Number 51 June 2002
After spending most of the winter working on manuscripts, in
April we enjoyed three weeks touring Italy and were shown the remarkable
Ciona
rearing facility at the Stazione Zoologica in Naples by Dr. Elisabetta
Tosti and her associates. We were very much impressed by the facility
and their success in rearing Ciona for many different studies.
Not only do they maintain genetically defined stocks but also their installation
will solve the supply problem. In early June we participated in a
rapid assessment survey for nonindigenous species on both sides of the
Panama Canal, led by Dr. Andrew Cohen. The team consisted of 11 specialists
in various marine groups. We sampled numerous sites and worked at
Smithsonian Tropical Research Institute labs on both the Pacific and Caribbean
sides. There were many more ascidians on the Caribbean side especially
on marina floats and pier pilings, nearly all of which could be considered
introduced. Ascidia sydneiensis, Styela canopus, Herdmania
pallida, Microcosmus exasperatus and several other species were
very abundant. We also greatly enjoyed seeing the canal, its locks
and its complex flooding system, which periodically reverses the flow of
rivers. However, the bugs were quite vicious on the Atlantic coast and
caused a great deal of discomfort to many of the crew.
We will spend a week identifying ascidian voucher specimens
at the Smithsonian Environmental Research Center in Maryland in late June.
In July we’ll return to Roscoff in Brittany, France, to work for 6 weeks.
It has been 5 years since our last visit. Gretchen will assist Dr.
Billie Swalla on the localization of gene products in ascidian embryos
and Charley will work on germinal vesicle breakdown in ascidian oocytes.
We will be at the Friday Harbor Labs for most of September and early October.
We hope to meet many of you and renew old acquaintances in the near future.
*Ascidian News is not part of the scientific literature and should not be cited as such.
NEWS AND VIEWS
1. It is with a very great deal of sadness that we report the death of Dr. R.H. Millar on 22 May. He was a tireless investigator of ascidians who contributed greatly to many aspects of their lives. Dr. Millar always published using only his initials but his first name was Robin. Dr. Millar received his Ph.D. from the University of Glasgow under the guidance of Prof. C.M. Yonge and worked at the Marine Biological Station in Millport until about 1970, when he moved to the Dunstaffnage Marine Research Laboratory at Oban, Scotland. From the 1940’s to the 1980’s he published numerous papers on many aspects of the lives of ascidians including structure, reproduction, development, systematics and distribution. In 1953 he published his Ph.D. thesis, an invaluable work simply titled “Ciona”, a thorough analysis of the biology of Ciona intestinalis that served many of us going back to our undergraduate days and is still a widely used classic reference. He had an encyclopedic knowledge of numerous taxa and his keen-eyed dissections made his taxonomic monographs particularly useful to all who have followed him. His review “The Biology of Ascidians” (Adv. Mar.Biol. 1971 vol. 9:1-100) summarizes most of what was known about the lives of ascidians up to that time and will certainly reward you to re-read it if only to see how far we have come. We look at this every few years and always find something we did not know or had forgotten. In addition to his work on ascidians Dr. Millar made many contributions to oyster research and was also involved in administration of the Millport and Oban laboratories. Unfortunately, we never had the opportunity to meet him but we did correspond with him occasionally and we both consult his publications frequently. The community of ascidiologists has lost a key contributor and resource and we all mourn his passing.
Dr. Patricia Mather knew Robin Millar very well and sent us this
beautiful tribute:
I knew him as a wonderful colleague, a careful, thorough
and objective observer of ascidians, an innate taxonomist and a dear friend.
He was a large man in every sense of the word - generous and kind, with
a wonderful, dry sense of humour. I met him first in 1950, when, as a graduate
student at Plymouth I made a visit to Millport. He took me out dredging
in the Clyde, it was a rough day and very uncomfortable and I'll never
forget his kindness. We had started working on ascidians at more or less
the same time. Robin published his Ciona monograph after he returned
from the war, and at about the same time, I had started working on Harold
Thompson's collection of Australian ascidians when, as a very young woman,
I was appointed to CSIRO Fisheries Division in New South Wales. I missed
him so much after he retired from working on ascidians - suddenly I was
very alone. A lot of the things that were to occupy a lot of my attention
(like the resolution of the clearly polyphyletic Euherdmaniinae) were things
that we had discussed, and often subjects that he had raised. I never lacked
a response from him during the greater part of the years I worked on the
Ascidiacea and I respected his opinion enormously. His principal contribution
to the understanding of the Ascidiacea is in their taxonomy, and he had
a vast experience of them-from those in his own Scottish waters, to collections
from South Africa, the Brazilian coast, Caribbean, Antarctic, western Pacific
and the deep water ascidians in the Galathea and the Vema collections.
He also leaves with me his concern for the precise use of words- he hated
the use of "to" when "or" is intended (such as in "2 or 3"); he intensely
disliked the use of the word endemic, when indigenous is intended (thereby
leaving me with a life long battle with most of my biological colleagues).
He served as deputy Director of the Dunstaffnage Laboratory at Oban for
a number of years. After his retirement he excluded ascidians from
his interests- Joan, his garden and his water colours occupying him entirely.
I was so pleased when he sent me his water colour of a Scottish loch that
now hangs on my study wall, where it will remain in memory of a gifted
scientist and dear friend who established a precise and scholarly basis
for modern studies on the taxonomy of the Ascidiacea.
Further interesting reminiscences of Dr. Millar from Dr. Ivan Goodbody,
Univ. of the West Indies, Kingston, Jamaica:
I was deeply saddened to receive the news that Robin Millar
had died. I first met Robin in the early 1950s when he was still at the
Millport laboratory. I immediately recognised in him a kindred spirit with
enthusaism for marine biology and indeed natural history in general. He
quickly introduced me to oysters and induced me to eat them raw as he collected
them from the sea. He took me to some coastal rapids (at Loch Fyne, I think),
to see large aggregations of Ascidiella aspersa living in the fast
flowing water. Around 1968 I found myself in possession of several undescribed
species of ascidian from Jamaica but I lacked the experience to describe
them unaided. I took them to Oban and sought help from Robin. He was only
too willing to share his knowledge and expertise with me and we spent many
useful and rewarding days together in his laboratory and in the process
formed a lasting friendship. In the paper we wrote afterwards on New Species
of Ascidian from the West Indies, all the line drawings were made by Robin.
During the day as we worked on the specimens, Robin made sketches with
a ball-point pen and next morning would come in with ready-to-publish line
drawings. I soon learnt that outside marine biology Robin had two other
great interests - art and gardening. He and Joan lived in a small house
north of Oban where Robin engaged in these two pursuits surrounded by a
lovely garden and with rooms decorated with his paintings. Nevertheless
it came as a shock when Robin retired and declared that he was taking no
further interest in ascidians but was going home to paint and grow his
garden - in both of which he was supremely happy. His large and valuable
collection of reprints he donated to the marine laboratory at Millport.
I do not think he was ever content working at Dunstaffnage and missed the
smaller and more intimate surroundings of the old Millport lab. We have
lost a very good friend and a great ascidian taxonomist, but his legacy
remains in the papers he published and is there for the next generation's
benefit.
