Search

This is the first study of the metabolic enzyme activity and energy state of the dwarf oysterOstrea stentina(Payraudeau, 1826) in Tunisia and in the Mediterranean region. The main purpose of this study was to examine the modulation of the physiological status of the oysterO. stentinadepending on the season and the presence of parasiteMarteilia refringens. The prevalence of bonamiosis and marteiliosis were established by PCR.Bonamia exitiosawas detected only in 2.91% of oysters while the prevalence ofM. refringenswas 100% (30 Ind./30) in February and 93.93% (28 Ind./30) in March. A 42–87% mortality rate was reported during the study period. Biochemical analyses were carried out to evaluate the management of the energy resources in regard to the biochemical changes of lipids and carbohydrates. The enzyme activity of pyruvate kinase (PK), citrate synthase (CS) and cytochrome c oxidase (CCO) were measured and the mean level of enzyme activity was respectively 20.31 mU mg−1protein; 12.06 mU mg−1protein and 3.59 mU mg−1protein. Carbohydrate contents ofO. stentinawere very low all year round with an average of 15.18% in dry weight, and lipid contents remain similar (11.77% in dry weight) compared with the values reported for most other temperate bivalves. Enzyme activity significantly decreased over time (P< 0.001). The energy resources of the oysterO. stentinawherein much of the energy was devoted to reproduction seem to affect the defence system.

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.

In the natural environment bivalve larvae are exposed to variable conditions and can therefore face periods of food shortage. To understand the resistance of early life stages of Pacific oyster (Crassostrea gigas), larvae were experimentally starved for 4 days at different periods of their development, and the resulting variations in their lipid reserves then analysed using image analysis and biochemical techniques. Faced with a temporary lack of food, the larvae halted their growth in size and weight and started to live off their lipid reserves; this effect was highly marked when the period of food deprivation was at the beginning of larval rearing, but was less marked when food deprivation was after day 14. The larvae conserved their developmental capacity despite these starvation treatments: when feeding was resumed, the larvae started to grow once more, rebuilt their reserves and showed a survival rate between 45 and 91% to the end of the experiment. Apart from the treatment where larvae were starved between days 6 and 10, high percentages became competent (56–85%) leading to a metamorphosis rate of 58–93%. Coloration of neutral lipids with Nile Red, followed by their quantification using image analysis to calculate lipid surface relative to total larval surface (OLI: overall lipid index) was particularly suited to studying the evolution of reserves through larval development. Results collected in this way supported those obtained in parallel using biochemical tests: triacylglycerols/sterols ratio (TAG/ST). The image analysis method offers the advantage of requiring less biological material to obtain a result; the correlation coefficient between OLI and TAG/ST ratio was 0.75.

Mortality of oysters Crassostrea gigas occurred along the coasts of France during summer 2008, causing a serious crisis in French oyster production. During spring to summer 2008, 5 groups of 1-yr old oysters of different origins and ploidy levels deployed in the Thau lagoon (France) were sampled for biometrical and biochemical analyses; environmental parameters were monitored in the same area. Mortality occurred in 2 episodes: 13 May to 10 June and 24 June to 9 July. Wild-collected oysters showed mortality sooner than other groups but total overall mortality was 85% in all groups. Mortality occurred when seawater temperature was >19 degrees C during the reproductive season when energetic reserves of oysters were at their lowest. Energy reserve levels and mortality in oysters seemed to be independent of reproductive effort, and most likely reflected an unusual deficit in phytoplankton. The level of bacterial fatty acids in oysters increased markedly before the second mortality event, probably indicating an enhancement of the microbial loop or decomposition processes. At the same time, unsaturation of oyster fatty acids decreased, suggesting a diet change, acclimatization to increasing temperature or, most likely, oxidative damage clue to an enhanced production of reactive oxygen species, which are often associated with stressful situations. Wild-collected and hatchery-produced oysters, which exhibited different mortality patterns, showed persistent differences in fatty acid (20:5n-3 and 22:6n-3) content in their membrane, despite the fact that they were reared in the same environment. Mortality started when fatty acid 20:4n-6 content increased to similar to 3% in the polar lipids, suggesting that this ratio could make a useful indicator of mortality risk. Finally, the 2008 mortality event coincided with a nationwide increase of similar to 1.5 degrees C in winter seawater temperature, which may impact the oyster and its pathogens.

