Claudie Quéré

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.

Effect of food fatty acid and sterol quality on Pecten maximus gonad composition and reproduction process

Abstract 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.

Spatial and Temporal Dynamics of Mass Mortalities in Oysters Is Influenced by Energetic Reserves and Food Quality

Although spatial studies of diseases on land have a long history, far fewer have been made on aquatic diseases. Here, we present the first large-scale, high-resolution spatial and temporal representation of a mass mortality phenomenon cause by the Ostreid herpesvirus (OsHV-1) that has affected oysters (Crassostrea gigas) every year since 2008, in relation to their energetic reserves and the quality of their food. Disease mortality was investigated in healthy oysters deployed at 106 locations in the Thau Mediterranean lagoon before the start of the epizootic in spring 2011. We found that disease mortality of oysters showed strong spatial dependence clearly reflecting the epizootic process of local transmission. Disease initiated inside oyster farms spread rapidly beyond these areas. Local differences in energetic condition of oysters, partly driven by variation in food quality, played a significant role in the spatial and temporal dynamics of disease mortality. In particular, the relative contribution of diatoms to the diet of oysters was positively correlated with their energetic reserves, which in turn decreased the risk of disease mortality.

Proteomic identification of quality factors for oocytes in the Pacific oyster Crassostrea gigas

We used a 2-DE proteomic approach to identify abundant proteins linked to oocyte quality in the Pacific oyster Crassostrea gigas, an economically important bivalve. Oocyte quality of 14 females was estimated by recording fertilisation and early developmental success until D-larval stage under controlled conditions. Proteins that were differentially expressed between females showing high or low oocyte quality were identified by nano-liquid chromatography tandem mass spectrometry. Twelve up-accumulated spots associated with low quality oocytes revealed 10 distinct proteins, including vitellogenin - breakdown products and metabolic enzymes. Eight up-accumulated spots from high quality oocytes revealed 6 distinct proteins, including chaperone molecules and cell-cycle control proteins. This is the first proteomic study dedicated to oocytes in C. gigas. Our results improve current knowledge about protein factors associated with oocyte quality in this species, and our understanding of the proteomic processes involved in their developmental competence.

Tissue expression of two α-amylase genes in the Pacific oyster Crassostrea gigas. Effects of two different food rations

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

The use of lipid emulsions for sterol supplementation of spat of the Pacific oyster, Crassostrea gigas.

Abstract 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.

Changes in motility, ATP content, morphology and fertilisation capacity during the movement phase of tetraploid Pacific oyster (Crassostrea gigas) sperm

Changes in sperm features during the movement phase are especially interesting to study in external fertilization species whose sperm duration movement is long because this implies a significant adaptation of moving cells to the external medium. This study describes the changes in tetraploid Pacific oyster sperm characteristics in relation to time post activation. Sperm individually collected on three tetraploid males were activated in seawater. Their features were analysed over a 24h period and compared to a sperm pool collected on three diploid males as a reference. The percentage of motile spermatozoa, the intracellular ATP content, and the fine structure of spermatozoa were studied in relation to time post activation. Furthermore, the fertilisation capacity of sperm individually collected on five diploid males was assessed after 1 and 24h post activation. A forward progressive movement was maintained for at least a 20h duration. Compared to diploid males, the percentage of motile spermatozoa was lower in tetraploid males. The intracellular ATP concentration was higher in spermatozoa from tetraploid males than in spermatozoa from diploid males. A decrease in ATP content was observed in the first 6h post activation and severe alterations were observed in sperm morphology after 24h. Then, a lower fertilisation capacity of sperm from diploid males was observed at the end of the movement phase. The cessation of Pacific oyster sperm motility was unlikely caused by ATP consumption as ATP concentration was still high at the end of sperm movement but rather caused by drastic changes in sperm morphology. Compared to sperm collected on diploid males, the lower quality of sperm from tetraploid males was emphasized by a shorter movement duration and deeper morphological alterations at the end of the movement phase.