2. From Shigeki Fujiwara, Dept. of Biol., Fac. of Sci., Kochi
Univ., Kochi, Japan:
The Japan Ascidian Club (informal meeting of ascidian
biology, held at the annual meeting of the Zool. Soc. of Japan) has changed.
The new organizers since 2001 are Hiroshi Wada at Seto Marine Biol. Laboratory,
Kyoto Univ., Wakayama (hwada@seto.kyoto-u.ac.jp) and Yutaka Satou at Kyoto
Univ. Dept. of Zool., Graduate Sch. of Sci., Kyoto (yutaka@ascidian.zool.kyoto-u.ac.jp).
The name of the meeting is "Hoya no Seibutsugaku Danwa-kai" (Hoya=ascidian(s),
no=of, Seibutugaku=biology, Danwa=informal talk, kai=meeting), shortened
to Hoya-no-Kai (see AN #37, 1995). The meeting was established more than
15 years ago. It does not require definite membership; everyone who attends
the meeting are members that year.
3. Much ado about vanadium: Later in this newsletter there are several contributions by Hitoshi Michibata and his colleagues on vanadium in ascidians. Recently we heard a rumor that Dr. Donald P. Abbott was asked by the war dept. during WWII to work on a project to extract vanadium from tunicates to use in making an atomic bomb. We wondered if there was the slightest shred of truth in that so we asked his widow Izzie Abbott and she replied, “Such a request was made of Don, but he showed them how much vanadium was in the tunicates that took it up, and it was just too small to bother with, and as I remember that was the end of it.”
WORK IN PROGRESS
1. A molecular analysis of ascidian metamorphosis reveals activation
of an innate immune response. Brad Davidson and Billie J.
Swalla, Zool. Dept., Univ. of Washington, Seattle WA. Development (in
Press).
Ascidian metamorphosis represents a powerful model for
comparative work on chordate development that has remained largely unexplored.
We isolated transcripts differentially expressed during metamorphosis in
the ascidian Boltenia villosa by suppressive PCR subtractions of
staged larval and juvenile cDNAs. We employed a series of three subtractions
to dissect gene expression during metamorphosis. We have isolated 132 different
protein coding sequences, and 65 of these transcripts show significant
matches to Genbank proteins. Some of these genes have putative functions
relevant to key metamorphic events including the differentiation of smooth
muscle, blood cells, heart tissue, and adult nervous system from larval
rudiments. In addition, a significant fraction of the differentially expressed
transcripts match identified genes from the innate immune system. Innate
immunity confers a rapid response to pathogen-specific molecules and/or
compromised self-tissues. The activation of innate immunity genes during
metamorphosis may represent the programmed maturation of the adult immune
system. In addition, this immune response may be necessary for phagocytosis
and re-structuring of larval tissues. An innate immune-related inflammatory
response may also underlie two waves of trans-epidermal blood cell migration
that occur during the swimming larval period and immediately upon settlement.
We characterized these trans-epidermal migrations and discovered that some
migratory cells leave the animal entirely through an anterior tunnel in
the tunic. We show that these cells are positioned to detect external settlement
cues and hypothesize that the innate immune system may also be employed
to detect and rapidly respond to environmental settlement cues.
2. Control of maturation of ascidian oocytes. Charles Lambert, Univ. of Washington Friday Harbor Labs, Friday Harbor, WA. Last summer I continued work on oocyte maturation using oocytes of Boltenia villosa. Boltenia oocytes spontaneously undergo germinal vesicle breakdown (GVBD) when dissected into natural pH 8.2 seawater but can be blocked at pH 4. In most chordates GVBD is initiated by the synthesis of cyclin which complexes with a cyclin dependent kinase to form the active maturation promoting factor (MPF). However, in some chordates and many echinoderms this is triggered by the dephosphorylation of a preexisitng MPF comprised of cyclin and a cyclin dependent kinase. This can often be inhibited by cAMP. Curiously, in ophiuroids, nemerteans and brachiopods cAMP actually induces GVBD rather than inhibiting it. Intracellular cAMP levels can be raised by the direct addition of a permeant form of cAMP, stimulation of adenylyl cyclase by forskolin or inhibition of the phosphodiesterase by methyl xanthines such as isobutyl methyl xanthine, hypoxanthine, caffeine or theophylline. In Boltenia oocytes the permeant 8-bromo cAMP activated GVBD in a dose dependent manner 4 mM gave a maximal response. Adenylyl cyclase was activated by forskolin, in a dose dependent manner with concentrations as low as .002 µm giving a maximal response. In addition, inhibition of c AMP phosphodiesterase with theophylline caused GVBD in a dose dependent manner with a maximal response at 0.5 µm, caffiene, hypoxanthine and isobutyl methylxanthine also caused GVBD at very low concentrations. Thus it appears that c-AMP elevation is important in GVBD in ascidian oocytes. Studies are now in progress to determine the role of the inner follicle cells to this process.
3. Gretchen Lambert spends most of her time on pesky squirts these days, identifying collections of mostly nonindigenous species in many parts of the world. The latest trip was a week in Panama with Charley surveying both sides of the canal with a team of 11 taxonomists. Other work includes a huge collection from a number of California sites for the Moss Landing Marine Lab and ongoing identifications for the Smithsonian Environmental Research Center in Maryland. I am also in contact with numerous aquaculturists involved in mussel and oyster culture in eastern Canada, Alaska, New Zealand, Puget Sound in Washington state, and elsewhere because of the overgrowth and smothering of the cultures by huge numbers of ascidians, primarily Ciona intestinalis and Styela clava in eastern Canada and C. intestinalis in New Zealand, and Botrylloides violaceus in the Sitka, Alaska and Puget Sound areas. The newest threat is an unidentified Didemnum sp. in NE U.S. and northern New Zealand, apparently the same species in both locations, whose populations have exploded in the past couple years. If you are plagued by pesky squirts and would like a willing ear to complain to, please send me an email at glambert@fullerton.edu. I am very interested in keeping track of these “outbreaks” worldwide.
THESIS ABSTRACTS
1. A morphological and genetic characterization of metamorphosis
in the ascidian Boltenia villosa. Bradley Justin Davidson
Ph.D. dissertation, Dept. of Zoology, Univ. of Washington, Seattle, WA
98195. bjd@u.washington.edu Chair
of the Supervisory Committee: Dr. Billie Swalla.
Since Kowalevsky’s insight into the chordate nature of
the ascidian larvae, numerous researchers have focused their efforts on
characterizing ascidian development in order to gain insight into chordate
evolution. Recent molecular analyses of solitary ascidian embryogenesis
have made it clear that the genetic framework for patterning of chordate
tissues is highly conserved between ascidians and vertebrates. This remarkable
level of genetic homology among chordates allows for the use of ascidian
development to elucidate the fundamental genetic networks that underlie
more complex vertebrate developmental events. However, the almost exclusive
focus on solitary ascidian embryogenesis has led researchers to neglect
the majority of ascidian development. Ascidians have a complex life history
in which free-swimming larvae settle and metamorphose into sessile adults.
