ASCIDIAN NEWS*
Charles and Gretchen Lambert
12001 11th Ave. NW, Seattle, WA 98177
206-365-3734
glambert@fullerton.edu or clambert@fullerton.edu
home page: http://depts.washington.edu/ascidian/
Number 55 July 2004
In this issue there are announcements of two upcoming ascidian symposia in 2005, one in April and the other in July. At the end of this issue are listed 145 new publications (!) on ascidians. Please send us reprints (preferably, for our permanent collection) or PDFs to assure inclusion of your papers in AN.
In May we spent 3 wonderful weeks in Britain. We visited Peter Dyrynda, Andrew Rowley, and John Ryland in Swansea, Wales and John Bishop in Plymouth. Although the trip was a vacation, mostly spent touring southern England, we did look at some interesting Plymouth harbor ascidians (mostly invaders) with John Bishop. At the end of May we and about 10 others participated in a nonindigenous species rapid assessment survey of San Francisco Bay organized by Dr. Andrew Cohen. Previously recorded introduced ascidians were all still present but we found no new ascidian invaders. We are at the Friday Harbor Labs this summer: Gretchen is writing two papers and Charlie is working on maturation in ascidian oocytes. We were pleased to see many colleagues at the excellent Comparative Developmental Biology Symposium: Insights into Embryos, Cells and Evolution, held at the Friday Harbor Labs in June and organized by Drs. Billie Swalla, Richard Strathmann, Victoria Foe, Garrett Odell, George von Dassow, and Volker Schmid, part of this year’s FHL centennial celebration. Nori Satoh gave the keynote address and there were several other presentations on ascidians (see the titles and abstracts below).
*Ascidian News is not part of the scientific
literature and should not be cited as such.
1. Upcoming ascidian conference:
INTERNATIONAL
INVASIVE SEA SQUIRT CONFERENCE (IISSC): The biology, bio-geography, and ecology
of invasive ascidians. Subphylum Tunicata
(Urochordata), Class Ascidiacea. April 21-22, 2005, Woods Hole Oceanographic Institution
(WHOI), Woods Hole, Massachusetts, Quissett Campus, Clark 507 & 509. Conference Organizer/contact person: Mary Carman, Geology & Geophysics, Woods
Hole Oceanographic Institution, Woods Hole, MA 02543. Phone 508-289-2987; email
mcarman@whoi.edu Abstract
deadline: December 1, 2004.
Sponsored by: WHOI Ocean Life Institute, NOAA Northeast Fisheries Science Center, MIT Sea Grant, WHOI Sea Grant, Northeast Aquatic Nuisance Species (NEANS) Panel. Editor of Conference Proceedings: Dr. Robert Whitlatch, University of Connecticut. To be published in Journal of Experimental Marine Biology and Ecology (JEMBE).
Conference Sessions:
OPENING AND PLENARY TALKS
Larry Madin, director WHOI Ocean Life Institute, “Welcoming Remarks”
Gretchen Lambert, Univ. of Washington Friday Harbor Laboratories, “Invasive ascidians: a growing global problem”
James T. Carlton, Prof. of Mar. Sci. and Director, Maritime Studies Program of Williams College and Mystic Seaport, Mystic, Connecticut, “Setting ascidian invasions on the global stage: retrospectives, perspectives, and prospectives”
I. BIOLOGY, SYSTEMATICS, AND BIOGEOGRAPHY
Biology (including physiochemical tolerances, feeding, reproduction, and physiology of invasive ascidians); pollution responses; taxonomy and systematics; biogeography; dispersal vectors; predicting future invasions; and related topics.
II. ECOLOGY AND GENETICS
Experimental ecology, natural history, and genetics of invasive ascidians (including their role in structuring benthic communities); responses to disturbance regimes; patterns of abundance over time and space; predators; predation (impact on prey- plankton communities); regional and global aspects of molecular and population genetics; topics may also include exploring reasons why ascidian invasions appear to have increased globally in the last half of the 20th century.
III. IMPACTS ON COASTAL RESOURCES
Quantitative studies on the economic impact of invasive ascidians on previously resident communities (including impact on native species of fisheries); impact on aquaculture and mariculture facilities; impact on water intake and other industrial facilities; impact on vessel fouling (large ocean-going vessels to small recreational traffic); other fouling impacts and related topics.
IV. RISK ASSESSMENT, MANAGEMENT, CONTROL, AND EDUCATION
The potential management and control of invasive ascidian populations (including mechanical, chemical and bio-control techniques); prevention of invasions and prevention of spread; education of the public, the press, and the political world; societal responses to invasive ascidians; risk assessment studies; local, state, and federal agency involvement.
V. PANEL SESSION
Panel composition will include 4 members, including a NEANS representative and a Sea Grant representative, and a 5th member as Panel Moderator; panel members will each make a 3 to 5 minute opening statement, followed by questions and discussion from and with the audience.
2. Mark your calendars! and send in your application for the International Tunicata meeting, July 8-13, 2005 at Univ. of California Santa Barbara. Organized by Bill Smith (UCSB) w_smith@lifesci.lscf.ucsb.edu and Billie Swalla (Univ. of Washington Biol. Dept.) bjswalla@u.washington.edu . More details and updates about this meeting will be sent in separate emails, or contact the organizers directly if you wish to make a presentation there. Sessions currently planned are: Genome Projects and ESTs; Developmental Programs and Genetic Pathways; Mechanisms of Metamorphosis; Evolution of the Immune System; Taxonomy and Phylogeny of Tunicates; Development and Evolution of Thaliaceans; Evolution of Coloniality.
