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International audience; The purpose of the present study was to provide a flow-cytometric (FCM) approach evaluating A. minutum cellular responses to mechanical, thermal and chemical stresses. Coupling SYBR-Green I and SYTOX-Green staining, FCM analysis and sorting, and microscopic observations permitted identification and characterization of five cellular states/forms of A. minutum; 1) vegetative cells, 2) pellicle cysts, 3) degraded cells, 4) empty theca and 5) dead cells. Storage on ice resulted in the transformation of a portion of vegetative cells into dead cells, pellicle cysts, and "degraded" cells; however, centrifugation resulted mostly in pellicle cysts and a few degraded cells. After these thermal and mechanical treatments, control and stressed cultures were monitored for 14 days. Stressed A. minutum cultures appeared to grow at the same rates as control cultures during the first seven days. During the last week of monitoring, however, cell densities of stressed cultures reached their stationary phase earlier than control cultures, suggesting incomplete recovery. Additional experiment assessing kinetics of excystment indicated that it can occur less than 9 hours following mechanical stress (centrifugation) and that 75% of the culture can excyst within 24 hours. Upon 30 min of exposure to chemical stressors (saponine and H2O2), only vegetative cells, pellicle cysts, and dead cells were detected. For both chemicals, encystment was dose-dependent. Counts of pellicle cysts increased with increasing saponine or H2O2 concentration. Pellicle cysts were the most-predominant cell type at chemical treatments above 0.05 g.L-1 saponine and above 0.015% H2O2. Occurrence of dead cells appeared to follow an all-or-none response as dead-cell percentage increased from 3% at 0.015% H2O2 to 81% at 0.03% H2O2 without pellicle cyst formation. Overall, encystment-excystment of A. minutum upon changes of environmental conditions can occur very rapidly but can be monitored using FCM and SYBR-Green I staining.

International audience; Sydney rock oysters (SRO) Saccostrea glomerata suffer mass mortalities during summer and autumn as a result of infection by a protozoan parasite Marteilia sydneyi (QX disease). Mass selected disease resistant (QXR) lines have been used with some success in affected estuaries in recent years, with resistance attributed to oxidative defense systems. However, the role of hemocytes in resistance to QX by SRO has not been fully explored. In the present study, fifty QXR and fifty wild caught (WC) oysters were collected from a lease at Pimpama River during a QX outbreak in January 2011. Hemocytes characteristics (type, morphology) and functions (mortality, phagocytosis and oxidative activity) from both oyster lines were analyzed by flow cytometry in the context of infection intensity and parasite viability (determined histologically). Amongst the QXR oysters, 20% were diseased containing viable parasite, 74% had killed M. sydneyi and 6% were uninfected. In contrast, 86% of WC oysters were diseased, 2% had killed M. sydneyi and 12% were healthy. Significant differences in hemocyte number and physiology between the two oyster lines were found (ANOVA). Phagocytosis rate and the mean oxidative activity per cell were similar between both oyster lines. Higher numbers of infiltrating and circulating hemocytes, higher percentage of circulating granulocytes, their higher size and complexity in QXR oysters, and the production of reactive oxygen species were associated with the ability to kill the parasite. High abundance of M. sydneyi in the digestive tubule epithelium of both oyster lines implied inability to kill the parasite at the beginning of the infection. However, QXR oysters had the ability to kill M. sydneyi at the stage of sporangiosorae in the epithelium of digestive tubules. The similar phagocytic ability of hemocytes from both oyster lines, the size of the parasite at this infection stage, and its localization suggested that encapsulation is likely to be the main process involved in the eradication of M. sydneyi by QXR oysters.

International audience; Most experimental procedures on molluscs are done after acclimatization of wild animals to lab conditions. Similarly, short-term acclimation is often unavoidable in a field survey when biological analysis cannot be done within the day of sample collection. However, acclimatization can affect the general physiological condition and particularly the immune cell responses of molluscs. Our aim was to study the changes in the hemocyte characteristics of the Pacific oyster Crassostrea gigas and the carpet shell clam Ruditapes decussatus acclimated 1 or 2 days under emersed conditions at 14 +/- 1 degrees C and for 1, 2, 7, or 10 days to flowing seawater conditions (submerged) at 9 +/- 1 degrees C, when compared to hemolymph withdrawn from organisms sampled in the field and immediately analyzed in the laboratory (unacclimated). The hemocyte characteristics assessed by flow cytometry were the total (THC) and differential hemocyte count, percentage of dead cells, phagocytosis, and reactive oxygen species (ROS) production. Dead hemocytes were lower in oysters acclimated both in emersed and submerged conditions (1%-5%) compared to those sampled in the field (7%). Compared to oysters, the percentage of dead hemocytes was lower in clams (0.4% vs. 1.1%) and showed a tendency to decrease during acclimatization in both emersed and submerged conditions. In comparison to organisms not acclimated, the phagocytosis of hemocytes decreased in both oysters and clams acclimated under submerged conditions, but was similar in those acclimated in emersed conditions. The ROS production remained stable in both oysters and clams acclimated in emersed conditions, whereas in submerged conditions ROS production did not change in both the hyalinocytes and granulocytes of oysters, but increased in clams. In oysters, the THC decreased when they were acclimated 1 and 2 days in submerged conditions and was mainly caused by a decrease in granulocytes, but the decrease in THC in oysters acclimated 2 days in emersed conditions was caused by a decrease in hyalinocytes and small agranular cells. In clams, the THC was significantly lower in comparison to those not acclimated, regardless of the conditions of the acclimatization. These findings demonstrate that hemocyte characteristics were differentially affected in both species by the tested conditions of acclimatization. The phagocytosis and ROS production in clams and phagocytosis in oysters were not different in those acclimated for 1 day under both conditions, i.e. emersed and submerged, and those sampled in the field (unacclimated). The THC was significantly affected by acclimatization conditions, so the differences between clams and oysters should be considered in studies where important concentrations of hemocytes are required. The difference in the immune response between both species could be related to their habitat (epifaunal vs. infaunal) and their ability of resilience to manipulation and adaptation to captivity. Our results suggest that functional characteristics of hemocytes should be analyzed in both oysters and clams during the first 1 or 2 days, preferably acclimated under emersed rather than submerged conditions.

Growth and spirolide production of the toxic dinoflagellate Alexandrium ostenfeldii (Danish strain CCMP1773) were studied in batch culture and a photobioreactor (continuous cultures). First, batch cultures were grown in 450 mL flasks without aeration and under varying conditions of temperature (16 and 22 degrees C) and culture medium (L1, f/2 and L1 with addition of soil extract). Second, cultures were grown at 16 degrees C in 8 L aerated flat-bottomed vessels using L1 with soil extract as culture medium. Finally, continuous cultures in a photobioreactor were conducted at 18 degrees C in L1 with soil extract; pH was maintained at 8.5 and continuous stirring was applied. This study showed that A. ostenfeldii growth was significantly affected by temperature. At the end of the exponential phase, maximum cell concentration and cell diameter were significantly higher at 16 degrees C than at 22 C. In batch culture, maximum spirolide quota per cell (approx. 5 pg SPX 13-desMeC eq cell(-1)) was detected during lag phase for all conditions used. Spirolide quota per cell was negatively and significantly correlated to cell concentration according to the following equation: y = 4013.9x(-0.858). Temperature and culture medium affected the spirolide profile which was characterized by the dominance of 13,19-didesMeC (29-46%), followed by SPX-D (21-28%), 13-desMeC (21-23%), and 13-desMeD (17-21%). Stable growth of A. ostenfeldii was maintained in a photobioreactor over two months, with maximum cell concentration of 7 x 10(4) cells mL(-1). As in batch culture, maximum spirolide cell quota was found in lag phase and then decreased significantly throughout the exponential phase. Spirolide cell quota was negatively and significantly correlated to cell concentration according to the equation: y = 12,858x(-0.8986). In photobioreactor, spirolide profile was characterized by higher proportion of 13,19-didesMeC (60-87%) and lower proportions of SPX-D (3-12%) and 13-desMeD (1.6-10%) as compared to batch culture. (C) 2011 Elsevier B.V. All rights reserved.