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.

Seasonal variations in fatty acid composition in several tissues of a 1-year-old population of giant lion's-paw scallop Nodipecten subnodosus were analyzed during gonad development. Samples were taken bimonthly from December 1999 to June 2000, a period in which gonad development was occurring. Fatty acid accumulation in neutral and polar lipids of the female gonad was concomitant to the gonad development and presented a maximum in June. Among essential fatty acids, a specific accumulation was observed only for 20:5n − 3 in the female gonad, as suggested by an increase in its proportion in the neutral and the polar fractions. However, no specific accumulation was observed for 22:6n − 3, as its proportion remained relatively constant in both fractions. We suggest that a specific increase of 22:6n − 3 is not necessary during gametogenesis because its proportion was high enough for reproductive purposes (20-30%). Although some data suggest a partial mobilization of polyunsaturated fatty acids from the muscle and digestive gland to the female gonad during gonad development, the main supply came directly from the diet. The maximum increase in total and specific fatty acids in the digestive gland occurred in April and was maintained in June. These changes reflect either a higher lipid availability of the food, or the beginning of storage process in the digestive gland. However, in the female gonad, the maximum increase was observed in June, whereas gonad development and spawning were observed from April. It is suggested that different patterns of lipid accumulation in the first (April) and second (June) maturation processes could be related to the reproductive cycle of this species, which exhibits an initial facultative maturation during spring and the main reproductive activity during summer.

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.

The influence of nutrition, in terms of both quantity and quality, upon innate immune system competence (cellular defense mechanisms) was explored experimentally in eastern oysters, Crassostrea virginica . Oysters (ca. 50 mm shell height) were fed diets of two cultured microalgal strains, Tetraselmis chui strain PLY429 and Skeletonema costatum strain LB1077/1B, known to differ in biochemical composition—both alone and as a 50:50 mix—at two daily rations. Unfed controls also were included. After 5 weeks of feeding at 20 °C, the temperature was increased to 28 °C over 2 days, and the oysters were fed the same algal diets for one additional week. Dependent variables measured by flow cytometry, both before and after temperature increase, were hematological characteristics (relative percentages, sizes and granularities of four hemocyte types) and hemocyte defense-related activities (viability, aggregation and adherence, phagocytosis, and respiratory burst). Main effects and interactions of temperature increase, food quantity, and food quality upon each immunological variable were determined by ANOVA. Furthermore, Discriminant Analysis (DA) was applied to identify a suite of immunological parameters capable of differentiating oysters from the separate dietary treatments, and Principal Component Analysis (PCA) was used to characterize relationships between immunological variables. ANOVA of individual immunological variables revealed few significant main effects or interactions of food quantity, quality, and temperature increase; however, trends were apparent. A DA model, incorporating several immune function variables, differentiated oysters according to the food quantity and quality treatments from which they were taken. PCA revealed positive relationships between hemocyte phagocytosis and aggregation, which had a negative relationship with respiratory burst and mortality. Accordingly, we characterized “healthy” oyster immune capability as (1) highly aggregating and (2) phagocytic hemocytes with (3) low mortality and (4) low respiratory burst, and “unhealthy” immune function as the opposite. Temperature increase had a significant ( p