3. From Patrick Lamaire: - New website for the programme of the Urochordate Meeting Oct. 2003 in France. The website contains Abstracts from the meeting [also included in AN#54 newsletter Dec. 2003]; - Useful links; -Libraries; - White Pages.
http://www.lgpd.univ-mrs.fr/more_info/lemaire/index.htm
4. From Christian Sardet, CNRS, Station Zoologique, Villefranche-sur-Mer, France http://biodev.obs-vlfr.fr/biomarcell/ Please visit our large and frequently updated website, where you will find many items on Fertilization and Development of Ascidians:
Pdf’s of recent
publications
Films of ascidian fertilization and development including several on calcium signals and calcium wave pacemakers in ascidian eggs
Downloadable color poster of embryo comparisons on Polarity in Eggs and Embryos
Photos and brief description of some local species used in our research
Methods for: Collecting biological material, Fertilization and culture of embryos, Making chamber for time lapse observation of development, In vivo labelling of ascidian eggs and embryos, Fixations for light and electron microscopy, Labelling cytoskeletal elements of fixed ascidian eggs and embryos, Treatment of eggs and embryos with cytoskeletal inhibitors, Micromanipulations of eggs and zygotes, Isolation and Characterization of cortices isolated from ascidian eggs and embryos, Solutions.
Links to many other relevant websites
*[Note from the Lamberts: This is an amazing website and we’re sure that many of you will find a great deal of interesting and useful information here, plus photos and history of the lab and its surroundings.]
1. Biochemical and functional characterization
of protein kinase CK2 in ascidian Ciona intestinalis oocytes at
fertilization. Gian Luigi RUSSO1,2, Mariarosaria TOSTO1, Annalisa
MUPO1, Immacolata CASTELLANO1, Annunziata CUOMO1,
and Elisabetta TOSTI1 1Stazione
Zoologica “Anton Dohrn”, 80121, Napoli, Italy; 2Istituto di Scienze
dell’Alimentazione, Consiglio Nazionale delle Ricerche, 83100 Avellino,
Italy. tosti@szn.it
Protein kinase CK2 is a ubiquitous and
pleiotropic kinase that has been studied and characterized in many organisms,
from yeast to mammals. The enzyme is a tetramer composed by two catalytic (a) and two regulatory (b) subunits. CK2 is a dual specificity
kinase, since it is able to phosphorylate both serine/threonine and tyrosine
residues located on its target substrates. The function of CK2 has been
associated to fundamental biological processes such as signal transduction,
cell cycle progression, cell growth, apoptosis, and transcription. Less known
is the role of CK2 during meiosis and early phase of embryogenesis. In this
work, we reported that CK2 is constitutively active in unfertilized and
fertilized oocytes in ascidian Ciona intestinalis. The enzymatic
activity oscillates through meiosis showing three major peaks: soon after
fertilization (metaphase I exit), before metaphase II and at metaphase II exit.
Biochemical analysis of CK2 subunit composition in activated oocytes indicated
that CK2-a is
catalytically active as a monomer, independently from its regulatory subunit b. On the opposite, CK2-b was only detectable in unfertilized
oocytes where it was associated to a bona fide identified ascidian MAP
kinase. Protein sequence analysis of CK2-a and -b cDNA indicated a high identity compared to vertebrate homologs. In
addition, the absence of putative phosphorylation sites for Cdc2 kinase on both
a and –b subunits suggested an important
role of CK2 in regulating meiotic cell cycle in Ciona intestinalis
oocytes. The data presented in this
work suggest a new and unexplored function of CK2 during meiosis. that
increases the broad range of biological processes where CK2 is, in some way,
involved. The publication of Ciona genome will certainly accelerate
studies on early phase of embryogenesis of this organism, and will probably
better clarify the role of CK2 in meiosis completion. [in press J. Biol. Chem.)
2. From Gretchen Lambert: update on invasive ascidians: Perophora japonica was recorded from Humboldt Bay in northern California in August 2003, the first North American record for this species. A number of large healthy colonies with well-developed bright yellow stellate terminal buds were growing on a Smithsonian Environmental Research Center Invasions Lab settling panel. It was first observed in Europe, on the French side of the English Channel, in 1982 (Monniot, C. and Monniot, F. 1985. Apparition de l'ascidie Perophora japonica sur les côtes et dans les ports de la Manche. Comptes Rendus de la Societé de Biogéographie 61: 111-116). It invaded southern England a few years ago (Nishikawa, T., Bishop, J. D. D. and Sommerfeldt, A. D. 2000. Occurrence of the alien ascidian Perophora japonica at Plymouth. J. Mar. Biol. Ass. U.K. 80: 949-950; Baldock, J. and Bishop, J. D. D. 2001. Occurrence of the non-native ascidian Perophora japonica in the Fleet, southern England. J. Mar. Biol. Ass. U.K. 81: 1067).
The nonindigenous Didemnum now growing rampant on both coasts of the U.S., in New England on the Atlantic side and in northern California on the Pacific side, has been identified by me as Didemnum cf. lahillei, a native of Europe, based on a comparison with samples from France. Though long known from various locales in France, it has recently become much more abundant in several French harbors; the French samples were kindly identified by Dr. Françoise Monniot. The earliest well-documented New England records are from about 1977; the first documented San Francisco Bay record is 1993. Molecular studies should soon be completed, comparing the genetics of the U.S. populations with those from France and the recently described Didemnum vexillum from New Zealand (Kott, P. 2002. A complex didemnid ascidian from Whangamata, New Zealand. J. Mar. Biol. Ass. U.K. 82: 625-628) which we believe is a recent invader to that country. Until the sequencing studies are complete, we are referring to the U.S. populations as Didemnum cf. lahillei. The following website, created by the U.S. Geological Survey, contains many photos and distribution locations: http://woodshole.er.usgs.gov/project-pages/stellwagen/didemnum/index.htm. Although the U.S. listings on this website are given as Didemnum lahillei, this is as yet an unpublished unofficial designation even though all morphological aspects of the adult zooids, colony, spicules and larvae are identical with the French specimens.
