Number 49                                                                                                                       May 2001

Greetings!  We are getting ready for another summer at the Friday Harbor Laboratories where Charley will teach comparative invertebrate embryology with Mark Martindale and Gretchen will continue with her huge monograph on the ascidians of Guam.  We hope to see many of you if you pass through Friday Harbor this summer. We will also be in Seattle during the Society for Developmental Biology meetings in Seattle this July.  Give us a call if you will be at the meetings.

This winter we made a brief trip to Maryland to the Smithsonian Environmental Research Center where Gretchen identified several hundred ascidians from SERC settling panels from harbors on both coasts as well as Alaska.  Gretchen’s recent taxonomic projects have also included ascidians from Baja California green sea turtle guts and offshore oil platforms near Santa Barbara.  Later this summer we will participate in an expedition to monitor invasive marine invertebrates in the major harbors of Kodiak Alaska and its surroundings.  This Smithsonian-sponsored survey will include about 14 specialists in different taxa.

There are 105 new references in this issue; keep up the splendid work. Please send us reprints; thanks very much.  We use them constantly, not only in our own work but also in answering the many queries we get.

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

1.  If you have not yet joined the new email Tunicata listserv you are missing a very interesting and valuable means of communication on a fascinating variety of topics.  Perhaps you have a question you would like to ask the group at large. There are about 200 or more subscribers now.  To join, go to the website http://www.jiscmail.ac.uk/lists/tunicata.html and follow the simple directions.  Also on the website is an archive of previous email dialogues since the listserv was created.

2.  The symposium proceedings volume resulting from the June 2000 ascidian meeting in Sapporo (the First International Symposium on the Biology of Ascidians) should reach those who ordered copies any day now.  The title is "The Biology of Ascidians" edited by Hitoshi Sawada, Hideyoshi Yokosawa, and Charles C. Lambert, issued by Springer-Verlag Tokyo.  It contains 69 chapters with 57 figures (photographs) including 13 in color, 465 pages total.  This book will be an invaluable resource for many years to come; we urge you to purchase copies for yourself or request that your institution’s library purchase one.  For more information please go to http://www.hokudai.ac.jp/pharma/seika/ISOBA.htm.  There you will find an order form and Table of Contents in .pdf form that can be downloaded.

3.  Bill Jeffery (Dept. of Biol., Univ. of Maryland; wj33@umail.umd.edu) has just published an excellent new review on molecular aspects of developmental biology of ascidians: Jeffery, W. R. 2001. Determinants of cell and positional fate in ascidian embryos. Int Rev. Cytol. 203: 3-62.

4.  Yukiko Yamazaki (Center for Genetic Resource Information, Natl. Inst. of Genetics, Yata 1111, Mishima, Shizuoka 411-8540, Japan; yyamazak@lab.nig.ac.jp) has a new website containing “Information of Genetic Resource Databank” which contains many useful URLs and contacts for databases and stocks.  The full list of the collection can be viewed at http://www.shigen.nig.ac.jp/grd.   If you have information to add to this valuable website, please contact Dr. Yamazaki.

5.  Judy D’Amore (Univ. of Washington Biol. Dept. and Friday Harbor Labs; jdamore@macaid.com) has developed a fantastic website for teaching field techniques on marine invasions at the middle school and high school level, using Ciona savignyi as an example.  Entitled Exotic Tunicates on the Move:  a summer marine biology investigation for middle or high school students of Puget Sound, the web address is http://depts.washington.edu/fhlk12/StudentProjects/TunIndex.html.

1. Dr.V.K. Meenakshi, Dept. of Zoology, APC Mahalaxmi College for Women, Tuticorin 628 002, Tamil Nadu, India.  vkmeenakshi@yahoo.com
    Our department has an ongoing project on "Marine Biodiversity-Taxonomy of Indian Ascidians" sponsored by the Ministry of Environment & Forests, Government of India, New Delhi. Under this project during the past year, we have collected several hundred ascidians from many uninvestigated areas along the Indian coast, including intertidal rocky areas, materials used for aquaculture operations, hulls of ships, harbour installations, marine aquarium tanks, trawls, chunk, pearl paars, coral reefs etc. This is the first time in India that such an extensive collection effort is being carried out. Collection of literature and identification of the collected ascidians are underway.

