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International audience; 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.

Summer mortality of the Pacific oyster Crassostrea gigas is the result of a complex interaction between oysters, their environment, and pathogens. Heredity appears to be a major factor determining the sensitivity of oysters to summer mortality, allowing resistant (R) and susceptible (S) lines to be produced. We conducted genome-wide expression profiling of R and S gonads during the 3-month period preceding a summer mortality event, using a cDNA microarray that we designed. ANOVA analysis revealed that 34 genes were differentially expressed between R and S lines on four dates preceding the mortality event. Annotation of some of these genes highlights reproduction and its allocation and antioxidant defenses as the main pathways that operate differentially between R and S lines. This transcriptional analysis provides new indications to define markers for quantitative trait loci searches and functional studies and evaluate the potential role of each gene in the resistance to summer mortality.

This study investigated the non-neutrality of genetic polymorphism in two alpha-amylase genes (AMYA and AMYB) in the oyster Crassostrea gigas. Bi-parental oyster families, bred to be polymorphic for markers in these genes, were monitored for growth and survival for 1 year under standard culture conditions in two French production sites. Within-family genotype frequencies indicated that the two amylase genes were closely linked (c. 1.7 cM). Within two of three families, significant differences in growth were observed between genotypes at one of the two production sites, suggesting that this polymorphism is not neutral and might be under selection because of its role in digestive function. Estimated daily yields were different between amylase genotypes, indicating the potential value of amylase markers in selective breeding programmes to improve oyster growth.

Summer mortality has been reported in the Pacific oyster, Crassostrea gigas, for many years in different parts of the world. The causes of this phenomenon are complex. The multidisciplinary program "MOREST", coordinated by IFREMER, was initiated to understand the causes of summer mortality of Crassostrea gigas juveniles in France and to reduce its impact on oyster production. Within this program, three successive groups of bi-parental families were bred in a hatchery in 2001 and placed in the field during summer in three sites (Ronce, Riviere d'Auray, and Baie des Veys). This paper reports the relative importance of family, site and field placement timing for three characters of major importance for oyster production: survival, growth, and yield. At the end of the summer period, significant differences for the three characters were observed among sites and families for each group. Family effect was the largest variance component for survival, representing 46% of the total. Variance component analysis revealed that variation in yield among families depended either on survival or on growth according to the site. Significant family x environment interactions were observed for yield and survival but not for growth. No difference in survival was found among groups in the three sites at the end of the experiment, but a critical period of mortality was identified from late July until early September. The influence of environmental conditions, notably on reproductive allocation and its relationship with the studied traits, is discussed. (c) 2005 Elsevier B.V. All rights reserved.

To investigate the control at the mRNA level of glycogen metabolism in the cupped oyster Crassostrea gigas, we report in the present paper the cloning and characterization of glycogen phosphorylase and synthase cDNAs (Cg-GPH and Cg-GYS, respectively, transcripts of main enzymes for glycogen use and storage), and their first expression profiles depending on oyster tissues and seasons. A strong expression of both genes was observed in the labial palps and the gonad in accordance with specific cells located in both tissues and ability to store glucose. Cg-GPH expression was also found mainly in muscle suggesting ability to use glycogen as readily available glucose to supply its activity. For seasonal examinations, expression of Cg-GYS and Cg-GPH genes appeared to be regulated according to variation in glycogen content. Relative levels of Cg-GYS transcripts appeared highest in October corresponding to glycogen storage and resting period. Relative levels of Cg-GPH transcripts were highest in May corresponding to mobilization of glycogen needed for germ cell maturation. Expression of both genes would likely be driven by the oyster's reproductive cycle, reflecting the central role of glycogen in energy storage and gametogenic development in C. gigas. Both genes are useful molecular markers in the regulation of glycogen metabolism and reproduction in C. gigas but enzymatic regulation of glycogen phosphorylase and synthase remains to be elucidated.