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

Thiamin and riboflavin (vitamins B1 and B2) were measured in different organs of the Pacific oyster Crassostrea gigas over an annual cycle in Marennes Oleron (France), to study the natural accumulation and their possible transfer to gonads and eggs. We observed an annual cycle of vitamin concentration with low levels in winter and high levels in spring. The first increase was observed in the digestive gland (DG) in March with no change in the mantle plus gonad tissue or muscle, suggesting no immediate transfer to gonad tissue or a strong utilisation of these vitamins in the gonad in winter. A second peak was observed in the DG in spring, coinciding with an increase in the gonad plus mantle and muscle, suggesting that vitamin transfer was immediate in spring. The DG could be an intermediate compartment in vitamin transfer. Riboflavin was mainly concentrated in eggs and disappeared from the gonad during spawning. Thiamin was also concentrated in eggs but also in the gonad–mantle tissue. By artificial conditioning, it was possible to increase the thiamin concentration 2.5-fold in eggs in spring, but riboflavin concentrations remained similar to those in nature. No difference was evident in D-larval production from natural or hatchery spawnings, nor in larval growth rate in spring. Vitamins B1 and B2 in eggs were probably above D-larval requirements in spring (9 and 20 fg/egg (dry weight) for thiamin and riboflavin, respectively). Low vitamin concentrations observed in winter coinciding with gonadal mitoses and glycogen metabolism, could be explained by a high vitamin utilisation for these biological processes. These results may explain low levels of vitamins observed in eggs from winter conditioning of oysters as natural vitamin concentration in this period is low, and transfer to gonads seems limited.

Vitamins B1(thiamin) and B2 (riboflavin) in eggs of the scallop Pecten maximus are highly utilized during larval development. Possible storage of these vitamins, for later transfer to gonads and eggs, was studied over the natural reproductive cycle of the scallop. Vitamin concentrations were measured during hatchery conditioning at three periods of the year. Vitamin B1 was found mainly in muscle, gonad and eggs, while B2 was detected in the digestive gland, gonad and eggs. They varied simultaneously in the three organs during the annual cycle. Two main peaks were observed for digestive gland and gonads in spring and summer. The first peak corresponded to vitellogenesis and decreased in gonads in June as the scallops spawned and the vitamins were lost with the eggs. The second peak was interpreted as vitamin storage in gonad tissue in fall. The large vitamin decrease occurring from November to February coincided with gonial mitoses observed by histology. Possible high utilisation of thiamin and riboflavin during winter was supported by hatchery conditioning at three periods during the year: during the same winter period, when broodstock were fed a high-vitamin diet, vitamin levels stayed low in all organs, as vitellogenesis was in progress. However, in spring and summer vitamin incorporation increased. We concluded that attention should be paid to winter-conditioning diets, as riboflavin and thiamin, even when provided at high levels in the food, did not accumulate in organs, suggesting intense utilisation when mitoses in the gonads take place.

In this study, lipid classes and fatty acid composition were determinedin eggs of P. vannamei as a function of survival to zoeaIII stage. Spawns were reared individually to zoea III and grouped, accordingtotheir final survival to this larval stage, into spawns of high and lowsurvival.Eggs of individual spawns were analyzed for lipid and fatty acid composition ofneutral and polar lipids and the results were then grouped according tosurvivalto zoea III. The lipids within each group (high and low survival to zoea III)were pooled for the separation of phospholipids and the analysis of their fattyacid composition.Higher levels of triglycerides, carotenoids, and linoleic (18:2n-6) acid ineggs were associated with improved survival to zoea III. Linoleic acid washigher in spawns from the high survival group in both neutral and polarfractions and in most of the phospholipid classes analyzed. Docosahexanoic acid(DHA) was not related to survival to zoea, probably because its content washighenough (> 15% of total fatty acids) to satisfy embryo and early larvaldevelopment needs. A high content of eicosapentaenoic acid (EPA) was found inphosphatidylethanolamine, lysophosphatidylethanolamine, phosphatidylserine, andphosphatidylinositol compared to other phospholipid classes, suggesting aspecific role of EPA in these lipids. These results describe the specificphospholipid composition of penaeid eggs and could (potentially) be used aspredictive indicators of larval quality for research and production purposes.

