larval CNS is made up of fewer than 400 cells, and forms in the absence of extensive migration or cell death. These features suggest that cells can be followed through embryogenesis to determine the origin and mitotic history of all cells within the larval CNS. The invariant cleavage patterns allow such an analysis to be carried out on fixed tissue, because cells can be identified reliably in individuals of subsequent stages of development. Such an analysis was done previously on the ascidian Ciona intestinalis for the cells of the neural plate through to the end of neurulation. The current study details these cells from neurulation through to hatching.
    Confocal scanning laser microscopy has been used to image wholemount embryos, thus creating 3-D image stacks of entire embryos. To accomplish this, embryos were stained with a nucleic acid probe, BOBO-3, which allows for imaging of cell nuclei. In addition, cytoplasmic RNA stains with this dye, giving images of some structural details of the cells. Such confocal image stacks were used to reconstruct the relative positions of the cells which constitute the CNS. Maps of successive stages were then used to catalogue the mitotic history of these cells, thus creating cell-lineage diagrams from neurulation to hatching for each cell. TUNEL staining was used to verify the absence of cell death in the development of the nervous system.
    There was no evidence for cell death from TUNEL staining in any tissues throughout embryogenesis. Cell death was apparent in 12hr larvae and metamorphosing animals. Cell lineage analysis confirms these findings, at least for the region of the caudal nerve cord and visceral ganglion. The entire nervous system is formed from 10th- to 13th-generation cells. Three bilateral pairs of 10th-generation cells in the region of the visceral ganglion are presumed to be previously described ventro-lateral motor neurons. There are two additional bilateral pairs of cells in this region also thought to be motor neurons, a 11th-generation pair and a 12th-generation pair. All other cells of the visceral ganglion are in their 12th and final generation at hatching, with most mitotic activity ceasing around 85% of embryonic development. In contrast, cells of the caudal nerve cord are 13th generation cells, as are many cells of the sensory vesicle. It has not been possible to catalogue fully the cells of the sensory vesicle because the high density of nuclei precluded individual identification of cells and their progeny. Although there is some variation in cell positioning, lineage is invariant in cells derived from A-line blastomeres, forming the caudal nerve cord and visceral ganglion. In total 274 of the approximate 331 cells of the CNS have their later lineage now documented.
    Cell maps from the current study could not be matched to previously reported cell maps, and some discrepancies with previously reported lineage data were found. It is likely that the inability to match the different cell maps is due to the different methodological approaches used, and not necessarily errors in either report. Nonetheless, the result of these differences is that the cell-lineage tree for the neural plate will need to be re-mapped in order to root the trees created here. The applicability of this lineage data and possible approaches for the further analysis of the sensory vesicle are discussed.

2. The Evolution of Larval Morphology in Ascidians: A Phylogenetic Analysis of Speciation in the Molgulidae. Jennifer Lynn Huber M.S. thesis, Zool. Dept., Univ. of Washington, Seattle, WA. B. J. Swalla, thesis advisor.
    The Molgulidae family of ascidians has several species which have independently evolved anural, or tailless, larvae. This is in contrast to the typical urodele larva that develops in all other families, and contains a notochord, muscle cells and a dorsal hollow nerve cord. Molecular phylogenies using the 18S rRNA gene and the hypervariable D2 loop of the 28S rRNA gene indicate that species in the family Molgulidae fall into at least four distinct clades, three of which have multiple anural members. For at least two of these clades, we present clear evidence of a circumpolar tailed ancestor whose distribution has subsequently diminished. Furthermore, we show that complex life history characters such as viviparity, loss of tail, and ability to self-fertilize are polyphyletic within the Molgulidae, and appear to have evolved independently. Speciation of the Molgulidae and evolution of tailless larvae appears to have occurred primarily at northern latitudes. Our analyses suggest that the evolution of tailless larvae may be correlated with the biogeography, rather than the ecology, of adult molgulids.
    This work was written as partial fulfillment of a M.S. degree awarded to Jennifer Huber from Penn State University in the laboratory of Dr. Billie J. Swalla. A manuscript has been submitted to Evolution describing in detail the molgulid clades. Meanwhile, Jenn is planning to continue her education at the University of Hawaii, pursuing a Ph.D. in the laboratory of Dr. Mark Martindale.

