Exposure to toxic Alexandrium minutum activates the detoxifying and antioxidant systems in gills of the oyster Crassostrea gigas.

Harmful algal blooms of Alexandrium spp. dinoflagellates regularly occur in French coastal waters contaminating shellfish. Studies have demonstrated that toxic Alexandrium spp. disrupt behavioural and physiological processes in marine filter-feeders, but molecular modifications triggered by phycotoxins are less well understood. This study analyzed the mRNA levels of 7 genes encoding antioxidant/detoxifying enzymes in gills of Pacific oysters (Crassostrea gigas) exposed to a cultured, toxic strain of A. minutum, a producer of paralytic shellfish toxins (PST) or fed Tisochrysis lutea (T. lutea, formerly Isochrysis sp., clone Tahitian (T. iso)), a non-toxic control diet, in four repeated experiments. Transcript levels of sigma-class glutathione S-transferase (GST), glutathione reductase (GR) and ferritin (Fer) were significantly higher in oysters exposed to A. minutum compared to oysters fed T. lutea. The detoxification pathway based upon glutathione (GSH)-conjugation of toxic compounds (phase II) is likely activated, and catalyzed by GST. This system appeared to be activated in gills probably for the detoxification of PST and/or extra-cellular compounds, produced by A. minutum. GST, GR and Fer can also contribute to antioxidant functions to prevent cellular damage from increased reactive oxygen species (ROS) originating either from A. minutum cells directly, from oyster hemocytes during immune response, or from other gill cells as by-products of detoxification.

Cellular and biochemical responses of the oyster Crassostrea gigas to controlled exposures to metals and Alexandrium minutum.

Effects of simultaneous exposure of Pacific oyster, Crassostrea gigas, to both a harmful dinoflagellate that produces Paralytic Shellfish Toxins (PST), Alexandrium minutum, and cadmium (Cd) and copper (Cu), were assessed. Oysters were exposed to a mix of Cd-Cu with two different diets (i.e. A. minutum or Tisochrysis lutea) and compared to control oysters fed A. minutum or T. lutea, respectively, without metal addition. Metals and PST accumulations, digestive gland lipid composition, and cellular and biochemical hemolymph variables were measured after 4 days of exposure. Oysters exposed to Cd-Cu accumulated about thirty-six times less PSTs than oysters exposed to A. minutum alone. Exposure to Cd-Cu induced significant changes in neutral lipids (increase in diacylglycerol - DAG - and decrease in sterols) and phospholipids (decreases in phosphatidylcholine, phosphatidylethanolamine, cardiolipin and ceramide aminoethylphosphonate) of digestive gland suggesting that lipid metabolism disruptions and/or lipid peroxidation have occurred. Simultaneously, concentrations, percentages of dead cells and phenoloxidase activity of hemocytes increased in oysters exposed to metals while reactive oxygen species production of hemocytes decreased. Feeding on the harmful dinoflagellate A. minutum resulted in significant decreases in monoacylglycerol (MAG) and DAG and ether glycerides (EG), as well as significant increases in hemocyte concentration and phagocytic activity as compared to oysters fed T. lutea. Finally, the present study revealed that short-term, simultaneous exposure to Cd-Cu and A. minutum may induce antagonistic (i.e. hemocyte concentration and phagocytosis) or synergic (i.e. DAG content in digestive gland) effects upon cellular and tissular functions in oysters.

Genetic and genotoxic impacts in the oyster Crassostrea gigas exposed to the harmful alga Alexandrium minutum.