2. M.Tamil Selvi, Dept. of Zoology, V.O.Chidambaram College, Tuticorin 628 008, India. tsseellvi@usa.net
 My Ph.D. thesis is on "Ecological studies on ascidians of the Tuticorin coast" in India. I am mainly focussing on: population, distribution, biochemical components, physico-chemical characteristics of the water, associated flora & fauna, associated bacteria and metal accumulation under Dr.V.Sivakumar,

3.  Chris Cameron (Dept. of Mar. Sci., Univ. of Texas at Austin Mar. Sci. Institute, Port Aransas, TX; cameron@utmsi.utexas.edu) and colleagues have a number of new papers in press and in preparation, as well as quite a few recently published that you will find in this and the previous issues of AN:  Cameron, C.B. (2001) Particle retention and flow in the pharynx of the enteropneust worm Harrimania planktophilus: The filter feeding pharynx evolved prior to the divergence of the hemichordates and the chordates. Biol. Bull. In press.  Cameron, C.B. (2001) The anatomy, life habits and later development of a new species of enteropneust, Harrimania planktophilus (Hemichordata: Harrimaniidae) from Barkley Sound. Biol. Bull. In press.   Articles submitted to refereed journals: Winchell, C.J., J. Sullivan, B.J. Swalla, C.B. Cameron and J. Mallatt (0000) Phylogeny of the deuterostome animals: Evaluating competing theories with new LSU and SSU ribosomal DNA data. Molecular Biology and Evolution. 52 pp, 8 figs + 6 tables

3. Gretchen and Charles Lambert continue their work on  the invasive ascidians of southern California harbors and the individual marinas within each harbor.  Presently we are investigating the relationship between the distribution of ascidian species, currents and the density of microbes attributed to anthropogenic sources.  Our data includes all major harbors from San Diego to Santa Barbara from 1994-2000.  We hope to complete a major publication this year.  In additon to ongoing work on ascidian oocyte maturation, Charley is continuing work on germ-cell warfare.  New experiments suggest that styelid  sperm are not able to elicit glycosidase release or block fertilization of Ascidia columbiana (formerly A. callosa) eggs as are pyurid and heterologous phlebobranch sperm.  These experiments will be continued this summer. Gretchen continues work on her monograph on the ascidians of Guam and is also planning to write a brief paper on the now worldwide spread of Botrylloides violaceus.  Many of the recent sightings are not yet in any publications.  She would greatly appreciate receiving any information you have about the presence of this species in your area.  If B. violaceus exists in your local harbor or collecting sites please drop her an email (glambert@fullerton.edu) as she would like this paper to be as inclusive as possible.

1. Stimulation of the beta adrenergic receptor releases the enzyme N-acetylglucosaminidase in Ascidia ceratodes eggs.  Sabrina Sunghe Hahn, M.S. thesis, Laboratory for Sperm Cell Biology and Gamete Ultrastructure, Dept. of Biol. Science, California State Univ., Fullerton, CA 92834. Advisor Dr. Robert A. Koch.
    The study of the beta adrenergic receptor (betaAR) and the signaling pathway that leads to the early block to polyspermy, glycosidase release, in the Ascidia ceratodes eggs was undertaken.  The betaAR agonist consistently resulted in high glycosidase activity and was inhibited by the competitive betaAR antagonist propranolol.  Propranolol also had a significant affect on fertilization.  The glycosidase assay using the fluorescent substrate 4 MU was used to measure glycosidase activity.  The cholinergic agonist acetylcholine did not have any effect on glycosidase release, which indicates that cholinergic receptors are not present in significant amounts.  The ability of the alphaAR antagonist, pheoxybenzamine, to significantly inhibit glycosidase release in both isoproterenol-treated and fertilized eggs suggests there there exists some alpha character on egg surfaces.  However, the predominant receptor types in the acsidian eggs are the betaARs.  PTX, a Gi protein sensitive agent, also showed some inhibition in glycosidase release.  The secondary messenger system, cAMP also showed high levels of glycosidase release.  This betaAR-mediated signaling model exists as a parallel pathway to the tyrosine kinase pathway as suggested by others.