International audience; The detailed sterol (free sterol proportions and compositions) and phospholipid (PL) compositions (relative proportions of PL classes and subclasses and their respective fatty acid (FA) compositions) of hemocyte membranes were investigated in two bivalve mollusks: the Pacific oyster Crassostrea gigas and the Manila clam Ruditapes philippinarum. Hemocyte membrane lipids of both species revealed similar general composition: i) their free sterol/PL ratio was above 0.4 and ii) their PL were predominated by the diacyl + alkyl forms of glycerophosphatidylcholine (PC), the plasmalogen form of glycerophosphatidylethanolamine (PE) and ceramide aminoethylphosphonate (CAEP). Free sterols were predominated by cholesterol in both species. Plasmalogen forms of PE and glycerophosphatidylserine (PS) represented 82-83% and 46-55% of total PE and PS, respectively. When compared to their respective diacyl + alkyl forms, plasmalogen forms of PE and PS were specifically enriched in non-methylene-interrupted (NMI) FA and 20:1n-11, suggesting a functional significance of these PL molecular species in bivalve hemocytes. Lysoglycerophosphatidylcholine (LysoPC) levels were found to be fairly high in hemocytes, accounting for about 8% of the PL Some species-specific features were also found. LysoPC and glycerophosphatidylinositol (PI) FA compositions differed between Ruditapes philippinarum and Crassostrea gigas. CAEP proportion was higher in R. philippinarum than in C. gigas (14.5% and 27.9% of the PL respectively). Hemolymph cell monolayer observations and flow-cytometric analyses revealed species-specific hemocyte morphology and sub-populations which could account for some of the observed species-specific membrane lipid compositions.

International audience; 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.

International audience; 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.

This study assessed the effects of an artificial bloom of the toxin-producing dinoflagellate, Alexandrium minutum, upon nutrition related processes and reproductive output of the Pacific oyster, Crassostrea gigas. Oysters were exposed to A. minutum, Paralytic Shellfish Toxins (PST) producer and compared to a control batch of oysters fed Isochrysis galbana clone Tahitian (T.Iso). The experiment was performed in June 2008, when oysters were found ripe. Several physiological variables of oysters, such as PSTs accumulation, digestive gland and histological observations as well as spermatozoa quality, were measured at the end of the exposure. Results indicate that the digestive gland was greatly impacted upon A. minutum exposure. Monoacylglycerol and diacylglycerol contents as well as phospholipids (mainly phosphatidylcholine) drastically decreased in the digestive gland of oysters exposed to A. minutum compared to control oysters. At the same time, many oysters exposed to the harmful microalga presented a strong inflammatory response in different tissues of the digestive gland: in intestine as well as in digestive ducts and tubules. Spermatozoa in oysters exposed to A. minutum were morphologically and functionally modified compared to spermatozoa of control oysters. Indeed, spermatozoa were less motile and had lower ATP content in oysters exposed to A. minutum. Meanwhile, spermatozoa produced by control oysters showed higher percentage of mortality and relative DNA content than those produced by A. minutum exposed oysters. Finally, the characteristics of the mitochondria of spermatozoa also appeared to be modified upon A. minutum exposure. The results of this study suggests that an exposure of oysters to A. minutum, reducing energy status and motility of spermatozoa associated to morphological changes at the cellular and sub-cellular levels, can have consequences on spermatozoa fertility and reproduction success. (C) 2010 Elsevier B.V. All rights reserved.

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. (C) 2009 Elsevier B.V. All rights reserved.

International audience; Two feeding systems for maturing oysters were compared, one a continuous feeding system and the other a batch system in which the whole microalgal ration was supplied once daily. The maturation diet consisted in Isochrysis galbana (T-ISO) complemented with an enriched lipid emulsion. Survival and growth did not differ between the feeding systems after 3 weeks of conditioning. Maturation, biochemical composition, fatty acids in membranes and reserves, digestive enzymes activities and immune parameters in Crassostrea corteziensis were analysed. Only oysters fed using the once-daily system had vitellogenic oocytes, whereas the gonad of oysters fed using a continuous-drip system remained immature. Total and differential haemocyte counts were similar between both the systems, but respiratory burst was significantly higher in oysters fed using the once-daily system. Amylase, lipase and trypsin activities in oyster's digestive gland were similar between both the feeding systems. Total lipids, however, differed significantly in oyster tissue in relation to feeding system, with highest level in those fed using the once-daily system, but fatty acid composition in reserves and membrane were similar. No differences were found for biochemical parameters in haemolymph. These results suggest that feeding oysters using a batch, once-daily system allows more rapid initial gonad maturation without affecting general physiological condition and growth.

International audience; The present report presents the first evidence of disseminated neoplasia (DN) in cockles Cerastoderma edule from Arcachon Bay (France). Aneuploidy of neoplastic cells allowed the use of flow cytometry (FCM) to diagnose and stage DN. A 1 year survey (2007) of the prevalence and intensity (% of aneuploid circulating cells in neoplastic cockles) was conducted. Prevalences ranged from 2.2% (June) to 13.6% (May), and disease intensity ranged from 18.7% (June) to 95.5% (September). These percentages were not correlated with seawater temperature, but rather showed unexplained oscillations over the year. Prevalence and intensity of DN were higher in cockles found at the surface of sediment compared to those buried normally (11.8% vs. 6.7% and 53.0% vs. 40.6%, respectively, p < 0.05). DN could thus be one mechanism leading to unexplained presence of cockles at the surface of the sediment in Arcachon Bay. Ploidy characteristics of neoplastic cells were also investigated using FCM, revealing an unusual, broad continuum of ploidy distribution from 1.6 to 9.6n. Ploidy values were not in whole numbers in contrast to the rounded values reported in other studies. Ploidy varied according to DN intensity, with the ploidy distribution of neoplastic cells from lightly-diseased cockles being unimodal (3.7n median). In contrast, highly-diseased cockles showed a bimodal ploidy distribution (3.0n and 4.7n medians). This suggests that, in cockles from Arcachon Bay, mechanisms leading to aneuploidy are complex, developing during disease progression.

International audience; Introduction Within the last decade, numerous studies have investigated the role of environmental history on tolerance to stress of many organisms. This study aims to assess if Manila clams Ruditapes philippinarum may react differently to cadmium exposure and trematode parasite infection (Himasthla elongata) depending on their origin and environmental history in Arcachon Bay (France). Materials and methods Clams were exposed to Cd (15 µg L−1) and parasites (25 cercariae per clam), alone or in combination, at 15°C under controlled laboratory conditions for 7 days. Metal accumulation and success of parasite infestation were examined, also physiological parameters such as metallothionein response and hemocyte counts and activities (phagocytosis, oxidative burst, viability, and adhesion). Results and Discussion Sensitivity of Manila clams to both stressors differed from one site to another, suggesting local adaptation of populations. Clams from the more parasitized site presented better resistance to trematodes than the others in terms of first line defense, i.e., avoidance of infection. On the other hand, clams that adapted to chronic Cd contamination showed better detoxification mechanisms, both in a faster transfer of metal from gills to visceral mass and in a higher metallothionein baseline, than clams which had never experienced Cd contamination. Finally, hemocyte concentration and viability differed between clam origin site, highlighting the fact that populations living in different environments may adapt their physiological and biochemical responses to environmental stressors. Conclusion It is therefore important to be cautious when extrapolating results from field studies of one species and one site, if the life history of the organisms is not taken into account.