Genotoxic, genetic and behavioral impacts of the paralytic shellfish toxin (PST)-producing alga Alexandrium minutum on the oyster Crassostrea gigas were assessed using RAPD-PCR, qPCR and valve activity recording. Oysters were exposed to a dose mimicking an algal bloom (≈1600 cells ml(-1)) for 48 h. Results indicate a rapid and sustained behavioral disturbance. Animals remained open but exhibited reduced valve-opening amplitude, correlated to the amount of toxin accumulated in the digestive gland. They also exhibited increased micro-closures. In the gills, gene transcription levels were modified: a transcriptional repression of genes involved in oxidative and mitochondrial metabolism, endogenous clock, immunity and detoxification processes was observed. DNA impacts, both quantitative and qualitative, were observed as well. Indeed, both the PCR product profile and the number of hybridization sites for the RAPD probe OPB7 were modified. These results indicate genotoxic effects and gene repression in C. gigas following behavioral disturbance by A. minutum.

An integrated environmental approach to investigate biomarker fluctuations in the blue mussel Mytilus edulis L. in the Vilaine estuary, France.

Estuarine areas represent complex and highly changing environments at the interface between freshwater and marine aquatic ecosystems. Therefore, the aquatic organisms living in estuaries have to face highly variable environmental conditions. The aim of this work was to study the influence of environmental changes from either natural or anthropogenic origins on the physiological responses of Mytilus edulis. Mussels were collected in the Vilaine estuary during early summer because this season represents a critical period of active reproduction in mussels and of increased anthropogenic inputs from agricultural and boating activities into the estuary. The physiological status of the mussel M. edulis was evaluated through measurements of a suite of biomarkers related to: oxidative stress (catalase, malondialdehyde), detoxication (benzopyrene hydroxylase, carboxylesterase), neurotoxicity (acetylcholinesterase), reproductive cycle (vitelline, condition index, maturation stages), immunotoxicity (hemocyte concentration, granulocyte percentage, phagocytosis, reactive oxygen species production, oxidative burst), and general physiological stress (lysosomal stability). A selection of relevant organic contaminant (pesticides, (polycyclic aromatic hydrocarbons, polychlorobiphenyls) was measured as well as environmental parameters (water temperature, salinity, total suspended solids, turbidity, chlorophyll a, pheopigments) and mussel phycotoxin contamination. Two locations differently exposed to the plume of the Vilaine River were compared. Both temporal and inter-site variations of these biomarkers were studied. Our results show that reproduction cycle and environmental parameters such as temperature, organic ontaminants, and algal blooms could strongly influence the biomarker responses. These observations highlight the necessity to conduct integrated environmental approaches in order to better understand the causes of biomarker variations.

Influence of Crassostrea gigas (Thunberg) sexual maturation stage and ploidy on uptake of paralytic phycotoxins.

The purpose of this study was to assess paralytic phycotoxin uptake in diploid and triploid oysters at two stages of their sexual cycle corresponding to their status in early summer (June) and winter (November). Samples of diploid and triploid oysters were exposed to a toxic culture of Alexandrium minutum for 4 days in each season. No significant differences in filtration or clearance rates were observed during either November or June experiments. When diploid oysters were at resting stage (November), toxin uptake showed no significant difference between the ploidy classes. In contrast, when the diploid oysters were at the peak of their sexual maturation (June), the triploid oysters were seen to accumulate almost double the amount of paralytic toxins as the diploid ones.

A new insight into allelopathic effects of Alexandrium minutum on photosynthesis and respiration of the diatom Chaetoceros neogracile revealed by photosynthetic-performance analysis and flow cytometry.

The allelopathic effects of Alexandrium minutum, a toxic dinoflagellate, on the diatom Chaetoceros neogracile were evaluated using unialgal cultures evaluated by flow cytometry (FCM) and photosynthetic-performance analysis. Using FCM, we demonstrated that red chlorophyll fluorescence, relative cell size (Forward scatter of blue laser light, FSC) and cell complexity (Side scatter, 90°-angle scatter of blue laser light, SSC) significantly and rapidly decreased in C. neogracile cells exposed to A. minutum. Cells of C. neogracile exposed to A. minutum had fewer active photosynthetic reaction centers and sharply decreased photosynthetic efficiency. These effects were intensified with advancing A. minutum batch culture age and cell density. The supernatant of A. minutum contained the majority of the putative allelopathic compounds, and the biological activity of these compounds remained active less than 9 h after release. This paper describes for the first time specific effects of allelochemicals produced by A. minutum on the photosynthetic apparatus of microalgal target cells. The biochemical composition of A. minutum allelopathic agents, however, remains unknown and still needs to be investigated.