Aniello, F., A. Locascio, M.G. Villani, A. Di Gregorio, L. Fucci, and M. Branno 1999. Identification and developmental expression of Ci-msxb: a novel homologue of Drosophila msh gene in Ciona intestinalis. Mech. Dev. 88: 123-126.

Bassham, S., and J. Postlethwait 2000. Brachyury (T) expression in embryos of a larvacean urochordate, Oikopleura dioica, and the ancestral role of T. Dev. Biol. 220: 322-332.

Brock, R., J.H. Bailey-Brock, and J. Goody 1999. A case study of efficacy of freshwater immersion in controlling introduction of alien marine fouling communities: the USS Missouri. Pac. Sci. 53: 223-231.

Burighel, P., and G.B. Martinucci. 2000. 7. Urochordata. In Progress in Male Gamete Ultrastructure and Phylogeny. B.G.M. Jamieson, editor. John Wiley & Sons, Ltd. Pp. 261-298.

Butler, D.M., K.M. Allen, F.E. Garrett, L.L. Lauzon, A. Lotfizadeh, and R.A. Koch 1999. Release of Ca(2⁺) from intracellular stores and entry of extracellular Ca(2⁺) are involved in sea squirt sperm activation. Dev. Biol. 215: 453-464.

Byrd, J., and C.C. Lambert 2000. Mechanism of the block to hybridization and selfing between the sympatric ascidians Ciona intestinalis and Ciona savignyi. Mol. Repro. Dev. 55: 109-116.

Cameron, C.B., J.R. Garey, and B.J. Swalla 2000. Evolution of the chordate body plan: new insights from phylogenetic analyses of deuterostome phyla. PNAS 97: 4469-4474.

Carballo, J.L., A. Hernandez-Zanuy, S. Naranjo, B. Kukurtzu, and A.G. Cagide 1999. Recovery of Ecteinascidia turbinata Herdman, 1880 (Ascidiacea: Perophoridae) populations after different levels of harvesting on a sustainable basis. Bull. Mar. Sci. 65: 755-760.

Cima, F., and L. Ballarin 2000. Tributyltin induces cytoskeletal alterations in the colonial ascidian Botryllus schlosseri phagocytes via interaction with calmodulin. Aquatic Toxicology 48: 419-429.

Clarke, M., V. Ortiz, and J.C. Castilla 1999. Does early development of the Chilean tunicate Pyura praeputialis (Heller, 1878) explain the restricted distribution of the species? Bull. Mar. Sci. 65: 745-754.

Coles, S.L., R.C. DeFelice, L.G. Eldredge, and J.T. Carlton 1999. Historical and recent introductions of non-indigenous marine species into Pearl Harbor, Oahu, Hawaiian Islands. Mar. Biol. 135: 147-158.

Connell, S.D. 2000. Floating pontoons create novel habitats for subtidal epibiota. J. Exp. Mar. Biol. Ecol. 247: 183-194.

Crenshaw, H.C., C.N. Ciampaglio, and M. McHenry 2000. Analysis of the three-dimensional trajectories of organisms: estimates of velocity, curvature and torsion from positional information. J. Exp. Biol. 203: 961-982.

Dawson, M.N., K.A. Raskoff, and D.K. Jacobs 1998. Field preservation of marine invertebrate tissue for DNA analyses. Molec. Mar. Biol. Biotech. 7: 145-152.

Degnan, B.M., and C.R. Johnson 1999. Inhibition of settlement and metamorphosis of the ascidian Herdmania curvata by non-geniculate coralline algae. Biol. Bull. 197: 332-340.

Di Gregorio, A., and M. Levine 1999. Regulation of Ci-tropomyosin-like, a Brachyury target gene in the ascidian, Ciona intestinalis. 126: 5599-5609.

Duran, R., E. Zubia, and J. Salva 1999. Novel alkaloids from the red ascidian Botryllus leachi. Tetrahedron 55: 13225.

Edwards, M., A.W.G. John, H.G. Hunt, and J.A. Lindley 1999. Exceptional influx of oceanic species into the North Sea late 1997. J. Mar. Biol. Ass. U.K. 79: 737-739.

