Two methods were used to estimate the reproductive output of female Pacific oysters reared in Normandy: histology with image analysis and ELISA (Enzyme-Linked ImmunoSorbent Assay) which allowed the quantification of egg protein. Condition indices, gonad area and gametogenetic stages of the oysters were determined in the entire population (males and females) between May and October 2005. All investigations were performed in 3 age classes: oysters in their first, second or third years (corresponding to spat, half-grown and market-sized oysters, respectively). Both quantitative histology and ELISA provided similar results in terms of reproductive effort (illustrated by the gonado-somatic index, GSI) except during the GSI drop, corresponding to spawning, which was less marked with the ELISA method. Growth depended on oyster age, the sex ratio was well balanced and the reproductive cycle was synchronized in all age classes. In the 3 age classes, most of the oysters were ripe and ready to spawn on August 8, and ten days after the post-spawning stage was observed in 40% of spat oysters and 70% of half-grown and market-sized bivalves. The major difference between age classes was observed in the reproductive investment, with spat having a lower reproductive output. For example, in males and females, the gonad area reached 78-79% in the median animal section at full maturity (August 8) in half-grown and marketable oysters while it attained only 59% in spat. At the same time, GSI in females was, respectively in spat and the 2 oldest age classes, 33% (quantitative histology)-36% (ELISA) and 55% (quantitative histology)-60% (ELISA). The mean assessed gonad weight and fecundities increased with the age of the oysters: 1.3 g and 12 million eggs, 7.8 g and 135 million eggs, and 11.5 g and 146 million eggs in spat, half-grown and market-sized oysters, respectively. Marked differences thus appear between 2 and 3-year-old oysters and spat. As early as their first reproductive cycle, the young oysters not only showed the reproductive features of the species in Normandy, but also a pronounced lower reproductive effort. This lower energy demand could explain their higher survival rate.

Arachidonic acid (20:4n-6, ArA) and its eicosanoid metabolites have been demonstrated to be implicated in immune functions of vertebrates, fish, and insects. Thus, the aim of this study was to assess the impact of ArA supplementation on the FA composition and hemocyte parameters of oysters Crassostrea gigas. Oyster dietary conditioning consisted of direct addition of ArA solutions at a dose of 0, 0.25, or 0.41 microg ArA per mL of seawater into tanks in the presence or absence of T-Iso algae. Results showed significant incorporation of ArA into gill polar lipids when administered with algae (up to 19.7%) or without algae (up to 12.1%). ArA supplementation led to an increase in hemocyte numbers, phagocytosis, and production of reactive oxygen species by hemocytes from ArA-supplemented oysters. Moreover, the inhibitory effect of Vibrio aestuarianus extracellular products on the adhesive proprieties of hemocytes was lessened in oysters fed ArA-supplemented T-Iso. All changes in oyster hemocyte parameters reported in the present study suggest that ArA and/or eicosanoid metabolites affect oyster hemocyte functions.

Brest harbor (Bay of Brest, Brittany, France) has a severe past of anthropogenic chemical contamination, but inputs tended to decrease, indicating a reassessment of its ecotoxicological status should be carried out. Here, native and caged mussels (Mytilus spp.) were used in combination to evaluate biological effects of chronic chemical contamination in Brest harbor. Polycyclic aromatic hydrocarbon (PAH) contamination was measured in mussel tissues as a proxy of harbor and urban pollution. Biochemical biomarkers of xenobiotic biotransformation, antioxidant defenses, generation of reducing equivalents, energy metabolism and oxidative damage were studied in both gills and digestive glands of native and caged mussels. In particular, activities of glutathione-S-transferase (GST), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), NADP-dependent isocitrate dehydrogenase (IDP), pyruvate kinase (PK) and phosphoenolpyruvate carboxykinase (PEPCK) were measured and lipid peroxidation was assessed by malondialdehyde (MDA) quantification. In addition, a condition index was calculated to assess the overall health of the mussels. Moderate PAH contamination was detected in digestive glands of both native and caged individuals from the exposed site. Modulations of biomarkers were detected in digestive glands of native harbor mussels indicating the presence of a chemical pressure. In particular, results suggested increased biotransformation (GST), antioxidant defenses (CAT), NADPH generation (IDP) and gluconeogenesis (PEPCK), which could represent a coordinated response against chemically-induced cellular stress. Lipid peroxidation assessment and condition index indicated an absence of acute stress in the same mussels suggesting metabolic changes could, at least partially, offset the negative effects of contamination. In caged mussels, only GR was found modulated compared to non-exposed mussels but significant differences in oxidative stress and energy-related biomarkers were observed compared to native harbor mussels. Overall, these results suggested mussels chronically exposed to contamination have set up metabolic adaptation, which may contribute to their survival in the moderately contaminated harbor of Brest. Whether these adaptive traits result from phenotypic plasticity or genetic adaptation needs to be further investigated.

Manila clams, Venerupis philippinarum (Adams and Reeve, 1850), were experimentally challenged with two Vibrio tapetis strains: CECT4600T, the causative agent of Brown Ring Disease (BRD); and LP2 supposedly non-pathogenic in V. philippinarum. Changes in phenoloxidase (PO) and superoxide dismutase (SOD), two major enzymes involved in immunity, were studied in two tissues, the mantle and hemolymph for 30 days after infection in the extrapallial cavity. Bacterial infection in V. philippinarum resulted in modulation of PO and SOD activities that was both tissue- and time-dependent. A response at early times was detected in the mantle and was associated with significant increases in PO and SOD activities in LP2- and CECT4600T-challenged clams 36 h post injection. This first response in the mantle could be explained by the proximity to the injection region (extrapallial cavity). In the hemolymph the response occurred at later times and was associated with an increase in PO activity and a decrease in SOD activity. As hemolymph is a circulating fluid, this response delay could be due to an "integration time" needed by the organism to counteract the infection. Injections also impacted PO and SOD activities in both tissues and confirmed a difference in pathogenicity between the two V. tapetis strains.