cited By 38; International audience; Two bivalve species Crassostrea gigas and Ruditapes philippinarum were fed eight weeks with three mono-specific algae diets: T-Isochrysis galbana, Tetraselmis suecica, Chaetoceros calcitrans, selected on the basis of their polyunsaturated fatty acid (PUFA) composition. The incorporation and the modification of dietary fatty acids in C. gigas and R. philippinarum gill lipids were analysed and compared. Essential PUFA (20:4n-6, 20:5n-3 and 22:6n-3) and non-methylene interrupted PUFAs (known to be synthesised from monounsaturated precursors) contents of gill polar lipid of both species were greatly influenced by the dietary conditioning. Interestingly, oysters and clams responded differentially to the mono-specific diets. Oysters maintained higher 20:5n-3 level and higher 22:2j/22:i and n-7/n-9 ratio in gill polar lipids than clams. To better discriminate dietary and species influences on the fatty acid composition, a Principal Component Analysis followed by a MANOVA on the two most explicative components was performed. These statistical analyses showed that difference in fatty acid compositions attributable to species were just as significant as the diet inputs. The differences of gill fatty acid compositions between oysters and clams are speculated to result of an intrinsic species characteristic and perhaps of a group characteristic: Fillibranch vs. Eulamellibranch. © 2005 Elsevier Inc. All rights reserved.

A semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) assay was developed to estimate tissue-specific expression of two amylase genes in five different tissues of the Pacific oyster Crassostrea gigas and the experimental effects of food level on the cellular, enzymatic and gene expression changes of the digestive gland. Results indicated that both genes were transcribed at different levels depending on tissue type. A strong preferential expression of both amylase genes was observed in the digestive gland leading to high amylase activities, in accordance with the digestive function of this organ. Traces of gene expression and activity were observed in other analysed tissues. These appeared higher in labial palps than in other non-digestive tissues. After 4 weeks of feeding two groups of oysters on either a high or low food level, cellular growth of the digestive gland (+56%) and increase in the total activity of amylase enzymes and in specific amylase activity (+50%) were observed under high food conditions compared to low food conditions. A significant increase (+18%) in the expression of gene A was observed in the digestive gland of oysters fed the high ration compared to that of oysters fed on the low food level whereas no changes in the expression of gene B were observed. Gene A was expressed at a higher level than gene B in all tissues and at all conditions. A role of the amylase gene A in digestive processes and changes in its expression according to food availability is demonstrated in this study. The functional role of the α-amylase B gene remains to be elucidated. Author keywords: α-Amylase; Digestive enzyme activity; Food regulation; Gene expression; Oyster

International audience; The lipid composition of Crassostrea gigas was analyzed during the reproductive phase in natural as well as under artificial conditions. The lipid content increased and accumulated in the gonads, but to a higher extend in the naturally conditioned animals. The percentage of neutral lipid in total lipid of the gonad plus mantle was stable, high (>70%) and equal under both conditions, underscoring that the lipid reserves were preferentially located in that organ. The composition of the polar lipid classes was stable with little variations. The fatty acid (FA) composition of the neutral lipids in all organs is influenced by diet. However, there is a different response according to organs. A high dietary impact occured in the digestive gland whereas the muscle was less affected. The polyunsaturated fatty acid (PUFA) level of the neutral and polar lipids in the gonads changed little despite the dietary conditions but the respective proportions of (n-3) and (n-6) PUFA differed drastically as a result of diet composition. There was clear evidence in all organs for a specific accumulation of 22:6(n-3) and 20:5(n-3) in the polar lipids under both conditioning diets. The proportions of 22:6(n-3) and 20:5(n-3) from neutral and polar lipids of oysters conditioned artificially were significantly lower than of those that were naturally conditioned. Copyright (C) 1999 Elsevier Science Inc.

