Number 48                                                                                                                      December 2000

   This issue marks our completion of 25 years of producing Ascidian News.  The newsletter was started by Don Abbott and Lu Eldredge in 1966; we took over after the third issue.  Far from tiring of it or getting “burned out”, we feel more enthusiastic than ever about the exciting new discoveries coming from so many laboratories.  It is your contributions that fill these pages; please continue to send them.  As long as AN is useful we hope to continue with it for many more years.  There are more than 90 new papers listed in this issue of AN.  Thanks very much to all of you for sending us your reprints.
   We greatly enjoyed the ISOBA meetings in Sapporo at the end of June.  The breadth and quality of the talks and posters were excellent and we look forward to seeing the published version soon in the Proceedings volume, which Hitoshi Sawada assures us is progressing splendidly.  It will be published by Springer (Tokyo) early in 2001.  After the meetings we visited a number of interesting places in southern Hokkaido.  Dr. Billie Swalla has the thanks of all ascidologists for generously volunteering to organize the next ascidian meeting in 3 years, tentatively planned to be held in Honolulu.
   In July we attended a symposium at Scripps in honor of Vic Vacquier where Charley presented a poster on sperm competition.  In August we participated in 3 nonindigenous species surveys on both US coasts, of which more will be found in the Work in Progress section.  In October Gretchen presented a paper on nonindigenous ascidians in tropical waters at the Intl. Coral Reef Symposium in Bali.  The weather was sunny and warm, quite a contrast to Seattle; we spent a week traveling around the island after the meetings.
   George Mackie and Mike Thorndyke have initiated an exciting new electronic discussion list for all ascidiologists.  This makes it possible to enter into all sorts of interesting dialogues via email on ascidians.  We are most grateful to George and Mike for setting this up.  Judging from the spirited flow of letters so far, it is a fantastic success (more than 150 subscribers already).  For those of you who have not yet joined, please see the instructions below.  And don’t worry about having missed anything; already-sent letters are in an archive on the website.
   We will be at the Friday Harbor Labs from mid-December until mid-January and then again next summer when Charley will teach the comparative invertebrate embryology class with Mark Martindale.  As always, our email addresses remain the same wherever we go, and we always enjoy hearing from you.

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

1. George Mackie and Michael Thorndyke have created an internet discussion forum for topics on the Tunicata. It has already proven to be extremely useful, and we congratulate George and Mike on this accomplishment. The list is intended for discussion of all aspects of the biology of tunicates, including questions concerning early chordate evolution.  The list is an "open" one. That means anyone can join and leave, send messages, see who else is on the list or search archived messages on the web page. The list server recently changed but all the archives have been transferred to the new site.
To join the list send a message to jiscmail@jiscmail.ac.uk saying:  join tunicata first name last name
You will receive an acknowledgment giving information on how to send messages and commands.
You can also join or leave the list or browse the archives etc. by going to: http://www.jiscmail.ac.uk/lists/tunicata.html

2. From Karen Sanamyan, Kamchatka Institute of Ecology, Russia (terra@mail.kamchatka.ru), who has an exciting new software ascidian taxonomic database for sale that works in Windows 95/98.
   ZooBase is a tool for professional taxonomists and systematists designed for navigating through literature, taxonomic descriptions and synonymies of nominal species. It provides information on who has described this species as valid and in what publications (with complete citations), complete synonymies of every species in the database and so on.  With cross-referencing, the user can search all references that include a particular species, or all references by a particular author, etc.  There are currently several systematics software programs on the market; some of them are simply electronic keys for species identification (useful for students, but not for specialists), others require drawing up a list of valid species. But how do you proceed if one specialist says that a certain species is invalid, while others continue to use this name as valid? This problem does not exist in ZooBase. ZooBase is a collection of headings of species descriptions compiled from taxonomic papers, accompanied by the synonyms given in these papers.  The search system allows the user to find the necessary records and display them in the proper format.
   ZooBase contains a large database on Ascidiacea of about 17,000 records and a smaller Actiniaria database of 6800 records.  To use the databases, a customer should purchase both the program shell (3Mb; $120.00) and the Ascidiacea/Actiniaria databases (958KB; $390.00).  Thus the total price is $510.00.  The databases will not work without the program shell (like an Excel spreadsheet will not work without the Microsoft Excel program).  On the other hand, if someone purchases only the program shell, he can build his own database - (I have sold one copy to an ichthyologist). The Ascidiacea database (958KB) is a compressed file with 17000 records, while the program shell is an engine that retrieves and shows the records from the database.  All updates will be free for registered users.
   You can view a sample page from the database on my website, or download a small trial version of ZooBase to try it before purchasing. Although the unregistered version has many restrictions (it is read-only and the demo database is small) it is still useful, because it contains about 1000 taxonomic papers on Ascidiacea.
   To purchase the full version, please visit http://www.taxon.newmail.ru and follow the order link, which will direct you to Secure Order Form for ZooBase on the http://www.RegSoft.com server. This server allows online ordering via credit card, FAX, postal mail ordering, phone ordering, etc.  After registering, you will receive password and registration info to remove all limitations of the unregistered version. BUT BETTER, please contact me directly - sanamyan@newmail.ru.  If this address does not work or you have difficulty accessing the Zoobase homepage, please contact me at terra@mail.kamchatka.ru.

