Jean-Francois Samain

The effect of monospecific algal diets on growth and fatty acid composition of Pecten maximus (L.) larvae

Abstract 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.

Changes in the fatty acid composition of Pecten maximus (L.) during larval development

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).

Effect of a mono-specific algal diet on immune functions in two bivalve species - Crassostrea gigas and Ruditapes philippinarum

SUMMARY The impact of diets upon the fatty acid composition of haemocyte polar lipids and consequently upon immune parameters has been tested in the oyster Crassostrea gigas and the clam Ruditapes philippinarum. Oysters and clams were fed each of three cultured algae: Chaetoceros calcitrans, which is rich in 20:5(n-3) and 20:4(n-6) and poor in 22:6(n-3) fatty acids; T-Iso (Isochrysis sp.), which is rich in 22:6(n-3) and deficient in 20:5(n-3) and 20:4(n-6); and Tetraselmis suecica, which is deficient in 22:6(n-3) and contains only small amounts of 20:5(n-3) and 20:4(n-6). Fatty acid composition of haemocyte polar lipids was greatly affected by the diet. Oysters and clams fed C. calcitrans maintained a higher proportion of 20:5(n-3) and 20:4(n-6) in their haemocyte polar lipids, while these polyunsaturated fatty acids decreased drastically for animals fed T-Iso. However, the T-Iso diet maintained 22:6(n-3) in haemocyte polar lipids of both species. Higher 20:5(n-3) and 20:4(n-6) contents in diets appeared to have a positive effect upon total haemocyte count, granulocyte percentage, phagocytic rate and oxidative activity of clam haemocytes. Similarly, a positive effect of 20:5(n-3) on oxidative activity of oyster haemocytes was observed but to a lesser extent than in clams. Interestingly, when oyster haemocytes are submitted to a stressful condition, a positive effect of a higher dietary 22:6(n-3) content on the phagocytic rate was noticed.

The Benefit of a Roseobacter Species on the Survival of Scallop Larvae

Abstract A marine strain (BS107), identified as a Roseobacter species, was antagonistic to Vibrio species on agar plates. Results suggested that the inhibitory effect was displayed only in the presence of another bacterium. Quantification of the antibacterial activity showed that 48-hour-coculture supernatants from BS107 and another bacterial strain (V. anguillarum 408) reached the highest titers of bacterial inhibition. The antibacterial substance was also liberated when supernatants from V. anguillarum 408 were added to pure cultures of the inhibition-productive bacterium. The presence of a proteinaceous molecule may induce BS107 to display the inhibitory effect. The antibacterial substance was sensitive to trypsin (8000 U/ml) and stable at 100°C. Cell extracts of the isolate BS107 (106 cells/ml) significantly enhanced scallop larval survival, thus being beneficial to the rearing process.

Growth and lipid class composition of Pecten maximus (L.) larvae grown under hatchery conditions

Abstract 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.

Impact of the quality of dietary fatty acids on metabolism and the composition of polar lipid classes in female gonads of Pecten maximus (L.)

Abstract 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.

Effects of algal diet on digestive enzyme activity in Calanus helgolandicus

Adult female Calanus helgolandicus were transferred, immediately after collection in the English Channel in June 1984, to two unialgal diets one of which, the flagellate Cryptomonas maculata, was rich in starch, while the other, the diatom Thalassiosira weissflogii, contained no starch. The activity of the digestive enzymes amylase, trypsin and laminarinase was measured in these two populations under foodsaturating conditions over an acclimation period of eight days. Ingestion rates were measured on a daily basis and the results confirmed, together with a constant level of body protein, that the experimental conditions were above the incipient limiting concentration. In the long-term (4 to 8 d), the activities of all three enzymes were significantly elevated in the C. maculata-fed copepods, whereas ingestion rates were lower than those on T. weissflogii. These results observed under food-saturating conditions indicate a compensatory mechanism between digestive enzymes and the substrate ingested. They are consistent with previous work on Artemia sp. suggesting that the rate of assimilation is a function of the digestive enzyme activity and ingestion rate. Enzyme activity exhibited differing shortterm responses (<48 h) on transfer to the two algal diets, which are interpreted in relation to the in situ activity of the field population.

Incorporation of Microalgae Sterols by Scallop Pecten maximus (L.) Larvae

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 appeared able to limit the incorporation of methylpavlovol, ethylpavlovol, and 4alpha-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.

Impact of 20:4n-6 supplementation on the fatty acid composition and hemocyte parameters of the pacific oyster Crassostrea gigas

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 μg 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.

Relation between lipid and fatty acid composition of eggs and larval survival in white pacific shrimp (Penaeus vannamei, Boone, 1931)

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.