2. Ultrastructure of the visceral ganglion in the ascidian larva Ciona intestinalis: cell circuitry and synaptic distribution. S. Stanley MacIsaac, M.Sc. Thesis, Dept. of Biology, Dalhousie University, 1999. Thesis advisor Dr. I.A. Meinertzhagen.
   The main goal of this project is to decipher the ultrastructure of the visceral ganglion using serial transmission electron microscopy (EM), and to construct a circuit diagram similar to that already achieved for the invertebrate nematode worm Caenorhabditis elegans.  Ascidian larvae are among the most numerically simple organisms organized along a vertebrate body plan, and were chosen for this investigation primarily because of their phylogenetic position, lying close the presumed ancestor of all chordate groups.  The dispersive stage of the life-cycle in the ascidian Ciona intestinalis, the swimming tadpole larva, is only 1.5 mm in length, and has a dorsal central nervous system (CNS) with a small total cell number which is probably eutelic.  A series of 2100 ultrathin sections was obtained from a single specimen, two hours post-hatching.  Results are mainly from this series, however, two other EM series exist from a previous investigator and were used to corroborate many results.  The region analyzed extends from the caudal sensory vesicle or neck region through the visceral ganglion, and ends at the rostral commencement of the caudal nerve cord.  In Ciona  a total of 66 cells are identified in the visceral ganglion, comprising 37 glial/ependymal cells, 10 motoneurons and 19 interneurons.  The interneurons are of two types, those with either ipsilateral or contralateral fibres, the second category consisting of two pairs of decussating interneurons.  The muscle innervation consists of nerves to the dorsal and middle muscle bands only and does not exhibit any rostrocaudal pattern.  This pattern differs from that described in previous investigations describing larval ascidians, granted these studies concentrated primarily on another species. The ganglion has a central neuropile region containing 80-90 fibres surrounded by a cortex of cell bodies. Caudally this neuropile region splits longitudinally, forming two ventrolateral fibre bundles. Within the ganglion are the five pairs of motoneurons, so defined on the basis of tracing their axons out to neuromuscular endplates.  Innervation is multiterminal, with the same motoneuron forming multiple endplates.  In some cases motoneuron axons also form direct synaptic connections with the muscle bands, without forming any terminal endplates.  The motoneurons tend to lack dendrites.  The two decussating cell pairs are situated at either end of the ganglion, the rostral pair send descending fibres, and the caudal pair send ascending fibres, through the ganglion.  All synapses are monads with clear vesicles.  There are 503 chemical synapses within the ganglion, as well as structures resembling gap junctions that are localized specifically to the caudal end of the ganglion.  The visceral ganglion neurons synapse rarely with each other, rather they mainly receive input from fibres descending from the sensory vesicle.  The exception to this is the decussating neuron pair with ascending axons, which have the most synaptic contacts onto other identified ganglion neurons but with few synaptic inputs.  Such circuit data, based solely on structural indications of pathways can certainly not address fully the functional complexities of the working nervous system.  The results did not provide conclusive evidence of a morphologically distinct central pattern generator subserving swimming movements of the tail.  They did, however, identify certain cell pairs that are likely key mediators of locomotion, primarily the rostral decussating cell pair 1/2, midline cell pair 3/4 and the decussating ascending interneurons 26/27 all of which should be targeted in future studies.

3. Cell-lineage of the larval nervous system in the ascidian Ciona intestinalis: neurula stage through to hatched larva. Alison G. Cole, M.Sc. Thesis, Dept. of Biology, Dalhousie University, 2000. Thesis advisor Dr. I.A. Meinertzhagen.
   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.

