Deleterious effects of a nonPST bioactive compound(s) from <Emphasis Type="Italic">Alexandrium tamarense</Emphasis> on bivalve hemocytes

The known negative effects of shellfish toxin-producing dinoflagellates on feeding, burrowing and survival of some bivalve mollusks has prompted questions concerning whether they might also impair the internal defense system of affected bivalves and make them more susceptible to disease agents. The primary components of the cellular defense system are hemocytes. Many toxic dinoflagellates are too large to be ingested whole by hemocytes and would most likely be exposed to intracellular toxins only after the algae are consumed, broken down, and the water-soluble toxins, released. Therefore, we conducted a series of experiments in which hemocytes from two suspension-feeding bivalves—the Manila clam, Ruditapes philippinarum, and the softshell clam, Mya arenaria—were exposed in vitro to filtered extracts of one highly toxic paralytic shellfish toxin (PST)-producing and one nonPST-producing strain of Alexandrium tamarense (isolates PR18b, 76 ± 6 STXeq cell⁻¹ and CCMP115, with undetectable PST, respectively). We measured adherence and phagocytosis, two hemocyte attributes known to be inhibited by bacterial pathogens and other stressors. We found no measurable effect of a cell-free extract from a highly concentrated suspension of the PST-producing strain on hemocytes of either bivalve species. Instead, extract from the nonPST-producing strain had a consistent negative effect on both clams, resulting in significantly lower adherence and phagocytosis compared to strain PR18b and filtered seawater controls. The bioactive compound produced by strain CCMP115, which has yet to be characterized, may be similar to the PST-independent allelopathic compounds described for Alexandrium spp., which act on other plankters. These compounds and those produced by other harmful algae are known to cause immobilization, cellular deformation and lysis of co-occurring target organisms. Thus, nonPST producing Alexandrium spp., which do not cause paralysis and burrowing incapacitation of clams, may still produce a compound(s) that has negative effects not only on hemocytes, but on other molluscan cell types and their functions, as well.     

Toxin accumulation and feeding behaviour of the planktonic copepod Calanus finmarchicus exposed to the red-tide dinoflagellate Alexandrium excavatum

The planktonic copepod Calanus finmarchicus is a dominant member of the zooplankton community in the lower St. Lawrence Estuary in eastern Canada. Blooms of the toxic marine dinoflagellate Alexandrium excavatum which produces high cellular levels of paralytic shellfish poisoning (PSP) toxins, occur during the period of high C. finmarchicus production in summer in this region. To study the feeding behaviour of C. finmarchicus in the presence of Alexandrium spp., experiments were conducted in which female adult copepods collected from the St. Lawrence Estuary between May and September 1991 were exposed under controlled conditions to two toxic isolates of A. excavatum (Pr18b and Pr11f) from the estuary and to a non-toxic control (PLY 173) of a closely related species, A. tamarense isolated from the Tamar Estuary, Plymouth, U.K. Clearance rates on non-toxic A. tamarense cells averaged 5.5 ml ind^-1 h^-1 but were nearzero with either toxic isolate. When presented with a mixture of A. excavatum and the non-toxic diatom Thalassiosira weissflogii in varying proportions, C. finmarchicus fed upon the diatom but avoided the toxic dinoflagellate. Although feeding rates on A. excavatum were very low, toxin analysis by high-performance liquid chromatography with fluorescence detection (HPLC-FD) revealed that the PSP toxins were accumulated in copepods exposed to toxigenic dinoflagellates.The toxin composition in copepods was similar to that of the toxic dinoflagellate, but not necessarily identical, particularly after short-term (2-h) exposure, when relatively elevated levels of N-sulfocarbamoyl toxins were detected. The evidence suggests that C. finmarchicus ingests toxic dinoflagellate cells, either mistakenly or during exploratory bouts of feeding, and accumulates PSP toxins in its gut system and perhaps in other tissues.     