Eri, R., J.M. Arnold, V.F. Hinman, K.M. Green, M.K. Jones, B.M. Degnan, and M.F. Lavin 1999. Hemps, a novel EGF-like protein, plays a central role in ascidian metamorphosis. Development 126: 5809-5818.

Fontana, A., M.C. Gonzalez, and G. Cimino 2000. Structure and absolute stereochemistry of stolonoxide A, a novel cyclic peroxide from the marine tunicate Stolonica socialis. Tetrahedron Lett. 41: 429.

Forward, R.B.J., J.M. Welch, and C.M. Young 2000. Light induced larval release of a colonial ascidian. J. Exp. Mar. Biol. Ecol. 248: 225-238.

Frizzo, A., L. Guidolin, L. Ballarin, and A. Sabbadin 1999. Purification and partial characterisation of phenoloxidase from the colonial ascidian Botryllus schlosseri. Mar. Biol. 135: 483-488.

Fu, X., M.L. Ferreira, and F.J. Schmitz 1999. Longithorols A and B, novel prenylated paracyclophane- and metacyclophane-type hydroquinones from the tunicate Aplidium longithorax. J. Nat. Prod. 62: 1306-1310.

Fujii, K., and M. Fukumoto 1999. Multiple acrosomal vesicles and their differentiation during spermiogenesis in Ascidia zara and Ascidia gemmata (Ascidiacea, Tunicata). J. Morph. 242: 101-106.

Fujimura, M., and K. Takamura 2000. Characterization of an ascidian DEAD-box gene, Ci-DEAD1: specific expression in the germ cells and its mRNA localization in the posterior-most blastomeres in early embryos. Dev Genes Evol 210: 64-72.

Fukumoto, M. 2000. Acrosome reaction in spermatozoa of the ascidian Styela plicata (Ascidiacea, Tunicata). Invert. Repro. Dev. 37: 89-94.

Glasby, T.M. 1999. Interactive effects of shading and proximity to the seafloor on the development of subtidal eipbiotic assemblages. Mar. Ecol. Prog. Ser. 190: 113-124.

Glasby, T.M. 2000. Surface composition and orientation interact to affect subtidal epibiota. J. Exp. Mar. Biol. Ecol. 248: 177-190.

Gorbman, A. 1999. Brain-Hatschek's pit relationships in amphioxus species. Acta Zool. 80: 301-305.

Hirose, E. 1999. Pigmentation and acid storage in the tunic: protective functions of the tunic cells in the tropical ascidian Phallusia nigra. Invert. Biol. 118: 414-422.

Hopcroft, R.R., and B.H. Robison 1999. A new mesopelagic larvacean, Mesochordaeus erythrocephalus, sp. nov., from Monterey Bay, with a description of its filtering house. J. Plankton Res. 21: 1923-1938.

Hotta, K., H. Takahashi, A. Erives, M. Levine, and N. Satoh 1999. Temporal expression patterns of 39 Brachyury-downstream genes associated with notochord formation in the Ciona intestinalis embryo. Dev. Growth Differ. 41: 657-664.

Itaya, T., Y. Hozumi, and T. Ohta 1999. Synthesis and structure of the marine ascidian 8-oxoadenine aplidiamine. Chemical & pharmaceutical bulletin. 47: 1297.

Jackman, W.R., J.A. Langeland, and C.B. Kimmel 2000. islet reveals segmentation in the amphioxus hindbrain homolog. Dev. Biol. 220: 16-26.

Kanamori, K., M. Sakurai, and H. Michibata 1999. Direct reduction from vanadium(V) to vanadium(IV) by NADPH in the presence of EDTA. A consideration of the reduction and accumulation of vanadium in the ascidian blood cells. J. Inorg. Biochem. 77: 157-161.

Kawashima, T., S. Kawashima, M. Kanehisa, H. Nishida, and K.W. Makabe 2000. MAGEST: MAboya gene expression patterns and sequence tags. 28: 133-5.

Lacalli, T.C., and S.J. Kelly 2000. The infundibular balance organ in amphioxus larvae and related aspects of cerebral vesicle organization. Acta Zool. 81: 37-47.

Lambert, C.C. 2000. Germ-cell warfare in ascidians: sperm from one species can interfere with the fertilization of a second species. Biol. Bull. 198: 22-25.