Relationship between valve activity, microalgae concentration in the water and toxin accumulation in the digestive gland of the Pacific oyster Crassostrea gigas exposed to Alexandrium minutum.

The complexity of the relationships between Alexandrium minutum (A.m.) concentration in the water ([A.m.](w)), Paralytic Shellfish Poisoning contamination in the digestive gland ([PSP](dg)) and valve behavior was explored in oysters Crassostrea gigas. Two experiments were conducted, during which oysters' valve behaviour were analyzed. Oysters, first acclimated for 10-days with the non harmful microalgae Heterocapsa triquetra (H.t.), were exposed to four microalgae mixtures at constant total concentrations of 10×10(3)cells ml(-1) (experiment-1) and 5×10(3)cells ml(-1) (experiment-2): 100% A.m.; 50% A.m.-50% H.t.; 25% A.m.-75% H.t.; 100% H.t. At the end of experiment-2, [PSP](dg) were measured. At 10×10(3)cells ml(-1), the microalgal ingestion decreased (p<0.05) with increasing [A.m.](w) but not at 5×10(3)cells ml(-1) (p>0.05). The frequency of microclosures specifically increased with [A.m.](w) (p<0.05) and the opening duration with [PSP](dg) (p<0.0001). Oysters exhibiting the maximum increase in opening duration also exhibited the highest [PSP](dg). The results are discussed in terms of oyster physiology and origin of the behavioral response.

In vitro interactions between several species of harmful algae and haemocytes of bivalve molluscs.

Harmful algal blooms (HABs) can have both lethal and sublethal impacts on shellfish. To understand the possible roles of haemocytes in bivalve immune responses to HABs and how the algae are affected by these cells (haemocytes), in vitro tests between cultured harmful algal species and haemocytes of the northern quahog (= hard clam) Mercenaria mercenaria, the soft-shell clam Mya arenaria, the eastern and Pacific oysters Crassostrea virginica and Crassostrea gigas and the Manila clam Ruditapes philippinarum were carried out. Within their respective ranges of distribution, these shellfish species can experience blooms of several HAB species, including Prorocentrum minimum, Heterosigma akashiwo, Alexandrium fundyense, Alexandrium minutum and Karenia spp.; thus, these algal species were chosen for testing. Possible differences in haemocyte variables attributable to harmful algae and also effects of haemolymph and haemocytes on the algae themselves were measured. Using microscopic and flow cytometric observations, changes were measured in haemocytes, including cell morphology, mortality, phagocytosis, adhesion and reactive oxygen species (ROS) production, as well as changes in the physiology and the characteristics of the algal cells, including mortality, size, internal complexity and chlorophyll fluorescence. These experiments suggest different effects of the several species of harmful algae upon bivalve haemocytes. Some harmful algae act as immunostimulants, whereas others are immunosuppressive. P. minimum appears to activate haemocytes, but the other harmful algal species tested seem to cause a suppression of immune functions, generally consisting of decreases in phagocytosis, production of ROS and cell adhesion and besides cause an increase in the percentage of dead haemocytes, which could be attributable to the action of chemical toxins. Microalgal cells exposed to shellfish haemolymph generally showed evidence of algal degradation, e.g. loss of chlorophyll fluorescence and modification of cell shape. Thus, in vitro tests allow a better understanding of the role of the haemocytes and the haemolymph in the defence mechanisms protecting molluscan shellfish from harmful algal cells and could also be further developed to estimate the effects of HABs on bivalve molluscs in vivo.

Effects of Alexandrium minutum exposure upon physiological and hematological variables of diploid and triploid oysters, Crassostrea gigas.