Reversed-phase HPLC was applied to obtain a sensitive and efficient means for quantitating nucleotides in the mussel Mytilus galloprovincialis. We obtained a good separation of adenylic, guanylic, uridylic and cytidylic nucleotides. Adenine nucleotides play a critical role in the regulation and integration of cellular metabolism; particularly in the mantle tissue in the mussel, they are involved in the regulation of the enzyme glycogen phosphorylase, a key enzyme in the transfer of bioenergetic reserves (glycogen) to gametogenic development; it is of great importance to have a measure of the concentrations in vivo during the reproductive cycle of the organism. Different elution conditions were tested: isocratic versus step gradient elution, different mobile phase pH and the type and proportion of ion-pairing agent added to the mobile phase. The best method was selected and the separation and accurate determination of adenine, citidine, guanine and uridine nucleotides was accomplished within a 20-min run, with UV-Vis detection (254 nm).

A Crassostrea gigas digestive gland copy DNA (cDNA) library constructed in the lambda phage ZapII (Stratagene, La Jola, USA) was screened with an amylase heterologous proble. To get access to the complete cDNA, a polymerase chain reaction extension was conducted using DNA extracted from the phages. The complete cDNA sequence is 1688 base pairs (EMBL = Y08370). The deduced protein sequence is 519 aminoacids long with a 19 aminoacid signal peptide. Similarity with Pecten maximus amylase is 72%. A 3-day nutrition experiment with a cyclic algal food supply was carried out. Amylase enzyme activities and mRNAs were individually measured on five animals, nine times a day. Messenger RNAs were quantified by dot hybridization using the previously characterized cDNA as probe. Variation of amylase mRNA was observed, in relation with the level of activity of the enzyme. Coordinated changes in RNA and enzyme levels suggested a possible transcriptional regulation of amylase in C. gigas as in vertebrates.

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.

Changes in sterol composition of Pecten maximus larvae during the larval development stage with standard algal mixtures and unialgal diets were analysed. The sterol composition of four microalgae currently used in mollusc hatchery were also examined. Under standard algal conditions, the larvae quickly use the steryl ester from larvae reserves during the endotrophic and the mixotrophe phases. The preferential incorporation of Pavlova lutheri and T-Isochrysis sterols, rather than Skeletonema costatum sterols, during the larval development stage would indicate that S. costatum cells were poorly ingested and digested by larvae. Among the ingested sterols, cholesterol and stigmasterol were preferentially incorporated by the larvae. Conversely, the larvae ap- peared able to limit the incorporation of methylpavlovol, ethylpavlovol, and 4α-methylporiferasterol. In the unialgal experiment, the best growths were obtained with the diet richest in cholesterol (Chaetoceros calcitrans) and the best compromise of good growth and settlement rate was observed with the diet richest in C24 ethyl sterol. The selective incorporation of the cholesterol was confirmed by the larval rearing with C. calcitrans. The strong sterol dietary imprint in larvae corroborated the absence of an important capacity in P. maximus larvae to convert or biosynthesise sterol. comp biochem physiol 119A;2:451-457, 1998. © 1998 Elsevier Science Inc.

The impact of dietary deficiency in the essential fatty acids 20:5(n−3), 22:6(n−3) and 20:4(n−6) from three different microalgal diets for broodstock conditioning on polar lipid classes of Pecten maximus male gonads was studied. The relative percentage of polar lipid classes was not influenced by the composition of the microalgae. Diet did influence the fatty acid composition of all phospholipid classes with an increasing proportion (1) glycolipid (GLY), (2) plasmalogen (PLSM), phosphatidylethanolamine non plasmalogen (PE), phosphatidylinositol (PI) and phosphatidylserine (PS), (3) phosphatidylcholine (PC). The main differences of male gonads compared to female gonads were in the fatty acid composition of phosphatidylinositol (PI) and plasmalogen (PLSM). In PI, 20:4(n−6), which was the major PUFA and which was stable in female gonads whatever the food composition, was found in similar quantities to 20:5(n−3) and 22:6(n−3) in male gonads and was more influenced by diet composition. This result corroborates the specific importance of 20:4(n−6) in oocytes. In PLSM, 20:5(n−3) was the predominant polyunsaturated fatty acid in male gonads, independent of the three diets tested whereas 22:6(n−3) and 20:5(n−3) were at equivalent levels in female gonad PLSM. This reflects a specific importance of PLSM for spermatozoa.