International audience; A new fatty acid isolated from the scallop Pecten maximus was determined to be a 22:4 geometrical isomer [Δ4?,7?,10?,13?] (X) using GC-MS analysis of the 2-alkenyl-4,4-dimethyloxazoline derivative. The number of trans double bonds was determined on silver ion HPLC by comparison with the mono-trans geometrical isomers of 22:4[Δ4cis,7cis,10cis,13cis] (S), obtained through partial hydrazine reduction of all-cis-22:6(n-3) followed by a p-toluenesulfinic isomerization of the derivatives. Comparing the trienes obtained through partial hydrazine reduction of (X) and (S) determined a cis-4,7,10,trans-13-docosatetraenoic fatty acid. This fatty acid has not been reported previously in any organisms and is believed to be specific to the pectinid family. Copyright (C) 1999 Elsevier Science B.V.

International audience; Wild female Crassostrea corteziensis oyster (n=245) were analyzed over one year to understand the main ecophysiological events associated to gonad development. Different indicators (mainly biochemical) were analyzed to infer: i) utilization and accumulation of energy reserves (e.g. neutral lipids, carbohydrates, proteins; vitellogenin), ii) membrane components provided by the diet as essential nutrients and indicative of cell proliferation (e.g. highly unsaturated fatty acids linked to phospholipids, sterols), iii) indicators of food availability (chlorophyll a in water, pigments in tissues, specific fatty acids and sterols), iv) gonad development (e.g. gonad coverage area, vitellin). A PCA analysis was applied to 269 measured variables. The first PC (PC1) was composed of total carbohydrate and lipid concentration, percentage of esterified sterols, fatty acids specific of diatoms; 16:1n-7/16:0, 20:5n-3 in neutral lipids with positive loadings and non methylene-interrupted fatty acids (NMI) in neutral lipids with negative loadings. The second PC (PC2) was composed of 18:4n-3 in lipid reserves and the concentration of zeaxanthin, a pigment typical of cyanobacteria with positive loadings and the proportion of 20:4n-6 in polar lipids with negative loading. The third PC (PC3) was composed of gonad coverage area (GCA) and the concentration of vitellin. Variation in GCA confirms that gonad development began in April with an extended period of spawning and rematuration from April to November. The PCA further shows that a second period of minimal maturation from November to March corresponds to the accumulation of reserves (PC1) together with an initial high availability of food (PC2) at the beginning of this period. These two periods are in accordance with the classical periods of allocation of energy to reserves followed by gonad development reported for several mollusks.

International audience; In the oyster Crassostrea gigas consumption-related traits, amylase properties and growth were found to be linked through genotypes that differed for polymorphism in the two amylase genes AMYA and AMYB. Modulation of AMYA mRNA level had already been observed in response to food availability, whereas the functional role of AMYB was still unknown. To improve knowledge about the regulation of amylase expression in C. gigas and the respective roles of the two genes, we made an assay of amylase expression at mRNA and enzymatic levels in the digestive gland of oysters that had received dietary supplements of starch. After 18 days, a significant increase of translatable mRNA for AMYB was observed, with a correlated increase in Michaelis-Menten constant Km values and a decrease in total amylase activity. This modulation is the first evidence of observable functioning of AMYB in digestive processes. Amylase B is suggested to display a higher Km than amylase A, offering a means of adapting to high substrate concentrations. The highest starch supplement level (10 mgL(-1)) induced alteration in oyster physiology. The 1 mgL(-1) treatment should be tested as a practical food supplement that could lead to growth benefits for oysters.

The effect of four temperatures, ranging between 17 degrees C and 32 degrees C, was studied on development and lipid reserve management of Crassostrea gigas larvae. No effect of temperature was found on larval mortality, as high survival (>90%) was recorded before competence at all temperatures studied. Temperature did, nonetheless, have a strong effect on growth and settlement success. At low temperature (17 degrees C), larvae competent to metamorphose were only observed from day 23 and only a low percentage finally achieved metamorphosis (12%). The opposite was seen at temperatures >= 27 degrees C: larval competence appeared at day 18 and led to high rates of metamorphosis (60-90%). This difference at settlement seemed to be linked to larval growth, which showed rates of 7 mu m d(-1) at 17 degrees C vs. 10.5 mu m d(-1) at 32 degrees C. In addition, a higher accumulation of lipid reserves at low temperature was revealed by both biochemical (TAG/ST) and colorimetric (OLI) methods. In fact, the lower the temperature, the higher the mean TAG/ST levels recorded (6-9 at 17 degrees C vs. 2-4 at 32 degrees C). In the same way, larvae reared at 17 degrees C had a percentage lipid surface coverage between 19 and 29% (at sizes between 80 and 230 mu m), while lipids covered only 5 to 16% of the surface of larvae reared at 32 degrees C (at size