Note from Gretchen: I have used the database and am very enthusiastic about it.  It is amazingly easy to use, has a large number of entries and the cross-referencing has been very well thought out.  All commands are menu driven like other programs in Microsoft’s Windows.  AN subcriber Imène Meliane, a taxonomist working on the ascidians of Oman, describes it as “excellent” and is finding it invaluable in her own work. “What I most appreciate in this database,” she says, “is that it gives you the synonymies of the species and that's very important when you don't know that a species has changed.”   We think this will be an indispensable tool for many ascidiologists.

3.  Congratulations to Dr. John Faulkner, Professor of marine chemistry at Scripps Institution of Oceanography in La Jalla, California, who was awarded the Paul J. Scheuer Award in Marine Natural Products for outstanding contributions to the field. The award was presented on March 2 at the Gordon Research Conference on Marine Natural Products held in Ventura, Calif. Much of his work has been funded by the Calif. Sea Grant College Program and this announcement was published in their May newsletter.

4.  Here is another old silly song for those who like that sort of thing (see Man and the Ascidian by Andrew Lang in AN44 and Amphioxus by Philip Pope in AN47). This one is titled “A Sea Squirt Problem” by Ivar Haglund, who was a very well-known character around Seattle for many years. He started the Ivar’s Acres of Clams restaurants.  (We’ll ignore the possibility that he may have been referring to a clam in this song.)

A deep sea problem is troubling me
It may sound as silly as silly can be.
How far, oh how far can a sea squirt squirt?
Will a sea squirt squirt if his feelings are hurt?

The sea-squirt means harm to nobody,
But that doesn’t answer what’s bothering me—
How far, oh how far can a sea squirt squirt?
Will a sea squirt squirt if his feelings are hurt?

I asked sand dollars to tell me how,
But they’re making change and can’t answer me now—
How far, oh how far can a sea squirt squirt?
Will a sea squirt squirt if his feelings are hurt?

He hides in the sand by the ocean blue
Just waiting his chance for a squirt at you.
He loves his work and his eyes simply gleam
When he lets you have it with a salty stream.
No one’s ever figured how far he could
Squirt if he had to, but his aim’s darn good.

5.  The 3rd Intl. Symposium on Chemistry and Biological Chemistry of Vanadium will be held in Osaka, Japan Nov. 26-29, 200l.  For more information contact Prof. Toshikazu Hirao,  hiraov@chem.eng.osaka-u.ac.jp

6.  From Bill Austin (baustin@island.net)  Director, Mar. Ecol. Sta., Cowichan Bay, BC, Canada: http://mareco.org/
 “I found a number of Styela clava [based on size 10-11cm, short stalk, triangular form, vertical orientation of siphons, tubercles] on floats in Birds Eye Cove, Maple Bay, Vancouver Island yesterday (lat. 48 deg. 47.2'N, 123 deg. 36.00'W).  A number of transient vessels dock here, including from WA and CA. Lots of Botrylloides there too.”  We [Lamberts] have followed a population of S. clava at French Creek (north of Maple Bay on Vanc. Is.) for at least 8 years but had not found it anywhere else in British Columbia though it is common at the marina at Blaine, Washington, on the U.S./Canada border.  The Botrylloides is undoubtedly B. violaceus, now abundant throughout the Pacific NW.