International audience; Hemocytes are the immune effectors in clams, directing cellular but also humoral defences. In the Manila clam (Ruditapes philippinarum) and the carpet shell clam (Ruditapes decussatus), two phenotypically similar venerid species, fishery and aquaculture importance motivated scientific studies to improve knowledge about the participation of hemocytes in immune response. Currently, gaps still persist concerning the classification of hemocyte sub-populations as well as their non-immune activities. This review summarizes current knowledge about hemocytes of the Manila clam and the carpet shell clam, suggests some probable misinterpretations arising from extrapolations from other bivalves and vertebrates, and suggests future research prospects. The susceptibility of hemocytes to environmental modifications as well as biological and chemical toxicants is also re-evaluated. Hemocytes of bivalves are not limited in function to immunity and should be considered in a wider scheme including the environment and whole-animal physiology.

International audience; Thirty-five species of marine mollusk bivalves were analyzed for their fatty acid (FA) composition of cardiolipin (Ptd(2)Gro). All species showed a Ptd(2)Gro with strong selectivity for only a few polyunsaturated fatty acids, but three characteristic FA profiles emerged, with clear parallels to bivalve phylogeny. A first group of 12 species belonging to the Eupteriomorphia subgroup (Filibranchia) was characterized by a Ptd(2)Gro almost exclusively composed of 22:6n-3, whereas in the four Filibranchia Pteriomorph species analyzed, this FA was combined with substantial proportions of 18:2n-6 and 18:3n-3. Finally, a third group of 20 species, all belonging to the Heterodonta subclass, possessed Ptd(2)Gro containing predominantly both 22:6n-3 and 20:5n-3. Polyunsaturated FA moieties and arrangements in the Ptd(2)Gro of some marine species investigated in other classes of the mollusk phylum (Gastropoda, Polyplacophora) were found to be different. The present results suggest that the specific Ptd(2)Gro FA compositions in bivalves are likely to be controlled and conserved in species of the same phylogenetic group. Functional significances of the evolution of this mitochondrial lipid structure in bivalves are discussed.

The present study evaluated the possible effects of a toxic dinoflagellate, Karenia selliformis , upon immunological hemocyte functions of the Manila clam Ruditapes philippinarum , and on the progression of infection by Perkinsus olseni . Clams with variable levels of perkinsosis were exposed for 6 weeks to simulated blooms of cultured the K. selliformis (10 2 and 10 3 cell ml −1 ). Samples were collected after 0, 2, 3, and 6 weeks of exposure. The following hemocyte parameters were measured by flow cytometry: percentage of dead cells, cell size and complexity, apoptosis, phagocytosis, and production of reactive oxygen species. Agglutination activities of K. selliformis on horse erythrocytes, serum protein concentration, and condition index of clams were also assessed. The harmful alga K. selliformis caused a significant decrease in hemocyte size and percentage of apoptotic cells. In contrast, P. olseni did not affect clams strongly; the only significant effect was an increase in hemocyte size in heavily infected clams. After 2 and 3 weeks, the prevalence and burden of P. olseni decreased in clams exposed to K. selliformis , but after 6 weeks, and a diminution in K. selliformis cell density in the exposure, this effect disappeared. In vitro tests exposing P. olseni to K. selliformis showed direct algal toxicity to the parasite (increased percentage of dead cells and altered morphology). Initial exposure of P. olseni -infected clams to K. selliformis appeared to modify the host–parasite interaction by causing effects in both organisms.

International audience; The kinetics and efficiency of sterol production and bioconversion of phytosterols in two heterotrophic protists Oxyrrhis marina and Gyrodinium dominans were examined by feeding them two different algal species (Rhodomonas salina and Dunaliella tertiolecta) differing in sterol profiles. R. salina contains predominantly brassicasterol (≅99%) and

Within the framework of a national scientific program named "MORtalites ESTivales de l'huitre creuse Crassostrea gigas" (MOREST), a family-based experiment was developed to study the genetic basis of resistance to summer mortality in the Pacific oyster, Crassostrea gigas. As part of the MOREST project, the second generation of three resistant families and two susceptible families were chosen and pooled into two respective groups: "R" and "S". These two groups of oysters were conditioned for 6 months on two food levels (4% and 12% of oyster soft-tissue dry weight in algal dry weight per day) with a temperature gradient that mimicked the Marennes-Oleron natural cycle during the oyster reproductive period. Oyster mortality remained low for the first two months, but then rapidly increased in July when seawater temperature reached 19 degrees C and above. Mortality was higher in "S" oysters than in "R" oysters, and also higher in oysters fed the 12% diet than those fed 4%, resulting in a decreasing, relative order in cumulative mortality as follows; 12% "S" > 12% "R" >4% "S" >4% "R". Although the observed mortality rates were lower than those previously observed in the field, the mortality differential between "R" and "S" oysters was similar. Gonadal development, estimated by tissue lipid content, followed a relative order yielding a direct, positive relationship between reproductive effort and mortality as we reported precedently by quantitative histology. Regarding hemocyte parameters, one of the most striking observations was that reactive oxygen species (ROS) production was significantly higher in "S" oysters than in "R" oysters in May and June, regardless of food level. The absence of known environmental stress under these experimental conditions suggests that the ROS increase in "S" oyster could be related to their higher reproductive activity. Finally, a higher increase in hyalinocyte counts was observed for "S" oysters, compared to "R" oysters, in July, just before mortality. Taken together, our results suggest an association of genetically based resistance to summer mortality, reproductive strategy and hemocyte parameters. (C) 2007 Elsevier B.V. All rights reserved.

Bivalve hemocyte competence has been measured by quantifying functional characteristics, including reactive oxygen intermediate (ROI) production after activation with zymosan or phorbol myristate acetate (PMA). However, untreated oyster hemocytes also produce ROI and RNI (reactive nitrogen intermediates) after bleeding even if not stimulated by Zymosan or PMA. Extensive investigation of this parameter by flow cytometry showed that, in vitro, ROI/RNI production by untreated hemocytes maintained in seawater appeared to be independent of both bacterial burden in the serum and non-self particle phagocytosis. ROI/ RNI production in granulocytes was higher than in hyalinocytes and could be intensified when activated by zymosan but not by PMA. Both cell types used NADPH-oxidase- and NO-synthase-like pathways to produce these molecules; the NO-synthase pathway seemed relatively more dominant in hyalinocytes and NADPH-oxidase appeared more effective in granulocytes. These results provide new insights for interpreting the modulation of ROI/RNl production by untreated hemocytes shown by other studies, relative to environmental conditions or physiological status of the oysters. (c) 2007 Elsevier B.V. All rights reserved.

The aim of this study was to assess the effect of dietary eicosapentaenoic acid (20:5n-3) on hemocyte parameters such as hemocyte concentration, phagocytosis, and non-stimulated reactive oxygen species (ROS) production in Pacific oysters Crassostrea gigas, as well on proximate biochemical and fatty acid compositions. One-year-old oysters (C. gigas) were fed T-Isochrysis aff. galbana(T-Iso), which is low in 20:5n-3, either alone or with supplements of a lipid emulsion rich in 20:5n-3 at 1%, 10% or 50% (dry weight of the algal ration) for up to 7 weeks. Changes in gill fatty acid composition demonstrated that the lipid emulsion was well ingested by oysters during the dietary conditioning. Biochemical analysis indicated that oysters fed supplements of 50% and, to a lesser extent, 10% lipid emulsions had a higher total lipid content compared with oysters fed other diets, suggesting a more advanced reproductive status for the oysters fed high doses of lipid emulsion. Moreover, some oysters in these two treatment groups spawned during the last three weeks of the seven-week feeding experiment. Lipid supplements had a significant influence on hemocyte concentration, phagocytic index and non-stimulated hemocyte ROS production. After 4 weeks, highest hemocyte concentrations were found in oysters fed on a supplement of 50% lipid emulsion compared with those fed on other diets but the hemocytes derived from these oysters had the lowest short-term phagocytic index. After 7 weeks of dietary conditioning, the ROS production in non-stimulated hemocytes of oysters fed 10% and 50% lipid emulsion declined. These results suggested that 20:5n-3, and perhaps its eicosanoid metabolites, affected oyster hemocyte functions; however, the reproductive status of oysters may also have interfered with the 20:5n-3 dietary effect. (c) 2007 Elsevier B.V. All rights reserved.