The impact of dietary deficiency of the essential fatty acids 20:5(n-3), 22:6(n-3) and 20:4(n-6) was studied at the level of different polar lipid classes that constitute the membrane structure of female gonads of Pecten maximus by using three microalgal diets. After 11 weeks of conditioning of the broodstock, the relative percentages of polar lipid classes were very little, or not at all, influenced by the composition of the microalgae. On the other hand, the imprint of dietary fatty acid composition was reflected in the relative composition of fatty acids specific to some classes but not all of them. The dietary fatty acid composition thus clearly influenced that of phosphatidylcholine (PC), the plasmalogens and to a smaller extent the phosphatidylserine (PS) and phosphatidylethanolamine (non plasmalogen PE), but not of phosphatidylinositol (PI) and the glycolipid (GLY). The PI showed a very stable composition and was rich in 20:4(n-6) (>30%) irrespective of the diet. Likewise, the 22:6(n-3) content was consistently higher than 76% in the glycolipid. The maintenance of the specific fatty acid composition of these classes is probably a physiological necessity for gametogenesis and embryogenesis.

Bacterial nutrition of great scallop, Pecten maximus, larvae was investigated using the radioactive tracer technique. The bacterial labelling was studied initially to obtain a high and stable specific radioactivity (14C) of bacterial cells. A higher bacterial specific ~4C activity was obtained when the tracer (amino acid) was introduced in the culture medium at the beginning of the exponential growth phase. After a 12 h labelling period in a rich nutritive medium, the bacteria were depurated in seawater for 5 h (chase) to prevent further 14C excretion and then added to larval rearing vessels. The larval labelling was followed for 12 h and then larvae were placed in new vessels without radioactive bacteria. The depuration of larvae was followed for 3 days. Data obtained on ingestion and assimilation efficiency show that bivalve larvae are able to ingest and digest bacteria.

Spawning individuals of the scallop Pecten maximus were conditioned on three test diets: Tahiti Isochrysis , a mixture (PTSC) and Chaetoceros calcitrans . The scallops fed T- Isochrysis showed a better hatching rate and lower atresia than those fed the other two diets. Proximate composition of the female gonads was not modified by the differences in the diets. Enrichment of gonads with sterol esters and triglycerides, characteristic of storage of lipids, observed with the broodstock fed the diatoms, did not result in successful gametogenesis and spawning. The monounsaturated fatty acid (MUFA) and polyunsaturated fatty acid (PUFA) composition of neutral and polar lipids of gonads was related to the fatty acid composition of the diet. However, the 20 and 22 carbon PUFA were maintained at levels relatively independent of those of the diet; when these fatty acids were low in the diet, their concentration in the lipids of the gonads decreased but still remained significant. This effect was more pronounced in the polar than the neutral lipids. The preferential incorporation of 22:6 n − 3, 20:4 n − 6 and 20:5 n − 3 in the polar lipids indicates their role in gametogenesis and embryogenesis. The sterol composition of the gonads (free sterols and sterol esters) reflected that of the diet; however, the cholesterol, mainly found in the esterified fraction, was maintained at stable levels independent of dietary supply. Two sterols (22-dehydrocholesterol and 24-methylenecholesterol) that were either absent from, or present only in trace amounts in the diets, were found to occur at constant levels in the female gonads, regardless of the nature of the diet.

An easy method for the separation of major polar lipid classes by HPLC is described. Maximum resolution was achieved by an automated combination of a silica gel column and a diol column. Polar lipid analysis of the larvae and gonads of Pecten maximus showed the presence of a particular glycolipid especially rich in 22:6(n - 3) and the predominance of 20:4(n - 6) in the phosphatidylinositol. The phosphatidylcholine and phosphatidylethanolamine (diacyl form + alkenylacyl) were the major fractions. The plasmalogen form (25% in larvae, 34% in gonads) was essentially composed of polyunsaturated fatty acids of 20 and 22 carbons in the sn-2 position.