Levasseur, M., and A. McDougall 2000. Sperm-induced calcium oscillations at fertilisation in ascidians are controlled by cyclin B1-dependent kinase activity. Development 127: 631-41.

Lindsay, B.S., A.M. Almeida, C.J. Smith, R.G. Berlinck, R.M. da Rocha, and C.M. Ireland 1999. 6-Methoxy-7-methyl-8-oxoguanine, an unusual purine from the ascidian Symplegma rubra. J. Nat. Prod. 62: 1573-5.

Lopez-Gonzalez, P.J., C. Megina, and M. Conradi 1999. Ascidioxynus ibericus n. sp. (Copepoda: Poecilostomatoida: Lichomolgidae) associated with the ascidian Clavelina dellavallei from the Strait of Gibraltar. Hydrobiologia 400: 205.

Lopez-Urrutia, A., and J.L. Acuna 1999. Gut throughput dynamics in the appendicularian Oikopleura dioica. Mar. Ecol. Prog. Ser. 191: 195-205.

Loya, S., A. Rudi, Y. Kashman, and A. Hizi 1999. Polycitone A, a novel and potent general inhibitor of retroviral reverse transcriptases and cellular DNA polymerases. Biochem. J. 344 Pt 1: 85-92.

Matsumura, K., S. Mori, and N. Fusetani 1999. Induction of larval metamorphosis in the ascidian, Halocynthia roretzi by excess potassium ion and by reduced calcium ion. J. Mar. Biol. Ass. UK 79: 1143-1144.

Matsumura, K., S. Tsukamoto, M. Nagano, H. Kato, H. Hirota, and N. Fusetani 1999. Inhibition of hatching in the ascidian, Halocynthia roretzi, by (Z)- and (E)- narains isolated from a marine sponge, Jaspis sp. Invert. Repro. & Dev. 35: 19-25.

Miralto, A., A. Ianora, S.A. Poulet, G. Romano, I. Buttino, and S. Scala 1999. Embryonic development in invertebrates is arrested by inhibitory compounds in diatoms. Mar. Biotechnol. 1: 401-402.

Monniot, F., and C. Monniot 1997. Ascidians collected in Tanzania. J. E. Afr. Nat. Hist. 86: 1-35.

Nishikawa, T. 1999. Ascidians of the Döderlein collection. In Preliminary Taxonomic and Historical Studies on Prof. Ludwig Döderlein's Collction of Japanese Animals Made in 1880-81 and Deposited at Several European Museums [grant report; not an official publication]. T. Nishikawa, editor. Nagoya University, Nagoya. 144-146.

Nishino, A., K. Kubokawa, M. Sekifuji, N. Azuma, and M. Morisawa 1999. A survey of Amphioxus (Cephalocordata: Branchiostoma belcheri) in the offing of Misaki. Benthos Res. 54: 29-35.

Ogasawara, M., R. Di Lauro, and N. Satoh 1999. Ascidian homologs of mammalian thyroid transcription factor-1gene are expressed in the endostyle. Zool. Sci. 16: 559-565.

Ohkuma, M., and M. Tsuda 1999. Visualization of retinal proteins in the cerebral ganglion of ascidian, Halocynthia roretzi. Zool. Sci.: .

Okada, T., and M. Yamamoto 1999. Differentiation of the gonad rudiment into ovary and testis in the solitary ascidian, Ciona intestinalis. Dev. Growth Differ, 41: 759-768.

Paffenhofer, G.-A., and D.M. Gibson 1999. Determination of generation time and asexual fecundity of doliolids (Tunicata, Thaliacea). J. Plankton Res. 21: 1183-1189.

Parker, L.E., S. Culloty, R.M. O'Riordan, B. Kelleher, S. Steele, and G. Van der Velde 1999. Preliminary study on the gonad development of the exotic ascidian Styela clava in Cork Harbour, Ireland. J. Mar. Biol. Ass. UK 79: 1141-1142.

Patton, S.J., G.N. Luke, and P.W.H. Holland 1998. Complex history of a chromosomal paralogy region: insights from amphioxus aromatic amino acid hydroxylase genes and insulin-related genes. Mol. Biol. Evol. 15: 1373-1380.

Raftos, D.A., D.L. Stillman, and E.L. Cooper 1998. Chemotactic responses of tunicate (Urochordata, Ascidiacea) hemocytes in vitro. J. Invert. Pathol. 72: 44-49.