Perkinsus marinus, a protozoan parasite of the Eastern oyster Crassostrea virginica, has severely impacted oyster populations from the Mid-Atlantic region to the Gulf of Mexico coast of North America for more than 30 yr. Although a chemotherapeutic treatment to reduce or eliminate P. marinus from infected oysters would be useful for research and hatchery operations, an effective and practical drug treatment does not currently exist. In this study, the antimicrobial drug triclosan 5-chloro-2-(2,4 dichlorophenoxy) phenol, a specific inhibitor of Fab1 (enoyl-acyl-carrier-protein reductase), an enzyme in the Type II class of fatty acid synthetases, was tested for its effects on viability, proliferation and fatty acid synthesis of in vitro-cultured P. marinus meronts. Treatment of P. marinus meront cell cultures with concentrations ofor = 2 microM triclosan at 28 degrees C (a temperature favorable for parasite proliferation) for up to 6 d stopped proliferation of the parasite. Treatment ator = 5 microM at 28 degrees C greatly reduced the viability and fatty acid synthesis of meront cells. Oyster hemocytes treated withor = 20 microM triclosan exhibited no significant (p0.05) reduction in viability relative to controls for up to 24 h at 13 degrees C. P. marinus meronts exposed toor = 2 microM triclosan for 24 h at 13 degrees C exhibited significantly (p0.05) lower viability relative to controls. Exposure of P. marinus meronts to triclosan concentrations ofor = 20 microM resulted in50% mortality of P. marinus cells after 24 h. These results suggest that triclosan may be effective in treating P. marinus-infected oysters.

International audience; The distribution of cis-4,7,10,trans-13-docosate-traenoic (c4,7,10,t13-22∶4), a peculiar FA previously isolated in the glycerophospholipids of some pectinid bivalves, was investigated in glycerophospholipid classes and subclasses of separated organs (gills, mantle, gonads, and muscle) of the queen scallop Aequipecten opercularis and the king scallop Pecten maximus. Plasmalogen (Pls) and diacyl + alkyl (Ptd) forms of serine, ethanolamine, and choline glycerophospholipids were isolated by HPLC and their FA compositions analyzed by GC-FID. Pls and Ptd forms of serine glycerophospholipids (PlsSer and PtdSer), and to a lesser extend the Pls form of ethanolamine glycerophospholipids (PlsEtn), were found to be specifically enriched with c4,7,10,t13-22∶4. This specificity was found to decrease in the tested organs in the following order: gills, mantle, gonad, and muscle. In gills, c4,7,10,t13-22∶4 was shown to be the main unsaturated FA of serine glycerophospholipids in both Pls and Ptd forms (23.8 and 19.4 mol%, respectively, for A. opercularis, and 21.0 and 26.2 mol% for P. maximus). These results represent the first comprehensive report on the FA composition of plasmalogen serine subclass isolated from pectinid bivalves. The specific association of the PlsSer with the c4,7,10,t13-22∶4 for the two pectinid species can be paralleled to the specific association of the PlsSer with the non-methylene interrupted (NMI) FA and 20∶1(n−11) observed in mussels, clams, and oysters (Kraffe, E., Soudant, P., and Marty, Y. (2004) Fatty Acids of Serine, Ethanolamine and Choline Plasmalogens in Some Marine Bivalves, Lipids 39, 59-66.) This, led us to hypothesize a similar functional significance for c4,7,10,t13-22∶4, NMI FA, and 20∶1(n−11) associated with PlsSer subclass of bivalves.

The strategies used by bacterial pathogens to circumvent host defense mechanisms remain largely undefined in bivalve molluscs. In this study, we investigated experimentally the interactions between the Pacific oyster (Crassostrea gigas) immune system and Vibrio aestuarianus strain 01/32, a pathogenic bacterium originally isolated from moribund oysters. First, an antibiotic-resistant V. aestuarianus strain was used to demonstrate that only a limited number of bacterial cells was detected in the host circulatory system, suggesting that the bacteria may localize in some organs. Second, we examined the host defense responses to V. aestuarianus at the cellular and molecular levels, using flow-cytometry and real-time PCR techniques. We showed that hemocyte phagocytosis and adhesive capabilities were affected during the course of infection. Our results also uncovered a previously-undescribed mechanism used by a Vibrio in the initial stages of host interaction: deregulation of the hemocyte oxidative metabolism by enhancing the production of reactive oxygen species and down-regulating superoxide dismutase (Cg-EcSOD) gene expression. This deregulation may provide an opportunity to the pathogen by impairing hemocyte functions and survival. These findings provide new insights into the cellular and molecular bases of the host-pathogen interactions in C. gigas oyster. (c) 2006 Elsevier Masson SAS. All rights reserved.

Le texte intégral est accessible via Archimer: http://archimer.ifremer.fr/doc/2006/publication-1549.pdf; International audience; The aim of this study was to test the effect of food quantity on energy storage and defense capacities of oysters during a reproductive cycle. One-year-old Crassostrea gigas oysters were fed two different dietary rations (4% and 12% of oyster dry weight in algal dry weight per day) in controlled experimental conditions over an annual cycle. Oyster dry weights, carbohydrate and lipid contents, energetic adenylate charge, and hemocyte parameters of oysters were significantly affected by reproductive processes related to seasonal temperature variation and, to a lesser extent, by the dietary rations. Energy parameters decreased during gametogenesis as gonads developed then increased during the gonad resorption phase. The additional energy provided to oysters fed the 12% diet compared to oysters fed the 4% diet was allocated mainly to the development of more gonad tissue. Regardless of diet, hemocyte concentrations were also seasonally affected. Hemocyte concentrations were low during gametogenesis and significantly increased during the gonadal resorption and tissue restoration phase. Phagocytic activity and adhesive capacity of hemocytes were temporarily inhibited during gametogenesis and were at their lowest levels in June. Oysters fed the 12% diet had significantly higher hemocyte concentrations and lower phagocytosis activity and reactive oxygen species production compared to those fed the 4% diet.

International audience; 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 mu g 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.

International audience; Epizootiological outbreaks of disseminated neoplasia (DN) have been reported in association with mass mortalities in various bivalve species including the cockle Cerastoderma edule. A flow cytometric (FCM) procedure to study DNA content was successfully adapted and tested in haemolymph cells (haemocytes and neoplastic cells) of the cockle. The FCM results were similar to those obtained by histological analysis (DN diagnosis and haemolymph cell features). FCM analysis revealed differences in DNA content among normal haemocytes (diploid) and neoplastic cells. Four types of cells with abnormal DNA content were found in the haemolymph of affected animals: hypodiploid, hyperdiploid, triploid-sesploid and pentaploid. Our results suggest that the flow cytometric DNA content analysis can be applied to identify neoplastic cell types and to study the association between different cell types and the DN progression or remission in this edible and commercially important bivalve species.

International audience; 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 cl, 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.