1. Comparative Developmental Biology: Insights into Embryos, Cells and Evolution. June 6-11, 2004, Univ. of Washington Friday Harbor Laboratories. Organized by Dr. Billie Swalla, UW Dept. of Biology. A symposium to celebrate the FHL centennial 1904-2004.
A. Ciona intestinalis: Molecular embryology and the genome. Noriyuki Satoh, Kyoto Univ., Japan (keynote address). No abstract. satoh@ascidian.zool.kyoto-u.ac.jp
B. Cell biology and physiology of early development in the ascidians Ciona intestinalis, Halocynthia roretzi and Phallusia mammillata. Christian Sardet, Janet Chenevert, François Prodon, Rémi Dumollard, Chakib Djediat, Solenn Patalano, and Philippe Dru. BioMarCell, CNRS, Univ Paris 6, Station Zoologique, Villefranche sur Mer, France. http://www.obs-vlfr.fr/biomarcell sardet@obs-vlfr.fr
Ascidian eggs acquire a primary
animal-vegetal (a-v) polarity during maturation. At fertilization, the a-v
gradient of cortical rough Endoplasmic Reticulum (cER) with its associated messenger RNAs (PEM 1, 3 and Macho1) and
the subcortical gradient of myoplasm (a mitochondria-rich layer) are amplified
through a microfilament(MF)-driven cortical contraction triggered by the calcium wave initiated at
the sperm entry site. This first major phase of reorganization concentrates the cER and
5 determinants (including the muscle determinant Macho1 mRNA) in a protrusion (vegetal-contraction pole) which
defines the future site of gastrulation and dorsal side of the embryo. This contraction pole also contains a stable
calcium wave pacemaker located in the concentrated cER layer . This calcium
wave pacemaker is essential for the completion of meiosis and the stimulation
of mitochondrial respiration.
A second
major phase of reorganization starts just after meiosis completion during the
meeting of pronuclei via extensive interactions between the sperm aster
microtubules (MT) and the cortex.
MT-driven translocations relocate the
cER with its associated mRNAs and the bulk of the myoplasm towards the future
posterior pole of the embryo. Two important determinants - Macho1 for muscle, and the yet unidentified determinant(s) for
asymmetric division - are thus relocated posteriorly and distributed evenly in
the first 2 blastomeres. Unequal cleavages
starting at the 8 cell stage
dissociate the cER and associated PEMs mRNAs
from the myoplasm in distinct cell lineages with different fates. The larger
cells inherit myoplasm and differenciate into primary muscle cells, while the
smaller cells which contain many PEM RNAs
and Vasa are thought to be the
precursor of germ cells. Our recent observations show that the macroscopic
cortical structure (called the CAB for Centrosome Attracting Body) implicated
in asymmetric division and PEM RNA
localization is made of a submembranous layer of PAR protein complex
(PAR3/PAR6/aPKC) associated with the cortical layer of ER which at that stage
has been highly compacted. Refs.-
Dumollard R, Carroll J, Dupont G, Sardet C (2002) “Calcium wave pacemakers in eggs” J. Cell Sci. 115: 3557-3564. - Sardet C, Prodon F., Dumollard R., Chang
P., Chenevert J.(2002), Structure and function of the egg cortex. Dev. Biol.
241: 1-23. - Dumollard R, Hammar M, Porterfield M, Smith P. J.,
Cibert C., Rouvière C & Sardet C (2003) Mitochondrial
respiration and calcium waves are linked during fertilization and meiosis
completion. Development 130: 5839-5849.- Sardet C, Nishida H, Prodon F, Sawada
K.( 2003) Maternal mRNAs of PEM and macho 1, the ascidian muscle determinant,
associate and move with a rough endoplasmic reticulum network in the egg
cortex. Development 130: 5839-5849.
C. Evolution of the chordate: worms or squirts? Billie Swalla, Univ. of Washington Biol. Dept., Seattle, WA. bjswalla@u.washington.edu
We study the development and evolution of the chordate body plan within the deuterostomes, by studying development of hemichordates and tunicates. The four major clades of deuterostomes are echinoderms, hemichordates, urochordates (tunicates) and chordates (vertebrates and lancelets). Echinoderms and hemichordates are sister groups as has been shown by rRNA phylogenies, mitochondrial data and larval morphology, but the monophyly of the chordates and tunicates is hard to recover by molecular analysis. Using developmental characters to compare the four major deuterostome groups it appears that echinoderm and urochordate adult body plans appear to have become highly derived at the time of their divergences from the other major clades. Previous theories of chordate origins propose that chordates evolved from a sessile urochordate tadpole larva. However, our results suggest that the deuterostome ancestor was likely to have been a motile, benthic, filter-feeding worm with gill slits, an organized cartilaginous skeleton, coelom, and perhaps even neural crest cells. Therefore, the chordate ancestor was likely to have been a motile worm, instead of a sessile urochordate. This scenario would suggest that tunicates evolved from a motile worm-like chordate ancestor by dissociation of the ancestral adult and larval developmental modules. The non-feeding larva contains the notochord but no gut, heart, blood or gonads. The internal organs all develop after metamorphosis in solitary ascidians. Our results suggest that the deuterostome and chordate ancestors are similar benthic filter-feeding worms.