Depuration of twelve trace metals in tissues of the oysters Crassostrea gigas and C. virginica

The depuration of 12 trace metals in the mantle, gill, digestive gland, and kidney of Crassostrea gigas and C. virginica was investigated under natural field conditions; oysters from a relatively contaminated environment (Redwood Creek in south San Francisco Bay) were transplanted to a relatively clean environment (Tomales Bay). In the transplanted oysters, the digestive gland and kidney depurated Cd, Cu, Hg, Ag, and Zn more readily than the mantle and gill. Other trace metals As, Fe, Mn, Ni and Se showed varying depuration patterns. The results for Cr and Pb were inconclusive, since initial concentrations were too low to follow any losses. Interspecific differences in trace metal depuration were observed. Biological half-lives for most trace metals were on the order of 23 to 60 d for C. gigas and on the order of 70 to 180 d for C. virginica.     

Environmental stress,competition and dominance of Crassostrea virginica near Beaufort,North Carolina,USA

Observations and experiments were made at 2 intertidal areas near Beaufort, North Carolina, USA from May 1977 to July 1978 to determine why the oyster Crassostrea virginica dominated the community in areas protected from wave action but not in areas directly exposed to waves. Barnacles, oysters, the green alga Enteromorpha sp. and the mussel Brachiodontes exustus were the main occupiers of primary space at the mid and low intertidal levels of exposed areas. The intertidal community at the protected site consisted of a mid intertidal occupied by the barnacles Balanus amphitrite and Chthamalus fragilis, and the oyster C. virginica, and a low intertidal dominated by C. virginica. The exposed area was highly variable with high colonization and mortality for all species producing large seasonal changes in structure. The protected site remained constant throughout the year; there was no evidence of further colonization of either barnacles or oysters and mortality was very low. Recolonization experiments, selective removal of species, and growth and survival data demonstrated that C. virginica does not become dominant at exposed locations because (1) the constant wave shock at the ocean site reduces growth and increases mortality of young and adult oysters and (2) oysters are outcompeted by the mussel B. exustus. The monopolization of space by C. virginica at protected sites contrasts with studies north of Cape Hatteras where the abundance of predators produces a more diverse and heterogeneous community. Predation was unimportant in Beaufort because predators were absent at the exposed areas and the oyster drill Urosalpinx cinerea was restricted to the subtidal zone at the protected site. This absence of predators indicates a higher level of environmental stress near Beaufort compared to areas farther north.     

Electrophoretic identification and genetic analysis of bivalve larvae

Taxonomic identification and genetic analysis of larval marine invertebrates have been vexing problems. We describe a polyacrylamide mini-gel electrophoresis technique for resolving proteins from individual larval bivalves (shell length 250 to 350 m) and apply this technique to three species of laboratory-cultured larval oysters [Ostrea edulis L., 1758, Crassostrea gigas (Thunberg, 1793) and c. virginica (Gmelin, 1791)] reared during summer 1989. Electrophoretic patterns of proteins clearly discriminate among the three species and allow genetic analysis of a polymorphic allozyme locus (Pgi) in field-collected larvae and juveniles of C. virginica. This technique provides an economical tool for largescale taxonomic, ecologic, and genetic studies of meroplanktonic stages of various species.     

Particle sorting in bivalves: in vivo determination of the pallial organs of selection

Benthic particle feeders are exposed to a food supply varying in both quantity and quality. Previous studies have shown that bivalve molluscs deal with such fluctuating particle regimes in a variety of ways, including adjustments in pumping and ingestion rates, and selective rejection of non-nutritive particles as pseudofeces. The actual site of particle selection within the pallial cavity, however, has remained a topic of speculation. During August 1995 and January and August 1996, we exposed the oysters Crassostrea virginica (Gmelin) and C. gigas (Thunberg), and the mussel Mytilus trossulus Gould to a mixture of ground, aged Spartina alterniflora Loisel and similar-sized phytoplankton at three concentrations (10³, 10⁴, 10⁵ particles ml⁻¹). We then examined the ctenidia and labial palps by means of endoscopy and sampled, in vivo, the particulate material from various ciliated tracts, and analyzed the samples with a flow cytometer. We found that in oysters, the ctenidia are responsible for particle sorting, whereas the labial palps play an accessory role in particle selection, or function to control the volume of material to be ingested. In mussels, however, the ctenidia play little role in particle selection and simply transport particulate matter to the palps for further processing. We suggest that selection by the ctenidia of oysters is a function of their architecture (plicate, heterorhabdic). Received: 4 June 1997 / Accepted: 23 January 1998     