Although tail structures are fully differentiated within the larvae, these
structures are resorbed upon settlement and make no contribution to the
adult body plan. Instead, rudiments within the larval head differentiate
to form juvenile organs such as the pharyngeal gill slits, endostyle and
heart. In most solitary ascidians, juvenile differentiation occurs only
after embryogenesis of the larva is complete. Thus, solitary ascidian post-embryonic
development represents a crucial untapped resource for evolutionary and
comparative work on chordate development. Here I present an analysis of
post-embryonic development in the solitary ascidian Boltenia villosa.
I begin with a general introduction to ascidian metamorphosis. Next, I
present my work on a morphological and genetic characterization of post-embryonic
development in Boltenia. The third chapter focuses on the acquisition
of metamorphic competence in Boltenia larvae. In the fourth chapter,
I present our characterization of innate immune activation during Boltenia
metamorphosis. This includes the up-regulation of a number of innate immune
related genes as well as a series of intriguing cell migrations that may
be coordinated by immune signaling. The final chapter contains an evolutionary
analysis of solitary/ colonial life history transitions, focusing primarily
on the ascidians.
2. Ascidiacea (Chordata: Tunicata) from the tropical Brazilian
coast. Tito M. C. Lotufo, Ph.D. Thesis. Dept. of Zool., Instit.
of Biosci., Univ. of São Paulo, Brazil. Advisor Dr. Sergio de Almeida
Rodrigues. Current address Univ. Fed. do Ceará, Centro de
Ciências Agrárias, Dept. de Engenharia de Pesca, C. P. 12168,
60455-760 Fortaleza-CE, Brazil. tmlotufo@ufc.br
Although ascidians are well known in many regions of the
globe, information about the group on the Brazilian coast is very scanty.
Most of the Brazilian coastline is in the tropical region, which is the
poorest known. In order to obtain an inventory of ascidian species on the
Brazilian tropical coast, surveys were conducted at different points, ranging
from the intertidal to shallow subtidal depths. Another goal of the present
work was to organize all available information through a revision of bibliography
and visits to institutions that held representative collections.
61 visits were conducted in places along the coast of the states of Rio
de Janeiro, Espírito Santo, Bahia, Alagoas, Pernambuco, Paraíba,
Rio Grande do Norte e Ceará. Specimens were collected, examined
and identified to the species level. An extensive taxonomic revision was
made for every species, by means of literature as well as examination of
types and other specimens deposited in different institutions. The present
work includes synonymy lists, descriptions, pictures and remarks for each
species studied. Keys for all taxa an every category were also included.
Up to the present work, 90 species of ascidians had been
recorded for Brazil, of which 54 are listed for the State of São
Paulo. The surveys revealed a total of 67 species, expanding the list to
98 Brazilian species. Those species are distributed in 2 orders and 3 suborders
of the class, with a total of 31 genera included in 14 of the 23 families
currently accepted. As an immediate result, 9 new records were registered
for the Brazilian coast, along with the description of 1 new genus and
10 new species. Furthermore, 8 species have had their taxonomic status
altered by synonymy or separation. The present results, together
with data from literature, generated tables that were submitted to cluster
analysis and a parsimony analysis of endemicity. These analyses revealed
a distribution pattern similar to others observed for different benthic
taxa. The region studied comprises two provinces, Brazilian Province and
Paulista Province.
The full version of this thesis (in Portuguese) will be available
in PDF format on the thesis/dissertations digital library (http://www.teses.usp.br).
MEETINGS ABSTRACTS
1. 5th International Larval Biology Meeting, ,Vigo, Spain, Sept 15-20, 2002.
The effect of nutritional stress on solitary and colonial ascidian
juveniles: a comparison of temperate and tropical species. M.W. Jacobs*
and K.M. Sherrard** . *Friday Harbor Labs, Univ. of Washington,
Seattle, WA. **Univ. of Chicago, Chicago, IL. mwjacobs@u.washington.edu
Colonial ascidians produce large, well-differentiated
larvae which quickly metamorphose into functional feeding juveniles, while
solitary ascidians produce small, simple tadpoles which must undergo an
extended period of metamorphosis after settlement before gaining the ability
to feed. In both the nutrient-rich, cold water of San Juan Island near
Puget Sound, USA, and the relatively nutrient-poor, warm water of the Great
Barrier Reef, Australia, colonial ascidians are often dominant competitors
for space in fouling communities and on the undersides of coral rubble.
We examined the effect of food levels on early growth rates of seven species
of tropical and temperate colonial and solitary ascidians. Despite large
initial size and and advanced developmental state at settlement, colonial
ascidians at San Juan Island were slower than solitary ascidians to respond
to food treatment. Preliminary results from the Great Barrier Reef in the
tropics indicate the opposite trend: colonial ascidians responded quickly
to high food levels with elevated growth rates, while solitary ascidians
showed no difference in juvenile size between treatments for the first
three weeks. At the temperate site, solitary and colonial species were
about equally vulnerable to starvation/food stress, but at the tropical
site solitary species were more vulnerable. This variation in the relationship
between embryonic provisioning and early juvenile growth and survival is
surprising, and causes us to question our assumptions about the selective
advantages and disadvantages of having large or small eggs. A larger sample
of colonial species is required to determine whether the observed differences
in response to food regime are related to environmental differences between
sites or due simply to variation between species.
2. 48th meeting of the Italian Embryological Group, Grottammare, Italy June 4-7, 2002.
Effects of antagonists of 5-HT1A receptor on ascidian
embryos development. F. De Bernardi, , S. Gropelli, R. Pennati,
U. Fascio*, C. Sotgia. Dpto. di Biol.,*Centro Interdipartimentale
di Microscopia Avanzata, Univ. di Milano, Milano, Italy. fiorenza.debernardi@unimi.it
Ascidian embryos form a tubular CNS in a similar way to
that of vertebrates, by folding of the neural plate. At least four different
regions are distinguishable by morphology and cell composition from rostral
to caudal: the anterior sensory vesicle, containing the two pigmented sensory
organs, the otolith and the ocellus; the neck; the visceral ganglion and
the spinal cord (Meinertzhagen and Okamura, 2001, Trends Neurosci. 24:
101-410). There are evidences that early embryos, at cleavage and gastrulation
stage, use 5-HT, together with GABA and acetylcholine, to regulate cell
division and morphogenetic cell movements (Buznikov et al., 2001, Cell
Tissue Res. 305: 177-186). At this moment serotonin was detected in ascidian
only from swimming larva onward, but there are suggestions about the presence
of serotonin and of its multiple receptors in earlier embryonic stages
(Pennati et al., 2001, Develop. Growth Differ.43:647-656)
Embryos of the ascidian Phallusia mammillata has been treated
for three hours with three different 5-HT1A antagonists: pindolol, spiperone
and WAY-100635 (N[2-[4-(2-Methoxyphenyl)-1-piperazinyl]ethyl]-N-pyridinyl-cycloexane-
carboxamide maleate) at concentrations from 0.1 to 20 µM in
Millipore filtered sea water (MFSW) starting at different developmental
stages When control reached the swimming larva stage , about 18 hours after
fertilization, all treated and control specimens were collected and processed
for immunofluorescence and histology. Among the tested antagonist,
only WAY-100635 caused malformations, in a dose dependent manner, in larvae
developed from treated embryos, but the incidence and the type of malformations
caused by the treatment at different stages, from gastrula to early tailbud
stage, were not significantly different. The larvae showed a shorter trunk
region: the adhesive papillae were fused to form a single structure, were
displaced more dorsally than in controls and AChE-positive cells were lacking.
