Number 44 December 1998
After retiring at the end of spring semester, after
28 years at Calif. State Univ. Fullerton, we moved to Seattle, Washington
in June. In September we joined a group of about 14 invertebrate specialists
on an 8 day expedition to survey a large number of marinas in Puget Sound,
the San Juan Islands, and north to the Canadian border, to document
recent introductions of non-indigenous marine invertebrates to the U.S.
Pacific Northwest coast. There are a large number of new records
for this state; see the WORK IN PROGRESS section for details of the ascidian
results and information on how to receive a copy of the full report. Gretchen
received a small grant from the Bishop Museum in Honolulu to identify their
ascidian collection, so we spent the month of November in Honolulu. Charles
investigated aspects of fertilization in Phallusia nigra at the
Univ. of Hawaii Kewalo Marine Biol. Lab. We put on three ascidian workshops
this year, the first in May at CSUFullerton, the second in October at the
Paulsbo marine station near Seattle, and the third at the Kewalo lab in
Honolulu in November. All were focussed on helping people identify the
local species, introduced as well as indigenous, in each of these localities,
with many live and preserved specimens to examine. Gretchen was also invited
to the Univ. of Guam to help them identify their local ascidians, with
another modest grant to defray expenses, so in February we will spend several
weeks there and Charles will continue his research on ascidian fertilization.
We are pleased to report that there are 84 citations
in the New Publications section. Keep in touch, and send us an article
for Work in Progress, abstracts from recent meetings attended, or your
students' thesis abstracts on ascidian topics. Please send us a copy
of your new publications to our Seattle address. This will assure that
they are included in the Recent Publications section at the end of each
AN. We use our ascidian reprints every day and they are very valuable
to us.
*Ascidian News is not part of the scientific literature and should
not be cited as such.
1. Sea squirt die off - Korea: Request for information:
Albert Kwang-Sik Choi, Dept. of Aquaculture, Cheju National University,
1 Ara 1 Dong, Cheju City, Cheju, 690-756 Korea (skchoi@cheju.cheju.ac.kr)
& Jeffrey Shields (jeff@vims.edu) http://shellfish.cheju.ac.kr
The sea squirt Halocynthia roretzi is commercially
cultured in the southern coast of Korea. Due to an unidentified disease,
large numbers of sea squirts in the farming areas died this summer and
the industry is now in bad shape. Water temperature during last summer
was abnormally high due to the El Nino. The dying sea squirts usually exhibited
thinning of the skin and the meat become quite soft and dissolving. We
do not know the agents involved in this disease and if anybody has any
information on Sea Squirt disease, please help us.
2. While searching for new references for this newsletter, we found an extremely useful website maintained by the University of Washington library in Seattle. http://www.lib.washington.edu/ While not all databases are accessible to the public, many are, such as CarlUncover, in which you can search for papers using keywords as well as author or journal.
3. Our colleague Alan Kohn recently brought to our attention a long-forgotten poem which we reproduce here from The Poetical Works of Andrew Lang (1923) by Mrs. Lang. vol. 3, pp. 133-135.
'The ancestor remote of man,'
Says Darwin, 'is th' ascidian,' A scanty sort of water-beast That, ninety million years at least Before gorillas came to be, Went swimming up and down the sea. Their ancestors the pious praise,
Th' ascidian tadpole, young and gay,
He's sensitive to grief and pain,
|
And we, his children, truly we
In youth are, like the tadpole, free; And where we would we blithely go; Have brains and hearts, and feel and know. Then age comes on! To habit we Affix ourselves and are not free. The’ ascidian’s rooted to a rock,
Ah, scarce we live, we scarcely know
---------Andrew Lang |
1. The following is taken, with minor changes, from a recently prepared document, Report of the Puget Sound Expedition September 8-16, 1998: A Rapid Assessment Survey of Non-indigenous Species in the Shallow Waters of Puget Sound. Washington State Dept. of Natural Resources, Olympia, WA and U.S. Fish and Wildlife Service, Lacey, WA. For a copy of the complete report please contact Helen Berry at the Washington State DNR. helen.berry@wadnr.gov
Ascidians in Puget Sound and the San Juan Islands, September 1998. Gretchen
Lambert, Charles Lambert and Claudia Mills.