D. All about ascidian fertilization. Charles C. Lambert, Univ. of Washington Friday Harbor Laboratories, Friday Harbor, WA. clambert@fullerton.edu
Ascidian oocytes are enclosed by a single layer of follicle cells (FC) surrounding a vitelline coat (VC) outside the test cells and egg. The sperm with its eccentric mitochondrion, binds to the VC between the follicle cells by means of surface N-acetylglucosaminidase (NAGase) binding to a VC N-acetylglucosamine group. After binding, sperm surface proteases aid in penetration of the VC that is accomplished by the egg binding, sperm NAGase, anchoring the mitochondrion to the VC as it slides down the tail, driving the sperm head into the egg. Chymotrypsin-like activity is essential for fertilization, while trypsin-like activity accelerates the rate. The mitochondrion remains outside the VC during fertilization. Actin-myosin interactions controlled by intracellular calcium release drive the mitochondrion down the tail. To insure that only a single sperm enters the egg, ascidians have evolved at least two blocks to polyspermy. The first involves the release of NAGase from the FC within a few seconds of insemination, this binds to VC N-acetylglucosamine groups, inhibiting sperm binding. The affinity of the FC enzyme for substrate is much higher than that of the sperm enzyme, insuring that the egg enzyme will exclude sperm binding. The second requires a few minutes for the sperm to reach the egg surface and trigger an electrical change in the egg membrane that excludes supernumerary sperm. The first block is triggered by sperm contacting follicle cells and is not species specific, the second block is species specific and entails sperm contact with the egg surface.
E. Functional screen of novel genes with developmental function identifies genes that are essential for beta-catenin-mediated signaling pathway in the ascidian Ciona intestinalis. Mayuko Hamada1, Kenji Kobayashi1, Nori Satoh12, and Shuichi Wada2 1Dept. Zool., Graduate Sch. of Sci., Kyoto Univ., Sakyo-ku, Kyoto 606-8502, Japan; 2CREST, Japan Sci. & Technol. Agency, Tokyo, Japan. [poster] hamada-m@ascidian.zool.kyoto-u.ac.jp
Ascidians belong to the subphylum Urochordata, which is considered to be the most primitive group in the phylum Chordata. Recently draft genome sequence of the ascidian Ciona intestinalis was determined and thereby it was shown that the ascidian genome contains a basic set of chordate genes with less redundancy compared to the vertebrate genome. In order to identify novel genes essential for the chordate embryogenesis, we conducted a functional screening of Ciona genes. Here we report results of the screening, together with analyses of identified genes. Translation of a total 518 genes, selected from a collection of genes discovered through the Ciona intestinalis cDNA project, was inhibited by specific morpholino antisense oligonucleotides. Most of these genes were chosen because they are transcribed during early developmental stages and have vertebrate counterparts whose functions are unknown. Translational suppression of 113 genes among them caused morphological abnormalities in manipulated embryos. Expression analyses of specific differentiation makers for major tissues that consist of the ascidian embryo suggested that 55 genes out of the 113 genes are involved in tissue differentiation. In ascidian embryos, beta-catenin acts in the first step of endoderm determination. In our screening, five genes were found to be associated with phenotypes that are similar to those of beta-catenin knockdown embryos. These genes were subjected to subsequent detailed analyses. We named these five genes vegetal hemisphere defective (vhd), since suppression of translation of each of these genes resulted in embryos lacking most of the vegetal hemisphere-derived tissues including endoderm and notochord. We provided lines of evidence that vhd genes play essential roles in beta-catenin-mediated signaling pathway in endoderm formation; vhd1, 2, 3 and 4 genes act upstream of beta-catenin while vhd5 functions downstream of beta-catenin. Since vertebrate genomes contain homologues of the vhd genes, these findings provide novel clues to understand factors involved in beta-catenin-mediated signaling pathway in vertebrates.
F. Microarray analysis of gene expression profiles during Ciona intestinalis metamorphosis. Akie Nakayama, Kyoto Univ. Dept. of Zool., Graduate School of Science, Kyoto, Japan. akie@ascidian.zool.kyoto-u.ac.jp [poster]
Metamorphosis is one of the most attractive phenomena of life, in which the shape of an organism change dynamically. It contains a lot of questions not only from developmental biology but also from ecology, physiology and evolutionary biology, and thus various studies have been performed to investigate this important biological phenomenon. Together with a lot of information of genome, ESTs and cDNA sequences, recent studies of Ciona intestinalis enable us genome-wide study of gene expression and function. We constructed oligo nucleotide microarray based on sequence information of ESTs and genome, on which about 21,000 types of oligo nucleotides were spotted. Microarray analysis is useful to recognize the framework of gene expressions, which was difficult to approach by other methods. Because metamorphosis is very complex process and hard to understand the outlines, microarray would be very useful to identify and analyze the expression of genes that control this complex process. In the present study, we performed a series of DNA microarray using 8 time points, during a period from newly hatched larvae through swimming larvae to juveniles 24 hours after initiation of metamorphosis in C. intestinalis. We report here the expression profiles of genes during this period. Genes were grouped into several categories based on GO (Gene Ontology) and KEGG Pathway Database. We also report that several genes that are shown to be involved in the metamorphosis in other animals would also be related to the Ciona metamorphosis. This study would provide a lot of fundamental information for further study of metamorphosis.