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.

Four batches of Pecten maximus (L.) larvae were grown under hatchery conditions and fed the monospecific diets Pavlova lutheri Droop, Isochrysis aff. galbana Green (clone T-iso; termed Tahitian Isochrysis), Chaetoceros calcitrans Takano and Dunaliella tertiolecta Butcher which were selected on their very different polyunsaturated fatty acid (PUFA) profiles. Neutral and polar lipid PUFA compositions of larvae were influenced by diets. The constant level of (n-3) PUFA in neutral lipids of larvae although very different in the diet indicated that neutral lipid composition was partially controlled by the larval metabolism. Similarly, the stability of total saturated, monounsaturated and PUFA levels (25, 15 and 55% of total fatty acids), as well as total (n-3) PUFA level and the preferential incorporation of the 22:6(n-3) in polar lipids clearly indicated a metabolic regulation of the fatty acid composition. The 20:4(n-6) and 22:5(n-6) acids were also preferentially incorporated. The accumulation of 18:3(n-3) or 20:5(n-3) with depletion of 22:6(n-3) in neutral and polar lipids of larvae fed diets with high levels of the first two fatty acids but little or no 22:6(n-3), indicated that elongation-desaturation of dietary fatty acids was too low in P. maximus larvae to maintain high 22:6(n-3) levels in polar lipids. The selective incorporation of dietary long chain PUFA into neutral and polar lipids by acyltransferases may be the main mechanism controlling the fatty acid composition of larvae. These results indicate that P. maximus larvae have essential requirements for long chain (n-3) and (n-6) PUFA, similar to many other marine species.

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.

The fatty acid composition of Pecten maximus (L.) larvae grown under hatchery conditions was determined during development from the oocyte stage to the pediveliger stage (Day 23). The levels of total fatty acids in neutral and polar lipids increased 3 days after the first feeding and were ≈10 times higher in pediveligers than in D-larvae (32.46 vs. 3.14 ng·larva−1 and 11.38 vs. 1.23 ng·larva−1 in neutral and polar lipids, respectively). The proportions of total saturated, monounsaturated and polyunsaturated fatty acids in neutral and polar lipids remained stable throughout the development. The fatty acid composition of P. maximus larvae was typical of that described for other marine bivalve larvae since the (n-3) polyunsaturated fatty acids (PUFA) formed 34.4–39.6% of the neutral lipids and 43.0–50.7% of the polar lipids. The major PUFA identified in oocytes and larvae were similar to those supplied by the diet [18:2(n−6), 18:4(n−3), 20:5(n−3) and 22:6(n−3)]. We observed that significant changes of the PUFA composition occurred in neutral and polar lipids during embryogenesis and the subsequent nutritional transition. These changes were expressed by the relative decline of the 20:5(n−3) and 20:4(n−6) percentage levels in both lipid fractions while the 22:6(n−3) increased in neutral lipids and the 22:5(n−6) increased in both neutral and polar lipids. We observed also that polar lipids exhibited a 22:6(n−3) level considerably higher than that of the diet (26.8 vs. 6.8%) and constant throughout development, indicating a preferential incorporation and a major structural role. These results suggest that a metabolic control of the fatty acid composition of neutral lipids and mainly polar lipids takes place in developing larvae. With reference to previous studies on fatty acid metabolism of molluscs, it is probable that this control results from a selective incorporation of dietary fatty acids by acyltransferases rather than a desaturation of dietary precursors. P. maximus larvae seem therefore to have a high nutritional requirement for long chain PUFA and especially the 22:6(n−3).