Ascidians are prominent members of the fouling community
and are often present in very high densities on marina floats and boat
hulls. We found that, after mussels and barnacles, they comprised
perhaps the most important group of dock inhabitants in Puget Sound. Boat
harbors were surveyed from a northern extreme of Blaine at the Canadian
border to a southernmost point near Olympia on the Washington mainland,
as well as sites on Fidalgo and Whidbey Islands, and San Juan,Orcas and
Lopez Islands in the San Juan Islands. Sampling was accomplished
by reaching under floating docks to remove adherent ascidians and by raising
ropes, hoses and wires to examine attached organisms. Salinity and
temperature were measured at each collection site.
Summarizing our findings from north to south, the
harbor at Blaine contained the native species Distaplia occidentalis
and Corella inflata along with the introduced Botrylloides violaceus
and Styela clava. This is the first report of S. clava
in American waters north of San Francisco Bay in the NE Pacific, although
we observed a population on Vancouver Island several years ago that
is still flourishing. Bellingham marina with reduced salinity had only
a few colonies of Botrylloides violaceous. The Edmonds marina
contained numerous colonies of Botrylloides violaceus as well as
the natives Distaplia occidentalis, Corella inflata, Styela
gibbsii and Diplosoma listerianum. Similar ascidians were encountered
in the Elliott Bay Marina. At the Des Moines Marina several native
species were common along with the non-natives Botryllus schlosseri
and Botrylloides violaceus, but the floats were dominated by huge
numbers of Ciona savignyi, a species introduced into California
in 1985 but never before noted north of San Francisco. In Tacoma we found
numerous Botryllus schlosseri and a few native S. gibbsii
and Distaplia occidentalis. At the Shelton Yacht Club both
B. violaceus and B. schlosseri were very numerous as was
a third introduced ascidian Molgula manhattensis, together covering
nearly all submerged objects. At the Brownsville Yacht Club we found a
number of native species along with B. violaceus and a few Ciona
savignyi. Port Townsend marina had all the previously mentioned
native species and also the native Perophora annectens and Ascidia
callosa. In addition B. violaceus was present in large
numbers.
Ascidians are thus an important component
of nearly every float-fouling community in Puget Sound. Non-indigenous
species comprise a substantial part of this ascidian load, in places forming
the dominant biota. At the Des Moines Marina (south of the Seattle-Tacoma
airport), Ciona savignyi occupied an estimated 90% of the concrete
float surface area under covered (shaded) docks, although it was much less
abundant in uncovered areas and limited to float bottoms. At many sites,
the non-indigenous B. violaceus accounted for at least 10-20% of
the float cover, often overgrowing other organisms.
We further examined 7 boat harbors on San Juan Island
and 6 on Orcas Island and found only the native species in most harbors
but B. violaceus was in Roche Harbor, Mitchell Bay and Westcott
Bay on San Juan Island, in most harbors on Orcas Island, and at Fisherman's
Bay on Lopez Island. Ciona savignyi, Molgula manhattensis
and Styela clava seem to be absent from the San Juan Archipelago
at this time.
There is thus an indication of a north-south gradient
in the number of introduced ascidian species on the U.S. Pacific coast,
with 5 recorded so far for Puget Sound, 8 in San Francisco Bay and 14 in
southern California [see Lambert and Lambert 1998, Mar. Biol. 130:675-688].