-Sessile animals that live on the foreshore undergo tidal cycles, and have to face variations in physical and chemical parameters such as oxygen concentration. During emersion, availability of dissolved oxygen can be lowered for bivalves, which have only a small reserve of seawater inside their closed shell. Differences in oxygen concentration are thus expected to lead to modifications of the metabolism, including changes in mitochondrial activity. Previous studies investigated air exposure under extreme conditions, which do not always reflect environmental conditions these invertebrates have to cope with. In this study, oxidative capacities of gill mitochondria of the oyster Crassostrea gigas were studied during a tidal cycle period, by comparing oysters collected after emersion and immersion. Only minor differences were found in state 3 (oxidative phosphorylation) or state 4 (non-phosphorylating oxygen consumption) rates between the two conditions. Similarly, no difference was observed in cytochrome c oxidase activity or in oxygen consumption related to maximal electron flux through complexes I-IV, II-IV and IV. While capacities of substrate oxidation were maintained in both emersion and immersion conditions, capacity of mitochondria to produce adenosine triphosphate (ATP) was significantly lower in oysters sampled during emersion. These results suggest that although C. gigas could maintain aerobic metabolism during emersion period within a tidal cycle in its environment, energy producing mechanisms are affected.

Vitamins were analysed in food (microalgae) and larvae of great scallop, Pecten maximus, during larval development. Microalgae used to feed larvae in hatcheries show great variability in their vitamin composition depending on both the species and culture condition (phase of growth). The microalgae used to feed Pecten maximus larvae were rich in vitamins; their content compared with diets used in fish culture appeared sufficient, with the possible exceptions of pyridoxine, biotin and pantothenic acid. Vitamins in bacteria, isolated from the larval rearing tank were also analysed, as they can also contribute to the diet. Vitamin B12, tz-tocopherol and t~-carotene were detected in very low concentration in bacteria; however, some bacterial strains were rich in pantothenic acid, and the pattern of other vitamins was similar to that from microalgae. The presence of bacteria can complement the diet in panthothenic acid, as it has been demonstrated that bacteria are ingested by larvae. The vitamin content of Pecten maximus larvae was analysed from the second day after hatching to just before metamorphosis. The content of some vitamins, ascorbic acid (C), ~x-tocopherol and l~-carotene, increased during larval development, suggesting that their requirement was satisfied. However, thiamin and riboflavin decreased during larval development and further studies, possibly using microencapsulated vitamins supplements, are needed to determine whether these vitamins are limiting during larval development.

The present study tested two techniques for dietary supplementation of Crassostrea gigas spat with PUFA, such as arachidonic acid (AA). The first technique consisted of a preliminary enrichment and growth of an algal concentrate (T-ISO, Isochrysis sp.) with AA dissolved in an ethanol solution, the whole culture then being fed to the spat. This enrichment increased the AA weight percentage in T-ISO neutral and polar lipids from 0.6 to 22.4% and from 0.4 to 6.8%, respectively. The second delivery technique was direct addition separately of free AA dissolved in ethanol solution and algal concentrate (T-ISO + AA) to the spat-rearing tank. To test the efficiency of these delivery techniques, oyster spat were supplemented with AA-enriched T-ISO, T-ISO + AA, and T-ISO alone. The possible biological impacts of these dietary treatments were assessed by measuring growth, condition index, and TAG content of oyster spat. Dry weight and condition index of spat fed AA-enriched T-ISO decreased by 24 and 49%, respectively, after 26 d of feeding; basically, TAG content declined 88% after 34 d of conditioning. When AA was added directly to seawater, spat growth and condition index were comparable with those of oysters fed T-ISO alone. AA incorporation in oyster tissues was assessed by analysis of the FA compositions in both neutral and polar lipid fractions. After 34 d, AA content in neutral lipids reached 7 and 11.7% in the spat fed, respectively, AA-enriched T-ISO and T-ISO + AA, as compared with 1.1% in spat fed only T-ISO. AA incorporation was greater in polar lipids than in neutral lipids, reaching 7.8 and 12.5% in spat fed AA-enriched T-ISO and T-ISO + AA, respectively. A direct addition of PUFA along with the food supply represents an effective and promising means to supplement PUFA to oyster spat.

In a previous study, dietary supplementation with arachidonic acid (ARA) to oysters Crassostrea gigas increased haemocyte numbers, phagocytosis, and production of reactive oxygen species level (ROS) by haemocytes (Delaporte et al., 2006). To assess if the observed stimulation of these cellular responses resulted from changes of ARA-related prostaglandin (PG) production, we analysed prostaglandin E2 metabolite (PGEM) content on the same oysters fed three levels of ARA. Dietary supply of polyunsaturated fatty acids (PUFA) could also induce an oxidative stress that could similarly increase cellular responses; therefore, two indicators of oxidative stress were analysed: peroxidation level and antioxidant defence status. Together the observed positive correlation between ARA and PGEM levels and the absence of lipid peroxidation and antioxidant activity changes supports the hypothesis of an immune stimulation via PG synthesis. Although ARA proportion in oyster tissues increased by up to 7-fold in response to ARA dietary supplementation, peroxidation index did not change because of a compensatory decrease in n-3 fatty acid proportion, mainly 22:6n-3. To further confirm the involvement of PG in the changes of haemocyte count, phagocytosis and ROS production upon ARA supplementation, it would be interesting to test cyclooxygenase and lipooxygenase inhibitors in similar experiments.