G. Cellular mechanisms of ascidian endoderm invagination. Team Squirt: Maria-Jose Bravo, Ryan Gile, James Lee, Tammie Robinson. Mentors: Drs. Garrett Odell, Edwin Munro, George von Dassow, Kristin Sherrard, Billie Swalla. Univ. of Washington Biol. Dept., Seattle, WA. [poster]
Invagination is a dynamic process in which a sheet of cells bends inward, moving its component cells into the interior of the embryo. Despite considerable experimental and theoretical attention, the cellular and mechanical basis for invagination is still not known. We have studied ascidian endoderm invagination, the first step of gastrulation, which leads to the formation of the gut. We used time-lapse microscopy to document the pattern of cell shape changes that accompany endoderm invagination within intact embryos. In particular, we observed a pattern of nuclear descent within endoderm cells (from the apical surface to the basal interior) during invagination that has not been described previously. We also examined the behavior of isolated vegetal plates (that contain mostly endoderm) and individual isolated endoderm cells. We found that vegetal plates invaginate normally, while isolated endoderm cells displayed patterns of cell shape change and nuclear movement reminiscent of endoderm cells during invagination in whole embryos, suggesting that the forces driving invagination are intrinsic to these cells. To explore the cytoskeletal basis for these changes, we used confocal microscopy to examine embryos that were fixed during invagination and stained for filamentous actin and microtubules. Our observations contradict, rather than support, existing theories of invagination. However, we did observe prominent clouds of cytoplasmic F-actin associated with the microtubule organizing centers that flank nuclei. These clouds appear to connect to one another through points of contact between neighboring cells to form a meshwork that spans the endoderm plate. We speculate that this meshwork could generate components of the forces that contribute to invagination. Finally, we used a computer model to explore how various hypothesized interactions among different cellular components could account for the invagination of a sheet of cells. Future studies will incorporate our observations of ascidian endoderm cells into this model.
A. Developmental expression of a POU gene of class IV in the ascidian Ciona
intestinalis
1Candiani S., 2Pennati R., 1Oliveri D., 3Locascio A.M. 3Branno M., 2De Bernardi F., and 1Pestarino M. 1Dip. di Biologia Sperimentale, Ambientale e Applicata, Università di Genova. 2Dip. di Biologia, Università di Milano. 3Stazione Zoologica “Anton Dohrn” di Napoli.
The POU family of transcription factors
was originally defined on the sequence homology of POU domain identified in the
mammalian transcription factors Pit-1, Oct-1 and Oct-2 and the nematode unc86 protein.
Sequence-specific DNA binding of POU-domain genes is mediated by a bipartite
motif, which consists of POU homeodomain and POU-specific domain. The POU
homeodomain is a 60 amino acid segment with similarity to the classic
homeodomain transcription factors. The POU-specific domain is an additional
motif of 75 amino acid approxymately, that cooperates with the POU homeodomain
to enhance the binding affinity and specificity of DNA binding. At present,
POU-domain transcription factors have been classified into seven classes based
on their amino acid sequences. Several lines of evidence suggest that members
of the POU genes family may regulate invertebrates and vertebrates
neurogenesis. In particular, POU IV genes appears to be neuron-specific
regulating the differentiation of specific neuron cell types, including a range
of sensory neurons in molluscs, Drosophila,
Caenorhabditis elegans and vertebrates. Recently, 83 homeobox genes have
been identified in the Ciona genome
(Wada et al. 2003), and at least 3 genes belong to the POU family. A
cDNA sequence for a POU IV-like gene, containing the entire ORF as well as 3’
and 5’ UTR, was identified from the database of the Ciona cDNA project database.
In the
present work, we describe the expression pattern of a homologue of class POU IV
genes, Ci-POU4, in the ascidian Ciona intestinalis by whole-mount in
situ hybridization on stages from neurula to early larva. Ci-POU4 is expressed exclusively in neural precursor cells during.
In particular transcripts are prevalently localized in the peripheral nervous
system, with the expression in the central nervous system restricted to the
posterior sensory vesicle. Then, the evolution of a complex sensory system
seems to be on the control of a common genetic mechanism. (Work supported by grant from MIUR-FIRB).
B. In the ascidian Phallusia mammillata the onset of
metamorphosis is regulated by dopamine and serotonin. Pennati R.,
Zega G., Groppelli S., Sotgia C. and De Bernardi F. Dept. of Biology, Univ. of Milano, Italy fiorenza.debernardi@unimi.it
Ascidian larvae settle within minutes,
hours or days after hatching, depending on the species, and then start to
metamorphose. Metamorphosis occurs after the larva develops the ability to
respond to external inducing signals: the responsive state is termed
competence. Many environmental factors which can influence the setllement are
known, but nothing is known about how these agents can trigger metamorphosis.
Neurotransmitters play important roles during morphogenetic events in different
organisms. In this work, the role of dopamine and serotonin during
metamorphosis of the ascidian Phallusia mammillata larvae was examined. By
immunofluorescence experiments dopamine was localized in some neurons of the
central nervous system and in the adhesive papillae of the larvae. Dopamine and
serotonin signaling was inhibited by means of antagonists of these
neurotransmitters receptors (R (+)-SCH-23390, a D1 antagonist;
clozapine, a D4 antagonist; WAY-100635, a 5-HT1A
antagonist) and by sequestering the neurotransmitters with specific antibodies.
Moreover, dopamine synthesis was inhibited exposing 2-cell embryos to a-methyl-L-tyrosine. Dopamine
depletion, obtained by these different approaches, caused an early
metamorphosis, while serotonin depletion delayed the onset of metamorphosis.
The opposite effects were obtained using agonists of the neurotransmitters:
lisuride, a D2 agonist, inhibited metamorphosis while DOI
hydrochloride and 8-OH-DPAT HBr, two serotonin agonists, promoted it. So it is
possible to suppose that dopamine signaling prevents metamorphosis while
serotonin signaling triggers it; in young larvae high levels of dopamine are
necessary to promote the swimming behavior and are sufficient to prevent
metamorphosis; when larvae become competent, they start to produce serotonin in
response to external stimuli (light, chemical nature of the substratum), and
when a threshold level is reached, the cascade of events that triggers metamorphosis
is activated.
3. North Atlantic
Chapter of the Soc. of Environmental Toxicology & Chemistry 2004
meeting. June 9-11, 2004, Roger Williams University,
Portsmouth, Rhode Island.