All the larvae showed a dramatic reduction of the anterior sensory vesicle
and of the pigment of the two sensory organs: the ocellus and the otolith.
Immunofluorescence experiments with an anti-tubulin monoclonal antibody
specific for the neural system showed that the most anterior part of the
nervous system, anterior to the visceral ganglion, was affected in a dose-dependent
way. At the higher dose, it was not possible to localize the primary sensory
neurons of the adhesive papillae and the papillary nerves. Moreover the
neural fibers that in the control larvae connect the sensory organs to
the visceral ganglion were absent or hardly detectable and also part of
the anterior sensory vesicle appeared reduced or absent. On the other hand,
the tail appeared always normal, with a normal spinal cord running along.
In order to understand if the reduction of the sensory
vesicle was due to a failure in differentiation or to a defect in cell
proliferation, we performed nuclear staining with DAPI: the counting of
cell nuclei gave an average of 992 nuclei in the trunk region of the control
larvae, whereas an average of 402 in the treated larvae. This suggests
that the reduction of the anterior sensory vesicle can be partially due
to an alteration of cell proliferation. However, the absence of papillary
nerves in the larvae developed from treated embryos would also suggest
that the terminal neural differentiation may be affected by the drug. A
small number of larvae metamorphosed in a rather normal way: the juveniles
showed a smaller oral siphon, with altered innervation. Taken together,
these results suggest that 5-HT plays a role in the development of
the neural system in ascidians and receptors similar to the members of
5-HT1A receptor subtype of mammals mediate the development of the most
anterior nervous system of the larvae.
3. Annual meeting of the Amer. Soc. for Cell Biology, Washington, DC, 8-12 Dec. 2001.
Do D-3 phosphoinositides signal actin polymerization during ascidian
sperm activation? Lamar Blackwell, Dave Bolger, Mohammad Hanizavareh,
Emily Zebadua, and Robert A. Koch. Calif. State Univ. Fullerton,
Dept. of Biol. Sci., Fullerton, CA. rkoch@fullerton.edu
Sperm activation in the sea squirt Ascidia ceratodes
is characterized by mitochondrial translocation (MTL), an actin:myosin-dependent
movement known to require elevation of both intracellular pH (pHi) and
free calcium ion concentration ([Ca]i). Previously, we have shown
that myosin activation requires a G protein-mediated pathway involving
inositol 1,4,5-trisphosphate-mediated internal Ca release and a PKC-dependent
internal alkalization that precedes external Ca entry. Here, we explore
signaling elements that are involved in triggering actin polymerization.
In MTL assays, the actin polymerization inhibitor latrunculin (10µM)
completely blocked high pH artificial sea water (ASW)-induced sperm activation
(positive
control), and the actin polymerization inducer jasplakinolide (7.4µM)
stimulated sperm activation equal to positive controls, an action blocked
by latrunculin. Dual labeling with fluorescently tagged phalloidin
and DNaseI revealed that filamentous actin was distributed most heavily
on the mitochondrion whereas monomeric actin was also found along
the length of the tail. Sperm activation appears to increase filamentous
actin on the mitochondrion. In MTL assays, the phosphatidylinositol
3-kinase (PI3K) inhibitor LY294002 (50µM) blocked sperm activation
induced by pH 9.4 ASW but not that induced by the G protein activator mas7
(3.5µM) or the PKC activator OAG (50µM), agents shown to be
part of the myosin activation pathway. Liposomes that incorporated
phosphatidylinositol 3,4,5-trisphosphate (PIP3) stimulated levels of sperm
activation similar to positive controls. Indirect immunofluorescence
using anti-profilin antibodies showed profilin to be present on the mitochondrion,
providing a possible connection between PI3K-induced PIP3 production and
actin polymerization. (Funded by CSUF University Student Research Initiative
grant to MH; NIH R25-GM56820 to RAK for LB, DB & EZ; NIH R15HD36500
to RAK.) Molec. Biol. Cell 12:117a.
4. Programme IV "Anton Dohrn" Workshop New Perspectives in Tunicate
Biology, Stazione Zoologica, Punta San Pietro, Ischia Sept. 29-Oct. 2,
2001
Aiming to draw the entire picture of the accumulation of vanadium
in ascidians. H. Michibata, Marine Biol. Lab., Grad. Sch. Sci., Hiroshima
Univ., Hiroshima 722-0073, Japan. hmichi@sci.hiroshima-u.ac.jp
(no abstracts)
5. The 2002 Gordon Research Conference on Marine Natural Products
Chemistry, Ventura, California, Feb. 24 - March 1, 2002.
The unusual mechanism of accumulation and reduction of vanadium
by ascidians. H. Michibata, Marine Biol. Lab., Grad. Sch. Sci.,
Hiroshima Univ., Hiroshima, Japan (no abstracts)
6. The 3rd Intl. Symp. on Chemistry & Biol. Chemistry of Vanadium. Osaka Univ., Osaka, Japan. Nov 26-29, 2002.
a) Molecular biological approaches to the accumulation and reduction
of vanadium by ascidians. H. Michibata, Marine Biol. Lab., Grad. Sch.
Sci., Hiroshima Univ., Hiroshima, Japan
About 90 years ago, M. Henze discovered high levels of
vanadium in the blood (coelomic) cells of an ascidian collected from the
Bay of Naples (Henze, 1911). His discovery attracted the interdisciplinary
attention of chemists, physiologists, and biochemists. Two decades ago,
we quantified the vanadium levels in several ascidian tissues definitively
using neutron-activation analysis and revealed that some species in the
family Ascidiidae accumulate vanadium at concentrations in excess of 350
mM, corresponding to about 107 times that found in seawater. Vanadium accumulated
is reduced to the +3 oxidation state via the +4 oxidation state and stored
in vacuoles of vanadocytes (vanadium-containing blood cells) where high
levels of protons and sulfate are also contained. To investigate
this unusual phenomenon, we isolated several proteins and genes that are
expressed in vanadocytes. To date, three types of vanadium-binding protein,
designated as Vanabins, have been isolated, with molecular masses of 12.5,
15, and 16 kDa, along with the cDNAs encoding these proteins. In addition,
four types of enzyme related to the pentose phosphate pathway that produces
NADPH were revealed to be located in vanadocytes. The pentose phosphate
pathway participates in the reduction of vanadium(V) to vanadium(IV). The
cDNA for each of the vacuolar-type H+-ATPase (V-ATPase) A, B, C, and D
subunits, which are located on the vacuolar membranes of vanadocytes, has
been isolated and analyzed. V-ATPase generates a proton-motive force, and
is thought to provide the energy for vanadium accumulation.