Several quality indices have been defined for marine bivalve larvae, including the coloration of whole larvae and the Triacylglycerols/Sterols ratio (TAG/ST), putting emphasis on the role of lipids in larval development. Using an image analysis system, epifluorescence microscopy and Nile Red, to specifically colour the reserves, we improved and quantified the overall physiological index based on the ratio of the lipid surface to the total larval surface. This Overall Lipid Index (OLI) was then tested in two experiments on the influence of nutritional parameters on larval development and metamorphosis in Crassostrea gigas, following the parallel evolution of the indices. The higher the input of microalgae, the quicker larvae developed, leading to a maximum development rate of 16.5 µm d− 1. This rate was accompanied by the highest lipid reserves. At the end of rearing, the lipid reserves occupied 30% of the larval surface in the batches with the highest food ration, while they only covered 12% in the larvae on the lowest ration. In the same way, the TAG/ST ratio increased over the rearing period and reached its highest levels in larvae with the highest food ration. We also noted that the decrease in lipids over the first week of rearing was higher when the nutritional input was lower. Finally, deficient reserves and poor performances were recorded in larvae fed only the flagellates Pavlova lutheri (P), Isochrysis affinis galbana (T) or a mixture of these species (PT). In contrast, a high lipid accumulation and better performances were shown when the diet was based on the diatom C. calcitrans f. pumilum (Cp), whether alone or in combination with other species.

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.

Background Although bivalves are among the most-studied marine organisms because of their ecological role and economic importance, very little information is available on the genome sequences of oyster species. This report documents three large-scale cDNA sequencing projects for the Pacific oyster Crassostrea gigas initiated to provide a large number of expressed sequence tags that were subsequently compiled in a publicly accessible database. This resource allowed for the identification of a large number of transcripts and provides valuable information for ongoing investigations of tissue-specific and stimulus-dependant gene expression patterns. These data are crucial for constructing comprehensive DNA microarrays, identifying single nucleotide polymorphisms and microsatellites in coding regions, and for identifying genes when the entire genome sequence of C. gigas becomes available. Description In the present paper, we report the production of 40,845 high-quality ESTs that identify 29,745 unique transcribed sequences consisting of 7,940 contigs and 21,805 singletons. All of these new sequences, together with existing public sequence data, have been compiled into a publicly-available Website http://public-contigbrowser.sigenae.org:9090/Crassostrea_gigas/index.html. Approximately 43% of the unique ESTs had significant matches against the SwissProt database and 27% were annotated using Gene Ontology terms. In addition, we identified a total of 208 in silico microsatellites from the ESTs, with 173 having sufficient flanking sequence for primer design. We also identified a total of 7,530 putative in silico, single-nucleotide polymorphisms using existing and newly-generated EST resources for the Pacific oyster. Conclusion A publicly-available database has been populated with 29,745 unique sequences for the Pacific oyster Crassostrea gigas. The database provides many tools to search cleaned and assembled ESTs. The user may input and submit several filters, such as protein or nucleotide hits, to select and download relevant elements. This database constitutes one of the most developed genomic resources accessible among Lophotrochozoans, an orphan clade of bilateral animals. These data will accelerate the development of both genomics and genetics in a commercially-important species with the highest annual, commercial production of any aquatic organism.