Nitrogen
contamination influence on invasive and native ascidian species diversity. Mary
R. Carman, Woods Hole Oceanographic Institution, Woods Hole, MA 02543. mcarman@whoi.edu
Invasive and native ascidian diversity appears to correlate with excessive nitrogen levels in the coastal waters of Cape Cod and Martha’s Vineyard, Massachusetts. Considered to be a fouling organism, ascidians, also called tunicates or sea squirts, are soft-bodied marine chordates that attach to hard substratum in the intertidal and subtidal zones. The main contaminant causing the degradation of water quality in these coastal waters is nitrogen. Excessive nitrogen levels act as a nutrient for algal blooms, which in turn decrease eelgrass beds, the habitat for juvenile bay scallops. Toxic levels of nitrogen have caused eutrophication and fish kills. The effect of nitrogen loading on the ascidian populations has not been previously studied. In a survey of harbors, embayments and marine ‘ponds’ during the summer of 2003, nine different species of ascidians (four native and five invasive) were observed living at 26 sites. Nitrogen loading measurements were available for 14 of these sites. A moderate diversity of ascidians, both native and invasive species, live in fairly excessive nitrogen enriched, fair quality waters. The greatest diversity occurred in high levels of nitrogen, where water quality was poor. In areas of toxic levels of nitrogen and eutrophication, only two native species survived. Diversity has decreased by almost half since the late 1970’s, and may continue to decrease in the future if eutrophication continues to happen. Further monitoring and nitrogen reduction should be addressed.
4. 6th Intl. Larval Biology Conference, June 21-25, 2004, Hong Kong.
A. Metamorphosis in solitary ascidians: some surprising variations. Molly Jacobs, Friday Harbor Laboratories, University of Washington, Seattle, WA USA.
Solitary ascidians produce small, short-lived, non-feeding larvae which function primarily in habitat selection and dispersal. Attachment typically initiates a radical metamorphosis in which larval features are sloughed or resorbed and juvenile features begin to differentiate. Because all adult ascidians are sessile, dispersal is usually thought to be limited by larval lifespan (except in cases of rafting by adults). I show that the tropical solitary phlebobranch Phallusia julinea can undergo extensive differentiation of juvenile structures prior to attachment, a strategy previously described only for colonial ascidians. I also show that the tropical stolidobranch ascidian Herdmania curvata (formerly H. momus) is capable of detaching and dispersing after metamorphosis. Evidence from these and other species suggests that in solitary ascidians the different components of metamorphosis – tissue differentiation, attachment, and morphological rearrangement of existing tissues- are independent and can happen in different orders if environmental conditions demand it. These data have serious implications for our understanding of evolution and reproductive strategies in these common invasive species.
B. The ascidian, Herdmania curvata, as a model organism for ecological functional genomics. Kathrein E. Roper1,2, Rick G. Woods2, Mary J. Garson3, Martin F. Lavin2 and Bernard M. Degnan1
1Dept. Zool. & Entomol., Univ. of
Queensland, Brisbane 4072; 2Microarray Facility, Queensland Inst. of
Medical Res., Brisbane 4022; 3Dept. of Chem., Univ. of Queensland,
Brisbane 4072.
We have utilized our knowledge of the ascidian, Herdmania curvata, to study the processes involved in settlement and metamorphosis, particularly at a molecular level. H. curvata undertakes a rapid embryogenesis, hatching only 10 hours after fertilisation and becomes competent to settle 4 hours later. Besides exhibiting a spontaneous rate of metamorphosis, this ascidian responds to both natural and recombinant inducers and inhibitors making it an ideal organism in which to study the ecology of settlement. In addition, ascidian larvae are relatively simple, with the embryonic cell fates and fates of most larval tissues through metamorphosis being known. This background knowledge facilitates the study of molecular changes at in metamorphosis. For instance, a novel EGF-like protein, HEMPS, was first identified in H. curvata as being a key mediator of early metamorphosis. In order to examine global gene expression during metamorphosis in larvae and early postlarvae under varying experimental and ecological conditions, we constructed a 4800 clone H. curvata cDNA microarray chip. Firstly, we subjected larvae to an anti-HEMPS antibody to examine changes in gene expression when this signalling cascade is blocked. This approach has identified downstream targets of HEMPS and novel genes and expression patterns involved in metamorphosis. Second, we isolated compounds produced by two marine sponges which appear to act as allelochemicals. These chemicals induce H. curvata to settle but inhibit further progression of metamorphosis. Using the expression profile approach, we looked at the genes expressed in normally developing animals compared to those subjected to the sponge chemicals. By incorporating a stringent analytical model, we have been able to not only identify possible modes of action of the sponge allelochemicals, but also gain insight into the genes involved in metamorphosis initiation. Further studies are focused on using this information in a comparative approach in other marine invertebrate phyla to elucidate possible conserved targets of the sponge allelochemicals.
[Note from G.
Lambert concerning the above 2 abstracts: P. Kott has synonymized H. curvata
under H. momus so it is back to what it was. Kott, P. 2002. The genus Herdmania Lahille, 1888
(Tunicata, Ascidiacea) in Australian waters. Zool. J. Linn. Soc. 134:
359-374.]
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Dumollard, R., McDougall, A., Rouviere, C. and Sardet, C. 2004. Fertilisation calcium signals in the ascidian egg. Biol. Cell 96: 29-36.
Ebert, D. A. and Cowley, P. D. 2003. Diet, feeding behaviour and habitat utilisation of the blue stingray Dasyatis chrysonota (Smith, 1828) in South African waters. Mar. Freshwater Res. 54: 957-965.
Epel, D. 2003. Protection of DNA during early development: adaptations and evolutionary consequences. Evol. & Dev. 5: 83 - 88.
Faulkner, D. J., Newman, D. J. and Cragg, G. M. 2004. Investigations of the marine flora and fauna of the Islands of Palau. Nat. Prod. Rep. 21: 50-76.
