Oyster Gill Research Articles

Effects of cadmium exposure on expression and activity of P-glycoprotein in eastern oysters, Crassostrea virginica Gmelin

Heavy metal pollution is a worldwide problem, and cadmium (Cd) is one of the most noxious pollutants in aquatic environments. We studied P-glycoprotein (P-gp) expression and function in control and Cd exposed (50 μg L −1 Cd, 30–40 days) oysters Crassostrea virginica as a possible mechanism of cell protection against Cd. Our data show that P-gp is expressed on cell membrane and in mitochondria of oyster gills and hepatopancreas. Inhibitor studies with verapamil, cyclosporine A and JS-2190 suggest that in the gills, mitochondrial P-gp pumps substrates from cytosol into the mitochondria, while cell membrane P-gp pumps substrates from cytosol out of the cell. Cd exposure resulted in a 2–2.5-fold increase in P-gp protein expression in cell membranes and a 3.5–7-fold increase in transport activity measured as the inhibitor-sensitive rhodamine B extrusion rate. In contrast, p-gp mRNA levels were similar in control and Cd-exposed oysters. No difference in P-gp protein expression was observed between mitochondria of control and Cd-exposed oysters but the apparent transport activity was higher in mitochondria from Cd-exposed oysters. Overall, a stronger increase in substrate transport activity in Cd-exposed oysters compared to a relatively weaker change in P-gp protein levels suggests that P-gp activity is post-translationally regulated. Our data show that direct determination of P-gp transport activity may be the best measure of the xenobiotic-resistant phenotype, whereas p-gp mRNA levels are not a good marker due to the likely involvement of multiple post-transcriptional regulatory steps. Cd exposure resulted in a significantly elevated rate of oxygen consumption of isolated oyster gills by 46%. Specific inhibitors of ATPase function of P-gp (cyclosporine A and JS-2190) had no significant effect on tissue oxygen consumption indicating that P-gp contribution to energy budget is negligible and supporting indirect estimates based on the ATP stoichiometry of substrate transport that also suggest low energy demand for P-gp function.

Differential gene expression in oyster exposed to sewage

In order to investigate the influence of domestic sewage on the gene expression of Pacific oysters Crassostrea gigas, suppression subtractive hybridization (SSH) method was employed. Oysters were sampled at a farming area and, after 10 days of acclimation in the laboratory, were exposed to untreated domestic sewage diluted 33% for 48 h. Gills of male oysters were excised for total RNA extraction. mRNA was purified and the differential gene expression was analyzed by SSH. We obtained 61 cDNA sequences but only 15 were identified, which includes fatty acid binding protein, multidrug resistance protein, omega glutathione S-transferase, cytochrome P450 isoform CYP356A1, among others. The identified genes are associated with different metabolic functions like biotransformation, membrane transport, aerobic metabolism and translational machinery, evidencing the potential toxic effects elicited by these effluents.

Effects of cadmium on cellular protein and glutathione synthesis and expression of stress proteins in eastern oysters, Crassostrea virginica Gmelin

Cadmium (Cd) is an important toxicant in estuarine and coastal environments that can strongly affect energy balance of aquatic organisms by increasing the organism's basal energy demand and reducing its aerobic capacity. Mechanisms of cadmium-induced increase in basal metabolic costs are not well understood and may involve elevated detoxification costs due to the synthesis of cellular protective proteins and glutathione. We studied the short-term effects of cadmium exposure (4 h) on protein and glutathione (GSH) synthesis and expression of stress proteins (heat shock proteins HSP60, HSP70 and HSP90) and metallothioneins in isolated gill and hepatopancreas cells of the eastern oyster, Crassostrea virginica. Our study showed that exposure to cadmium resulted in a dose-dependent increase in the rate of protein synthesis in oyster cells, which reached 150% of the control at the highest tested Cd level (2000 micromol l(-1)). GSH synthesis was significantly inhibited by the highest Cd concentrations, especially in hepatopancreas, which resulted in a slight but significant decrease in the total GSH concentrations. Elevated protein synthesis was associated with the increased expression of metallothioneins and heat shock proteins. Interestingly, stress protein response differed considerably between gill and hepatopancreas cells. In hepatopancreas, expression of metallothionein mRNA (measured by real-time PCR) increased 2-8-fold in response to Cd exposure, whereas no significant increase in metallothionein expression was found in Cd-exposed gill cells. By contrast, HSP60 and HSP70 protein levels increased significantly in Cd-exposed gill cells (by 1.5-2-fold) but not in hepatopancreas. No change in HSP90 expression was detected in response to Cd exposure in oyster cells. These data indicate that metallothionein expression may provide sufficient protection against Cd-induced damage to intracellular proteins in hepatopancreas, alleviating the need for overexpression of molecular chaperones. By contrast, Cd detoxification mechanisms such as inducible metallothioneins and GSH appear to be insufficient to fully prevent protein damage in gill cells, thus necessitating induction of HSPs as a secondary line of cellular defense. Therefore, gills are likely to be among the most Cd-sensitive tissues in oysters, which may have important implications for impaired oxygen uptake contributing to energy misbalance and reduced aerobic scope in Cd-exposed oysters.