Five independent batches of Pecten maximus (L.) larvae, reared under the same standard hatchery conditions, showed different growth rates, ranging from 1.9 to 6.6 μm·day −1 . A standard lipid profile was defined based on the fast-developing larvae and variations to this standard were seen when abnormal growth performances occurred. The lipid class composition was determined during the development of D-larvae to Day 23 using chromarods and flame ionization detection. Phospholipids and triglycerides were the main lipid classes in P. maximus larvae as reported for other cultured bivalve larvae (accounting for 50 and 37% of total lipids, respectively). Sterols and free fatty acids formed 10 and 3% of the total lipids, respectively. These relative proportions of lipid classes were constant during development of fast developing larvae. The triglyceride:organic matter ratio (TG:OM) stabilized after an initial decline during 3 days after the first feeding and the increase of triglycerides after this time indicated that the catabolism of endogenous triglycerides was rapidly offset by the food intake during the nutritional transition. The comparison of this lipid class pattern with those from larvae which exhibited a lower growth rate revealed that: (i) no stability of the lipid class composition was apparent throughout development; (ii) the triglyceride:total lipid (TG:TL) ratio in D-larvae was not related to the subsequent growth; (iii) a low TG:OM ration in D-larvae was related to poor growth while higher levels did not correspond necessarily to better growth rates; (iv) both the degree and the duration of the TG:OM ration decline for all these batches during the 1st wk can be related to the ability of the larvae to overcome the nutritional transition from trochophore to veliger rapidly and allow for subsequent successful growth. The possible influence of uncontrolled genetic or environmental factors is suggested.

International audience

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.

Adenylatc Encrgy Charge (AEC) values of Crassostrea gigas (9 and 18 months old) cultured in ponds in Marennes-Oleron Bay (Atlantic coast, France) are reported. Two ponds were loaded with two different initial oyster biomasses in order to obtain different food conditions. The growth of oysters was different in the two ponds which corroborated the effectivences of attaining different food conditions. AEC level was different depending on the pond and was therefore related to food availability per unit oyster biomass. Well-fed oysters exhibited greater AEC values than poorly-fed oysters, throughout thc study. Information on nutritional situations for wild or cultured populations can therefore be obtained by AEC. In cach pond, the seasonal AEC variations were similar for both age-groups and were characterized by a minimum value in summer and a maximum value in wintcr. The influence of reproduction on these variations cannot be ruled out as for al1 the batches maturation processes were evidenced. Research will be done on a temperature effect to try to distinguish these seasonal variations. A strong correlation between adenylatc energy charge (AEC) and guanylatc encrgy charge (GEC) was demonstrated; it was similar for both ponds. The levecs of nucleotide concentration were relatively stable during the survey cxcept in September and December for oysters in the low density pond. Among nucleotides, adenylate pool represented the major part (80%). A decrease in the total concentration of adenylates was observed in August. The best growth correlated with the lowest adenylate perccntage and a better GTP/ATP ratio, suggesting that this ratio could be a useful index for growth.

1. 1. O. edulis and C. gigas both exhibit a seasonal variation in AEC with minimum values in summer. Two factors, food and temperature, were examined to explain these low summer values. 2. 2. The AEC level varied with food level but a seasonal pattern was still observed. Two age groups of oysters were tested, giving a similar response. 3. 3. The effect of temperature on the seasonal variations in AEC was confirmed by a significant correlation between AEC and temperature. This relationship allows us to calculate an AEC standard that only retains the trophic information. 4. 4. Different trophic levels were identified in Marennes-Oleron Bay with AEC standard but growth rate was not related to them. So, AEC may inform on the carrying capacity of a given area but does not predict growth performances which will depend on other parameters.

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.