Fernández, D., López-Urrutia, Á., Fernández, A., Acuña, J. L. and Harris, R. 2003. Retention efficiency of 0.2 to 6 µm particles by the appendicularians Oikopleura dioica and Fritillaria borealis. Mar. Ecol. Prog. Ser. 266: 89-101.
Fofonoff, P. W., Ruiz, G. M., Steves, B. and Carlton, J. T. 2003. In ships or on ships? Mechanisms of transfer and invasion for nonnative species to the coasts of North America. In Invasive Species: Vectors and Management Strategies. Ruiz, G. M. and Carlton, J. T. Island Press, Washington DC. Pp. 152-182.
Fujita, T., Endo, Y. and Nonaka, M. 2004. Primitive complement system--recognition and activation. Molec. Immunol. 41: 103-111.
Fukumoto, M. and Zarnescu, O. 2003. Acrosome differentiation and the acrosome reaction in ascidian spermatozoa: Ascidiella aspersa and Ascidia mentula with some implications for tunicate phylogeny. Mar. Biol . 143: 1151 - 1160.
Gehring, W. J. 2004. Precis of Edwin G. Conklin's JEZ article, "Mosaic Development in Ascidian Eggs". Journal of Experimental Zoology A 301: 461-463.
Gili, J. M., Coma, R., Orejas, C., López-González, P. J. and Zabala, M. 2001. Are Antarctica suspension-feeding communities different from those elsewhere in the world? Polar Biol. 24: 473-485.
Gissi, C., Iannelli, F. and Pesole, G. 2004. Complete mtDNA of Ciona intestinalis reveals extensive gene rearrangement and the presence of an atp8 and an extra trnM gene in ascidians. J. Mol. Evol. 58: 376-389.
Godeaux, J. 2003. History and revised classification of the order Cyclomyaria (Tunicata, Thaliacea, Doliolida). Bull. Inst. Roy. Sci. Nat. Belgique 73: 191-222.
Godeaux, J. and Harbison, G. R. 2003. On some pelagic doliolid tunicates (Thaliacea, Doliolida) collected by a submersible off the eastern North American coast. Bull. Mar. Sci. 72: 589-612.
Gompel, M., Leost, M., Joffe, E. B. D., Puricelli, L., Franco, L. H., Palermo, J. and Meijer, L. 2004. Meridianins, a new family of protein kinase inhibitors isolated from the ascidian Aplidium meridianum. Bioorg. & Med. Chem. Lett. 14: 1703-1707.
Goodbody, I. 2003. The ascidian fauna of Port Royal, Jamaica - I. Harbor and mangrove dwelling species. Bull. Mar. Sci. 73: 457-476.
Groppelli, S., Pennati, R., Scari, G., Sotgia, C. and De Bernardi, F. 2003. Observations on the settlement of Phallusia mammillata larvae: effects of different lithological substrata. Ital. J. Zool. 70: 321-326.
Guo, D., Holmlund, C., Henriksson, R. and Hedman, H. 2004. The LRIG gene family has three vertebrate paralogs widely expressed in human and mouse tissues and a homolog in Ascidiacea. Genomics 84(1): 157-165.
Hewitt, C. L., Campbell, M. L., Thresher, R. E., Martin, R. B., Boyd, S., Cohen, B. F., Currie, D. R., Gomon, M. F., Keough, M. J., Lewis, J. A., Lockett, M. M., Mays, N., McArthur, M. A. and et.al. 2004. Introduced and cryptogenic species in Port Phillip Bay, Victoria, Australia. Mar. Biol . 144: 183 - 202.
Hino, K., Satou, Y., Yagi, K. and Satoh, N. 2003. A genomewide survey of developmentally relevant genes in Ciona intestinalis. VI. Genes for Wnt, TGFß, Hedgehog and JAK/STAT signaling pathways. Dev. Genes & Evol. 213: 264-272.
Hirose, E. and Akahori, M. 2004. Comparative morphology of the stolonic vessel in a didemnid ascidian and some related tissues in colonial ascidians. Zool. Sci. 21: 445-455.
Holland, N. D., Venkatesh, T. V., Holland, L. Z., Jacobs, D. K. and Bodmer, R. 2003. AmphiNk2-tin, an amphioxus homeobox gene expressed in myocardial progenitors: insights into evolution of the vertebrate heart. Dev. Biol. 255: 128-137.
Hozumi, A., Satouh, Y., Ishibe, D., Kaizu, M., Konno, A., Ushimaru, Y., Toda, T. and Inaba, K. 2004. Local database and the search program for proteomic analysis of sperm proteins in the ascidian Ciona intestinalis. Biochemical and Biophysical Research Communications 319: 1241-1216.
Immesberger, A. and Burmester, T. 2004. Putative phenoloxidases in the tunicate Ciona intestinalis and the origin of the arthropod hemocyanin superfamily. J. Comp. Physiol. B 174: 169-180.
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Ishikawa, M., Tsutsui, H., Cosson, J., Oka, Y. and Morisawa, M. 2004. Strategies for sperm chemotaxis in the siphonophores and ascidians: a numerical simulation study. Biol. Bull. 206: 95-102.
James, N. P., Feary, D. A., Betzler, C., Bone, Y., Holbourn, A. E., Li, Q. Y., Machiyama, H., Simo, J. A. T. and Surlyk, F. 2004. Origin of late pleistocene bryozoan reef mounds; Great Australian Bight. J. Sediment. Res. 74: 20-48.
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Johnson, S. L. and Yund, P. O. 2004. Remarkable longevity of dilute sperm in a free-spawning colonial ascidian. Biol. Bull. 206: 144-151.