Changes in the contents of ATP and its related breakdown compounds in various tissues of oyster during frozen storage

The changes in the contents of adenosine 5’-triphosphate (ATP) and its related breakdown compounds were investigated in the adductor muscle, mantle, gill and body trunk of oyster (Crassostrea gigas) during frozen storage at −20°C and −30°C and compared with that of the fresh oyster. The investigation was performed using an HPLC system. Different extents of ATP decomposition were found in various tissues frozen at the two temperatures. The K, K’ and A.E.C values were calculated as the chemical freshness indices. Considering the results of sensory evaluation, the A.E.C. value in body trunk at −20°C and −30°C could be used as the best freshness index for frozen oyster.

Change in lipid composition in eastern oyster ( Crassostrea virginica Gmelin) exposed to constant or fluctuating temperature regimes

A temperature decrease usually induces an ordering effect in membrane phospholipids that can lead to membrane dysfunction. Ectotherms typically counteract this temperature effect by remodeling membrane lipids as stipulated in the homeoviscous adaptation theory (HVA). Previous studies mostly focused on the remodeling of membrane lipids during long-term acclimatization or acclimation at constant temperature regimes, whereas in nature, many organisms experience variations in temperature on a daily basis and must react to this changing thermal environment. The objective of this study was to examine the composition of membrane lipids in oysters subjected to long-term acclimation at constant temperatures (12 or 25 °C) or to environmentally realistic daily fluctuations in temperature between 12 and 25 °C for 7 d. The lipid composition of gill in oysters subjected to long-term acclimation at a constant temperature or to daily temperature fluctuations varied in a way consistent with HVA: oysters adjusted their phospholipid to sterol ratio in response to long-term acclimation to a constant temperature but not to daily temperature fluctuations. In contrast, the unsaturation index of polar lipids in oysters varied in response to both long-term acclimation to a constant temperature and to daily temperature fluctuations, mainly due to changes in 22:6n-3 and 20:5n-3. The 20:4n-6 levels in oyster gills increased as temperature rose, suggesting an increasing availability of this fatty acid for eicosanoid biosynthesis during stress responses.