To clarify the entire mechanism involved in the accumulation and reduction,
many more genes and proteins expressed in the blood cells needed to be
systematically identified. Thus, we performed an expressed sequence tag
(EST) analysis of blood cells and obtained 300 cDNA clones from a blood
cell library. We have, furthermore, isolated and cloned cDNA of ascidian
Nramp (natural resistance-associated macrophage protein), known to transport
several heavy metals, from the blood cells. Now, we are examining whether
the cultured CHO-K1 cell line overexpressed a fusion protein of ascidian
Nramp accumulates vanadium.
b) Analysis of metal-binding activity of vanadium-binding proteins
(Vanabins) of an ascidian Ascidia sydneiensis samea. T.
Ueki*, T. Uyama and H. Michibata, Marine Biol. Lab., Grad. Sch. Sci., Hiroshima
Univ., Hiroshima 722-0073, Japan *ueki@sci.hiroshima-u.ac.jp
Ascidians have been known to accumulate high levels of vanadium
ion in the vacuole of one or more type(s) of blood cells. From the signet
ring cells, which are vanadium-accumulating cells, of Ascidia sydneiensis
samea we previously identified several low molecular weight vanadium-binding
proteins, Vanabins, and cloned cDNAs for 12.5 kDa and 15 kDa Vanabins.
In the present experiment, we constructed plasmids expressing recombinant
Vanabins in E. coli. We have examined the activities of the recombinant
Vanabins to bind metal ions including vanadium (IV) and vanadium (V) by
Hummel-Dreyer's method. As a result, 15 kDa Vanabin bound to approximately
20 vanadium (IV) or 20 vanadium (V) ions in 10mM HEPES buffer at pH 7.2
containing 20 mM NaCl. In the same buffer, 15 kDa Vanabin bound to 5 copper
(II) ions, but not to iron (III) ions. In 20 mM sodium phosphate buffer
at pH 7.2, 15 kDa Vanabin bound one vanadium (V) ion while it bound to
7 vanadium (IV) ions. This may be due to the similarity of the structure
of phosphate and vanadate ions.
c) Hunting for vanadium-binding proteins from vanadium-accumulating
ascidian by metal-chelating column. M. Yoshinaga*, M. Aoshima,
T. Watanabe, N. Yamaguchi, T. Ueki, T. Uyama and H. Michibata, Marine Biol.
Lab., Grad. Sch. Sci., Hiroshima Univ., Hiroshima, Mukaishima 2445, Hiroshima
722-0073, Japan. * pyochi@hiroshima-u.ac.jp
Ascidians accumulate high levels of vanadium in their
vacuoles of vanadocytes from sea water. We have extracted vanadium-binding
proteins, designated as Vanabin, from the cytoplasm of vanadocytes, cloned
cDNAs encoding the proteins and prepared recombinant proteins. The analysis
of their metal binding activity is in progress. In order to solve the ultimate
mechanism of this unusual phenomenon, many more proteins, that are associated
with the phenomenon, need to be systematically identified. The present
experiment was newly planned to isolate exclusively vanadium-binding proteins
not only from the vanadocytes but also from the other organs of the ascidian,
Ascidia
sydneiensis samea, using a metal-chelating column coupled with imidiaceticacid
to which vanadium(IV) was immobilized. Soluble proteins of blood cells,
plasma membrane proteins and coelomic serum proteins were submitted to
the present experiments. As a result, several Vanabin-like proteins were
identified in the soluble fraction. Several proteins were also hunted by
the metal-chelating column when plasma membrane proteins solubilized by
urea-containing buffer were applied to the column. Among them, 4.5 kDa
protein sequence almost matched with that obtained by EST analysis of a
cDNA library derived from vanadocytes. The predicted protein was a novel
protein composed of 43 amino acids, including 4 histidine residues. Further,
several vanadium-associated proteins have been extracted from the coelomic
serum, A main component of the proteins is 14 kDa protein having basic
amino acid-rich in N-terminal amino acids. We are now trying to determine
inner amino acid sequence of this protein to examine their metal binding
activity by constructing recombinant proteins.
d) Observation of vanadocytes of ascidians by an x-ray microscope
using synchrotron radiation. K. Takemoto1*,T. Ueki2, B. Fayard3,
M. Salome3, J. Susini3, A. Yamamoto1, H. Kihara1, S. Scippa4 and H. Michibata2.
1Dept. Physics, Kansai Medical Univ., Uyamahigashi 18-89, Hirakata, Osaka
573-1136, Japan. 2Marine Biol. Lab., Grad. Sch. Sci., Hiroshima Univ.,
Hiroshima 722-0073, Japan. 3ESRF, BP220, 38043 Grenoble cedux, France.
4Dept. Genet. Gener. Mol. Biol., Fac. Sci., Univ. Naples, 80134 Naples,
Italy. * takemoto@makino.kmu.ac.jp
Several species of ascidians accumulate vanadium in one
or more type(s) of blood cells (coelomic cells), such as signet ring cells
and some other vacuolated cells. We applied a scanning X-ray microscope
at ID21 of the European Synchrotron Radiation Facility (ESRF, Grenoble,
France) to the observation of living blood cells of a Mediterranean ascidian
species Phallusia mammillata and a Japanese species Ascidia sydneiensis
samea. The vanadium-accumulating cells (vanadocytes) of these species
contain 60mM and 13 mM vanadium in their vacuoles, respectively. The high
levels of vanadium are therefore expected to give a high contrast by X-ray
microscopy. Using a scanning transmission X-ray microscopy equipped with
ESRF, we succeeded not only in observing living blood cells in a sealed
spatial sample holder at 1 micron resolution but also in visualizing of
vanadium in signet ring cells of both species and vacuolated amoebocytes
of Phallusia mammillata using a x-ray fluorescence probe. Morula
cells which had been thought to be vanadium-accumulating cells of both
species and type-I compartment cells of Phallusia mammillata did
not contain vanadium. These results are regarded as the first epoch-making
one as a direct observation of vanadium in living cells.
e) Expressed sequence tag analysis of blood cells in the vanadium-rich
ascidian, Ascidia sydneiensis samea. N. Yamaguchi*, T.
Ueki, T. Uyama and H. Michibata, Mar. Biol. Lab., Graduate Sch. of Sci.,
Hiroshima Univ., Mukaishima-cho 2445, Hiroshima 722-0073, Japan *
yam@sci.hiroshima-u.ac.jp
Some species in the family Ascidiidae accumulate vanadium
at concentrations in excess of 350 mM, which corresponds to about 107 times
that found in seawater. The vanadium ions are stored in vacuoles located
within vanadium-containing blood cells, vanadocytes. To investigate the
phenomenon, an expressed sequence tag analysis (EST) of a cDNA library
of Ascidia sydneiensis samea blood cells was carried out. Three hundred
clones were obtained and sequenced by EST analysis. A similarity search
revealed that 127 of the clones (42.3%) were known genes, and 173 of the
clones (57.7%) did not have any similarity to genes registered in the SwissProt
database. According to the functions of their genes the identified EST
clones were categorized into seven types of clones; these consisted of
genes related to nuclear proteins (21 clones), signal transduction (18
clones), the cytoskeleton (17 clones), metal ion transport or the redox
of metals (16 clones), energy conversion (8 clones), ribosomal proteins
(6 clones), and other proteins (41 clones). The H-subunit of ferritin has
a high degree of similarity to that of mammals; the iron-binding sites
of ferritin are well conserved including His-118 which is important for
capturing Fe2+, also works as a ligand for VO2+.
f) Detection of metal transport activity in cultured CHO-K1 cell-line,
which expressed AsNramp-GFP fusion protein. T. Uyama*, T. Ueki,
and H. Michibata, Marine Biol. Lab., Grad. Sch. Sci., Hiroshima Univ.,
Hiroshima., Mukaishima 2445, Hiroshima 722-0073, Japan. *tauyama@hiroshima-u.ac.jp
Ascidians belonging to Ascidiacea store vanadium ion in
vacuoles of vanadocytes, vanadium-containing blood cells. The concentration
of vanadium attains 350mM, which is 107 times higher than that in sea water.