Karabinos, A., Zimek, A. and Weber, K. 2004. The genome of the early chordate Ciona intestinalis encodes only five cytoplasmic intermediate filament proteins including a single type I and type II keratin and a unique IF-annexin fusion protein. Gene 326: 123-129.
Kasahara, M., T., S. and DuPasquier, L. 2004. On the origins of the adaptive immune system: novel insights from invertebrates and cold-blooded vertebrates. Trends Immunol. 25: 105-111.
Kehraus, S., Gorzalka, S., Hallmen, C., Iqbal, J., Muller, C. E., Wright, A. D., Wiese, M. and Konig, G. M. 2004. Novel amino acid derived natural products from the ascidian Atriolum robustum: Identification and pharmacological characterization of a unique adenosine derivative. J. Med. Chem. 47: 2243-2255.
Kicklighter, C. E., Kubanek, J., Barsby, T. and Hay, M. E. 2003. Palatability and defense of some tropical infaunal worms: alkylpyrrole sulfamates as deterrents to fish feeding. Mar. Ecol. Prog. Ser. 263: 299-306.
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Marchenkov, A. and Boxshall, G. A. 2004. The notodelphyid genus Doroixys Kerschner, 1879 (Crustacea: Copepoda: Cyclopoida), with the description of a new species from the Russian Far East. Syst. Parasitol. 58: 223-233.
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Nakajo, K. and Okamura, Y. 2004. Development of transient outward currents coupled with Ca2+-induced Ca2+ release mediates oscillatory membrane potential in ascidian muscle cells. Journal of Neurophysiology 92: [March online; probably Aug. issue].
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Pakhomov, E. A., Fuentes, V., Schloss, I., Atencio, A. and Esnal, G. B. 2003. Beaching of the tunicate Salpa thompsoni at high levels of suspended particulate matter in the Southern Ocean. Polar Biol. 26: 427-431.
Pearce, A. N., Appleton, D. R., Babcock, R. C. and Copp, B. R. 2003. Distomadines A and B, novel 6-hydroxyquinoline alkaloids from the New Zealand ascidian, Pseudodistoma aureum. Tet. Lett. 44: 3897-3899.
Pemberton, A. J., Sommerfeldt, A. D., Wood, C. A., Flint, H. C., Noble, L. R., Clarke, K. R. and Bishop, J. D. D. 2004. Plant-like mating in an animal: sexual compatibility and allocation trade-offs in a simultaneous hermaphrodite with remote transfer of sperm. J. Evol. Biol. 17: 506-518.
Phillippi, A., Hamann, E. and Yund, P. O. 2004. Fertilization in an egg-brooding colonial ascidian does not vary with population density. Biol. Bull. 206: 152-160.
Pinto, M. R., Chinnici, C. M., Kimura, Y., Melillo, D., Marino, R., Spruce, L. A., De Santis, R., Parrinello, N. and Lambris, J. D. 2003. CiC3-1a-mediated chemotaxis in the deuterostome invertebrate Ciona intestinalis (Urochordata). J. Immunol. 171: 5521-5528.
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Rabinowitz, C. and Rinkevich, B. 2004. In vitro delayed senescence of extirpated buds from zooids of the colonial tunicate Botryllus schlosseri. J. Exp. Biol. 207: 1523-1532.
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Raftos, D. A., Fabbro, M. and Nair, S. V. 2004. Exocytosis of a complement component C3-like protein by tunicate hemocytes. Dev. Comp. Immunol. 28: 181-190.
Ramasamy, M. S. and Murugan, A. 2003. Chemical defense in ascidians Eudistoma viride and Didemnum psammathodes in Tuticorin, southeast coast of India: Bacterial epibiosis and fouling deterrent activity. Indian J. Mar. Sci. 32: 337-339.
Rao, M. R. and Faulkner, D. J. 2004. Botryllamides E-H, four new tyrosine derivatives from the ascidian Botrylloides tyreum. Journal of Natural Products 67: 1064-1066.
Rennert, J., Coffman, J. A., Mushegian, A. R. and Robertson, A. J. 2003. The evolution of Runx genes I. A comparative study of sequences from phylogenetically diverse model organisms. BMC Evol. Biol. 3: 57-67.
Richardson, A. D. and Ireland, C. A. 2004. A profile of the in vitro antitumor activity of lissoclinolide. Toxicol. & Applied Pharmacol. 195: 55-61.
Rinkevich, B. 2004. Primitive immune systems: Are your ways my ways? Immunol. Rev. 198: 25-35.
Russo, G. L., Tosto, M., Mupo, A., Castellano, I., Cuomo, A. and Tosti, E. 2004. Biochemical and functional characterization of protein kinase CK2 in ascidian Ciona intestinalis oocytes at fertilization: Cloning and sequence analysis of cDNA for alpha and beta subunits. Journal of Biological Chemistry
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Sakai, N., Sawada, M. T. and Sawada, H. 2004. Non-traditional roles of ubiquitin-proteasome system in fertilization and gametogenesis. Int. J. Biochem. Cell Biol. 36: 776-784.
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Sanamyan, K. and Schories, D. 2003. Ascidians from the Strait of Magellan. aqua, J. Ichthyol. & Aquatic Biol. 7: 89-96.
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Satou, Y., Imai, K. S., Levine, M., Kohara, Y., Rokhsar, D. and Satoh, N. 2003. A genomewide survey of developmentally relevant genes in Ciona intestinalis. I. Genes for bHLH transcription factors. Dev. Genes & Evol. 213: 213-221.
Segraves, N. L., Robinson, S. J., Garcia, D., Said, S. A., Fu, X., Schmitz, F. J., Pietraszkiewicz, H., Valeriote, F. A. and Crews, P. 2004. Comparison of fascaplysin and related alkaloids: a study of structures, cytotoxicities, and sources. Journal of Natural Products 67: 783-792.
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