7.  It is with great sadness that we report the death of Professor Zen-Ichiro Hoshino, who passed away on Oct. 22.  A memorial service was held on Oct. 26 in Morioka.  His family, his many friends, and colleagues will miss him very much.  Dr. Takaharu Numakunai writes: “. Dr. Hoshino's most important contribution to ascidiology is that he clarified the difference between C. savignyi and C. intesinalis with Nishikawa-san [Hoshino, Z.-I. and Nishikawa, T. 1985. Taxonomic studies of Ciona intestinalis (L.) and its allies. Publ. Seto Mar. Biol. Lab. 30: 61-79].  Now it is very easy for us to distinguish them from each other, and we can use their gametes without confusion.”
   We also have used this important paper many times, most recently just in the past few weeks, and have given the reference to many colleagues because of the rapid spread of C. savignyi to new areas in recent years.

1. From: Imene Meliane (Imene.Meliane@ua.es) and Alfonso Ramos (alfonso.ramos@ua.es):
We are preparing an article "Records of ascidians (Chordata, Tunicata) from Oman, south-east of Arabian Peninsula".  This will be presented at the international conference organised by the Sultan Qaboos University (Oman): "Fisheries, Aquaculture and Environment in the NW Indian Ocean".  The conference will be held January 8-10, 2001.  This article is only a small part of my Ph.D. which will deal with the ascidians of Oman: taxonomy, ecology and biogeography.

2.  Non-indigenous ascidian research:  Gretchen and Charles Lambert.  This past summer we participated in rapid assessment nonindigenous species (NIS) surveys on both U.S. coasts.  We surveyed most of the Massachusetts coastline with a group headed by Judy Pederson of Mass. Sea Grant program at MIT that included taxonomic specialists in most marine groups.  The following week most of the group continued with an NIS assessment of Narragansett Bay in Rhode Island, headed by Kevin Cute, Marine Resources Specialist with the R.I. Coastal Resources Management Council.  The biota on most of the harbor structures was dominated by huge numbers of non-native ascidians including Ascidiella aspersa, Styela clava, Botrylloides violaceus, and Botryllus schlosseri. Ciona intestinalis and Diplosoma listerianum were also common at a few sites.  The east coast Ciona is somewhat different from the west coast C. intestinalis but nevertheless appears to be the same species. Nowhere in Massachusetts did we find Botrylloides diegensis although it was intentionally introduced to Eel Pond at Woods Hole some years ago for genetics studies.  A number of recent publications and reports list B. diegensis on the east coast, but the correct identification is B. violaceus.  Interestingly, although B. violaceus is now very abundant on the U.S. west coast with a range from Alaska to Baja Mexico, A. aspersa has not yet appeared on this side of the continent..
   After a few days of recuperation at home we joined an NIS survey of southern California and Ensenada (Baja Mexico) headed by Andy Cohen of the San Francisco Estuary Institute.  We visited many of the sites of our 1996-98 nonindigenous ascidian study and the ropes we installed 4 years ago were still there.  Some of these were now so heavy with Styela plicata that they could not be lifted from the water and had to be examined length by length under the surface.  It was interesting to see that most of the introduced ascidians listed in our 1998 paper have continued to flourish.  We are including this new data in a paper in preparation on site-specific analysis of introduced ascidians in California harbors 1994-2000.