2. Sulfur allocation and vanadium-sulfate interactions in whole blood
cells from the tunicate Ascidia ceratodes, investigated using X-ray
absorption spectroscopy. P. Frank, B. Hedman, and K.O. Hodgson. Dept. Chem,
Stanford Univ., Stanford, CA 94305. In press, Inorganic Chemistry
(1998). frank@SSRL01.slac.stanford.edu
Sulfur K-edge x-ray absorption spectroscopy (XAS)
has been used to investigate the distribution of sulfur types in two whole
blood cell samples, in selected subcellular blood fractions, and in cell-free
plasma from the tunicate Ascidia ceratodes. Whole blood cells are
rich in sulfate, aliphatic sulfonate, and low-valent sulfur. The sulfur
K-edge XAS spectrum of washed blood cell membranes revealed traces of sulfate
and low-valent sulfur, but no sulfate ester or sulfonate. Sulfonate
is thus exclusively cytosolic. Cell-free blood plasma contains primarily
sulfate sulfur. Gaussian fitting and sulfate quantitation yielded average
sulfur type concentrations for two whole blood cell samples, each representing
dozens of individuals from separate collections taken per annum, yielded
average sulfur type concentrations for the two populations (first year,
second year): [sulfate], 110 mM, 150 mM; [sulfonate], 99 mM, 70 mM, and;
[low-valent sulfur], 41 mM, 220 mM. On titration of sulfate with acid or
V(III) in aqueous solution the strong 2482.4 eV 1s-->(valence t2) sulfur
K-edge XAS transition of tetrahedral SO4(2-) splits into 1s-->a1 and 1s-->e
transitions because HSO4(-) and VSO4(+) are of C3v symmetry. Gaussian fits
and appropriate comparisons allow the following assignments: (compound/complex,
1s-->a1(eV), 1s-->e (eV)): myo-inositol hexasulfate, 2480.8, 2482.8; HSO4(-),
2481.4, 2482.7; VSO4(+), 2481.2, 2482.9. The energy separating the
a1 and e states of complexed sulfate appears to be solvation dependent.
From these studies an explicit inorganic spectrochemical model is derived
for biological V(III) and sulfate. The average endogenous equilomer concentrations
of sulfate complexed with V(III) and/or acid within the two blood cell
samples are calculated from the model. The results provide a natural explanation
for the observed biological broadening of A. ceratodes blood cell
sulfur K-edge XAS spectra.
3. Phenoloxidase and cytotoxicity in the compound ascidian Botryllus
schlosseri. L. Ballarin, F. Cima & A. Sabbadin, Dipartimento di
Biologia, Univ. di Padova. In press, Dev. Comp. Immunol., 22, 1998. ballarin@civ.bio.unipd.it
The vacuoles of morula cells (MC) of the colonial
ascidian Botryllus schlosseri contain phenoloxidase (PO). As the
release of their vacuolar content at the border of incompatible contacting
colonies is associated with the formation of necrotic masses which characterize
the rejection reaction, the role of PO in Botryllus cytotoxicity
was investigated. When hemocytes are incubated with blood plasma
from incompatible (heterologous) colonies, MC degranulate and, after 60
min, the cytotoxicity index becomes significantly greater than that observed
in controls incubated with autologous plasma. The rise in cell mortality
is completely inhibited by the addition of PO inhibitors sodium benzoate,
tropolone and phenylthiourea, and serine protease inhibitors phenylmethylsulfonyl
fluoride, benzamidine, N-tosyl-L-phenylalanine chloromethyl ketone and
N-tosyl-L-lysine chloromethyl ketone. The addition of either reducing agents
L-cysteine and ascorbic acid or reactive oxygen species scavenger enzymes
superoxide dismutase and catalase has a similar effect. Significant inhibition
of cytotoxicity is also observed with the quinone scavenger 3-methyl-2-benzothiazolinone
hydrazone. In the presence of sodium benzoate and phenylthiourea, there
is a significant reduction in the number, size and color intensity of necrotic
masses along the contact border of incompatible colonies. A significant
increase in superoxide anion production, completely inhibited by Na-benzoate,
is observed when hemocytes are incubated with heterologous blood plasma.
These results indicate that: i) PO is the enzyme responsible for the cytotoxicity
observed in both hemocyte cultures and rejection reactions; ii) PO is present
inside MC vacuoles as a proenzyme which is activated, upon release, by
humoral proteases; iii) cytotoxicity appears to be mainly due to oxidative
stress generated by PO during oxidation of polyphenols to quinones without
the involvement of other oxidases such as NADPH oxidase and peroxidase.