International audience; To examine further a previously reported association between amylase gene polymorphism and growth in the Pacific oyster Crassostrea gigas, ecophysiological parameters and biochemical and molecular expression levels of alpha-amylase were studied in Pacific oysters of different amylase genotypes. Genotypes that previously displayed significantly different growth were found to be significantly different for ingestion and absorption efficiency. These estimated parameters, used in a dynamic energy budget model, showed that observed ingestion rates (unlike absorption efficiencies) allowed an accurate prediction of growth potential in these genotypes. The observed association between growth and amylase gene polymorphism is therefore more likely to be related to ingestion than to absorption efficiency. Additionally, relative mRNA levels of the two amylase cDNAs were also strongly associated with amylase gene polymorphism, possibly reflecting variation in an undefined regulatory region, although no corresponding variation was observed in specific amylase activity. Amylase gene sequences were determined for each genotype, showing the existence of only synonymous or functionally equivalent non-synonymous polymorphisms. The observed associations among growth, food consumption-related traits and amylase gene polymorphism are therefore more likely to be related to variation in the level of amylase gene expression than to functional enzymatic variants.

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.

The purpose of this study was to examine the responses of the oyster C. gigas to oxygen levels at subcellular and whole-organism levels. Two experiments were carried out. The first experiment was designed to measure at 15°C, 20°C and 25°C for 20 hours the clearance (CR) and oxygen consumption (OC) rates of oysters exposed at different concentrations of oxygen. The goal of this first part was to estimate the hypoxic threshold for oysters below which their metabolism shifts toward anaerobiosis, by estimating the oxygen critical point (PcO2) at 15, 20 and 25°C. The second experiment was carried out to evaluate the metabolic adaptations to hypoxia for 20 days at three temperatures: 12°C, 15°C and 20°C. The metabolic pathways were characterized by the measurement of the enzymes pyruvate kinase (PK) and phosphoenolpyruvate carboxykinase (PEPCK) and the alanine and succinate content and the adenylate energy charge (AEC). The respiratory chain functioning was estimated by the measurement of the activity of the electron transport system (ETS). The values of PcO2 were 3.020.15, 3.430.20 and 3.280.24 mg O2 L-1 at 15, 20 and 25°C, respectively. In whole oysters, hypoxia involved the inhibition of PK whatever the temperature but PEPCK was not stimulated. Succinate accumulated significantly only at 12°C and alanine at 12 and 15°C. A negative relationship between the PK activity and the alanine content was found only in hypoxic oysters. Lastly, hypoxia increased significantly the activity of ETS. With high PcO2 values, the metabolic depression occurred quickly showing that oysters had a low capacity to regulate their respiration when oxygen availability is reduced, particularly in summer.

International audience; Summer mortality of Pacific oysters is known in several countries. However no specific pathogen has been systematically associated with this phenomenon. A complex combination of environmental and biological parameters has been suggested as the cause and is now starting to be identified. A high genetic basis was found for survival in oysters when a first generation (G1) was tested in three sites during summer. This paper presents a synthesis on physiological characteristics of two selected groups (‘R' and ‘S', from families selected for resistance and susceptibility to summer mortality respectively), of the second and third generations. R and S showed improvement or reduction of survival compared with the control in both field and laboratory trials confirming the high heritability of survival of juveniles

The generation of EST information is an essential step in the genomic characterisation of species. In the context of the European Network Marine Genomics, a common goal was to significantly increase the amount of ESTs in commercial marine mollusk species and more specifically in the less studied but ecologically and commercially important groups, such as mussel and clam genera. Normalized cDNA libraries were constructed for four different relevant bivalves species (Crassostrea gigas, Mytilus edulis, Ruditapes decussatus and Bathymodiolus azoricus), using numerous tissues and physiological conditions. In this paper, we present the analysis of the 13,013 expressed sequence tags (ESTs) generated. Each EST library was independently assembled and 1300-3000 unique sequences were identified in each species. For the different species, functional categories could be assigned to only about 16 to 27% of ESTs using the GO annotation tool. All sequences have been incorporated into a publicly available database and form the basis for subsequent microarray design, SNP detection and polymorphism analysis, and the placement of novel markers on genetic linkage maps. © 2007 Elsevier B.V. All rights reserved., The research presented in this paper was funded by the European Commission through the Marine Genomics Network of Excellence (GOCE-CT-2004-505403). C.S. wishes to acknowledge additional funding from the “Ramón y Cajal” Program and grants AGL2003–04143 and AGL2006–08944 from the Spanish government.