This is thought to be the highest levels of metal concentration in any
living organism. Nramp (natural resistance-associated macrophage protein)
family is known to transport several heavy metals including including Fe2+,
Zn2+, Mn2+, Co2+, Cd2+, Cu2+, Ni2+, and Pb2+ and is highly conserved among
mammals, nematodes, yeast, and bacteria. As a first step to search for
vanadium transporter in ascidians, we examined whether Nramp homolog is
expressed in the vanadocytes of the vanadium-rich ascidian, Ascidia
sydneiensis samea. As a result, we isolated and cloned cDNA of Nramp
from the blood cells. In situ hybridization showed that ascidian Nramp
homolog, named AsNramp, was expressed in the vanadocytes exclusively. To
examine whether AsNramp acts as a proton-coupled vanadium transporter in
ascidians, we constructed a plasmid expressing a fusion protein of AsNramp
and a green fluorescent protein (GFP) under the control of cytomegalovirus(CMV)
promoter. The fusion protein is overexpressed in cultured CHO-K1 cell line.
NEW PUBLICATIONS
Aizenberg, J., Lambert, G., Weiner, S. and Addadi, L. 2002. Factors involved in the formation of amorphous and crystalline calcium carbonate: a study of an ascidian skeleton. J. Amer. Chem. Soc. 124: 32-39.
Alvarado, J. L., Pinto, R., Marquet, P., Pacheco, C., Guiñez, R. and Castilla, J. C. 2001. Patch recolonization by the tunicate Pyura praeputialis in the rocky intertidal of the Bay of Antofagasta, Chile: evidence for self-facilitation mechanisms. Mar. Ecol. Prog. Ser. 224: 93-101.
Arai, M., Suzuki-Koike, M., Ohtake, S., Ohba, H., Tanaka, K. and Chiba, J. 2001. Common cell-surface antigens functioning in self-recognition reactions by both somatic cells and gametes in the solitary ascidian Halocynthia roretzi. Microbiol. Immunol. 45: 857-866.
Ballarin, L., Scanferla, M., Cima, F. and Sabbadin, A. 2002. Phagocyte spreading and phagocytosis in the compound ascidian Botryllus schlosseri: evidence for an integrin-like, RGD-dependent recognition mechanism. Dev. Comp. Immunol. 26: 345-354.
Bishop, C. D., Bates, W. R. and Brandhorst, B. P. 2002. HSP90 function is required for morphogenesis in ascidian and echinoid embryos. Dev. Genes Evol. 212: 70-80.
Candiani, S., Augello, A., Oliveri, D., Passalacqua, M., Pennati, R., De Bernardi, F. and Pestarino, M. 2001. Immunocytochemical localization of serotonin in embryos, larvae and adults of the lancelet, Branchiostoma floridae. Histochem. J. 33: 413-420.
Canestro, C., Albalat, R., Hjelmqvist, L., Godoy, L., Jornvall, H. and Gonzalez-Duarte, R. 2002. Ascidian and amphioxus Adh genes correlate functional and molecular features of the ADH family expansion during vertebrate evolution. J. Mol. Evol. 54: 81-89.
Castilla, J. C. and Camaño, A. 2001. El piure de Antofagasta, Pyura praeputialis (Heller, 1878): un competidor dominante e ingeniero de ecosistemas [in Spanish; English abstract]. In: Alveal, K. and Antezana, T. (ed.), Sustenabilidad de la biodiversidad…. Univ. de Concepcion, Chile, pp. 719-729.
Castilla, J. C. and Guiñez, R. 2000. Disjoint geographical distribution of intertidal and nearshore benthic invertebrates in the southern hemisphere. Revista Chilena de Historia Natural 73: 585-603.
Cerda, M. and Castilla, J. C. 2001. Diversity and biomass of macro-invertebrates in intertidal matrices of the tunicate Pyura praeputialis (Heller, 1878) in the Bay of Antofagasta, Chile [in Spanish; English abstract]. Revista Chilena de Historia Natural 74: 841-853.
Ciancio, A., Scippa, S. and Cammarano, M. 2001. Ultrastructure of trophozoites
of the gregarine Lankesteria ascidiae (Apicomplexa: Eugregarinida) parasitic
in the ascidian Ciona intestinalis (Protochordata). Europ. J. Protistol.
37: 327-336.
Cima, F., Dominici, D., Mammi, S. and Ballarin, L. 2002. Butylins and
calmodulin: which interaction? Applied Organometal. Chem. 16: 182-186.
Clarke, M. and Castilla, J. C. 2000. Dos nuevos registros de ascidias (Tunicata: Ascidiacea) para la costa continental de Chile [in Spanish; English abstract]. Revista Chilena de Historia Natural 73: 503-510.
Coma, R., Ribes, M., Gili, J.-M. and Hughes, R. N. 2001. The ultimate opportunists: consumers of seston. Mar. Ecol. Prog. Ser. 219: 305-308.
Davis, R. A., Carroll, A. R. and Quinn, R. J. 2002. Lepadins F-H, new cis-decahydroquinoline alkaloids from the Australian ascidian Aplidium tabascum. J. Nat. Prod. 65: 454-457.
Davis, S. W. and Smith, W. C. 2002. Expression cloning in ascidians: isolation of a novel member of the asctacin protease family. Dev. Genes Evol. 212: 81-6.
Di Gregorio, A., Harland, R. M., Levine, M. and Casey, E. S. 2002. Tail morphogenesis in the ascidian, Ciona intestinalis, requires cooperation between notochord and muscle. Dev. Biol. 244: 385-395.
Dolcemascolo, G., Alessandro, R. and Gianguzza, M. 2001. Ultrastructural and cytochemical investigations on the formation of chorion in oocyte of Ascidia malaca. J. Submicrosc. Cytol. Pathol. 33: 201-215.
Faulkner, D. J. 2002. Marine natural products. Nat. Prod. Rep. 19: 1-48.
Fujita, T. 2002. Evolution of the lectin-complement pathway and its role in innate immunity. Nature Rev. Immunol. 2: 346-353.
Fujiwara, S., Maeda, Y., Shin-i, T., Kohara, Y., Takatori, N., Satou, Y. and Satoh, N. 2002. Gene expression profiles in Ciona intestinalis cleavage-stage embryos. Mech. Dev. 112: 115-127.