4.  Tracking an exotic tunicate in Puget Sound, Washington. Judy D’Amore, Biology Dept. and Friday Harbor Laboratories, University of Washington. M.S. Degree in Biology Teaching.  Thesis advisor Dr. Trish Morse.
  Test ropes suspended in six Puget Sound Marinas were monitored monthly over a six-month growing season for presence of Ciona savignyi, an exotic tunicate first discovered in two separate parts of Puget Sound in 1998 and in a third in 1999.  Its arrival in a new location was documented in this study.  It was found to favor depths of 2.5 to 3.5 meters and is more common on shaded ropes than in full sunlight. Once fully established, it is easily the most conspicuous organism in the depth ranges that it prefers. Preliminary findings suggest it may be causing depressed population densities of other tunicate species in those places.  Applications of this kind of science study for secondary school students in and out of the classroom are discussed.
 

Society for Developmental Biology 60th Annual Meeting, Seattle, WA July 18-21, 2001.

1. A developmental role for the immune system in urochordate metamorphosis: molecular and morphological investigations of ascidian metamorphosis reveal elements of an innate immune response. B. J. Davidson and B. J. Swalla, Univ. of Washington, Seattle, WA, 98195, USA
    We have isolated transcripts differentially expressed during metamorphosis in the ascidian Boltenia villosa by suppressive PCR subtractions of staged larval and juvenile cDNAs. These differentially expressed mRNA transcripts include a set which match vertebrate innate immunity genes.  Innate immunity is the primitive counterpart to adaptive immunity and is conserved across the bilaterians.  Innate immune responses include inflammation and complement activation. During ascidian metamorphosis there is a migration of blood cells across the epidermis.  Our results indicate that this migration can be considered an innate-immunity related inflammatory response. Careful observations of Boltenia metamorphosis has led to the detection of a group of mesenchymal cells which migrate through a tube connecting the anterior epidermis to the outside of the juvenile tunic.  The function of this extra-somatic migration is currently under investigation.  Innate immunity confers a rapid response to pathogen-specific molecules. Therefore, an innate immune response during Boltenia metamorphosis may trigger and/or coordinate settlement in response to bacterial cues. Alternatively, innate immunity during metamorphosis could be involved in phagocytosis and re-structuring of larval tissues. We are currently conducting experiments to explore these hypotheses.  We are also interested in exploring the possibility that the innate immune system in vertebrates may have a related developmental role.

2. Germinal vesicle breakdown in ascidian oocytes is controlled by kinases and phosphatases.  Charles C. Lambert, Univ. of Washington Friday Harbor Laboratories, Friday Harbor, WA.
    Maturation of the animal egg involves controls within the oocyte and external signals which regulate the internal events.  Ascidians, the invertebrate deuterostome group that gave rise to the vertebrates, includes 3 orders, the most complex of which contains 3 families: Pyuridae, Styelidae and Molgulidae.  Thus far the onset of maturation (germinal vesicle breakdown = GVBD) has been investigated in a single pyurid species.  Here GVBD begins when the oocyte contacts sea water (SW) but nothing is known about the internal events.  Herdmania pallida (Pyuridae) functions like the other pyurid studied, with GVBD occurring in sea water. Oocytes of Cnemidocarpa irene (Styelidae) do not undergo GVBD in SW but must be activated.  GVBD in Herdmania oocytes can be inhibited by pH 4 SW but can also then be activated at pH 4 by specific triggers.  Mastoparan (a G-protein activator), A-23187 (a calcium ionophore) and dimethylbenzanthracene (a tyrosine kinase activator) can all activate GVBD in Herdmania and Cnemidocarpa oocytes.  This activation of GVBD involves cyclin and cyclin-dependent kinase (maturation promoting factor = MPF) activity, as the cyclin dependent kinase inhibitors roscovitine and olomoucine inhibit the process.  It also involves dephosphorylation of the MPF as demonstrated by the ability of the phosphatase inhibitor vitamin K3 to inhibit GVBD. This pathway is typical of echinoderms but not of most  vertebrates, which must synthesize cyclin to initiate GVBD.  GVBD can also be inhibited by tyrphostin A23, a tyrosine kinase inhibitor, and LY-294002, a phosphatidylinositol 3 kinase (PI3K) inhibitor.  LY-294002 inhibits strongly when activation is by mastoparan or ionophore but not when the tyrosine kinase pathway is activated.  Thus ascidian oocytes have preformed MPF which only has to be dephosphorylated to trigger GVBD.  This dephosphorylation involves activation of a phosphatase by phosphorylation.