Richter, C., and M. Wunsch 1999. Cavity-dwelling suspension feeders in coral reefs--a new link in reef trophodynamics. Mar. Ecol. Prog. Ser. 188: 105-116.

Rigby, G.R., G.M. Hallegraeff, and C. Sutton 1999. Novel ballast water heating technique offers cost-effective treatment to reduce the risk of global transport of harmful marine organisms. Mar. Ecol. Prog. Ser. 191: 289-293.

Riisgard, H.U. 1998. Filter feeding and plankton dynamics in a Danish fjord: a review of the importance of flow, mixing and density-driven circulation. J. Env. Mgement 53: 195-207.

Riisgard, H.U., and I. Svane 1999. Filter feeding in lancelets (amphioxus), Branchiostoma lanceolatum. Invert. Biol. 118: 423-432.

Rinehart, K.L. 2000. Antitumor compounds from tunicates. Med. Res. Rev. 20: 1-27.

Rinkevich, B., and M. Shapira 1999. Multi-partner urochordate chimeras outperform two-partner chimerical entities. Oikos 87: 315-320.

Sagarin, R.D., J.P. Barry, S.E. Gilman, and C.H. Baxter 1999. Climate-related change in an intertidal community over short and long time scales. Ecol. Monog. 69: 465-490.

Sanamyan, K. 1999. Ascidians from the North-western Pacific region. 6. Didemnidae. Ophelia 51: 143-161.

Sanamyan, K.E. 2000. Three related Aplidium species from the southern Kurile Islands (Ascidiacea: Polyclinidae). Zoosyst. Rossica 8: 211-216.

Sanamyan, K.E., and N.P. Sanamyan 1999. Some benthic Tunicata from the southern Indo-Pacific Ocean. J. Nat. Hist. 33: 1835-1876.

Sasakura, Y., M. Ogasawara, and K.W. Makabe 2000. Two pathways of maternal RNA localization at the posterior-vegetal cytoplasm in early ascidian embryos. Dev. Biol. 220: 365-378.

Sata, N.U., and N. Fusetani 2000. Amaminols A and B, new bicyclic amino alcohols from an unidentified tunicate of the Family Polyclinidae. Tetrahedron Lett. 41: 489-492.

Sato, Y., and M. Morisawa 1999. Loss of test cells leads to the formation of new tunic surface cells and abnormal metamorphosis in larvae of Ciona intestinalis (Chordata, Ascidiacea). Dev. Genes Evol. 209: 592-600.

Shirae, M., E. Hirose, and Y. Saito 1999. Behavior of hemocytes in the allorejection reaction in two compound ascidians, Botryllus scalaris and Symplegma reptans. Biol. Bull. 197: 188-197.

Tanaka, K.J., H. Kawamura, H. Matsugu, and T. Nishikata 2000. An ascidian glycine-rich RNA binding protein is not induced by temperature stress but is expressed under a genetic program during embryogenesis. Gene 243: 207-214.

Tsutsui, H., and Y. Oka 2000. Light-sensitive voltage responses in the neurons of the cerebral ganglion of Ciona savignyi. Biol. Bull. 198: 26-28.

van den Brenk, A.L., D.P. Fairlie, L.R. Gahan, G.R. Hanson, and T.W. Hambley 1996. A novel potassium-binding hydrolysis product of ascidiacyclamide: a cyclic octapeptide isolated from the ascidian Lissoclinum patella. Inorg. Chem. 35: 1095-1100.

Vervoort, H., W. Fenical, and R.A. Epifanio 2000. Tamandarins A and B: new cytotoxic depsipeptides from a Brazilian ascidian of the Family Didemnidae. J. Org. Chem. 65: 782.

Wada, H., J. Garcia-Fernàndez, and P.W.H. Holland 1999. Colinear and segmental expression of amphioxus Hox genes. Dev. Biol. 213: 131-141.

Yokobori, S., T. Ueda, G. Feldmaier-Fuchs, S. Paabo, R. Ueshima, A. Kondow, K. Nishikawa, and K. Watanabe 1999. Complete DNA sequence of the mitochondrial genome of the ascidian Halocynthia roretzi (Chordata, Urochordata). Genetics 153: 1851-62.

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