3. From Patrick Frank (frank@SSRL.slac.stanford.edu): The following is the abstract of a manuscript in preparation, describing work speciating vanadium in blood cells from Phallusia (Ascidia) nigra, and makes some comparisons with Ascidia ceratodes. The differences are profound.
   Unprecedented forms of vanadium observed within the blood cells of Phallusia (Ascidia) nigra using K-edge X-ray absorption spectroscopy.  P. Frank^a, W.E. Robinson^b, K. Kustin^c and K.O. Hodgson^a,d.
a. Stanford Synchrotron Radiation Lab., SLAC, Stanford Univ., Stanford, CA 94309. b. Envl., Coastal, & Ocean Sci. Dept., Univ. Mass. Boston, Boston, MA 02125-3393. c. Dept. Chem., Brandeis Univ., Waltham, MA 02254-9110. d. Dept. Chem., Stanford Univ., Stanford, CA 94305-5080.
   Fits to the vanadium K-edge x-ray absorption spectra (XAS) of five whole blood cell samples from the tunicate Phallusia (Ascidia) nigra revealed unprecedented forms of complexed intracellular vanadium. Endogenous vanadium was divided between V(III) ion (74.2 ± 5.1% of total V) and vanadyl ion (V^IV=O)²⁺ (25.2 ± 5.4% of total V). The V(III) fraction included both [V(H₂O)₆]³⁺ (36.7 ± 5.5 %) modeled as VCl₃ in 1M HCl, and three previously unprecedented chelated V(III) forms (37.5 ± 4.6 %). Two of these could be represented by the model ligand environments V( acetylacetonate)₃ (17.9 ± 3.2%) and K₃V(catecholate)₃ (13.1 ± 4.7%), implying DOPA-like complexation. The third chelated form, present in only four of the samples, was represented by the 7-coordinate N₂O₅ complex Na[V(EDTA)(H₂O)] (8.0 ± 1.8%) and is suggestive of a novel mononuclear V(III) protein site. It may have been induced by the DTT uniquely added to these four samples. Endogenous V(IV) (25.2 ± 5.4%) was principally modeled as VOCl₂ in 1M HCl. EPR spectra confirmed the predominance of aquated vanadyl ion. The fit to the XAS pre-edge energy region of blood cell sample five uniquely included the XAS spectrum of VOSO₄ in 0.1M H₂SO₄ solution (13.0%), and of [OV^V( pivalate)₃] (3.1%) which is also unprecedented. These results are compared with those of analogous fits to the blood cells of Ascidia ceratodes and support assignment of P.(A.) nigra to a different genus.
  We have a paper in press, due out 25 December in the journal Inorg. Chem.: 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.

Aspects of mate choice in the colonial ascidian Diplosoma listerianum.  Andrew J. Pemberton—Ph.D. thesis, Dept. of Zoology , Univ. of Aberdeen, Scotland, and Marine Biol. Assoc. of the UK.  anp@mba.ac.uk  Supervisors: Dr J.D.D. Bishop (MBA) and Dr L.R. Noble (Aberdeen).
   Sessile organisms that release sperm into the environment but retain their eggs may experience obligate female promiscuity because a mixture of gametes is received from various potential mates.  This lack of control may be typical of a phylogenetically diverse array of suspension-feeding aquatic invertebrates that release sperm.  Although promiscuity seems unavoidable it does not follow that females have no control over the extent or direction of polyandry.  I have investigated the extent to which sperm are actively selected by the colonial ascidian Diplosoma listerianum – an animal that appears to offer a relatively uncluttered terrain for the study of late-acting female discrimination.
   It seems unlikely that the female role of this hermaphrodite is sperm-limited.  D. listerianum appeared able to utilise highly dilute sperm to achieve significant rates of fertilisation.  The colonial ascidian achieved comparable levels of fertilisation at sperm concentrations two or three orders of magnitude lower than is commonly found with echinoderm species where both eggs and sperm are released.  At a sperm concentration of 100 sperm/ml D. listerianum had near maximum fertilisation whereas broadcast spawners would display near complete reproductive failure.
   An extensive series of pairwise reciprocal matings between twelve different clones revealed surprisingly high levels of sexual incompatibility. Fecundities of crosses varied widely and suggested a continuous scale of compatibility.  Although correlations of compatibility between reciprocal crosses appeared positive, considerable noise was present in the data and some crosses showed strongly asymmetrical compatibility.  Somatic:gametic trade-offs may have been detected.
   Patterns of sperm precedence in D. listerianum with a long (five day) interval between matings revealed a consistent initial advantage toward the first of two acting males.  This was thought to suggest a first-in-first-out utilisation of individual stored gametes.  The proportion of second-male paternity subsequently increased with time, possibly reflecting an ageing or passive loss of first-male sperm.  Patterns may have been enhanced by the modular nature of the organism, in that any recently budded zooids would have been available exclusively to second-male sperm.  Estimated P2 for entire progeny arrays was consistently greater than 0.5, but varied widely.
   Two aspects of frequency-dependent selection were studied. 1. In competition with an alternative sperm source, acting males fathered more progeny if previously mated to a particular female than if no mating history existed, an advantage probably derived from fertilisations by stored sperm.  2. When virgin acting female colonies were given mixtures of sperm at widely divergent concentrations, offspring were shared between the two sperm sources in approximately the ratio of each mixture.  However, there existed a small but statistically significant deviation from the fair raffle model, in that sperm at the lower concentration consistently achieved a greater than expected share of paternity.  Environmentally-determined fixed female preferences could be responsible for this negative frequency dependence (‘rare male effect’).