Abe, Y., Nagata, R., Hasunuma, Y. and Yokosawa, H. 2001. Isolation, characterization and cDNA cloning of a one-lobed transferrin from the ascidian Halocynthia roretzi. Comp. Biochem. Physiol. B 128: 73-79.

Abourriche, A., Charrouf, M., Chaib, N., Bennamara, A., Bontemps, N. and Francisco, C. 2000. Isolation and bioactivities of epidioxysterol from the tunicate Cynthia savignyi. Farmaco 55: 492-494.

Aiello, A., Carbonelli, S., Fattorusso, E., Iuvone, T. and Menna, M. 2001. New bioactive sulfated metabolites from the Mediterranean tunicate Sidnyum turbinatum. J. Nat. Prod. 64: 219-221.

Angel, M. V. and Pugh, P. R. 2000. Quantification of diel vertical migration by micronektonic taxa in the northeast Atlantic. Hydrobiologia 440: 161-179.

Azumi, K., Ishimoto, R., Fujita, T., Nonaka, M. and Yokosawa, H. 2000. Opsonin-independent and -dependent phagocytosis in the ascidian Halocynthia roretzi: galactose-specific lectin and complement C3 function as target-dependent opsonins. Zool. Sci. 17: 625-632.

Ben-Schlomo, R., Douek, J. and Rinkevich, B. 2001. Heterozygote deficiency and chimerism in remote populations of a colonial ascidian in New Zealand. Mar. Ecol. Prog. Ser. 209: 109-117.

Bresciani, J. and Lopez-Gonzalez, P. J. 2001. Arthurhumesia canadiensis, new genus and species of a highly transformed parasitic copepod (Crustacea) associated with an ascidian from British Columbia. J. Crustac. Biol. 21: 90-95.

Britton, R., de Oliveira, J. H. H. L., Andersen, R. J. and Berlinck, R. G. S. 2001. Granulatimide and 6-bromogranulatimide, minor alkaloids of the Brazilian ascidian Didemnum granulatum. J. Nat. Prod. 64: 254-255.

Camargo, A. J., Oliveira, J. H. H. L., Trsic, M. and Berlinck, R. G. S. 2001. Molecular orbital calculations, experimental and theoretical UV spectra of granulatimides and didemnimides, biologically active polycyclic heteroaromatic alkaloids from the ascidian Didemnum granulatum. J. Mol. Struct. 559: 67-77.

Cameron, C. B., Mackie, G. O., Powell, J. F. F. and Sherwood, N. M. 1999. Gonadotropin-releasing hormone in mulberry cells of Saccoglossus (Hemichordata: Enteropneusta). Gen. Comp. Endocrinol. 114: 2-10.

Carballo, J. L. 2000. Distribution of Ecteinascidia turbinata (Ascidiacea : Perophoridae) in mangroves of the Yucatan Peninsula, Mexico. Rev. Biol. Trop. 48: 365-369.

Castilla, J. C., Guinez, R., Alvarado, J. L., Pacheco, C. and Varas, M. 2000. Distribution, population structure, population biomass and morphological characteristics of the tunicate Pyura stolonifera in the Bay of Antofagasta, Chile. P.S.Z.N.: Marine Biology 21: 161-174.

Castilla, J. C. and Varas, M. A. 1998. A plankton trap for exposed rocky intertidal shores [does not include ascidians but could be used for this purpose]. Mar. Ecol. Prog. Ser. 175: 299-305.

Clarke, M. and Castilla, J. C. 2000. Two new records of ascidians (Tunicata: Ascidiacea) for the continental coast of Chile [Asterocarpa humilis and Molgula ficus; in Spanish with English abstract]. Revista Chilena de Historia Natural 73: 503-510.