International audience; Juvenile clams were distributed in four rearing sites selected for their varied ecological characteristics to assess the effects of environmental conditions on the physiological and immunological parameters, and Brown Ring Disease (BRD) status. Clams were sampled every 3 months for 15 months at each site. Brittany rearing sites, especially the Bay of Brest, showed the worst performances in terms of immunological and physiological indices and disease status, while the best were obtained in Marennes ponds. When the health of the clams was compared to assess seasonal effects, the winter clearly was a stressful period. A combination of bad rearing site and winter conditions led to major mortalities in the Bay of Brest in February. In other sites, winter mortalities were low. Condition index, total haemocyte count and haemocyte size were greatly affected by seasonal variation whereas haemocyte complexity and lysozyme content were more affected by the location of the site. Growth and haemolymph protein content were affected by both season and sites. Linear regressions between and within the physiological and immunological parameters indicated that large haemocyte size was related to low total haemocyte count (THC) and low haemocyte mortality. This relationship suggests a reduction in the cell division rate. Total haemocyte count and protein and lysozyme concentrations were positively correlated to the condition index. Seeded clams showed very low BRD prevalence in all sites and for all seasons; however, high prevalence was observed in natural stocks from one of the studied sites (Gulf of Morbihan), suggesting that hatchery-seeded clams may be more resistant to BRD and may be worthy of further studies.

The aim of this workshop was to review current issues posed by perkinsosis in Europe with particular emphasis on taxonomv. epidemiology and impact an mollusc stocks, and identify where new developments in research are likely to improve our knowledge and understandings.

International audience; A flow cytometric method to measure the production of oxidative metabolism products was adapted for use with Crassostrea gigas hemocytes. The method is based upon the oxidation, by hydrogen peroxide (H2O2), of intracellular 2',7'-dichlorofluorescin (DCFH) to green-fluorescent dichlorofluorescein. Activation of the respiratory burst (RB) was tested using phorbol myristate acetate with no success. By contrast, activation by zymosan particles increased oxidation of DCFH in C. gigas hemocytes, mainly granulocytes, and optimization tests showed a good response with 20 zymosan particles per hemocyte. Anti-aggregant solution, used to prevent hemocytes from clumping during bleeding, inhibited the RB activity measured by DCFH oxidation. The flow cytometric method developed during this work was used to evaluate the DCFH oxidation-inhibiting capacity of four strains of vibrio bacteria, known or suspected to be pathogenic for bivalves.

The occurrence of brown ring disease (BRD) in farmed Manila clams Ruditapes philippinarum is seasonal. Development of the disease is believed to require the presence of the infective agent Vibrio tapetis and particular environmental conditions. This paper studies the effect of salinity (20 to 40 per thousand) on measurable immune parameters of Manila clams, and the progression of BRD in experimentally infected individuals. At 20 per thousand salinity, the total haemocyte count was reduced and disease prevalence was highest. At 40 per thousand salinity significantly fewer clams presented signs of BRD, and this was correlated with increases in the total haemocyte count, hyalinocyte count, phenoloxidase levels and phagocytic activity of haemocytes. Inoculation of clams with V. tapetis did not have a significant effect on the immune parameters measured. Thus, this laboratory-based study relates environmental stress to disease development.

Reproductively active oysters were fed daily with 0.2 g algal paste containing 0, 0.1, and 1.0 microgram polychlorinated biphenyls (PCBs) (1:1:1 mixture of Aroclor 1242, 1254 and 1260) for either 15 or 30 days, and accumulation of PCBs in different organ tissues and eggs assessed. The effects of PCB exposure on lipid content, lipid class and fatty acid composition were also evaluated. PCBs were accumulated by the oysters and transferred to the eggs. PCB accumulation in oysters was dose, time and tissue dependent. Mean PCB contents were 3150, 1970, and 250 ng/g dry wt., respectively, in the visceral mass, gills + mantle and muscle of oysters fed algal paste containing 1.0 microgram PCBs for 30 days. The PCBs in the eggs from the same oysters reached 671 ng PCBs/g dry wt. Feeding oysters with PCB-sorbed algal paste for 30 days significantly increased phospholipid and free fatty acid contents in gills + mantle tissue compared to the same tissues in the undosed control.

This study examined the stress protein response (HSP70 family) of reproductively inactive oysters fed 0.7 g algal paste containing 0, 0.35 and 3.5 micrograms polychlorinated biphenyls (PCBs) daily. A second set of treatment groups investigated the combined effect of PCBs and sediments (0.3 g sediments daily per oyster) on HSP70 response. After 8 weeks of PCB exposure, oyster tissues (mantle and gill) were sampled and analyzed for HSP70. Preliminary results did not show a significant effect in HSP70 response in oysters fed PCB sorbed to algal paste, albeit PCBs accumulated up to 1342 ng/g dry weight in the mantle, and up to 180 ng/g dry weight in gill tissues. However, the addition of sediments caused a significant increase in HSP70 levels of gills and mantle, although the mantle was less sensitive to the sediments.

The meront stage of the oyster protozoan parasite, Perkinsus marinus, cultivated in two media with different fatty acid profiles was analyzed for its fatty acid and lipid class composition. The composition of fatty acids in the prezoosporangium stage of the parasite as well as that of the host oyster were investigated. Although the lipid class composition of meronts was dominated by phospholipids and triacylglycerol, there was no triaclgycerol detected in either culture medium. Despite the difference in fatty acid composition of the two media, the fatty acid composition of meronts in each medium was dominated by 14:0, 16:0, 18:0, 18:1(n-9), 20: (n-9), 18:2(n-6) and 20:4(n-6), a profile that differed from its host. The quantities of total lipids and fatty acids in meronts increased as the number of meronts increased and far exceeded the initial amounts in the media and in the initial cell inoculum. The meronts harvested 25 d post-inoculation, had about 3 to 6 times higher total lipids and 4 to 13 times higher fatty acids than the amounts contained in the media. The fatty acid profiles of both prezoosporangia and oysters resembled each other and consisted primarily of 16:0, 20:4(n-6), 20:5(n-3), 22:2delta7,15, and 22:6(n-3). These results indicate that during meront proliferation, the parasite synthesizes certain fatty acids and lipid classes. For development from meront to prezoosporangium, the parasite may rely on its host for lipid resources.

International audience; Perkinsus marinus is one of two important protozoan parasites of the eastern oyster, Crassostrea virginica. The other is Haplosporidium nelsoni. Lipids extracted from 7-d-old in vitro cultured P. marinus meronts, incubated with fluorescent-labeled phosphatidylcholine (FL PC) and nonincubated P. marinus meronts, were analyzed by a high-performance liquid chromatography (HPLC) system equipped with a diol phase column, in combination with thin-layer chromatography coupled with a flame-ionization detector (TLC/FID), and high-performance thin-layer chromatography (HPTLC). Various polar and neutral lipid classes were separated by HPLC using a two-gradient solvent system. Five polar lipid classes - phosphatidylcholine (PC), phosphatidylethanolamine (PE), cardiolipin (CL), sphingomyelin (SM), and phosphatidylserine (PS) - were identified from P. marinus extracts. Four neutral lipid classes - triacylglycerol (TAG), steryl ester (SE), cholesterol (CHO), and fatty alcohol - were distinguished. TLC/FID analysis of meront lipids showed that the weight percentages of PC, PE, CL, SM, PS/PI, TAG, SE, and CHO were 21, 10.7, 4, 2.3, 4.3, 48.7, 7.8, and 1.2%, respectively. HPLC and HPTLC analyses revealed the presence of two SM and PS isomers in P. marinus extracts. Perkinsus marinus effectively incorporated FL PC acquired from the medium and metabolized it to various components (i.e., free fatty acid, monoacylglycerol, diacylglycerol, TAG, PE, and CL). Uptake and interconversion of FL PC in P. marinus meronts increased with time. After 48 h the total uptake of fluorescence (FL) was 28.9% of the FL PC added to the medium, and 43% of the incorporated FL resided in TAG.