Alterations in cytometric characteristics of hemocytes from the American oyster Crassostrea virginica exposed to a polycyclic aromatic hydrocarbon (PAH) contaminated environment

The effects of residing in a polycyclic aromatic hydrocarbon (PAH) contaminated environment on the cytometric characteristics of hemocytes from the American oyster Crassostrea virginica (collected from the Rappahannock River, Virginia, USA in Spring 1991) were analyzed using a multichannel Coulter counter (10000 hemocytes oyster^-1). The percentage and relative volume of small-sized hemocytes (>2.5 to 5.1 m) was higher (P<0.01) in oysters from Hospital Point (HP, a PAH contaminated site) in the Elizabeth River (ER), Virginia, compared to oysters from the relatively unpolluted Rappahannock River (RR). On the contrary, a decrease (P<0.001) in the percentage and volume of larger hemocytes (>6.2 to 10 m) was observed in the HP-oysters. Maintaining the RR-oysters at the HP site for 8 wk induced statistically significant increases in the number and relative volume contribution of the >2.5 to 5.1 m hemocytes and sharp decreases in the occurrence and relative volume of >6.2 to 13 m cells (P<0.01). By depurating the HP-oysters in the York River (the control site), the number of, and volume contributed by the small hemocytes (>2.5 to 5.1 m) showed a significant decrease over time as compared to the baseline values. On the other hand, the relative number of >6.2 to 13 m hemocytes increased sharply within 8 wk (P<0.001). This indicates that these changes are both inducible and reversible and, at least in part, due to exposure to the ER-sediment. Based on these studies, oyster hemocyte cytometric characteristics could be developed as a sensitive biomarker of exposure to PAH.     

Fluxes of dissolved and particulate calcium in selected tissues of Crassostrea virginica

Rates of calcium incorporation by selected tissues of Crassostrea virginica increased in a step-wise fashion from lowest values among organisms exposed to ambient total calcium concentrations of 45 mg l^-1, intermediate values among oysters exposed to 135, 225, and 315 mg l^-1, to highest rates for oysters exposed to 360 mg l^-1. Although excised visceral mass tissue had highest rates of calcium incorporation relative to mantle, muscle, and both organic and inorganic portions of the shell, mantle tissue appeared to have the most dynamic response to changes in ambient calcium concentrations. Rates of dissolved calcium incorporation from ambient water were approximately two to three orders of magnitude higher than comparable rates from ingested algal food. Behavioral response to concentrations of selected inoic, species in the ambient environment may have been responsible for observed differences in rates of calcium incorporation.     

Potential effects of a non-indigenous predator in its expanded range: assessing green crab, <Emphasis Type="Italic">Carcinus maenas</Emphasis>, prey preference in a productive coastal area of Atlantic Canada

The non-indigenous green crab (Carcinus maenas) is an important predator on bivalve wild beds in coastal areas worldwide. This study explored size-dependent green crab prey preference on American oysters (Crassostrea virginica), blue mussels (Mytilus edulis), and soft-shell clams (Mya arenaria) in a productive coastal system of Atlantic Canada. Using two sizes of prey and three different experimental manipulations, small, medium, and large green crabs were given a choice among these three bivalves, and their daily feeding rates were monitored over the course of 3 days. For both prey sizes, green crabs showed an early feeding preference for soft-shell clams and, only as they declined in numbers, a switch toward mussels and subsequently toward oysters. We found that such changes in the timing (order) of prey preference are related to prey differences in shell thickness, a fairly reliable indicator of prey shell strength.     

Use of high-performance liquid chromatography to investigate the production of paralytic shellfish toxins by Protogonyaulax spp. in culture

