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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.

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.

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.

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.

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.

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.

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 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; 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

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; 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.

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.

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.

The amylase and trypsin activities of Artemia reared on synthetic axenic media were studied during development and compared with earlier results obtained with phytoplankton-fed Artemia. The enzymatic activity responses are also analysed with regard to the Provasoli growth index, protein growth and survival during starvation experiments or during ones involving specific deletions (starch or albumin). These experiments demonstrated a repressive regulatory mechanism for amylase and trypsin under substrate-saturating conditions. Differences in response of amylase and trypsin to starch and albumin deletions are discussed in terms of the existence of internal carbohydrate and protein pools in crustaceans. The ability of Artemia to shift from a high metabolism with exponential growth to a low metabolism without any growth but permitting survival is observed. The related enzyme responses are discussed. The importance of interactions between nutritional conditions, metabolic requirements, and the regulation of digestive enzymes is stressed. The response times and implications for the field studies on Zooplankton ecology are discussed.

An apparent repressive control of amylase and trypsin respectively by starch and proteins of the diet were precedently demonstrated on artificial axenic media [1]. Such a mechanism has been improved also on Artemia fed Platymonas sueccica in more usual rearing conditions. Artemia face a starvation period of 4–5 days during growth or at adult stage by increasing enzymatic activities when food was lacking. This compensatory mechanism was immediate for amylase but was delayed for trypsin depending on the duration of starvation and the decrease of internal protein content. This response time difference is hypothesized to be related to differences in circulating carbohydrates, proteins or amino acid pools in the hemolymph.

Nutrients can be used for induction of carbohydrate or protein metabolism of three unicellular algae, their chemical variations measured by c/N ratio. The C/N determination and evolution can be used as a control for growth phase and satured conditions. Cultures of routine production have been tested by such a method. The results are discussed. [NOT CONTROLLED OCR]

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