HEAT AND CADMIUM: A DAMAGING COCKTAIL

Permanently glued to the rocks of their estuary home, eastern oysters(Crassostrea virginica) can cope with most of the extremes of their environment: daily and seasonal variations in temperature, from 5°C to 35°C, plus the coming and going of the tides. Add environmental stressors like pollution into the mix, however, and the oysters suffer from oxidative stress, where their cells' mitochondria produce more molecule-damaging reactive oxygen species (ROS) than the cells can clean up. It is important to understand how temperature and pollution interact with each other to affect the survival of marine invertebrates, such as oysters, given the predicted rise in global temperatures, explains Inna Sokolova of the University of North Carolina, Charlotte. Sokolova and her colleagues show that as the temperature rises, oysters suffer more from oxidative stress when they are exposed to cadmium, a toxic heavy metal (p. 46).Cadmium is similar in structure to zinc and calcium and it messes up many cellular processes, such as calcium dependent cell signalling, by blocking enzyme active sites. The metal also increases ROS production, and therefore oxidative stress, in mitochondria. Directly measuring ROS production in a cell is tricky, so to measure the level of oxidative stress the team focussed on a Krebs cycle enzyme called aconitase. This enzyme has an iron atom in the middle, which is a bit loose. `If the iron becomes oxidised by ROS, it falls off, and the enzyme doesn't work properly', says Sokolova. So if a cell is producing larger quantities of ROS, the activity of the aconitase enzyme goes down.To investigate the effects of cadmium and temperature on the aconitase enzyme, the team carefully isolated mitochondria from oyster gill tissue and transferred them to culture for experiments. They exposed the mitochondria to cadmium and higher temperatures and measured aconitase enzyme activity. At 20°C, cadmium slightly reduced aconitase activity, but mitochondria could mop up most of the excess ROS before they caused too much damage. However,when they exposed mitochondria to cadmium and raised the temperature to 30°C, the team found that aconitase activity decreased by up to 52%. This was probably because cadmium induces the mitochondria to produce more ROS at higher temperatures, which inhibits aconitase.Having shown that cadmium increases oxidative stress at higher temperatures, the team wondered if a group of mitochondrial membrane proteins called uncoupling proteins, which are known to diminish the effects of ROS,would reduce oxidative stress and the effects of ROS on aconitase. They suggested that stimulating uncoupling protein activity with fatty acids would reduce oxidative stress caused by cadmium and high temperature, while inhibiting uncoupling protein activity with ATP would increase it. They found no evidence that uncoupling proteins help protect aconitase from ROS, and Sokolova explains that the team also found a result that they weren't expecting: ATP directly inhibits the aconitase enzyme. This suggests ATP might play an important role in regulating the activity of the Krebs cycle, and scientists will need to look at the phenomenon more closely to see if it is unique to oysters or not.While the team's unexpected result will give researchers plenty of food for thought, their study also suggests that rising global temperatures could have a serious effect on oyster survival: `moderately polluted areas could become graveyards', says Sokolova. In the future, researchers might have to adjust their criteria for water quality, to take into account the effects of rising temperatures.

Preliminary characterisation of the blue-green pigment “marennine” from the marine tychopelagic diatom Haslea ostrearia (Gaillon/Bory) Simonsen

Haslea ostrearia is a common marine tychopelagic diatom which has the particularity of synthesizing a blue-green hydrosoluble pigment called “marennine”. This pigment, when released into the external medium, is known to be responsible for the colour of oyster gills. Here we present results for main biophysical and biochemical characteristics of pure intra- and extracellular marennine. Tests for chemical determination show that the nature of the two forms of marennine cannot be distinguished and could be related to a polyphenolic compound. Nevertheless, based on spectral properties and the molecular weight, which is about 10751 ± 1 and 9893 ± 1 Da, for the intracellular and extracellular forms respectively, we assess that the pigment accumulated in the apex of the cell and the one released in the external medium have probably distinct molecular structures.

Purification of the blue-green pigment “marennine” from the marine tychopelagic diatom Haslea ostrearia (Gaillon/Bory) Simonsen

The diatom Haslea ostrearia that lives in oyster ponds has the distinctive feature of synthesizing “marennine”, a blue-green pigment of which the chemical nature still remains unknown. This pigment is responsible for the greening of oyster gills. Here, we report a new method for extraction and purification of intracellular (accumulated in the apex of the cell) and extracellular (released into the external medium) forms of the pigment. Intracellular marennine is obtained by extraction from blue algal pellets with a carbonate buffer. The extract is then centrifuged and filtered. Extracellular marennine is obtained by clarification of blue-coloured culture medium. Both extracts are then purified by a semi-preparative process, using ultrafiltration through membranes and anion-exchange chromatography. This procedure allows us to produce native pigment displaying the degree of purity required to enter upon the molecular characterisation of marennine. By this process, about 35% of the initial amount of pigment can be recovered. If necessary, this method could be easily scaled up to a larger production system to accommodate potential industrial applications.