Procedures have been developed for the extraction and high-performance liquid chromatography (HPLC) analysis of paralytic shellfish poisoning (PSP) toxins from Protogonyaulax spp. grown in batch culture. Using these procedures, the toxin content of two isolates of P. tamarensis (NEPCC 183 and 255) and one isolate of P. catenella (NEPCC 355) were examined. Total toxin and individual toxin concentrations were measured for each isolate during the exponential and stationary phases of growth in batch culture. The total toxicity of each isolate as measured by HPLC analysis was found to agree with toxicity as determined by the standard mouse bioassay. Two of the isolates (255 and 355) were found to be toxic and the third (183) was non-toxic. The toxic isolates (255 and 355) both showed higher average total PSP toxin content during the exponential phase (35 and 23 fmol toxin cell^-1, respectively) than during the stationary phase (21 and 8 fmol toxin cell^-1, respectively). These cultures differed dramatically in their toxin composition. P. tamarensis (255) contained a large proportion of the N(21) sulfo toxins (B₁, B₂, C₁, C₂) while P. catenella (355) contained primarily Gonyautoxins 1 through 4. The percent composition of individual toxins was found to be constant throughout the growth cycle for both toxic isolates, even though the total toxin concentration varied. Our results suggest that PSP toxin profiles might be useful as chemotaxonomic indicators.     

Accumulation,release and retention of petroleum hydrocarbons by the oyster Crassostrea virginica

Two Crassostrea virginica populations, differing in fat content, were experimentally exposed to a complex petroleum-hydrocarbon fraction. The hydrocarbons in this mixture were accumulated by both groups of oysters, and their lipid content, as well as the concentration of hydrocarbon in the water, were found to affect the rate and extent of accumulation. Hydrocarbons accumulated were rapidly, although incompletely, discharged when the oysters were transferred to an uncontaminated system. Amounts of hydrocarbons discharged and amounts retained after discharge are probably related to the level of contamination. The data can be interpreted as indicating that equilibration and the occurrence of multiple compartments where hydrocarbons can reside are factors involved in the uptake and retention of nonbiogenic hydrocarbons by oysters. The petroleum hydrocarbons contained in the oysters differed from the contaminating oil by displaying a greater aromatic content. In addition, gas-liquid chromatograms of aliphatic fractions of the hydrocarbons in the oysters rapidly showed a degraded appearance; the possibility that the oysters themselves are modifying the oil cannot be excluded.Contribution No. 3098 from the Woods Hole Oceanographic Institution.     

Effects of the polychlorinated biphenyl Aroclor® 1254 on the American oyster Crassostrea virginica

Young oysters (Crassostrea virginica) were continuously exposed to Aroclor^® 1254, a polychlorinated biphenyl (PCB), in flowing, unfiltered seawater. Growth rate (height and in-water weight) was significantly reduced (=0.05) in oysters exposed to 5 g/l (ppb) for 24 weeks. Growth rate was not affected in oysters exposed to 1 ppb for 30 weeks. Mortality was not significant in exposed and control groups. In oysters exposed to 5 ppb, greatest PCB residue (whole body) was 425 mg/kg (ppm), 85,000x the concentration in the water, and less than 0.3 ppm was retained after 28 weeks depuration in PCB-free water. In oysters exposed to 1 ppb, greatest residue was 101 ppm, 101,000x the concentration in the water, and less than 0.2 ppm was retained after 12 weeks depuration. Examination of oysters exposed to 5 ppb of this PCB for pathogenesis revealed atrophy of digestive diverticular epithelium and degeneration of vesicular connective tissues concomitant with leukocytic infiltration, but tissue recovery seemed excellent after 12 weeks depuration.Registered trademark, Monsanto Company, St. Louis, Missouri, USA. Mention of commercial products or trade names does not constitute endorsement by the Environmental Protection Agency.Contribution No. 146, Gulf Breeze Laboratory.     

Molecular phylogenies help resolve taxonomic confusion with Asian <Emphasis Type="Italic">Crassostrea</Emphasis> oyster species

Identification of oyster species is still largely based on phenotypic characters that are highly plastic. Prompted by the proposed introduction of the Asian oyster species Crassostrea ariakensis into the Chesapeake Bay region of the U.S.A., this study uses molecular genetic information to understand the taxonomic framework surrounding C. ariakensis and to confidently distinguish among various sympatric oyster species. Putative samples of C. ariakensis and other species of cupped oysters from across Asia were collected and phylogenetic analyses were conducted on DNA sequences of both nuclear (ITS-1) and mitochondrial (COI) regions. Trees generated based on the two independent molecular datasets were highly congruent, and indicate that many oysters collected for this study as C. ariakensis were originally misidentified. Results also indicate that C. ariakensis, C. hongkongensis and C. nippona are distinct, but closely related species. There is strong support in both analyses for a close relationship between C. gigas and C. sikamea, as well as between C. belcheri and C. gryphoides, and between C. iredalei and C. madrasensis. The parsimony analyses based on these DNA markers, however, did not provide evidence to support C. angulata as a distinct species from C. gigas. Overall, the results emphasize the need for rigorous species identification, and additional extensive and intensive sampling to more accurately determine relationships among Crassostrea species, define their geographic distributions, and establish existing sympatry patterns.     