International audience; To determine the ingestion and absorption of lipid emulsions, spat were fed algae deficient in stigmasterol and cholesterol and an emulsion containing these two sterols. The ingestion-absorption of the emulsion was estimated by measuring incorporation of these two sterols in oyster lipids during the 33-day feeding period. They were supplemented with 0%, 3%, 10% and 20% emulsion wet weight of the algae dry weight. The results showed that after only 7 days of emulsion supplementation, significant differences were observed in the sterol composition. The quantities of stigmasterol and cholesterol absorbed by the spat were time- and dose-dependent. Nevertheless, compared to algal sterols, the absorption rate of the sterols from the emulsion was quite low and decreased with an increasing supply of emulsion. Other emulsion formulations need to be tested to improve the emulsion absorption rate in oyster spat. However, the sterols appeared to be good markers for assessment of lipophilic artificial diet digestion and absorption. (C) 2000 Elsevier Science B.V.

The major phospholipid classes in the larvae of the scallop Pecten maximus were phosphatidylcholine (PC) and plasmalogens (PLSM) (35.5 and 32.1 mol%, respectively). The minor classes were glycosyldiacylglycerol-like (GLY), phosphatidylinositol (PI), phosphatidylserine (PS) and non-plasmalogen phosphatidylethanolamine (PE) at 6.7, 9.1, 8.5 and 8.1 mol%, respectively. Abundance of phospholipid classes and their content of saturated, monounsaturated and polyunsaturated fatty acids remained relatively conserved during the course of larval development. During larval development there was a decrease in the amount of 20-carbon polyunsaturated fatty acids (PUFA) in the polar lipids, while the amount of 22-carbon PUFA remained constant. These changes, which occurred mainly during the lecithotrophic phase, imparted a specificity in PUFA composition to the different classes of polar lipids. During this phase, 20:5(n 3) replaced 20:4(n 6) in PI but decreased sharply in PLSM and, to a lesser extent, in PC. After the initial phase of composition changes, the fatty acid composition of the polar lipid classes became stable, with specific associations of some of the PUFA with certain polar lipid classes: 22:6(n 3) with GLY, 20:5(n 3) with PE, 20:4(n6) with PI. The 22:6(n3):20:5(n3) ratio in PLSM during exotrophy was always twice as high as in the PC fraction. © 1998 Elsevier Science Inc. All rights reserved.

Polychlorinated biphenyls (PCBs) pose a severe ecological risk. They are hydrophobic and are efficiently sequestered in lipid-rich tissues. Lipids function as energy reserves and as membrane components, and are essential for growth and reproductive processes in bivalves. Accumulation of PCBs may affect the mobilization of lipid reserves and membrane lipid composition; thus they may also impair reproductive and developmental processes. To test whether there are any changes in lipid metabolism, the lipid class and fatty acid composition in structural and reserve lipids of visceral mass, gills and mantle, and eggs of reproductively active oysters were examined. Oysters were fed daily with 0.2 g algal paste containing 0, 0.1, or 1.0 μg PCBs for 15 and 30 days. PCBs in the oyster tissues and eggs were also quantified. After 30 days of exposure PCBs were highly accumulated, up to 2635 ng/g in the visceral mass, 1880 ng/g in gill+mantle, and 671 ng/g (dry wt. bases). No significant changes were noted in either lipid class composition or fatty acid composition of structural or reserve lipids from PCB-exposed oyster tissues and eggs.

International audience; Diatoms of the genus Pseudo-nitzschia are potentially toxic microalgae, whose blooms can trigger amnesic shellfish poisoning. The purpose of this study was to test and adapt different probes and procedures in order to assess the physiological status of Pseudo-nitzschia multiseries at the cell level using flow cytometry. To perform these analyses, probes and procedures were first optimized for concentration and incubation time. The percentage of dead Pseudo-nitzschia cells, the metabolic activity of live cells and their intracellular lipid content were then measured following a complete growth cycle. In addition, chlorophyll autofluorescence and efficiency of photosynthesis (quantum yield) were monitored. The concentration and viability of bacteria present in the medium were also assessed. Domoic acid (DA) was quantified as well. Just before the exponential phase, cells exhibited high metabolic activity, but low DA content. DA content per cell became most abundant at the beginning of the exponential phase when lipid storage was high, which provided a metabolic energy source, and when they were surrounded by a high number of bacteria (high bacteria/P. multiseries ratio). These physiological measurements tended to decrease during exponential phase and until stationary phase, at which time P. multiseries cells did not contain any DA nor store any lipids, and started to die.

Spawners of the great scallop, Pecten moximus, were conditioned on three microalgal diets: T-Isochrysis, a mixture of four species (PTSC) and Chaetoceros calcitrans. The polyunsaturated fatty acid (PUFA) composition of neutral and polar lipids of eggs was related to the fatty acid composition of the diet. However, the 20 and 22 carbon PUFAs were maintained at significant levels independent of those of the diet when these fatty acids were present in the diet at low levels. This effect was more pronounced in the polar than in the neutral lipids. A preferential incorporation of 22:6(Ω-3) and 20:4(Ω-6) was demonstrated in the polar lipids. This emphasizes their role in ovogenesis and embryogenesis. The 22:6(Ω-3) requirement of P. maximus was better satisfied by T-Isochrysis, which favoured the highest incorporation of this essential fatty acid in the eggs. The good success in reproduction obtained with this monospecies diet led us to modify multispecies diets by increasing the level of 22:6(Ω-3).

Separation of phospholipid classes in lipid extracts from the scallop Pecten maximus, the Pacific oyster Crassostrea gigas, and the blue mussel Mytilus edulis was conducted using HPLC. An isolated polar lipid fraction was found to contain a very high level of DHA, up to 80 mol% of the total FA. MS with electrospray ionization in the positive-ion mode, tandem MS (MS-MS) and multidimensional NMR spectroscopy were used to analyze the detailed chemical structure of this polar lipid fraction. The isolated fraction contained exclusively cardiolipin (CL) molecules, predominantly in a form with four docosahexaenoyl chains (Do4CL). To the best of our knowledge, this is the first time that such a CL form has been analytically characterized and described in these three bivalve species. This tetradocosahexaenoic CL is presumed to reflect a specific adaptation in bivalves that enhances the structural and functional mechanisms of biomembranes in response to variations in environmental conditions (temperature, salinity, emersion).

International audience; Vibrio tapetis is the causative agent of brown ring disease (BRD), which affects a species of clam, Ruditapes philippinarum. After incubation with V. tapetis, hemocytes lose filopods and become rounded, indicating cytotoxic activity of the bacterium. To rapidly quantify this cytotoxicity, a flow-cytometry test was developed based on the capacity of V. tapetis to inhibit adhesion of clam hemocytes to plastic. Several bacteria:hemocyte ratios, the cytotoxicity of other Vibrio spp. pathogenic to bivalves, and that of various V. tapetis isolates were tested. Inhibition of adherence is detectable with as few as 5 bacteria per hemocyte. The greater cytotoxic activity of V. tapetis compared to that of V. splendidus and V. pectenicida suggests a specific pathogenicity of V. tapetis to R. philippinarum hemocytes. Although all V. tapetis isolates inhibited adhesion, significant variations in cytotoxicity among isolates was demonstrated.