1. Intl. Conference on Environmental and Biological Aspects of Main group Organometals. Helnaes, Denmark, June 28 - July 1, 1998.
TBT-INDUCED APOPTOSIS IN TUNICATE HEMOCYTES. F. Cima1,
P. Fonti2 and L. Ballarin1
1Dept. of Biology, University of Padova, Via U. Bassi
58/B, 35131 Padova ballarin@civ.bio.unipd.it
2Centro Studi Ambientali, Via al Torrente 22, 47037 Rimini
- Italy
Early events in apoptosis include
chromatin condensation followed by DNA fragmentation as well as translocation
of phosphatidylserine (PS) in the plasma membrane. Organotin compounds
increase intracellular Ca2+ levels and cause apoptosis in mammalian cells.
In previous reports we demonstrated the immunotoxicity of TBT in the ascidian
Botryllus schlosseri, a filter-feeding marine invertebrate, as it
inhibits phagocytosis and respiratory burst. The effect is mediated by
an extensive cytosolic calcium increase resulting in volume loss and morphological
changes related to cytoskeletal alterations. In order to investigate whether
TBT can also induce apoptosis in ascidian hemocytes we exposed Botryllus
blood cells to 10 µM TBT - the dose which significantly changes cytoskeletal
structure and does not cause mortality - for 1, 2, 4 h. After 1 h we observed
a significant collapse and cleavage of nuclear chromatin following a post-treatment
with acridine orange or Pfitzner’s safranin, also revealed with TUNEL reaction
which is specific to oligonucleosome-sized DNA fragments. Moreover, cytoplasmic
blebbing occurred together with surface alterations triggered by PS translocation
from the inner side of the plasma membrane to the outer side as shown with
annexin-fluorescein method. Hemocyte mortality, detected with trypan blue,
took place only after 2 h. We suggest that all these apoptotic events are
strictly related with a TBT-triggered cytosolic calcium increase resulting
in inhibition of calmodulin and Ca2+-dependent enzyme activities, e.g.
Ca2+-ATPase and endonucleases.
2. 8th Intl. Congress on Invertebrate Reproduction & Development, Amsterdam, 10-14 August 1998.
ASCIDIANS HAVE A RELAXIN HORMONE AND AN IGF-I RELATED
PEPTIDE. D. Georges (Grenoble, France), M. Reinecke (Zürich, Switzerland).
DGeorges@europost.org
Ascidians have been considered, for
a long time, as very simple invertebrates. The first experiments demonstrated
the role of the nervous complex (nervous ganglion and neural gland) in
the regulation of spawning, during the tidal cycle. The main regulation
is the same as in vertebrates with a negative feed-back control by the
ovary, at the time of spawning (Georges,Gen.comp.Endocrinol.32, 454-473,
1977). Immunofluorescence techniques allowed then to show that vertebrate-like
peptides could be found in the nervous system, as well as in the digestive
tract or the ovary, peptides related to ACTH, LHRH, methionine-enkephalin,
serotonin, NGF, insulin, IGF-I and relaxin. Relaxin, a hormone from the
insulin family, is known in vertebrates, specially in mammals as a hormone
widening the birth canal. It has been localized only in the follicle cells
of mature oocytes of various ascidians (Georges et al., Gen.comp.Endocrinol.79,
429-438, 1990).The amino acid sequence of relaxin studied in most classes
of vertebrates shows a low level of homologies even between mammals themselves.