Comparing the effect of elevated pCO2 and temperature on the fertilization and early development of two species of oysters

This study compared the synergistic effects of elevated pCO₂ and temperature on the early life history stages of two ecologically and economically important oysters: the Sydney rock oyster, Saccostrea glomerata and the Pacific oyster, Crassostrea gigas. Gametes, embryos, larvae and spat were exposed to four pCO₂ (375, 600, 750, 1,000 μatm) and four temperature (18, 22, 26, 30°C) levels. At elevated pCO₂ and suboptimal temperatures, there was a reduction in the fertilization success of gametes, a reduction in the development of embryos and size of larvae and spat and an increase in abnormal morphology of larvae. These effects varied between species and fertilization treatments with S. glomerata having greater sensitivity than C. gigas. In the absence of adaptation, C. gigas may become the more dominant species along the south-eastern coast of Australia, recruiting into estuaries currently dominated by the native S. glomerata.     

Biogeography of toxic dinoflagellates in the genusAlexandrium from the northeastern United States and Canada

Twenty-eight strains of toxic dinoflagellates in the genusAlexandrium from the northeastern United States and Canada were characterized on the basis of morphology, bioluminescence capacity, mating compatibility, and toxin composition. The distributions of these characters were evaluated in the context of regional patterns of paralytic shellfish poisoning (PSP) and coastal hydrography. Two morphospecies were identified-A. tamarense Lebour andA. fundyense Balech. The two are interspersed geographically though there are areas, such as the Gulf of Maine, where apparently onlyA. fundyense occurs. Southern waters (Cape Cod, Connecticut, and Long Island) have especially diverse populations. The two species are sexually compatible. Virtually all northern isolates are bioluminescent, whereas southern isolates include bioluminescent and non-bioluminescent strains. Cluster analyses, based on high performance liquid chromatography (HPLC) determinations of the suite of toxins produced by each isolate, revealed two and perhaps three distinct groups. One is comprised almost exclusively of northern strains, and the other of southern strains. A Cape Cod cluster may be separable from the southern group. These analyses explain a previously reported north-to-south trend of decreasing toxicity, as the northern isolates produce greater proportions of the more potent toxins than do southern forms. The overall perspective is that the biogeography of toxicAlexandrium spp. in the study region is not that of a single, widespread, homogeneous population, but rather is comprised of several sub-populations, each with its own physiological characteristics and history. Two scenarios are considered with respect to this regional biogeography. The first invokes recent and continuing dispersal of isolates to the south from a center of origin in the north, followed by recombination and strong selection. The second holds that the northern and southern populations diverged from a common ancestor (vicariance), but now represent localized populations with little mixing of genotypes. Neither hypothesis can be completely refuted by the data presented here, though the weight of the evidence favors the latter. The correct scenario may be a combination of both, with recent and continuous speading occuring within the Gulf of Maine and perphaps the Gulf of St. Laerence, but with endemic localized populations persisting without genetic exchange in most southern locations. These data also indicate that although morphological criteria separate toxicAlexandrim isolates from the study region into two morphospecies, these assignments do not coincide with clusterings based on toxin composition or allozyme electrophoresis, and they are further violated by mating results. A revision of taxon designations to the varietal level could be justified.     