Experimental shift in diet δ13C: A potential tool for ecophysiological studies in marine bivalves

To test the potential of diet switching experiments in ecophysiological studies of marine invertebrates, stable carbon isotope ratios were measured at different seasons in the gonad, adductor muscle, digestive gland and gills of scallops ( Pecten maximus) and oysters ( Crassostrea gigas) held for 15 days on a constant diet of phytoplankton depleted in 13C. The aim of this study was to determine if differences in carbon incorporation could be detected among species, seasons and organs, and if so, whether it was consistent with their known energy-allocation patterns. After offering the new diet, isotope values of the different organs gradually shifted and significant differences among organs, seasons and species were found. A carbon incorporation index (CII) was calculated to compare the metabolic activity of each organ of the two species between day 0 and day 15. For both species, the digestive gland had the highest CII, the adductor muscle the lowest, while gonad and gills had intermediate values. The CII was generally much higher in P. maximus than in C. gigas, suggesting higher metabolic activity in this species. Seasonal differences in the CII were also observed for the two species and were interpreted as differences in metabolic activity in accordance with our energy allocation scenario. Therefore, stable isotope diet switching experiments appear to be of great value for assessing metabolic orientation in bivalves.

Effects of acclimation temperature and cadmium exposure on cellular energy budgets in the marine molluskCrassostrea virginica: linking cellular and mitochondrial responses

In order to understand the role of metabolic regulation in environmental stress tolerance, a comprehensive analysis of demand-side effects (i.e. changes in energy demands for basal maintenance) and supply-side effects (i.e. metabolic capacity to provide ATP to cover the energy demand) of environmental stressors is required. We have studied the effects of temperature (12, 20 and 28 degrees C) and exposure to a trace metal, cadmium (50 microg l(-1)), on the cellular energy budget of a model marine poikilotherm, Crassostrea virginica (eastern oysters), using oxygen demand for ATP turnover, protein synthesis, mitochondrial proton leak and non-mitochondrial respiration in isolated gill and hepatopancreas cells as demand-side endpoints and mitochondrial oxidation capacity, abundance and fractional volume as supply-side endpoints. Cadmium exposure and high acclimation temperatures resulted in a strong increase of oxygen demand in gill and hepatopancreas cells of oysters. Cd-induced increases in cellular energy demand were significant at 12 and 20 degrees C but not at 28 degrees C, possibly indicating a metabolic capacity limitation at the highest temperature. Elevated cellular demand in cells from Cd-exposed oysters was associated with a 2-6-fold increase in protein synthesis and, at cold acclimation temperatures, with a 1.5-fold elevated mitochondrial proton leak. Cellular aerobic capacity, as indicated by mitochondrial oxidation capacity, abundance and volume, did not increase in parallel to compensate for the elevated energy demand. Mitochondrial oxidation capacity was reduced in 28 degrees C-acclimated oysters, and mitochondrial abundance decreased in Cd-exposed oysters, with a stronger decrease (by 20-24%) in warm-acclimated oysters compared with cold-acclimated ones (by 8-13%). These data provide a mechanistic basis for synergism between temperature and cadmium stress on metabolism of marine poikilotherms. Exposure to combined temperature and cadmium stress may result in a strong energy deficiency due to the elevated energy demand on one hand and a reduced mitochondrial capacity to cover this demand on the other hand, which may have important implications for surviving seasonally and/or globally elevated temperatures in polluted estuaries.

Particle processing on the gill plicae of the oyster Crassostrea gigas: fine-scale mucocyte distribution and functional correlates

In order to resolve outstanding problems in the understanding of mechanisms of parti- cle processing in the oyster gill, the technique of mucocyte mapping was used to quantify mucocyte types and fine-scale distributions on the frontal ciliated tracts of the ordinary filaments comprising the gill plicae. The frontal tracts were divided into 1 frontal median tract (FMT) and 2 frontal lateral tracts (FLT) per filament, based on relative cilia lengths. Mucocyte counts within each tract were per- formed on periodic acid Schiff (PAS)-alcian blue-stained histological sections under oil immersion, and densities were recorded per linear µm (l µm) of tract width, after evaluation of artefactual shrink- age for exact dimensions of tracts. Total mucocyte volumes were obtained using mean mucocyte vol- umes. Two types of mucocytes were most frequently observed: acid mucopolysaccharide (AMPS) containing mucocytes, and mixed (acid + neutral) mucopolysaccharide (MMPS) containing muco- cytes. Neutral mucopolysaccharide (NMPS) containing mucocytes were rare. The apical filaments contained over twice the mucocyte densities and total mucocyte volumes, compared to the lateral filaments of the plicae (p ≤ 0.001), indicating that the apical filaments are the principal particle processing site of the plicae. The lateral filaments may accomplish functions more related to the establishment of the plicae and ensuing consequences (hydrodynamic particle capture, physical sep- aration of the dorsal and ventral gill particle groove functions, and enlargement of filtering area). On the apical filaments, the FMT showed much greater AMPS containing mucocyte densities and total mucocyte volumes, compared to the FLT. This, together with the respective cilia length differences, indicates that the FMT is the principal particle transport tract. Such a specialization was not observed on the lateral filaments of the plicae, again supporting the conclusion that the apical filaments are the active site of particle processing on the plicae. FLT appear to be uninvolved in particle transport, and probably participate in the establishment and maintenance of the dorsalward current flow in the pallial cavity.