For the first time, relaxin has been sequenced
in an ascidian. Surprisingly, the nucleotide sequence of relaxin in Ciona
intestinalis is about 98 % similar to that of porcine relaxin and is
the same as whale relaxin. The danger of a contamination was eliminated
as parallel experiments were running by C. Schwabe, in USA, who confirms
the results with the amino-acid sequence. Moreover, control experiments
using intestine tissue performed in the same time were negative. As results
were the same in genomic and cDNA, it appears that : 1) there is no intron
in the connecting peptide of the ascidian prorelaxin, 2) the exchanges
between pig and ascidian relaxin concern a change in the first or second
base in the codon. IGF-I occurs in the endocrine cells of the digestive
tract and in neurons of C. intestinalis (Reinecke et al., Histochemistry,
99, 277-285, 1993). Using gel chromatography and RIA, IGF-I immunoreactivity
was detected in two peaks with apparent molecular weights of approximately
16 kDa and 3 kDa both in the gastrointestinal tract and in ovary. Results
obtained by immunofluorescence techniques (antibodies raised in mammals
against insulin, IGF-I and relaxin)
demonstrate that an IGF-I related peptide coexists 1)
with an insulin-related peptide in the digestive tract and 2) with relaxin
in the ovary, but there, only in the medium-sized and mature follicle cells.
IGF-I exists alone in young oocytes.
In another ascidian, Chelyosoma productum,
McRory & Sherwood (DNA & Cell Biology, 16, 939-949, 1997) have
sequenced two insulin and IGF genes : the preproinsulin has 87 % identity
with the preproIGF, and the mature form of ascidian insulin shows 64% identity
with the human insulin. All these results suggest that an ancestral insulin/IGF/relaxin
molecule precedes the ascidians.
3. 19th Conference of European Comparative Endocrinologists,
Nimegen, 1-5 September 1998.
Ascidians have a relaxin hormone. Danielle Georges (Grenoble,
France). [similar to the above abstract]
4. 33rd EMBS meeting, Wilhelmshaven, Germany, September 1998.
ENERGY BUDGET OF TWO SPECIES OF BENTHIC
ANTARCTIC SUSPENSION FEEDERS. Jens Kowalke, Alfred Wegener
Institute for Polar and Marine Research, Bremerhaven, Germany
Although suspension feeders constitute
the majority of many Antarctic benthic communities, little information
about their biology or ecology is known. Complete energy budgets concerning
growth, reproduction, egestion or respiration are still lacking but are
essential for understanding ecosystems and interpreting communities over
time and space. In this work energy budgets during austral summer of both
the abundant ascidian Cnemidocarpa verrucosa (Lesson) and
the lamellibranch Laternula elliptica (King & Broderip) were
established. Based on measured data of energy contents in the water column,
filtration and egestion rates combined with respiration data taken from
the literature, the energy available for growth and reproduction was calculated.
The field work was carried out in Potter Cove, an inlet at King George
Island, South Shetlands. This subsytem is characterized by a low annual
primary production in the water column and enormous terrestrial sediment
input due to the inflow of glacial meltwater during summer and autumn months
as well as frequent disturbances through stranding icebergs and growlers.
These physical effects are the main structuring forces affecting
the soft bottom communities found in the cove, which are, in contrast to
less disturbed areas, dominated by solitary ascidians and bivalves. The
summer months are characterized by a low water column primary production,
resuspension being the major process of generating particles for suspension
feeding animals. The main nutritional components are benthic diatoms, protists
and bacteria. The overall energy budget is lower than described for species
of
temperate and tropical zones, but the net growth efficiency,
which is the energy disposable for growth and reproduction expressed in
percentage of the energy assimilated, is at the upper margin of ranges
measured for species of temperate regions. The values are 81% for C.
verrucosa and 79% for L. elliptica respectively (dry wt).
Field data of L. elliptica for somatic and gonad production show
a net growth efficiency of 42% on an annual basis. These findings reflect
the
important gonad production of this species during the
summer months, as L. elliptica spawns in the Antarctic autumn. During
the extended winter period most of the ingested energy is, in contrast,
used for respiration, thus reducing the yearly net growth efficiency to
42%. As field data for C. verrucosa are are not yet available, a
validation of the calculated production values remains open.