Cellular and biochemical responses of the oyster Crassostrea gigas to controlled exposures to metals and Alexandrium minutum

Effects of simultaneous exposure of Pacific oyster, Crassostrea gigas, to both a harmful dinoflagellate that produces Paralytic Shellfish Toxins (PST), Alexandrium minutum, and cadmium (Cd) and copper (Cu), were assessed. Oysters were exposed to a mix of Cd-Cu with two different diets (i.e. A. minutum or Tisochrysis lutea) and compared to control oysters fed A. minutum or T. lutea, respectively, without metal addition. Metals and PST accumulations, digestive gland lipid composition, and cellular and biochemical hemolymph variables were measured after 4 days of exposure. Oysters exposed to Cd-Cu accumulated about thirty-six times less PSTs than oysters exposed to A. minutum alone. Exposure to Cd-Cu induced significant changes in neutral lipids (increase in diacylglycerol - DAG - and decrease in sterols) and phospholipids (decreases in phosphatidylcholine, phosphatidylethanolamine, cardiolipin and ceramide aminoethylphosphonate) of digestive gland suggesting that lipid metabolism disruptions and/or lipid peroxidation have occurred. Simultaneously, concentrations, percentages of dead cells and phenoloxidase activity of hemocytes increased in oysters exposed to metals while reactive oxygen species production of hemocytes decreased. Feeding on the harmful dinoflagellate A. minutum resulted in significant decreases in monoacylglycerol (MAG) and DAG and ether glycerides (EG), as well as significant increases in hemocyte concentration and phagocytic activity as compared to oysters fed T. lutea. Finally, the present study revealed that short-term, simultaneous exposure to Cd-Cu and A. minutum may induce antagonistic (i.e. hemocyte concentration and phagocytosis) or synergic (i.e. DAG content in digestive gland) effects upon cellular and tissular functions in oysters.

Involvement of Mitochondrial Activity and OXPHOS in ATP Synthesis During the Motility Phase of Spermatozoa in the Pacific Oyster, Crassostrea gigas

ABSTRACT In the Pacific oyster, spermatozoa are characterized by a remarkably long movement phase (i.e., over 24 h) sustained by a capacity to maintain intracellular ATP level. To gain information on oxidative phosphorylation (OXPHOS) functionality during the motility phase of Pacific oyster spermatozoa, we studied 1) changes in spermatozoal mitochondrial activity, that is, mitochondrial membrane potential (MMP), and intracellular ATP content in relation to motion parameters and 2) the involvement of OXPHOS for spermatozoal movement using carbonyl cyanide m-chlorophenyl hydrazone (CCCP). The percentage of motile spermatozoa decreased over a 24 h movement period. MMP increased steadily during the first 9 h of the movement phase and was subsequently maintained at a constant level. Conversely, spermatozoal ATP content decreased steadily during the first 9 h postactivation and was maintained at this level during the following hours of the movement phase. When OXPHOS was decoupled by CCCP, the movement of spermatozoa was maintained 2 h and totally stopped after 4 h of incubation, whereas spermatozoa were still motile in the control after 4 h. Our results suggest that the ATP sustaining flagellar movement of spermatozoa may partially originate from glycolysis or from mobilization of stored ATP or from potential phosphagens during the first 2 h of movement as deduced by the decoupling by CCCP of OXPHOS. However, OXPHOS is required to sustain the long motility phase of Pacific oyster spermatozoa. In addition, spermatozoa may hydrolyze intracellular ATP content during the early part of the movement phase, stimulating mitochondrial activity. This stimulation seems to be involved in sustaining a high ATP level until the end of the motility phase.

Essential Fatty Acid Assimilation and Synthesis in Larvae of the Bivalve Crassostrea gigas

Essential fatty acids (EFA) are important for bivalve larval survival and growth. The purpose of this study was to quantitatively assess for the first time through a mass-balance approach dietary EFA incorporation and synthesis within Crassostrea gigas larvae. A first experiment was carried out using two microalgae, Tisochrysis lutea (T) and Chaetoceros neogracile (Cg), as mono- and bi-specific diets. A second experiment using a similar design was performed to confirm and extend the results obtained in the first. Flow-through larval rearing was used for accurate control of food supply and measurement of ingestion. Non-methylene-interrupted fatty acids were synthetized from precursors supplied in the diet: 16:1n-7 and 18:1n-9, mediated by Δ5 desaturase. Moreover, this Δ5 desaturase presumably allowed larvae to convert 20:3n-6 and 20:4n-3 to 20:4n-6 and 20:5n-3, respectively, when the product EFA were poorly or not supplied in the diet, as when larvae were fed T exclusively. Under our experimental conditions, none of the diets induced 22:6n-3 synthesis; however, 22:6n-3 incorporation into larval tissues occurred selectively under non-limiting dietary supply to maintain optimal levels in the larvae. This combination of flow-through larval rearing and biochemical analysis of FA levels could be applied to additional dietary experiments to precisely define optimal levels of EFA supply.