Phenotypic plasticity of reproductive effort in the colonial ascidian, Botryllus schlosseri.  A.W. Newlon, III M.Sc. thesis, Dept. Biological Sciences, Univ. of New Orleans, Louisiana, USA.  Dr. J. Stewart-Savage, thesis advisor (jssavage@uno.edu).
    Phenotypic plasticity, the capability of a genotype to produce alternative phenotypes depending on the proximate environment, is considered an adaptive response to temporal and spatial oscillations in the environment.  Since reproductive traits have a significant influence on overall fitness, and adaptation to environmental change within the span of a generation is not possible by genetic polymorphism, phenotypic plasticity of reproductive traits should be selected for in unstable environments.  Selection pressures on reproductive success would be expected to be more intense in natural populations of short-lived species where reproduction is limited to a single season.  In hermaphroditic species that also reproduce asexually and are capable of reproductive plasticity, selection would likely act differently on asexual, male and female reproduction, and the optimal combinations of traits should change according to the environment.  Previous studies have suggested the possibility for phenotypic plasticity of male, female and asexual reproductive effort in Damarascotta River populations of Botryllus schlosseri, a short-lived, hermaphroditic, colonial ascidian.  To test for plasticity in these life-history traits, clones derived from a down-river population were distributed between their native site (CI) and a site 10km up-river (DM).  Male reproductive effort was measured by sperm production, female reproductive effort by egg production, and asexual reproductive effort by growth rate.  Data was collected for the first and third generation of buds born in the field.  ANOVA results indicated plasticity in asexual reproductive effort for the first generation, and plasticity in all three traits for the third generation. Phenotypic plasticity in asexual reproductive effort varied significantly among genets, with a greater variation in the magnitude and direction of growth rate plasticity between sites during the first generation, than the third generation.  Although there was significant variation in asexual reproductive plasticity, the randomness of responses implies that selection is probably weak.  Sperm production in the third generation of buds was lower on average at DM, but there was little variation in the plasticity of male reproductive effort (i.e., responses were similar in direction and magnitude across genets), thus selection for large changes in testis size may be buffered by plasticity.  Plasticity in female reproductive effort in the third generation varied across genets in the magnitude of response, although the direction was the same (higher egg production at DM), therefore, selection on female reproductive effort has great potential for evolutionary change.  The cause of higher egg production at DM may be related to the greater phytoplankton abundance at that site.  Comparisons of the weighted frequency distributions for reproductive effort of individual buds at each site, demonstrated that the difference in sperm production between sites was due to an absolute difference in testis size, while higher egg production at DM was due to an increase in the proportion of reproductive buds within a colony.

Isolation of a new gene involved in nervous system of the ascidian Ciona intestinalis. Salvatore D'Aniello, Lab. Biochim. and Molec. Biol., Stazione Zool. A. Dohrn, Villa Comunale, 80121 Napoli, Italy.  Thesis advisor Dr. Margherita Branno (maggy@alpha.szn.it)
   In order to identify molecular markers of the ascidian cell-lineage and/or morphogenetic determinants that could be responsible for the early commitment in the typical "mosaic" embryo, I've carried out a survey of the homeobox-containing genes from the cosmopolitan ascidian Ciona intestinalis.  Between different cDNA clones isolated from our group I found one homeobox-containing gene that is specifically expressed in the neural system.  Sequence homology of the isolated cDNA clone demonstrates that it is a member of the paired-like family. The analysis of the sequence of CiPrx-1, as I named it following the literature usage, reveals its type of transcriptional factor by the occurrence of a well conserved homeodomain, consisting of 60 aa, that reveals a high homology of sequence with PAX and OTX family genes, some more with the Caenorhabditis elegans CEH-8 gene and with the well known murine Prx-2 gene (formerly called S8).  Northern blot analysis shows that the transcript of the Ciona gene, CiPrx-1, first appears at middle tailbud stage, peaks at late tailbud and then decreases during the larval stage. Preliminary in situ hybridization experiments show that CiPrx-1 mRNA first appears at middle tailbud stage in the four cells that are the precursor of the sensory vesicle; later, at the larva stage, the transcript is present in some cells around the ocellus in the sensory vesicle.