Mitochondrial cytochrome oxidase I gene sequences support an Asian origin for the Portuguese oyster Crassostrea angulata

The Portuguese oyster Crassostrea angulata (Lamarck, 1819) was long assumed to be native to the northeastern Atlantic, however, a number of lines of evidence now indicate that it is a close relative, or identical, to the Asian Pacific oyster C. gigas (Thunberg, 1793). Three hypotheses have been proposed to explain how this strikingly disjunct geographic distribution may have come about: ancient vicariance events, recent anthropogenic introduction to Asia and recent anthropogenic introduction to Europe. We have performed a molecular phylogenetic analysis of C. angulata based on mitochondrial DNA sequence data for a 579-nucleotide fragment of cytochrome oxidase I. Our results show that Portuguese oyster haplotypes cluster robustly within a clade of Asian congeners and are closely related, but not identical, to C. gigas from Japan. The mitochondrial data are the first to show that Portuguese oysters are genetically distinct from geographically representative samples of Japanese Pacific oysters. Our phylogenetic analyses are consistent with a recent introduction of C. angulata to Europe either from a non-Japanese Asian source population or from a subsequently displaced Japanese source population. Genetic characterization of Pacific oysters throughout their Asian range is necessary to fully reveal the phylogenetic relationships among Portuguese and Pacific oysters. Received: 15 April 1997 / Accepted: 12 February 1998     

The ecological energies of growth,respiration and assimilation in the intertidal American oyster Crassostrea virginica

Seasonal variations in the growth, respiration and assimilation of the intertidal oyster Crassostrea virginica (Gmelin) of different sizes were determined. The instantaneous growth rates for intertidal oysters decreased with increasing size and with lower temperatures. Q₁₀ values computed from instantaneous growth rates were approximately 2 during the warm growing season, but were higher in the colder months. Oxygen consumption increased with temperature and body size. A model was developed to predict oxygen consumption at any environmental temperature from 10° to 30°C for oysters ranging in weight from 0.1 to 100.0 g. Q₁₀ values computed from oxygen-consumption rates decreased with increasing temperature and increasing body size. Intertidal oysters utilize a large proportion of their assimilated energy in growth.Supported by a Belle W. Baruch Fellowship in Marine Ecology.     

Dietary protein levels and growth of the oyster Crassostrea virginica

The diatom Thalassiosira pseudonana in exponential phase or stationary phase and grown under 3 light regimes was fed to 6 groups of juvenile oysters, Crassostrea virginica Gmelin, for a 10-week period. Oysters fed diets from the stationary phase grew more rapidly and had a higher glycogen content than those fed diets from the exponential phase. The stationary phase, cool white-light diet produced the greatest increase in dry weight, shell height and glycogen content of the 6 diets examined. Oysters grew more rapidly when fed diets richer in carbohydrates than proteins.     

Comparative effects of two antifouling paints on the oyster Crassostrea gigas

The effects of two antifouling paints, one containing organotin compounds, the other copper oxide, were studied from September 1983 to October 1984 on Crassostrea gigas (Thunberg) grown under natural field conditions in the Bay of Arcachon, France. The organotin paint reduced growth rate expressed as weight, length and width, but did not affect shell height; it drastically decreased the dry condition factor and shell density, but did not affect the viability of embryos and larvae from exposed oysters. However, some decrease in larval growth rate was observed. The copper paint had no effect on oyster growth, but lowered the condition factor compared to controls. Neither viability of embryos or larvae nor larval growth were affected by this paint.     

Growth patterns in oysters, Crassostrea virginica, from different estuaries

We analyzed a data set collected over 15 yr, containing growth data from strains of eastern oysters, Crassostrea virginica (Gmelin, 1791), initiated from parent populations in Long Island Sound, Delaware Bay, and lower Chesapeake Bay. The long-term growth data proved to be a powerful tool for examining patterns of growth differentiation among separated populations of C. virginica. The oyster strains had been grown in a common environment in lower Delaware Bay for up to seven generations. We found that the oyster strains with origins in Long Island Sound were significantly larger over several generations than oyster strains from Delaware Bay and Chesapeake Bay. Chesapeake Bay oyster strains were larger than Delaware Bay oyster strains at 1.5 yr old, but Delaware Bay oysters were larger at 2.5 yr. Year-to-year variation in environmental conditions had a strong significant effect on absolute oyster size and the relative sizes of the oyster strains. Persistent differences between oyster strains from different origins over several generations support a hypothesis that these estuarine populations have experienced long-term genetically-based population differentiation. This result is consistent with hypotheses of population differentiation of oysters based on observations of local reproductive timing. Received: 12 August 1997 / Accepted: 26 May 1998