Pesticides represent a major proportion of the chemical pollutants detected in French coastal waters and hence a significant environmental risk with regards to marine organisms. Commercially-raised bivalves are particularly exposed to pollutants, among them pesticides, as shellfish farming zones are subject to considerable pressure from agricultural activities on the mainland. The aims of this study were to determine (1) the genotoxic effects of diuron exposure on oyster genitors and (2) the possible transmission of damaged DNA to offspring and its repercussions on oyster fitness. To investigate these points, oysters were exposed to concentrations of diuron close to those detected in the Marennes-Oleron Basin (two 7-day exposure pulses at 0.4 and 0.6 μg L(-1)) during the gametogenesis period. Genomic abnormalities were characterized using two complementary approaches. The Comet assay was applied for the measurement of early and reversible primary DNA damage, whereas flow cytometry was used to assess the clastogenic and aneugenic effect of diuron exposure. Polar Organic Chemical Integrative Samplers (POCIS) were used in exposed and assay tanks to confirm the waterborne concentration of diuron reached during the experiment. The results obtained by the Comet assay clearly showed a higher level of DNA strand breaks in both the hemocytes and spermatozoa of diuron-exposed genitors. The transmission of damaged genetic material to gamete cells could be responsible for the genetic damage measured in offspring. Indeed, flow cytometry analyses showed the presence of DNA breakage and a significant decrease in DNA content in spat from diuron-exposed genitors. The transmission of DNA damage to the offspring could be involved in the negative effects observed on offspring development (decrease in hatching rate, higher level of larval abnormalities, delay in metamorphosis) and growth. In this study, the vertical transmission of DNA damage was so highlighted by subjecting oyster genitors to short exposures to diuron at medium environmental concentrations. The analysis of POCIS showed that oysters were exposed to integrated concentrations as low as 0.2 and 0.3 μg L(-1), emphasizing the relevance of the results obtained and the risk associated to chemical contamination for oyster recruitment and fitness.

The work presented here reports the expression of immune-related genes during ontogenesis in the oyster Crassostrea gigas. Expression patterns of 18 selected genes showed that RNAs detected in oocytes and 2-4 cell embryos are of maternal origin and that gene transcription starts early after fertilization. The expression patterns of 4 genes (Cg-timp, Cg-tal, Cg-EcSOD and Drac3) suggested that hemocytes appear in the gastrula-trochophore stages. The localization of Cg-tal expression suggested that hematopoietic cells were derived from vessels and/or artery endothelia cells. Moreover, a bacterial challenge affected the level of expression of genes. Indeed, a change in expression levels was observed for Cg-LBP/BPI, Cg-timp, Drac3 and Cg-MyD88 genes in larval stages upon exposure to non-pathogenic bacteria. In early juveniles, a modulation was also observed for Cg-LBP/BPI, Cg-timp, Cg-MyD88 and for Cg-tal, according to the concentration of bacteria. Altogether, the results showed that studying the appearance of immunocompetent cells through their ability to express immune-related genes is a tool to gain insight the ontogenesis of the oyster immune system.

ABSTRACT. Vibriosis is a major disease problem in shrimp aquaculture. 'Syndrome 93' is a seasonal juvenile vibriosis caused by Vibrio penaeicida which affects Litopenaeus stylirostris in grow-out ponds in New Caledonia. This study assessed the toxic activities of extracellular products (ECPs) from V. penaeicida, l/. alginolyticus and V, nigripulchritudo using in vivo injections in healthy juvenile L. stylirostris (= Penaeus styljrostns) and in vitro assays on shrimp primary cell cultures and the fish cell line epithelioma papulosum cyprini (EPC). Toxic effects of ECPs were demonstrated for all pathogenic Vibrio strains tested both in vivo and in vitro, but for shrimp only; no effect was observed on the fish cell line. ECP toxicity for New Caledonian V. penaeicida was found only after cultivation at low temperature (20°C) and not at higher temperature (30°C). This points to the fact that 'Syndrome 93' episodes are triggered by temperature drops. The assays used here demonstrate the usefulness of primary shrimp cell cultures to study virulence mechanisms of shrimp pathogenic bacteria.

Changes in sperm features during the movement phase are especially interesting to study in external fertilization species whose sperm duration movement is long because this implies a significant adaptation of moving cells to the external medium. This study describes the changes in tetraploid Pacific oyster sperm characteristics in relation to time post activation. Sperm individually collected on three tetraploid males were activated in seawater. Their features were analysed over a 24h period and compared to a sperm pool collected on three diploid males as a reference. The percentage of motile spermatozoa, the intracellular ATP content, and the fine structure of spermatozoa were studied in relation to time post activation. Furthermore, the fertilisation capacity of sperm individually collected on five diploid males was assessed after 1 and 24h post activation. A forward progressive movement was maintained for at least a 20h duration. Compared to diploid males, the percentage of motile spermatozoa was lower in tetraploid males. The intracellular ATP concentration was higher in spermatozoa from tetraploid males than in spermatozoa from diploid males. A decrease in ATP content was observed in the first 6h post activation and severe alterations were observed in sperm morphology after 24h. Then, a lower fertilisation capacity of sperm from diploid males was observed at the end of the movement phase. The cessation of Pacific oyster sperm motility was unlikely caused by ATP consumption as ATP concentration was still high at the end of sperm movement but rather caused by drastic changes in sperm morphology. Compared to sperm collected on diploid males, the lower quality of sperm from tetraploid males was emphasized by a shorter movement duration and deeper morphological alterations at the end of the movement phase.

Vibriosis is a major disease problem in shrimp aquaculture, affecting all developmental stages, from larvae in hatchery tanks to juveniles and broodstock in growout ponds. However, bacterial strains responsible for vibriosis in the successive stages are usually considered to be different, and virulence specificity has been reported both at the species and at the stage levels. The so-called « Syndrome 93 » is a seasonal juvenile vibriosis caused by Vibrio penaeicida which affects Penaeus stylirostris in growout ponds and broodstock tanks in New Caledonia. This pathology does not cause any mortality in hatchery or nursery phases. An experimental infection design using balneation of postlarvae and early juveniles in V. penaeicida suspensions was used to evaluate the developmental stage at which shrimp become sensitive to this vibriosis. We demonstrated that the acquisition of susceptibility to this pathogen is very sudden and correlated with the acquisition of the definitive rostral formula, and from this draw conclusions regarding virulence mechanisms of V. penaeicida in P. stylirostris.