1. ENERGY BUDGETS OF BENTHIC SUSPENSION FEEDING ANIMALS
OF POTTER COVE (KING GEORGE ISLAND, ANTARCTICA). Reports on Polar Research
286, 1998; 147pp. Dr. Jens Kowalke, Alfred Wegener Institute for Polar
and Marine Research, Bremerhaven, Germany. jkowalke@awi-bremerhaven.de
The objective of this work was the
first comprehensive calculation of the energy budgets of dominating groups
of suspension feeders in Potter Cove, Antarctica. The field work was carried
out in the Dallmann Laboratory, King George
Island, during Antarctic summer field campaigns. 27 species
of sponges were recorded in the area, 13 of which were newly described
for the South Shetland Islands and the adjacent Antarctic peninsula. The
2 dominant species, Mycale acerata and Isodictya kerguelensis
were choosen for further ecological studies. Of the 15 ascidian species
already known, Ascidia challengeri, Cnemidocarpa verrucosa,
Corella eumyota and Molgula pedunculata were investigated,
as well as the bivalve Laternula elliptica. A quantification of
the amount of energy present in the particulate fraction in the water column
at 30m depth shows seasonal variation throughout the year, but no extreme
limitation of energy supply during the winter months. This is due to resuspension
of particulate matter from the seafloor, induced by currents and, during
no-ice conditions, by wave action. Bacteria and protista seem to play an
important role in the nutrition of these suspension feeders, especially
in winter, when photosynthesis plays a minor part. It is inferred that
at least in the coastal maritime Antarctic habitats, energy is provided
sufficiently throughout the year. The pumping rates of all Antarctic species,
reflecting reduced metabolism at low temperatures and higher viscosity
of the seawater, ranged below those of animals of temperate zones. The
bottom sediments receive a considerable amount of fecal material produced
by the ascidians and bivalves and thus support a variety of coprophag deposit
feeders. The energy budgets were calculated using respiration and production
data from the literature. All species showed a surplus energy uptake which
could be related to an underestimation of the biodeposition rates. The
results do not display a very harsh environment for the animals in general
but reveal different adaptations among the investigated taxa to the special
physical conditions of the Potter Cove, which make the ascidians and L.
elliptica dominant in contrast to other Antarctic sites, where sponges
dominate the communities.
2. Eudistoma carolinense VAN NAME, 1945 (TUNICATA:
ASCIDIACEA) AS A BIOLOGICAL SUBSTRATE, AND ITS ASSOCIATED MACROFAUNA. Tatiane
Regina Moreno Master's thesis. Advisor: Dr. Rosana Moreira da Rocha. Dept.
de Zool., Univ.Federal do Parana, Centro Politicnico, Cx.Postal 19020,
1531-990 Curitiba/ PR, Brasil. rmrocha@bio.ufpr.br
Eudistoma carolinense is a
colonial ascidian with vertical growth. The colony consists of groups of
digitiform expansions arising from an expanded incrusting base, which forms
a belt in the low intertidal. E. carolinense is a live substrate
and its type of growth and localization permit settlement of many invertebrates.
We studied the structural features of the colonies: belt width, colony
thickness, density of expansions, and the volume and grain size of accumulated
sediment, during one year. Fifteen samples were collected in February,
(Summer), May (Fall), August (Winter) and November (Spring) of 1996. The
abundance of the associated fauna was 2021 in Summer, 3077 in Fall, 2710
in Winter and 1872 in Spring. The following taxonomic groups were recorded:
Porifera, Cnidaria, Plathyhelminthes, Nemertinea, Nematoda, Polychaeta,
Sipuncula, Mollusca, Crustacea, Pycnogonida, Bryozoa, Echinodermata, and
Chordata and 107 species were identified. From these, Nicolea venustula,
Typosyllis hyalina, Pseudonereis gallapagensis, T. variegata,
Elasmopus pectenicrus, Sphenia antillensis and Molgula
phytophila were considered characteristic species, because these had
the greatest general Biological Index Values. The volume of sediment, total
number of individuals and associated species were all positively correlated
with the ascidian volume and density of expansions. Colony thickness appears
to be related to a decrease in diversity. A comparison of various biological
substrates with different architectures with patterns in the associated
fauna composition, abundance and diversity did not reveal any evident relationships.
Other important substrate characteristics should be considered, such as:
location of the substrate on the shore, presence of sediment or other inert
materials, chemical composition of the surface and the possibility that
E. carolinense eliminates alellopathic substances in the water.