Temporal variability of Cryptosporidium in the Chesapeake Bay.

Although Cryptosporidium has been found worldwide in molluscan shellfish from waters contaminated with human and animal feces, little or no related environmental data have been obtained. In the present study, oysters ( Crassostrea virginica) were collected eight times over 3 years from seven sites in the Chesapeake Bay or its tributaries, with accompanying data on water temperature, salinity, rainfall, and streamflow. Oyster gill washings were examined by immunofluorescence microscopy for Cryptosporidium oocysts. Of 1,590 oysters collected, 19.6% had detectable oocysts. Of 53 collections, oocysts were detected 81% of the time. The time when the greatest percentage of oysters at most sites had detectable oocysts coincided with the time of greatest weekly and monthly rainfall, greatest streamflow into the Bay, and lowest water temperatures. In 28% of 53 collections, C. parvum genotypes 1 and 2 and C. baileyi were identified by PCR and gene sequencing. Oocyst infectivity was confirmed from 37.5% of 40 collections by initiating C. parvum genotype 2 infections in mice.

Kinetic and toxicological characteristics of acetylcholinesterase from the gills of oysters (Crassostrea rhizophorae) and other aquatic species

The aim of this work was to characterize the cholinesterases from gills of Crassostrea rhizophorae in order to use them as biomarkers. Gills were homogenized and then centrifuged (9000×g, 4 °C, 30 min). S9 and Triton X-100 S9 treated (TX S9) fractions were employed as enzyme source. Kmap and Vmax were estimated, using acetylthiocholine iodide as substrate. Inhibition assays were performed with iso-OMPA and eserine. The Kmap for S9 and TX S9 fractions were 0.05 and 0.06 mM, whereas the Vmax were 1.92 and 5.84 nmol/min/mg protein, respectively. No inhibition was detected when the samples were incubated with iso-OMPA, suggesting the presence of acetylcholinesterases (AChE) in oyster gill homogenates. Sensitivity to eserine inhibition of AChE in the gills of oysters is intermediate when compared with other aquatic species.

Influence of metal exposure on metallothionein synthesis and lipid peroxidation in two bivalve mollusks: the oyster ( Crassostrea gigas) and the mussel ( Mytilus edulis)

The impact of metals (silver, cadmium, copper, mercury and zinc) on metallothionein (MT) and malondialdehyde (MDA) levels of the oyster ( Crassostrea gigas) and the mussel ( Mytilus edulis) was studied after 4 or 21 days of metal exposure. Moreover, total protein levels were determined. After 4 days of metal exposure, although C. gigas and M. edulis accumulated cadmium and mercury concentrations in the gills and digestive gland, no significant variation of total protein level was occurred. After 21 days of exposure, metals were bioaccumulated in the gills and the digestive gland of both mollusks. A decrease of total protein concentrations in the gills of oysters and the digestive gland of mussels and an increase on metallothionein concentrations in the gills of both mollusks were observed. An increase of MDA levels was noticed for the gills and the digestive gland of mussels exposed for 21 days to either cadmium, silver or mercury whereas a decrease of MDA levels was observed in the gills of the oysters exposed for the same time to the same metals. The levels of proteins, MDA and MT were metal, species or organ dependent.