Eukaryotic microparasites are essential component of ecosystems and have a considerable influence on host population dynamics and food web functioning. The Perkinsozoa lineage (Alveolata), composed of four groups of described parasitic protists, is a characteristic study case in marine environment. Some of them, e.g. the molluscan parasites Perkinsus spp., constitute serious ecological and economic threats because of their invasive character, pathogenicity and high phenotypic plasticity. The aim of this work was tocharacterise the ecological and functional role of Perkinsozoa at global and population scales. Within the global Ocean, Perkinsozoa clade is diverse (109 ASVs -6 clusters), low in abundance (< 1 %) and very localized in pelagic picoplanktonic communities. A new ribosomally active cluster, Cluster A, has been identified in the mesopelagic zone from Pacific, Atlantic and Indian oceans.However, it was not possible to determine whether these putative organisms are symbiotic or free-living because none of these have been characterised so far. At the population level, a duplex real-time quantitative PCR methodology has been developed for the in situ study of co-infection between Perkinsus olseni and P. Chesapeaki in Manila clam, Ruditapes philippinarum, population from the Arcachon bay (France). This coinfection is widespread in this area, ranging from 12 to36% of the clams sampled. Global infections are mainly dominated by mono-infection with P. olseni.Sporadic occurrence of P. chesapeaki in specific host organs suggests that this infection is secondary and could be influenced by environmental parameters affecting stages of the parasitic life cycle. In a global context of emerging diseases, an integrative view of host-parasite relationships is needed to resolve the ecological role of Perkinsozoa in different ecosystems., Les microparasites eucaryotes sont une composante essentielle des écosystèmes, dont l’influence est considérable sur la dynamique des populations hôtes et le fonctionnement des réseaux trophiques. Le clade des Perkinsozoa (Alveolata), composé de quatre grands groupes parasitaires, en est un exemple représentatif en milieu marin. Certains de ces membres emblématiques, comme Perkinsus spp., représentent de réelles menaces écologiques et économiques en raison de leur caractère invasif, de leur pathogénicité et de leur grande plasticité phénotypique. L’objectif de cette thèse était de caractériser le rôle écologique et fonctionnel des Perkinsozoa d'abord à une échelle globale puis à une échelle populationnelle. Au sein de l’Océan globale, le groupe des Perkinsozoa est diversifié (109 ASVs – 6 clusters), peu abondant (

Increase in plastic production since the 1950's in combination with plastic waste mismanagement lead to the presence of plastic particle in terrestrial and marine environment. Plastic particles measuring less than 5 mm, called microplastics, are ingested by a wide range of organisms. The goal of this PhD thesis was to evaluate the microplastic hazard for seafood consumers. firstly, method for digesting seafood tissue without degrading plastic polymer was selected and applications limits were determined for digestion of fish digestive tracts. Concurrently, a Pyrolysis-GC/MS method was optimized, limit of detection were determined and this method was applied on environmental samples. Both methods were used to study microplastics contamination in bivalve and fish species used for human consumption. Bivalve contamination was between 0.15 and 0.74 MP/g of tissue wet weight whereas contaminations in fish were low. Indeed, digestive tracts were contaminated by in mean 0.01 MP/g of tissue wet weight and muscles were contaminated by in mean 0.001 MP/g of tissue wet weight. Lately, leaching of a plastic additive was studied using an in vitro enzymatic model of digestion. However, such phenomenon was not highlighted. Overall, these results will help to standardize the study of microplastics in seafood products., L'augmentation de la production de matière plastique depuis les années 1950 combinée à une mauvaise gestion des déchets plastiques ainsi qu'aux mauvais comportements des citoyens conduit à la contamination des écosystèmes terrestres comme marins. L'ingestion par des organismes marins de particules plastiques mesurant moins de 5 mm, appelées microplastiques, a été décrite. L'objectif de ces travaux de thèse était d'évaluer le danger "microplastique" pour le consommateur de produits de la pêche. Afin de répondre à cet objectif, la sélection et la caractérisation d'une méthode permettant de digérer les tissus des produits de la pêche a été. En parallèle, une méthode de Pyrolyse-GC/MS a été optimisée afin d'identifier les microplastiques les plus petits possibles. Ses limites d'applications ont été calculées puis la méthode a été appliquée sur des microplastiques provenant de différents produits de la pêche. Ces deux méthodes ont été appliquées dans la recherche de microplastiques dans deux espèces de bivalves et trois espèces de poissons. Pour les bivalves, la contamination aux microplastiques était comprise entre 0.15 et 0.74 MP/g de chair humide alors que pour les poissons la contamination était assez faible : 0,01 MP/g de tractus gastro-intestinal et 0,001 MP/g de filet. Pour finir, une étude exploratoire de la lixiviation d'un additif plastique en utilisant un modèle de digestion in vitro a été entreprise. Globalement, ces travaux de thèse permettent de proposer des voies d'harmonisation des méthodes pour l'étude des microplastiques dans les produits de la pêche pour, in fine, évaluer le danger pour le consommateur.

Recent decades have witnessed the intensification and spread of harmful algal blooms (HAB). HAB are known to disrupt coastal ecosystems and to be toxic for marine organisms. These phenomena are also suspected to be responsible for recruitment failures of bivalves. The aim of this PhD was to study the consequences of blooms of toxic dinoflagellate Alexandrium minutum on the reproduction, development and recruitment of the oyster Crassostrea gigas, a species of major economic importance. A. minutum is known to produce paralytic shellfish toxins (PST) and bioactive extracellular compounds (BEC). Gametes and early life stages were the most sensitive, particularly to the bioactive extracellular compounds (BEC) produced by A. minutum, which inhibited fertilization and embryogenesis. A. minutum modified the behaviour of veliger larvae, decreased their filtration, growth and settlement. Exposure of adult oysters during gametogenesis affected the development of offspring, reflecting alterations in gamete content and/or vertical transfer of PST. Mode of action of PST and BEC are to further investigate. These oyster exposures, conducted at environmentally relevant concentrations of microalgae, suggest that recurrent blooms of A. minutum during oyster spawning and larval development could have long-term consequences on the structure of wild and cultured populations of C. gigas., Les dernières décennies ont été marquées par l’intensification et l’expansion des efflorescences de micro-algues toxiques (HAB). Connues pour perturber les écosystèmes côtiers et pour leur toxicité sur les organismes marins, les HAB sont suspectées d’être à l’origine de défauts de recrutement de bivalves. Cette thèse avait pour objectif d’étudier les conséquences des efflorescences du dinoflagellé toxique Alexandrium minutum, producteur de toxines paralysantes (PST) et des composés bioactifs extracellulaires (BEC), sur la reproduction, le développement et le recrutement de l’huître Crassostrea gigas, une espèce à l’importance économique majeure. Les gamètes libres et les jeunes stades de développement se révèlent être les plus sensibles, en particulier aux BEC produits par A. minutum qui inhibent la fécondation et l’embryogenèse. A. minutum modifie le comportement des larves véligères, provoque une diminution de leur filtration, de leur croissance et du taux de fixation. Une exposition des adultes, pendant la gamétogenèse, affecte le développement des descendants, traduisant des altérations du contenu gamétique et/ou un transfert vertical des PST. Les modalités d’action des PST et des BEC devront être précisées. Nos expérimentations, réalisées à des concentrations de micro-algues rencontrées dans l’environnement, suggèrent que des efflorescences récurrentes d’A. minutum lors des périodes de reproduction et de développement larvaire pourraient, sur le long terme, affecter la structure des populations naturelles et cultivées de C. gigas.