Structure, expression and transgenic analysis of the Cimshb gene in Ciona intestinalis. Monia Teresa Russo, Lab. Biochim. and Molec. Biol., Stazione Zool. A. Dohrn, Villa Comunale, 80121 Napoli, Italy.  Thesis advisor Dr. Margherita Branno (maggy@alpha.szn.it)
   This research investigates the regulation of the expression pattern of the Cimshb gene during the development of Cionaintestinalis (Aniello et al., 1999, Mech. Dev. 88, 123-126). Cimshb was isolated from a C.intestinalis cosmid library with a 4.0 Kb promoter region. I used this sequence to prepare constructs containing different promoter lengths (4.0 Kb, 3.0 Kb, 1.0 Kb, 0.9 Kb and 0.45 Kb) linked to a lacZ reporter gene. They were further utilised for electroporation experiments in which embryos developed until the stage of interest and were then assayed for ß-galactosidase activity. In larvae the constructs containing the 4.0 Kb, 3.0 Kb, 1.0 Kb and 0.9 Kb fragments showed the reporter gene in the primordial pharynx, the sensory vesicle and in the visceral ganglion, which are the endogenous expression territories. The smallest 0.45 Kb construct was negative in the primordial pharynx, suggesting that the region extending from position –450 bp to –900 bp seems to contain the regulatory elements driving the expression in the primordial pharynx. However this region apparently contains the elements responsible for expression in the sensory vesicle and visceral ganglion.  In neurulae and tailbud larvae the constructs containing the 4.0 Kb and the 3.0 Kb fragments induced expression along the neural folds in the neurula, the neural tube in the tailbud, in the precursors of otolith and ocellus and in the ventral epidermis (in both stages) which are endogenous expression territories. The 1.0 Kb fragment construct lacked expression in the ventral epidermis and in the precursors of otolith and ocellus, but the signal in the otolith and ocellus precursors reappeared once again with the shortest 0.9 Kb and 0.45 Kb constructs. Thus expression in the neural folds and neural tube is driven by regulatory elements contained in the smallest promoter region, while expression in the ventral epidermis is driven by elements contained in the region extending from position –1000 bp to –3000 bp. To interpret the expression in the sensory vesicle I propose a model requiring at least three regulatory regions with activating elements at –1000 bp to –3000 bp and the smallest promoter region; in contrast the region extending from position –900 bp to –1000 bp seems to contain a mild repressor in the presence of the upstream activator. When the region containing the repressor is absent, the signal appears after a few hours; but when the second upstream activator is present together with the repressor, the signal is seen only after some days.

Abourriche, A., Charrouf, M., Bennamara, A., Berrada, M., Chaib, N., Boudouma, M. and Francisco, C. 1999. Investigation of bioactivity of extracts from Moroccan solitary tunicate Cynthia savignyi. J. Ethnopharmacol. 68: 47-53.

Aiello, A., Carbonelli, S., Esposito, G., Fattorusso, E., Iuvone, T. and Menna, M. 2000. Novel bioactive sulfated alkene and alkanes from the Mediterranean ascidian Halocynthia papillosa. J. Nat. Prod. 63: 1590-1592.

Aiello, A., Fattorusso, E., Menna, M. and Iuvone, T. 2000. Sulcatin, a novel antiproliferative N-methylpyridinium alkaloid from the ascidian Microcosmus vulgaris. J. Nat. Prod. 63: 517-519.

Aknin, M., Dayan, T. L., Rudi, A., Kashman, Y. and Gaydou, E. M. 1999. Hydroquinone antioxidants from the Indian ocean tunicate Aplidium savignyi. J. Agric. Food. Chem. 47: 4175-4177.

Albrieux, M., Moutin, M. J., Grunwald, D. and Villaz, M. 2000. Calmodulin and immunophilin are required as functional partners of a ryanodine receptor in ascidian oocytes at fertilization. Dev. Biol. 225: 101-111.

Albrieux, M. and Villaz, M. 2000. Bilateral asymmetry of the inositol trisphosphate-mediated calcium signaling in two-cell ascidian embryos. Biol. Cell. 92: 277-284.

Ballarin, L., Tonello, C. and Sabbadin, A. 2000. Humoral opsonin from the colonial ascidian Botryllus schlosseri as a member of the galectin family. Mar. Biol. 136: 823-827.