Use of metallothionein in gills from oysters (Crassostrea gigas) as a biomarker: seasonal and intersite fluctuations

The use of oyster gills for the analytical determination of metallothionein (MT) concentration as a biomarker of metal exposure was investigated. Temporal variations in MT and metal concentrations (which can interfere with inter-site differences) were examined over a 7 month period (from spring to autumn) in Japanese oysters from a clean site (Bay of Bourgneuf, France) and a metal-rich site (Gironde estuary, France) as well as in individuals translocated from the clean to the contaminated area. The ratio between the annual average of MT concentrations in specimens from the clean and the metal-rich sites was 1.3. During the last 3 months of the experiment, significant differences were no longer registered between transplants and residents from the Gironde estuary. Metals concentrations in oyster gills differed consistently between the clean and the metal-rich sites (annual average ratios of 1.5, 2.7 and 9.8, respectively, for zinc, copper and cadmium) and a fast increase in metal concentrations (over a few months) was observed in transplants, mainly for cadmium. MT and soluble metal concentrations were found to be positively and significantly correlated over the period of the study. This relationship is a positive argument for a possible use of gill MT concentration as a biomarker of metal pollution in contrast to previous findings on the digestive gland, there being a smaller amount of seasonal variability in the weight of oyster gills.

Expression of P-glycoprotein in the gills of oysters, Crassostrea virginica: seasonal and pollutant related effects

The expression of p-glycoprotein (p-gp) in aquatic organisms has been proposed as a biomarker of pollution exposure. Previous research has provided evidence that p-gp is inducible by organic xenobiotics and that p-gp is overexpressed in mussels from degraded areas. However, seasonal changes in expression at polluted sites has not been described previously. The purposes of these studies were to evaluate the expression of p-gp in polluted and unpolluted sites in oysters ( Crassostrea virginica) and to determine if there were seasonal differences. P-gp expression and total protein concentrations were measured seasonally in the gills of southeastern oysters, Crassostrea virginica, at degraded and undegraded sites in Charleston Harbor, South Carolina, USA. At all sites, p-gp expression was generally higher during the warmer months and lower in the colder months. Polluted sites tended to show a decrease in p-gp expression in June and September, suggesting that p-gp inhibition may have occurred. P-gp expression was not significantly related to specific classes of sediment contaminants or to overall sediment contaminant loading. Total gill protein concentrations at all sites were lower during the warmer months and higher during the colder months. In general, all sites tended to show decreased total gill protein concentrations when compared to the control site. Total protein concentrations were significantly related to water temperature, specific classes of sediment contaminants and overall sediment contaminant loads, but there was no relationship with salinity.

Environmental bacteriophage-host interactions: factors contribution to natural transduction.

Over the past two decades the potential for the exchange of bacterial genes in natural environments through transduction (bacteriophage-mediated gene transfer) has been well established. Studies carried out by various laboratories throughout the world have demonstrated that both chromosomal and plasmid DNA can be successfully transduced in natural environments ranging from sewer plants to rivers and lakes. Transduction has been shown to take place in the gills of oysters and the kidneys of mice. Model studies have demonstrated the ability of transduction to maintain genetic material in bacterial gene pools that would otherwise be lost because of negative fitness. Thus, transduction may affect the course of bacterial evolution. Identification of natural transduction has led to the investigation of the dynamics of bacteriophage host interactions in natural aquatic environments and to the exploration of various environmental factors that affect virus-host interactions. Two important environmental factors which affect virus-host interactions are the metabolic state of the host and the exposure of the host to DNA-damaging stresses such as solar UV light. Recent researches on these two areas of virus-host relationships are reviewed.

Utilisation des métallothionéines comme biomarqueur de la contamination métallique : variabilité entre sites et organes chez l'huı̂tre Crassostrea gigas

The use of metallothioneins as biomarker of metal contamination: variability between sites and organs in the oyster Crassostrea gigas . Heavy metals (Cd, Cu, Hg and Zn) and metallothionein levels were determined in oysters Crassostrea gigas from two sites: the Gironde Estuary (contaminated site) and Bourgneuf Bay (uncontaminated site). Metallothionein levels led us to suspect the presence of cadmium in the environment when quantified in the gills. Soluble cadmium and metallothioneins were positively correlated in the digestive gland of oysters from Gironde (contaminated site), but metallothionein levels in this organ were not significantly different from levels observed in the digestive gland of oysters from Bourgneuf Bay (uncontaminated site). Conversely, no correlation was noticed between metals and metallothioneins in the gills of oysters from Gironde despite metallothionein levels significantly higher than levels observed in oyster gills from Bourgneuf Bay. This significant difference of metallothionein levels is attributed to a higher ratio of soluble metals (Cd, Cu, Zn) in the gills of oysters from Gironde. The generalization of the use of metallothioneins as biomarkers of metal contamination of aquatic environments is questioned. © 2000 Ifremer/CNRS/IRD/Éditions scientifiques et médicales Elsevier SAS metallothioneins / metals / Crassostrea gigas / biomarker