The effects of an artificial bloom of the toxin-producing dinoflagellate, Alexandrium minutum, upon physiological parameters of the Pacific oyster, Crassostrea gigas, were assessed. Diploid and triploid oysters were exposed to cultured A. minutum and compared to control diploid and triploid oysters fed T. Isochrysis. Experiments were repeated twice, in April and mid-May 2007, to investigate effects of maturation stage on oyster responses to A. minutum exposure. Oyster maturation stage, Paralytic Shellfish Toxin (PST) accumulation, as well as several digestive gland and hematological variables, were assessed at the ends of the exposures. In both experiments, triploid oysters accumulated more PSTs (approximately twice) than diploid oysters. Significant differences, in terms of phenoloxidase activity (PO) and reactive oxygen species (ROS) production of hemocytes, were observed between A. minutum-exposed and non-exposed oysters. PO in hemocytes was lower in oysters exposed to A. minutum than in control oysters in an early maturation stage (diploids and triploids in April experiment and triploids in May experiment), but this contrast was reversed in ripe oysters (diploids in May experiment). In the April experiment, granulocytes of oysters exposed to A. minutum produced more ROS than those of control oysters; however, in the May experiment, ROS production of granulocytes was lower in A. minutum-exposed oysters. Moreover, significant decreases in free fatty acid, monoacylglycerol, and diacylglycerol contents in digestive glands of oysters exposed to A. minutum were observed. Concurrently, the ratio of reserve lipids (triacylglycerol, ether glycerides and sterol esters) to structural lipids (sterols) decreased upon A. minutum exposure in both experiments. Also, several physiological responses to A. minutum exposure appeared to be modulated by maturation stage as well as ploidy of the oysters.

Comparative study of three analysis methods (TTGE, flow cytometry and HPLC) for xenobiotic impact assessment on phytoplankton communities.

The impacts of the fungicide Opus (epoxiconazole) on marine phytoplankton communities were assessed in a 12-day field experiment using in situ microcosms maintained underwater at 6 m depth. Three community analysis methods were compared for their sensitivity threshold in fungicide impact detection. When phytoplankton communities were exposed to 1 microg l(-1) of epoxiconazole, no effects could be demonstrated using TTGE (Temporal Temperature Gradient gel Electrophoresis), flow cytometry or HPLC. When exposed to 10 microg l(-1), TTGE fingerprints from PCR amplified 18S rDNA of communities exhibited significant differences compared with controls (ANOSIM, P = 0.028). Neither flow cytometry counts, nor HPLC pigment profiles allowed to show significant differences in microcosms exposed to 10 microg l(-1) of epoxiconazole. When exposed to 100 microg l(-1), all three methods allowed to detect significant differences in treated microcosms, as compared to controls. The TTGE analysis appears in this study as the most sensitive method for fungicide impact assessment on eukaryote microbial communities.

Impact of Roundup on the marine microbial community, as shown by an in situ microcosm experiment.

The effects of the herbicide Roundup (glyphosate) on natural marine microbial communities were assessed in a 7-day field experiment using microcosms. Bottles were maintained underwater at 6m depth, and 10% of their water content was changed every other day. The comparison of control microcosms and surrounding surface water showed that the microcosm system tested here can be considered as representative of the natural surrounding environment. A temporal temperature gradient gel electrophoresis (TTGE) was run on 16S and 18S rDNA-amplified extracts from the whole microbial community. Cluster analysis of the 16S gel showed differences between control and treatment fingerprints for Roundup at 1 microg L(-1) (ANOSIM, p=0.055; R=0.53), and 10 microg L(-1) (ANOSIM, p=0.086; R=0.40). Flow cytometry analysis revealed a significant increase in the prasinophyte-like population when Roundup concentration was increased to 10 microg L(-1). This study demonstrates that a disturbance was caused to the marine microbial community exposed to 1 microg L(-1) Roundup concentration, a value typical of those reported in coastal waters during a run-off event.