Ballaro, B. and Reas, P. G. 2000. Chemical and mechanical waves on the cortex of fertilized egg cells: a bioexcitability effect. Riv. Biol. 93: 83-101.

Bingham, B. L. and Reitzel, A. M. 2000. Solar damage to the solitary ascidian, Corella inflata. J. Mar. Biol. Ass. U.K. 80: 515-521.

Bishop, J. D., Manriquez, P. H. and Hughes, R. N. 2000. Water-borne sperm trigger vitellogenic egg growth in two sessile marine invertebrates. Proc. R. Soc. Lond. B 267: 1165-1169.

Bishop, J. D., Pemberton, A. J. and Noble, L. R. 2000. Sperm precedence in a novel context: mating in a sessile marine invertebrate with dispersing sperm. Proc. R. Soc. Lond. B 267: 1107-1113.

Caracciolo, A., Di Gregorio, A., Aniello, F., Di Lauro, R. and Branno, M. 2000. Identification and developmental expression of three Distal-less homeobox containing genes in the ascidian Ciona intestinalis. Mech. Dev. 99: 173-176.

Carballo, J. L. 2000. Larval ecology of an ascidian tropical population in a Mediterranean enclosed ecosystem. Mar. Ecol. Prog. Ser. 195: 159-167.

Castilla, J. C. 1998. Las comunidades intermareales de la Bahia San Jorge: estudios de linea base y el programa ambiental de Minera Escondida Ltda. en Punta Coloso. In: Arcos, D. (ed.), Minera del Cobre, Ecologia y Ambiente Costero. pp. 221-244.

Castilla, J. C. 1999. Coastal marine communities: trends and perspectives from human-exclusion experiments. Trends in Ecol. & Evol. 14: 280-283.

Cavagnari, B. M., Tatian, M., Sahade, R. J., Esnal, G. B. and Santome, J. A. 2000. A fatty acid-binding protein and a protein disulphide isomerase-related protein expressed in urochordate gonad cytosol. Int. J. Biochem. Cell Biol. 32: 769-777.

Cavalcante, M. C., Allodi, S., Valente, A. P., Straus, A. H., Takahashi, H. K., Mourao, P. A. and Pavao, M. S. 2000. Occurrence of heparin in the invertebrate Styela plicata (Tunicata) is restricted to cell layers facing the outside environment. An ancient role in defense? J. Biol. Chem. 275: 36189-36196.

Coma, R., Ribes, M., Gili, J.-M. and Zabala, M. 2000. Seasonality in coastal benthic ecosystems. Trends in Ecol. & Evol. 15: 448-453.

Connell, S. D. and Glasby, T. M. 1999. Do urban structures influence local abundance and diversity of subtidal epibiota? A case study from Sydney Harbour, Australia. Mar. Env. Res. 47: 373-387.

Cooper, E. L. and Bosch, T. C. 2000. Ontogeny recapitulates phylogeny: comparative immunology in Germany. Exp. Clin. Immunogenet. 17: 77-82.

Copp, B. R., Wassvik, C. M., Lambert, G. and Page, M. J. 2000. Isolation and characterization of the new purine 1,3, 7- trimethylisoguanine from the New Zealand ascidian Pseudodistoma cereum. J. Nat. Prod. 63: 1168-1169.

Dallman, J. E., Dorman, J. B. and Moody, W. J. 2000. Action potential waveform voltage clamp shows significance of different Ca²⁺ channel types in developing ascidian muscle. J. Physiol. (Lond) 524 Pt 2: 375-386.

Dassonneville, L., Wattez, N., Baldeyrou, B., Mahieu, C., Lansiaux, A., Banaigs, B., Bonnard, I. and Bailly, C. 2000. Inhibition of topoisomerase II by the marine alkaloid ascididemin and induction of apoptosis in leukemia cells. Biochem. Pharmacol. 60: 527-537.

Davies-Coleman, M. T., Gustafson, K. R., Cantrell, C. L., Beutler, J. A., Pannell, L. K. and Boyd, M. R. 2000. Stolonic acids A and B, new cytotoxic cyclic peroxides from an Indian Ocean ascidian Stolonica species. J. Nat. Prod. 63: 1411-1413.

Di Bella, M. A. and De Leo, G. 2000. Hemocyte migration during inflammatory-like reaction of Ciona intestinalis ( Tunicata, Ascidiacea). J. Invert. Pathol. 76: 105-111.

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