Cohen, B. F., Currie, D. R. and McArthur, M. A. 2000. Epibenthic community structure in Port Phillip Bay, Victoria, Australia. Mar. Freshwater Res. 51: 689-702.

Corrado, M., Aniello, F., Fucci, L. and Branno, M. 2001. Ci-IPF1, the pancreatic homeodomain transcription factor, is expressed in neural cells of Ciona intestinalis larva. Mech. Dev. 102: 271-274.

Craig, A. G. and Taylor, S. W. 2001. Fragmentation of a novel marine peptide, plicatamide, involves an unusual gas-phase intramolecular rearrangement. J. Amer. Soc. Mass Spectrometry 12: 470-474.

Davidson, B. and Swalla, B. J. 2001. Isolation of genes involved in ascidian metamorphosis: epidermal growth factor signaling and metamorphic competence. Dev. Genes Evol. 211: 190-194.

Delfourne, E., Bontemps-Subielos, N. and Bastide, J. 2000. Structure revision of the marine pentacyclic aromatic alkaloid: cystodamine. Tetrahed. Lett. 41: 3863-3864.

Delort, E., Watanabe, N., Etoh, H., Sakata, K. and Ceccaldi, H. J. 2000. Analysis of initial fouling process in coastal environment: Effects of settlement, attachment, and metamorphosis promoters. Mar. Biotechnol. 2: 224-230.

Di Gregorio, A., Corbo, J. C. and Levine, M. 2001. The regulation of forkhead/HNf-3 beta expression in the Ciona embryo. Dev. Biol. 229: 31-43.

Dionisio-Sese, M. L., Maruyama, T. and Miyachi, S. 2001. Photosynthesis of Prochloron as affected by environmental factors. Mar. Biotechnol. 3: 74-79.

Engel, S. and Pawlik, J. R. 2000. Allelopathic activities of sponge extracts. Mar. Ecol. Prog. Ser. 207: 273-281.

Erhardt, N. M., Fradinger, E. A., Cervini, L. A., Rivier, J. E. and Sherwood, N. M. 2001. Early expression of pituitary adenylate cyclase-activating polypeptide and activation of its receptor in chick neuroblasts. Endocrinology 142: 1616-1625.

Faulkner, D. J. 2000. Marine pharmacology. Antonie van Leeuwenhoek Intl. J. Gen. Mol. Microbiol. 77: 135-145.

Frank, P. and Hodgson, K. O. 2000. Defining chemical species in complex environments using K-edge X-ray absorption spectroscopy: vanadium in intact blood cells and Henze solution from the tunicate Ascidia ceratodes. Inorg. Chem. 39: 6018-6027.

Fresneda, P. M., Molina, P. and Bleda, J. A. 2001. Synthesis of the indole alkaloids meridianins from the tunicate Aplidium meridianum. Tetrahedron 57: 2355-2363.

Fu, X., Su, J. Y. and Zeng, L. M. 2000. Prepatellamide A, a new cyclic peptide from the ascidian Lissoclinum patella. Science in China ser. B: Chemistry 43: 643-648.

Fuke, M. 2001. Cell types involved in allogeneic contact reactions of the solitary ascidian, Halocynthia roretzi. Zool. Sci. 18: 195-205.

Gandra, M., Cavalcante, M. C. M. and Pavao, M. S. G. 2000. Anticoagulant sulfated glycosaminoglycans in the tissues of the primitive chordate Styela plicata (Tunicata). Glycobiology 10: 1-8.

Garcia, A., Lenis, L. A., Jimenez, C., Debitus, C., Quinoa, E. and Riguera, R. 2000. The occurrence of the human glycoconjugate C(2)-alpha-D- mannosylpyranosyl-L-tryptophan in marine ascidians. Org. Lett. 2: 2765-2767.

Gibson, D. M. and Paffenhofer, G. A. 2000. Feeding and growth rates of the doliolid, Dolioletta gegenbauri Uljanin (Tunicata, Thaliacea). 22: 1485-1500.