Groppelli, S., Pennati, R., Sotgia, C. and De Bernardi, F. 2001. AChE localization in adhesive papillae of ascidian larva: effects of citral, a retinoic acid synthesis inhibitor. Invert. Repro. Devel. 40: in press.
Guiñez, R. and Castilla, J. C. 2001. An allometric tridimensional model of self-thinning for a gregarious tunicate. Ecology 82: 2331-2341.
Harafuji, N., Keys, D. N. and Levine, M. 2002. Genome-wide identification of tissue-specific enhancers in the Ciona tadpole. Proc. Natl. Acad. Sci. 99: 6802-6805.
Harris, L. G. and Tyrrell, M. C. 2001. Changing community states in the Gulf of Maine: synergism between invaders, overfishing and climate change. Biol. Invasions 3: 9-21.
Heasman, J. 2002. Morpholino oligos: making sense of antisense? Dev. Biol. 243: 209-214.
Hirose, E., Ohshima, C. and Nishikawa, J. 2001. Tunic cells in pyrosomes (Thaliacea, Urochordata): cell morphology, distribution, and motility. Invert. Biol. 120: 386-393.
Holland, L. Z. 2002. Heads or tails? Amphioxus and the evolution of anterior-posterior patterning in deuterostomes. Dev. Biol. 241: 209-228.
Imai, K. S., Satoh, N. and Satou, Y. 2002. Early embryonic expression of FGF4/6/9 gene and its role in the induction of mesenchyme and notochord in Ciona savignyi embryos. Development 129: 1729-1738.
Jeffery, W. R. 2002. Role of PCNA and ependymal cells in ascidian neural development. Gene 287: 97-105.
Katano, M., Yamada, A., Tanaka, K. J., Murakami, A., Taira, K., Kawakami, J., Sugimoto, N. and Nishikata, T. 2001. Utilization of ribozymes for loss-of-function analyses of the early development of the ascidian Ciona intestinalis. Mem. Konan Univ., Sci. Ser. 48: 11-20.
Katsuyama, Y., Matsumoto, J., Okada, T., Ohtsuka, Y., Chen, L., Okado, H. and Okamura, Y. 2002. Regulation of synaptotagmin gene expression during ascidian embryogenesis. Dev. Biol. 244: 293-304.
Kawashima, T., Kawashima, S., Kohara, Y., Kanehisa, M. and Makabe, K. W. 2002. Update of MAGEST: Maboya gene expression patterns and sequence tags. Nucleic Acids Res. 30: 119-120.
Kodama, E., Baba, T., Kohno, N., Satoh, S., Yokosawa, H. and Sawada, H. 2002. Spermosin, a trypsin-like protease from ascidian sperm: cDNA cloning, protein structures and functional analysis. Eur. J. Biochem. 269: 657-663.
Kuramochi, T., Nishikawa, T., Hattori, M., Kanie, Y. and Akimoto, K. 1999. In situ observation of an ascidian Culeolus sp. from the Japan Trench. JAMSTEC J. Deep Sea Res. 15: 1-3.
Kusakabe, T., Yoshida, R., Kawakami, I., Kusakabe, R., Mochizuki, Y., Yamada, L., Shin-i, T., Kohara, Y., Satoh, N., Tsuda, M. and Satou, Y. 2002. Gene expression profiles in tadpole larvae of Ciona intestinalis. Dev. Biol. 242: 188-203.
Lacalli, T. C. 2002. Sensory pathways in amphioxus larvae I. Constituent fibres of the rostral and anterodorsal nerves, their targets and evolutionary significance. Acta Zool. 83: 149-166.
Lacalli, T. C. 2002. Vetulicolians--are they deuterostomes? chordates? BioEssays 24: 208-211.
Lacalli, T. C. 2002. The dorsal compartment locomotory control system in amphioxus larvae. J. Morph. 252: 227-237.
Lacalli, T. C. and Kelly, S. J. 2002. Floor plate, glia and other support cells in the anterior nerve cord of amphioxus larvae. Acta Zool. 83: 87-98.
Lambert, C. C., Someno, T. and Sawada, H. 2002. Sperm surface proteases in ascidian fertilization. J. Exp. Zool. 292: 88-95.
Makarieva, T. N., Dmitrenok, A. S., Dmitrenok, P. S., Grebnev, B. B. and Stonik, V. A. 2001. Pibocin B, the first N-O-methylindole marine alkaloid, a metabolite from the Far-Eastern ascidian Eudistoma species. J. Nat. Prod. 64: 1559-1561.
Marino, R., Kimura, Y., De Santis, R., Lambris, J. D. and Pinto, M. R. 2002. Complement in urochordates: cloning and characterization of two C3-like genes in the ascidian Ciona intestinalis. Immunogenetics 53: 1055-1064.
Matsumoto, M., Hirata, J., Hirohashi, N. and Hoshi, M. 2002. Sperm-egg binding mediated by sperm alpha-L-fucosidase in the ascidian, Halocynthia roretzi. Zool. Sci. 19: 43-48.
Meedel, T. H., Lee, J. J. and Whittaker, J. R. 2002. Muscle development and lineage-specific expression of CiMDF, the MyoD- family gene of Ciona intestinalis. Dev. Biol. 241: 238-246.
Michibata, H., Uyama, T., Ueki, T. and Kanamori, K. 2002. Vanadocytes, cells hold the key to resolving the highly selective accumulation and reduction of vanadium in ascidians. Microscop. Res. Tech. 56: 421-434.
Miya, T. and Nishida, H. 2002. Isolation of cDNA clones for mRNAs transcribed zygotically during cleavage in the ascidian, Halocynthia roretzi. Dev. Genes Evol. 212: 30-37.
Monniot, C. 2002. Stolidobranch ascidians from the tropical western Indian Ocean. Zool. J. Linn. Soc. 135: 65-120.
Monniot, C., Monniot, F., Griffiths, C. L. and Schleyer, M. 2001. South African ascidians. Ann. S. Afr. Mus. 108: 1-141.
Munro, E. M. and Odell, G. 2002. Morphogenetic pattern formation during ascidian notochord formation is regulative and highly robust. Development 129: 1-12.
Munro, E. M. and Odell, G. M. 2002. Polarized basolateral cell motility underlies invagination and convergent extension of the ascidian notochord. Development 129: 13-24.
Nieuwenhuys, R. 2002. Deuterostome brains: synopsis and commentary. Brain Res. Bull. 57: 257-270.
Nishida, H. 2002. Specification of developmental fates in ascidian embryos: molecular approach to maternal determinants and signaling molecules. Int. Rev. Cytol. 217: 227-276.
Okada, T., Katsuyama, Y., Ono, F. and Okamura, Y. 2002. The development of three identified motor neurons in the larva of an ascidian, Halocynthia roretzi. Dev. Biol. 244: 278-292.
Okuyama, M., Saito, Y. and Hirose, E. 2002. Fusion between imcompatible colonies of a viviparous ascidian, Botrylloides lentus. Invert. Biol. 121: 163-169.
Pavao, M. S. 2002. Structure and anticoagulant properties of sulfated glycosaminoglycans from primitive Chordates. An. Acad. Bras. Cienc. 74: 105-112.