Changes in the Electrophoretic Profiles of Gill Mucus Proteases of the Eastern Oyster Crassostrea virginica in Response to Infection by the Turbellarian Urastoma cyprinae

Urastoma cyprinae occurs on the gills of various bivalves species, including the eastern oyster Crassostrea virginica. While the worm is known to cause severe gill disruption in mussels, no evidence of this nature has been described for oysters. Nonetheless, high levels of U. cyprinae have been reported in oysters, which may, in turn, reduce the oyster's overall condition. U. cyprinae is strongly attracted to oyster gill mucus, which is suggested to play an active role in the worm's feeding activities. Furthermore, host mucus contains many active components, including proteases, which have been suggested to play a defensive role against invading organisms. It follows, therefore, that some of the interactions between U. cyprinae and oysters take place in host gill mucus. Studies were undertaken to determine whether the presence of U. cyprinae altered the electrophoretic profiles of oyster gill mucus, using proteases as indicators. Findings reveal that oyster gill mucus contains three proteases, a putative acid protease at 96 kDa, a zinc metalloprotease at 64 kDa, and a serine protease at 33 kDa. Results based on experiments using mucus preparations extracted from infected and noninfected oysters, along with those using lyophilized mucus incubated with live U. cyprinae, confirm that the presence of U. cyprinae alters the protease composition of gill mucus. The present data demonstrate that both U. cyprinae and host gill mucus actively secrete proteases. While the precise roles of these enzymes still need to be defined, one of their functions may be associated with digestion-related activities induced by the worm.

Feeding rate of the oyster Crassostrea gigas in a natural planktonic community of the Mediterranean Thau Lagoon

The Mediterranean Thau Lagoon is an important European oyster farming area. Oyster growth levels are among the highest in France, although chlorophyll a concentration is low. Previous studies have demonstrated that picophytoplankton, nano-microphytoplankton, dinoflagellates and loricate ciliates such as tintinnids are abundant in the Thau Lagoon. Moreover, heterotrophic flagellates and aloricate ciliates have not been investigated. The aim of this study was to assess picophytoplankton, protist and zooplankton abundances in water columns of the Thau Lagoon and to understand the particular structure of the Thau microbial food web, which may explain such a paradoxical oyster growth. In oligotrophic waters in the Thau Lagoon, the picoeukaryote Ostreococcus tauri is the dominant autotrophic picoplankter with a maximum Summer abundance. On 17 August 1998, the picophytoplankton and nanophytoplankton abundances were not as high as expected and we observed the development of large diatoms. At this time, available carbon resources arose from microphytoplankton (84.5 %) and picoplanktonic cells represented only 1.27 % in terms of carbon. The heterotrophic cells were few in abundance and constitued only 5 μm flagellates, microphytoplankton, dinoflagellates, ciliates and large zooplankton were retained by the oyster gills. Only < 5 μm flagellates and picoeukaryotic cells, Ostreococcus tauri, were not very well retained (45 % and 2 %). The high clearance rates of Crassostrea gigas found in this experiment can be explained by a low suspended particulate matter (0.65 mg l-1). Oysters adapted their retention mechanism when they lived in oligotrophic waters. These results indicate that, under the given experimental conditions, picophytoplankton did not represent a valuable trophic resource for farmed oysters because (1) Crassostrea can not retain picoparticles and (2) the picoplankton represented a poor available carbon resource to be transferred via a weak heterotrophic protist community. In the oyster pends of the Thau Lagoon during this study, which followed a rainfall event, microphytoplanktonic primary producers, in particular diatoms, could be considered as the main food sources for bivalve suspension feeders.