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.

International audience; The response of Crassostrea gigas to prolonged hypoxia was investigated for the first time by analyzing the metabolic branch point formed by pyruvate kinase (PK) and hosphoenolpyruvate carboxykinase (PEPCK). PK and PEPCK cDNAs were cloned and sequenced. The main functional domains of the PK sequence, such as the binding sites for ADP/ATP and phosphoenolpyruvate (PEP), were identified whereas the PEPCK sequence showed the specific domain to bind PEP in addition to the kinase-1 and kinase-2 motifs to bind guanosine triphosphate (GTP) and Mg(2+), specific for all PEPCKs. A C-terminal extension was detected for the first time in eukaryota PK. Separation of mitochondrial and cytosolic fraction showed that more than 92% of the PEPCK enzyme activity was cytosolic in gills, digestive gland, mantle and muscle. PK and PEPCK mRNAs and enzyme activities have been measured in muscle during prolonged hypoxia for 20 days. Adaptation of PK in hypoxic muscle at transcriptional level occurred lately by decreasing significantly the PK mRNA level at day 20 while PK enzyme activity was inhibited by the high content of alanine. The PEPCK mRNA ratio in hypoxic muscle significantly increased at day 10 simultaneously to the PEPCK enzyme activity. Succinate accumulation observed at day 10 and day 20 confirmed the anaerobic pathway of muscle metabolism in oyster subjected to hypoxia. Regulation of C. gigas PEPCK in muscle occurred at gene transcription level while PK was first regulated at enzyme level with alanine as allosteric inhibitor, and then at molecular level under a fast effect of hypoxia.

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; 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.

High variability among individuals is often encountered when hemocyte characteristics are measured in bivalves. Such variability is suspected to result partly from genetic factors. In this study, hemocyte characteristics of six families of Crassostrea gigas were compared by flow cytometry at one sampling date in October 2001. These families were obtained from a nested, half-sibling cross design, and reared from July to October 2001 at three sites distributed along the French Atlantic coast from north to south: Baie des Veys (Normandy), Riviere d'Auray (Brittany) and Ronce (Marennes-oleron Basin, Poitou Charentes). Among the 15 measured hemocyte characteristics, production of reactive oxygen species (ROS) of untreated hemocytes (maintained in filtered sterile seawater) and treated hemocytes (zymosan at 20 particles per hemocyte, and with vibrio sp. S322 at 50 bacteria per hemocyte) was the most notable differences between families. This supports the existence of a genetic basis, at least partly, for the hemocyte characteristics of oysters, and especially for ROS production. Among the six families analyzed, three have shown high survival during summer (named as "resistant", mean mortality 5.2%) and three experienced high mortality during summer (named as "susceptible", 30.6% mean mortality). Families showing high or low survival to summer mortality had similar hemocyte characteristics, regardless of the environmental conditions or reproductive state. Resistant families were observed to have higher total hemocyte counts and lower production of ROS than susceptible families. Moreover, ROS production of hemocytes from susceptible families was diminished significantly more by pathogenic vibrio than that of resistant families. However, this study demonstrates also that rearing site strongly affected the hemocyte characteristics of all families of oysters, most notably hemocyte concentration and morphology (size and granularity), production of reactive oxygen species (ROS), and susceptibility to the cytotoxic activity of the pathogenic vibrio sp. S322 (50 bacteria/ hemocyte). Food availability and reproductive state are the most probable explanations for the site differences observed. Finally, it appeared difficult to link oyster survival during summer mortality to hemocyte profiles evaluated at one sampling date; other relevant indicators would probably help explaining oyster survival during summer mortality events. (c) 2007 Elsevier B.V. All rights reserved.

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.