Pearce, A. N., Babcock, R. C., Lambert, G. and Copp, B. R. 2001. N2,N2,7-trimethylguanine, a new trimethylated guanine natural product from the New Zealand ascidian, Lissoclinum notti. Nat. Prod. Lett. 15: 237-241.
Pennati, R., Groppelli, S., Sotgia, C., Candiani, S., Pestarino, M. and De Bernardi, F. 2001. Serotonin localization in Phallusia mammillata larvae and effect of 5-HT antagonists during larval development. Dev. Growth & Differ. 43: 647-656.
Petersen, J. K. and Svane, I. 2002. Filtration rate in seven Scandinavian ascidians: implications of the morphology of the gill sac. Mar. Biol. 140: 397-402.
Runft, L. L., Jaffe, L. A. and Mehlmann, L. M. 2002. Egg activation at fertilization: where it all begins. Dev. Biol. 245: 237-254.
Saito, Y., Shirae, M., Okuyama, M. and Cohen, S. 2001. Phylogeny of botryllid ascidians. In: Sawada, H., Yokosawa, H. and Lambert, C. C. (ed.), The Biology of Ascidians. Tokyo, Springer-Verlag, pp. 315-320.
Salomon, C. E. and Faulkner, D. J. 2002. Localization studies of bioactive cyclic peptides in the ascidian Lissoclinum patella. J. Nat. Prod. 65: 689-692.
Salomon, C. E., Williams, D. H., Lobkovsky, E., Clardy, J. C. and Faulkner, D. J. 2002. Relative and absolute stereochemistry of the didemnaketals, metabolites of a Palauan ascidian, Didemnum sp. Org. Lett. 4: 1699-1702.
Sanamyan, K. E. and Sanamyan, N. P. 2002. Deep-water ascidians from the south-western Atlantic (RV Dmitry Mendeleev, cruise 43 and Academic Kurchatov, cruise 11). J. Nat. Hist. 36: 305-359.
Sato, S. and Yamamoto, H. 2001. Development of pigment cells in the brain of ascidian tadpole larvae: insights into the origins of vertebrate pigment cells. Pigment Cell Res. 14: 428-436.
Satou, Y., Takatori, N., Fujiwara, S., Nishikata, T., Saiga, H., Kusakabe, T., Shin-i, T., Kohara, Y. and Satoh, N. 2002. Ciona intestinalis cDNA projects: expressed sequence tag analyses and gene expression profiles during embryogenesis. Gene 287: 83-96.
Sawada, H. 2002. Ascidian sperm lysin system. Zool. Sci. 19: 139-151.
Sawada, H., Sakai, N., Abe, Y., Tanaka, E., Takahashi, Y., Fujino, J., Kodama, E., Takizawa, S. and Yokosawa, H. 2002. Extracellular ubiquitination and proteasome-mediated degradation of the ascidian sperm receptor. Proc. Natl. Acad. Sci. 99: 1223-1228.
Sawada, H., Takahashi, Y., Fujino, J., Flores, S. Y. and Yokosawa, H. 2002. Localization and roles in fertilization of sperm proteasomes in the ascidian Halocynthia roretzi. Mol. Reprod. Develop. 62: 271-276.
Schupp, P., Proksch, P. and Wray, V. 2002. Further new staurosporine derivatives from the ascidian Eudistoma toealensis and its predatory flatworm Pseudoceros sp. J. Nat. Prod. 65: 295-298.
Schwartsmann, G., Brondani da Rocha, A., Berlinck, R. G. and Jimeno, J. 2001. Marine organisms as a source of new anticancer agents. Lancet Oncol. 2: 221-225.
Seo, H. C., Kube, M., Edvardsen, R. B., Jensen, M. F., Beck, A., Spriet, E., Gorsky, G., Thompson, E. M., Lehrach, H., Reinhardt, R. and Chourrout, D. 2001. Miniature genome in the marine chordate Oikopleura dioica. Science 294: 2506.
Takada, N., York, J., Davis, J. M., Schumpert, B., Yasuo, H., Satoh, N. and Swalla, B. J. 2002. Brachyury expression in tailless molgulid ascidian embryos. Evol. Dev. 4: 205-211.
Takamura, K., Egawa, T., Ohnishi, S., Okada, T. and Fukuoka, T. 2002. Developmental expression of ascidian neurotransmitter synthesis genesI. Choline acetyltransferase and acetylcholine transporter genes. Dev. Genes Evol. 212: 50-53.
Takamura, K., Fujimura, M. and Yamaguchi, Y. 2002. Primordial germ cells originate from the endodermal strand cells in the ascidian Ciona intestinalis. Dev. Genes Evol. 212: 11-8.
Takamura, K., Oka, N., Akagi, A., Okamoto, K., Okada, T., Fukuoka, T., Hogaki, A., Naito, D., Oobayashi, Y. and Satoh, N. 2001. EST analysis of genes that are expressed in the neural complex of Ciona intestinalis adults. Zool. Sci. 18: 1231-1236.
Tanaka-Kunishima, M. and Takahashi, K. 2002. Cleavage-arrested cell triplets from ascidian embryo differentiate into three cell types depending on cell combination and contact timing. J. Physiol. 540: 153-176.
Taylor, S. W. 2002. Chemoenzymatic synthesis of peptidyl 3,4-dihydroxyphenylalanine for structure activity relationships in marine invertebrate polypeptides. Anal. Biochem. 302: 70–74.
Terakado, K. 2001. Induction of gamete release by gonadotropin-releasing hormone in a protochordate, Ciona intestinalis. Gen. & Comp. Endocrinol. 124: 277-284.
Thompson, E. M., Kallesoe, T. and Spada, F. 2001. Diverse genes expressed in distinct regions of the trunk epithelium define a monolayer cellular template for construction of the oikopleurid house. Dev. Biol. 238: 260-273.
Tincu, J. A. and Taylor, S. W. 2002. Tunichrome Sp-1: new pentapeptide tunichrome from the hemocytes of Styela plicata. J. Nat. Prod. 65: 377-378.
Tomioka, M., Miya, T. and Nishida, H. 2002. Repression of zygotic gene expression in the putative germline cells in ascidian embryos. Zool. Sci. 19: 49-55.
Ueki, T., Takemoto, K., Fayard, B., Salome, M., Yamamoto, A., Kihara, H., Susini, J., Scippa, S., Uyama, T. and Michibata, H. 2002. Scanning x-ray microscopy of living and freeze-dried blood cells in two vanadium-rich ascidian species, Phallusia mammillata and Ascidia sydneiensis samea. Zool. Sci. 19: 27-35.
Wessels, M., Konig, G. M. and Wright, A. D. 2001. New 4-methoxybenzoyl derivatives from the ascidian Polycarpa aurata. J. Nat. Prod. 64: 1556-1558.
Yoshida, T., Nishiyachi, M., Nakashima, N., Murase, M. and Kotani, E. 2002. New synthetic route to granulatimide and its structural analogues. Chem. Pharm. Bull. 50: 872-876.
Zaniolo, G., Lane, N. J., Burighel, P. and Manni, L. 2002. Development
of the motor nervous system in ascidians. J. Comp. Neurol. 443: 124-135.