Catherine Seguineau

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.

Vitamin requirements in great scallop larvae

Vitamins were analysed in food (microalgae) and larvae of great scallop, Pecten maximus, during larval development. Microalgae used to feed larvae in hatcheries show great variability in their vitamin composition depending on both the species and culture condition (phase of growth). The microalgae used to feed Pecten maximus larvae were rich in vitamins; their content compared with diets used in fish culture appeared sufficient, with the possible exceptions of pyridoxine, biotin and pantothenic acid. Vitamins in bacteria, isolated from the larval rearing tank were also analysed, as they can also contribute to the diet. Vitamin B12, α-tocopherol and Β-carotene were detected in very low concentration in bacteria; however, some bacterial strains were rich in pantothenic acid, and the pattern of other vitamins was similar to that from microalgae. The presence of bacteria can complement the diet in panthothenic acid, as it has been demonstrated that bacteria are ingested by larvae. The vitamin content of Pecten maximus larvae was analysed from the second day after hatching to just before metamorphosis. The content of some vitamins, ascorbic acid (C), α-tocopherol and Β-carotene, increased during larval development, suggesting that their requirement was satisfied. However, thiamin and riboflavin decreased during larval development and further studies, possibly using microencapsulated vitamins supplements, are needed to determine whether these vitamins are limiting during larval development.

Active and passive biomonitoring suggest metabolic adaptation in blue mussels (Mytilus spp.) chronically exposed to a moderate contamination in Brest harbor (France).

Brest harbor (Bay of Brest, Brittany, France) has a severe past of anthropogenic chemical contamination, but inputs tended to decrease, indicating a reassessment of its ecotoxicological status should be carried out. Here, native and caged mussels (Mytilus spp.) were used in combination to evaluate biological effects of chronic chemical contamination in Brest harbor. Polycyclic aromatic hydrocarbon (PAH) contamination was measured in mussel tissues as a proxy of harbor and urban pollution. Biochemical biomarkers of xenobiotic biotransformation, antioxidant defenses, generation of reducing equivalents, energy metabolism and oxidative damage were studied in both gills and digestive glands of native and caged mussels. In particular, activities of glutathione-S-transferase (GST), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), NADP-dependent isocitrate dehydrogenase (IDP), pyruvate kinase (PK) and phosphoenolpyruvate carboxykinase (PEPCK) were measured and lipid peroxidation was assessed by malondialdehyde (MDA) quantification. In addition, a condition index was calculated to assess the overall health of the mussels. Moderate PAH contamination was detected in digestive glands of both native and caged individuals from the exposed site. Modulations of biomarkers were detected in digestive glands of native harbor mussels indicating the presence of a chemical pressure. In particular, results suggested increased biotransformation (GST), antioxidant defenses (CAT), NADPH generation (IDP) and gluconeogenesis (PEPCK), which could represent a coordinated response against chemically-induced cellular stress. Lipid peroxidation assessment and condition index indicated an absence of acute stress in the same mussels suggesting metabolic changes could, at least partially, offset the negative effects of contamination. In caged mussels, only GR was found modulated compared to non-exposed mussels but significant differences in oxidative stress and energy-related biomarkers were observed compared to native harbor mussels. Overall, these results suggested mussels chronically exposed to contamination have set up metabolic adaptation, which may contribute to their survival in the moderately contaminated harbor of Brest. Whether these adaptive traits result from phenotypic plasticity or genetic adaptation needs to be further investigated.

Techniques for delivery of arachidonic acid to Pacific oyster, Crassostrea gigas, spat

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 d, 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.

Changes in tissue concentrations of the vitamins B1 and B2 during reproductive cycle of bivalves. Part 1 : The scallop Pecten maximus

Abstract 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.

Changes in tissue concentrations of the vitamins B1 and B2 during reproductive cycle of bivalves: Part 2. The Pacific oyster Crassostrea gigas

Abstract 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.

The influence of dietary supplementation of arachidonic acid on prostaglandin production and oxidative stress in the Pacific oyster Crassostrea gigas

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.