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

1. 1. The growth (dry weight, W), protein, lipid content and nucleotide composition (ATP, ADP, AMP, UTP and GTP) were estimated at 16°C at daily intervals for growing and starving zoea I of Carcinus maenas. 2. 2. Larvae fed with Artemia sp. during the entire development moulted at the 6th day to the next larval instar; the same developmental duration was found when larvae were starved after reaching the “D0 threshold” (“D0 starved”); no development to the next zoea was found when larvae were starved before this “D0 treshold”. 3. 3. The growth (W) and content in protein and lipids was strongly inluenced by the starvation periods. 4. 4. The amounts of nucleotides per individual were influenced by the moulting cycle and the nutritional status of the larvae: they increased in the Artemia-fed control until stage D0 and remained stable for the rest of the moulting cycle. Similar amounts were found in the Artemia-fed control group and “D0-starved” larvae; nucleotides decreased with the beginning of starvation in larvae starved with beginning of stage C of the moulting cycle (“C-starved”). In the starved control quantity of nucleotides increased only slightly during the first days before decreasing later. 5. 4. Similar patterns of nucleotide compositions were found in dry weight and protein related concentrations. 6. 5. The similarity in the nucleotide composition is reflected in the energy charge patterns, which were similar for all tested groups, showing that larvae can regulate their energetic pool as long as they can metabolize reserve substances such as protein and lipid; the individual nucleotide content decreased after using up these reserves.

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.

International audience; Knowledge on the energy distribution during the reproductive cycle of the giant lion-paw scallop, Nodipecten subnodosus is limited, but necessary for aquaculture purposes. In a previous work, we found that natural food availability can sustain the full cost of gametogenesis in optimal environmental conditions. The present study analyses the lipid and sterol classes' composition of several tissues during gametogenesis. In female gonads, the quantity of triacylglycerol (TG) increased parallel to gametogenesis from December (spawned gonads, 0.7 mg g(-1) or 7% of total lipids) to June (ripe gonads, 52 mg g(-1) or 66% of total lipids). In male gonads sampled in June, the concentration of TG (0.7 mg g(-1) or 3.5% of total lipids) and esterified sterols was significantly lower than in female gonads. Total phospholipids (PL) increased two-fold in female gonads of scallops sampled in June compared to the other months, which could be a result of oocyte production and growth, but no differences were found in the concentration of PL or in free sterol composition between male and female gonads. TG concentration in the digestive gland was similar in December and February (37-3 8 mg g- 1 or 68% of total lipids), but increased eight-fold by April and remained high in June (252-277 mg g(-1) or 90% of total lipids). Together with the increase in esterified sterols, these data suggest an increase in food availability during these months. A lack of decrease of TG levels in digestive gland during gonad development is in accordance to sufficient food availability in this location to satisfy the energetic demands of gametogenesis. Cholesterol was the principal sterol found in all tissues; its concentration, as well as the concentration of several phytosterols, remained constant throughout the maturation process, except in digestive gland where they increased from December to June, further supporting an increased availability of food. However, the presence of several phytosterols in substantial concentrations, mainly brassicasterol and 24-methylenecholesterol in gonad and somatic tissues, further supports a specific physiological role of these sterols in pectinids. (c) 2007 Elsevier B.V. All rights reserved.

The main objective of this study was to identify algal diets that maximize the survival and growth and alter the biochemical content of Pecten maximus larvae with the aim of improving metamorphosis. We also evaluated the potential of the cryptophyceae Rhodomonas salina as a food source for these larvae. Two flagellates, Isochrysis aff. galbana (T) and Pavlova lutheri (P), and two diatoms, Chaetoceros gracilis (C) or Skeletonema costatum (S), were tested as two ternary diets, namely PTC and PTS. PTC and PTS were compared with diets that also included R. salina (R). The addition of R. salina and the replacement of C. gracilis by S. costatum in the traditional hatchery diet seem to be two interesting alternatives for increasing the productivity of larval scallop culture and improving the metamorphosis rate. With these two diets, larval growth increased and metamorphosis was observed to occur more rapidly. Moreover, our results showed that the addition of R. salina significantly improved the overall condition of the larvae by promoting an increase in organic matter and total lipids. This accumulation of lipids during ontogeny seems to promote larvae to grow and to complete metamorphosis more rapidly than with the other diets. The level of polyunsaturated fatty acids in the algae could also at least partially explain the results obtained, since the PUFA level of C gracilis was about half those of S. costatum and R. salina. (c) 2006 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.