Hernandez-Zanuy, A., Garcia-Cagide, A., Esquivel, M. and Blanco, A. 2000. Reproduction and development of Ecteinascidia turbinata (Ascideacea : Perophoridae) in Cuba. Revista de Biologia Tropical 48: 193-199.

Huber, J. L., da Silva, K. B., Bates, W. R. and Swalla, B. J. 2000. The evolution of anural larvae in molgulid ascidians. Cell & Dev. Biol. 11: 419-426.

Hudson, C. and Lemaire, P. 2001. Induction of anterior neural fates in the ascidian Ciona intestinalis. Mech. Dev. 100: 189-203.

Jeffery, W. R. 2001. Determinants of cell and positional fate in ascidian embryos. Int Rev. Cytol. 203: 3-62.

Kamei, S., Yajima, I., Yamamoto, H., Kobayashi, A., Makabe, K. W., Yamazaki, H., Hayashi, S. I. and Kunisada, T. 2000.
Characterization of a novel member of the FGFR family, HrFGFR, in Halocynthia roretzi. Biochem. Biophys. Res. Commun. 275: 503-508.

Kano, S., Chiba, S. and Satoh, N. 2001. Genetic relatedness and variability in inbred and wild populations of the solitary ascidian Ciona intestinalis revealed by arbitrarily primed polymerase chain reaction. Mar. Biotechnol. 3: 58-67.

Kawamura, K. and Fujiwara, S. 2000. Advantage or disadvantage: is asexual reproduction beneficial to survival of the tunicate, Polyandrocarpa misakiensis? Zool. Sci. 17: 281-291.

Kenjo, A., Takahashi, M., Matsushita, M., Endo, Y., Nakata, M., Mizuochi, T. and Fujita, T. 2001. Cloning and characterization of novel ficolins from the solitary ascidian, Halocynthia roretzi. J. Biol. Chem. 20: 20.

Keough, M. J. 1998. Responses of settling invertebrate larvae to the presence of established recruits. J. Exp. Mar. Biol. Ecol. 231: 1-19.

Kimura, S., Ohshima, C., Hirose, E., Nishikawa, J. and Itoh, T. 2001. Cellulose in the house of the appendicularian Oikopleura rufescens. Protoplasma 216: 71-74.

Knight, R. D., Panopoulou, G. D., Holland, P. W. H. and Shimeld, S. M. 2000. An amphioxus Krox gene: insights into vertebrate hindbrain evolution. Dev. Genes Evol. 210: 518-521.

Levi-Kalisman, Y., Raz, S., Weiner, S., Addadi, L. and Sagi, I. 2000. X-Ray absorption spectroscopy studies on the structure of a biogenic "amorphous" calcium carbonate phase. J. Chem. Soc. Dalton Trans. 21: 3977-3982.

Mansueto, C., Puccia, E., Maggio, F., Di Stefano, R., Fiore, T., Pellerito, C., Triolo, F. and Pellerito, L. 2000. Organometallic complexes with biological molecules. XIV. Biological activity of dialkyl and trialkyltin(IV) [Meso-tetra(4-carboxyphenyl)porphinate] derivatives. Applied Organometal. Chem. 14: 229-235.

Manzanares, M., Wada, H., Itasaki, N., Trainor, P. A., Krumlauf, R. and Holland, P. W. H. 2000. Conservation and elaboration of Hox gene regulation during evolution of the vertebrate head. Nature 408: 854-857.

Meenakshi, V. K. 2000. Ecteinascidia venui sp. nov., a colonial ascidian (Perophoridae) from Tuticorin, southeast coast of India. Indian J. Mar. Sci. 29: 83-85.

Meenakshi, V. K. 2000. Trididemnum Della valle, 1881, an unrecorded genus of colonial ascidian from India. J. Bombay Nat. Hist. Soc. 97: 302-304.

Meenakshi, V. K. and Renganathan, T. K. 1997. On the occurrence of a rare simple ascidian, Rhodosoma turcicum (Savigny, 1816) from India. Geobios new Reports 16: 152-153.

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