The ecological functioning of coastal ecosystems is subjected to the synergistic action of climate change and anthropogenic pressures. In Arcachon Bay, the reproductive cycle of the oyster Crassostrea gigas has changed in recent years (e.g. weak gametogenesis, spawning delay). This pattern seems to be associated with global changes in thermal and trophic conditions. The aim of this work was to characterize the spatio-temporal variability in the reproductive investment of oysters in Arcachon Bay. This variability has been investigated in relation to the origin, quantity and quality of the trophic resources. The results revealed important spatial gradients of variability in gonadal development and trophic signal in C. gigas, between inner parts of the bay influenced by river inputs (south-east and north-east) and the external part of the bay, which is influenced by oceanic inputs. Two hydrodynamic factors explained most of the observed variability: immersion time and oceanic water renewal. A comparison of C. gigas gametogenesis and the nutritional value of available food sources between the south-eastern and north-eastern parts of the bay revealed temporal variability in the composition of phytoplankton. This variability was associated with variability in the amounts of essential fatty acids, necessary for gametogenesis. Changes in energy reserves and gamete release cycle were synchronized with the seasonal nutrient variability. These results constitute a first approach in the development of a model predicting the evolution of the energetic needs of C. gigas under the global change in environmental conditions., Le fonctionnement écologique des écosystèmes côtiers est soumis à l’action synergique des changements climatiques et des pressions anthropiques. Au sein du Bassin d’Arcachon, l’huître Crassostrea gigas, montre depuis quelques années une modification du cycle reproductif (i.e. faible formation des gamètes, retard de ponte), qui semble lié aux modifications globales des conditions thermiques et trophiques du milieu. L’objectif de ce travail consiste à réaliser un état des lieux de la variabilité spatio-temporelle de l’état reproductif des huîtres dans le Bassin d’Arcachon, en lien avec l’origine, la quantité et la qualité de la ressource trophique. Les résultats obtenus ont permis de révéler d’importants gradients spatiaux de variabilité du développement gonadique et du signal trophique chez C. gigas, entre les parties internes sous influences des rivières (sud-est et nord-est), et la partie externe sous influences océaniques. Deux facteurs clés se sont révélés en partie explicatifs de cette variabilité, le temps d’immersion et le temps de renouvellement océanique. Un comparatif de la gamétogenèse et du pool nutritif entre les parties sud-est et nord-est du bassin a permis de démontrer une variabilité temporelle de la composition phytoplanctonique associée à une variabilité des apports en acides gras essentiels à la formation et la maturité des gamètes. La dynamique de la mise en réserves ainsi que le déclenchement des pontes chez C. gigas se sont révélés synchrones à la variabilité saisonnière de la quantité et la qualité du pool nutritif. Ces résultats représentent une première étape vers le développement d’un modèle prédictif des besoins énergétiques de l’espèce sous l’effet des changements globaux des conditions environnementales.

During bloom of microalgae producing paralytic shellfish toxins (PST), filtering bivalves can bio-accumulate a large quantity of toxins and become toxic for human consumption. The amount of accumulated PST can greatly vary from one individual to another within a bivalve population. Indeed, under our experimental conditions, the amount of accumulated PST by Pacific oysters, Crassostrea gigas, exposed to the toxic dinoflagellate Alexandrium minutum, varied by a factor of 450. To explain such variability we hypothesized the existence of several forms of voltage-gated sodium channel (NaV), target of the PST, resulting in different sensitivities to PST. The main objective of this thesis was to understand whether there are relationships between nerve sensitivity to PST, the different forms of NaV and the amount of accumulated PST.The NaV was first characterized in C. gigas by a molecular biology approach. Two NaV genes were reported in C. gigas: CgNaV1, encoding a sodium channel and CgNaV2 encoding a channel potentially selective for sodium and calcium. Alternative splicing of CgNaV1 produced three variants (A, B and C) with different expression profiles: at the neuromuscular junctions for CgNaV1A, in the nerve cells for CgNaV1B and in both for CgNaV1C. The amino acid Q observed in the binding site of PST (domain II), of the sequence CgNaV1 for the 3 variants and in all individuals from the 4 studied populations possibly provide some PST resistance to oysters. Thus, the variants resulting from the genotyping/splicing of CgNaV1 would not therefore be the determining factor of the level of bioaccumulation in oysters.A second part allowed studying the nerve sensitivity to PST of C. gigas oyster in relation to the accumulation of PST by an electrophysiology approach. The sensitivity to saxitoxin (STX, a PST) of the cerebro-visceral nerves from oysters was assessed by studying their action potential (CNAP). C.gigas nerves have been shown to have sensitivity to STX of the micromolar range, which gives them intermediate sensitivity among bivalves. This nerve sensitivity may vary depending on the period at which the oysters were collected and potentially according to their physiological condition. A preexposure of oysters to A. minutum appears to increase nerve resistance to STX. However, there was no significant correlation between STX nerve sensitivity and PST content in the oyster digestive gland.Overall, it appears that the variability of the PST accumulation by oysters would result rather from a physiological plasticity, in terms of filtration, ingestion and assimilation, than from a differential sensitivity of the NaV., Lors des efflorescences de micro-algues productrices de toxines paralysantes (PST), les bivalves filtreurs peuvent bioaccumuler une grande quantité de toxines et devenir à leur tour toxiques, notamment pour l’homme. La quantité de toxines PST accumulée d’un individu à l’autre s’avère être très variable au sein même d’une population de bivalves. Ainsi, dans nos conditions expérimentales, la quantité de PST accumulées par des huîtres creuses, Crassostrea gigas, d’un même lot, exposées au dinoflagellé toxique Alexandrium minutum, variait d’un facteur 450. L’origine de cette variabilité est inconnue jusqu’alors mais l’une des hypothèses pour l’expliquer serait l’existence de plusieurs formes de canaux sodium voltage-dépendant (NaV), cible des PST, qui confèreraient aux bivalves des sensibilités différentes aux PST. L’objectif principal de cette thèse était de comprendre s’il existe une sensibilité individuelle aux PST différente entre les huîtres et si cette variabilité pouvait être due à des formes différentes de NaV.Une première partie a permis de caractériser le NaV chez C. gigas par une approche de biologie moléculaire. Deux gènes NaV ont été mis en évidence chez C. gigas : CgNaV1, codant un canal sodium et CgNaV2 codant un canal potentiellement sélectif du sodium et du calcium. L’épissage alternatif de CgNaV1 produits trois variants (A, B et C) avec des profils d’expression différents : au niveau des jonctions neuromusculaires pour CgNaV1A, dans les cellules nerveuses pour CgNaV1B et dans les deux pour CgNaV1C. L'acide aminé Q, observé dans le site de liaison aux PST (domaine II) de la séquence CgNaV1 pour les 3 variants et chez tous les individus des 4 populations étudiées, pourrait conférer aux huîtres une certaine résistance aux PST. Ainsi, les variants issus du génotypage/épissage de CgNaV1 ne seraient donc pas le point déterminant du niveau de bioaccumulation des huîtres.Une deuxième partie a permis d’étudier la sensibilité aux PST des nerfs de l’huître creuse C.gigas en relation avec l’accumulation de PST par une approche d’électrophysiologie. La sensibilité à la STX des nerfs cérébroviscéraux d'huîtres a été évaluée en étudiant leur potentiel d'action (CNAP).Il a été montré que les nerfs de C. gigas possédaient une sensibilité à la STX de l’ordre du micromolaire, ce qui leur confère une sensibilité intermédiaire parmi les bivalves. Cette sensibilité des nerfs peut varier selon la période à laquelle les huîtres ont été prélevées et potentiellement selon leur condition physiologique. Une pré-exposition des huîtres à A. minutum semble augmenter la résistance des nerfs à la STX. Cependant, aucune corrélation significative n'a été observée entre la sensibilité nerveuse à la STX et la charge en PST dans la glande digestive des huîtres.Il apparait donc que la variabilité de l’accumulation des PST par les huîtres résulterait plutôt d’une plasticité physiologique, en terme de filtration, d’ingestion et d’assimilation, que d’une sensibilité différentielle des NaV.