In vitro modulation of reactive oxygen and nitrogen intermediate (ROI/RNI) production in Crassostrea gigas hemocytes

International audienceBivalve 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/RNI production by untreated hemocytes shown by other studies, relative to environmental conditions or physiological status of the oysters

Generation and analysis of a 29,745 unique Expressed Sequence Tags from the Pacific oyster (Crassostrea gigas) assembled into a publicly accessible database: the GigasDatabase

Background: Although bivalves are among the most-studied marine organisms because of their ecological role and economic importance, very little information is available on the genome sequences of oyster species. This report documents three large-scale cDNA sequencing projects for the Pacific oyster Crassostrea gigas initiated to provide a large number of expressed sequence tags that were subsequently compiled in a publicly accessible database. This resource allowed for the identification of a large number of transcripts and provides valuable information for ongoing investigations of tissue-specific and stimulus-dependant gene expression patterns. These data are crucial for constructing comprehensive DNA microarrays, identifying single nucleotide polymorphisms and microsatellites in coding regions, and for identifying genes when the entire genome sequence of C. gigas becomes available. Description: In the present paper, we report the production of 40,845 high-quality ESTs that identify 29,745 unique transcribed sequences consisting of 7,940 contigs and 21,805 singletons. All of these new sequences, together with existing public sequence data, have been compiled into a publicly-available Website http://public-contigbrowser.sigenae.org:9090/Crassostrea_gigas/index.htm l. Approximately 43% of the unique ESTs had significant matches against the SwissProt database and 27% were annotated using Gene Ontology terms. In addition, we identified a total of 208 in silico microsatellites from the ESTs, with 173 having sufficient flanking sequence for primer design. We also identified a total of 7,530 putative in silico, single-nucleotide polymorphisms using existing and newly-generated EST resources for the Pacific oyster. Conclusion: A publicly-available database has been populated with 29,745 unique sequences for the Pacific oyster Crassostrea gigas. The database provides many tools to search cleaned and assembled ESTs. The user may input and submit several filters, such as protein or nucleotide hits, to select and download relevant elements. This database constitutes one of the most developed genomic resources accessible among Lophotrochozoans, an orphan clade of bilateral animals. These data will accelerate the development of both genomics and genetics in a commercially-important species with the highest annual, commercial production of any aquatic organism

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

cited By 30International audienceIn this study, lipid classes and fatty acid composition were determined in eggs of P. vannamei as a function of survival to zoca III stage. Spawns were reared individually to zoea III and grouped, according to their final survival to this larval stage, into spawns of high and low survival. Eggs of individual spawns were analyzed for lipid and fatty acid composition of neutral and polar lipids and the results were then grouped according to survival to 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 fatty acid composition. Higher levels of triglycerides, carotenoids, and linoleic (18:2n-6) acid in eggs were associated with improved survival to zoea III. Linoleic acid was higher in spawns from the high survival group in both neutral and polar fractions and in most of the phospholipid classes analyzed. Docosahexanoic acid (DHA) was not related to survival to zoea, probably because its content was high enough (> 15% of total fatty acids) to satisfy embryo and early larval development needs. A high content of eicosapentaenoic acid (EPA) was found in phosphatidylethanolamine. lysophosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol compared to other phospholipid classes, suggesting a specific role of EPA in these lipids. These results describe the specific phospholipid composition of penaeid eggs and could (potentially) be used as predictive indicators of larval quality for research and production purposes

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

International audienceIn 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

Environmental anomalies, energetic reserves and fatty acid modifications in oysters coincide with an exceptional mortality event

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

Effect of infection by the protistan parasite Marteilia refringens on the enzyme activity and energy reserves of oyster Ostrea stentina (Payraudeau, 1826) in Tunisia

WOS:000430473000014This is the first study of the metabolic enzyme activity and energy state of the dwarf oyster Ostrea stentina (Payraudeau, 1826) in Tunisia and in the Mediterranean region. The main purpose of this study was to examine the modulation of the physiological status of the oyster O. stentina depending on the season and the presence of parasite Marteilia refringens. The prevalence of bonamiosis and marteiliosis were established by PCR. Bonamia exitiosa was detected only in 2.91% of oysters while the prevalence of M. refringens was 100% (30 Ind./30) in February and 93.93% (28 Ind./30) in March. A 42-87% mortality rate was reported during the study period. Biochemical analyses were carried out to evaluate the management of the energy resources in regard to the biochemical changes of lipids and carbohydrates. The enzyme activity of pyruvate kinase (PK), citrate synthase (CS) and cytochrome c oxidase (CCO) were measured and the mean level of enzyme activity was respectively 20.31 mU mg(-1) protein; 12.06 mU mg(-1) protein and 3.59 mU mg(-1) protein. Carbohydrate contents of O. stentina were very low all year round with an average of 15.18% in dry weight, and lipid contents remain similar (11.77% in dry weight) compared with the values reported for most other temperate bivalves. Enzyme activity significantly decreased over time (P \textless 0.001). The energy resources of the oyster O. stentina wherein much of the energy was devoted to reproduction seem to affect the defence system

Impact of food availability on energy storage and defense related hemocyte parameters of the Pacific oyster Crassostrea gigas during an experimental reproductive cycle

Le texte intégral est accessible via Archimer: http://archimer.ifremer.fr/doc/2006/publication-1549.pdfInternational audienceThe aim of this study was to test the effect of food quantity on energy storage and defense capacities of oysters during a reproductive cycle. One-year-old Crassostrea gigas oysters were fed two different dietary rations (4% and 12% of oyster dry weight in algal dry weight per day) in controlled experimental conditions over an annual cycle. Oyster dry weights, carbohydrate and lipid contents, energetic adenylate charge, and hemocyte parameters of oysters were significantly affected by reproductive processes related to seasonal temperature variation and, to a lesser extent, by the dietary rations. Energy parameters decreased during gametogenesis as gonads developed then increased during the gonad resorption phase. The additional energy provided to oysters fed the 12% diet compared to oysters fed the 4% diet was allocated mainly to the development of more gonad tissue. Regardless of diet, hemocyte concentrations were also seasonally affected. Hemocyte concentrations were low during gametogenesis and significantly increased during the gonadal resorption and tissue restoration phase. Phagocytic activity and adhesive capacity of hemocytes were temporarily inhibited during gametogenesis and were at their lowest levels in June. Oysters fed the 12% diet had significantly higher hemocyte concentrations and lower phagocytosis activity and reactive oxygen species production compared to those fed the 4% diet

Oyster adenylate energy charge: response to levels of food

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

Effect of sediment nearness on the metabolic enzyme activity and energy state of the oyster

This study was designed to assess in situ the effects of sediment nearness on Crassostrea gigas metabolism. One year-old oysters were reared for 5 months (April-August) in plastic bags on metallic frames at 10 and 60 cm off the sediment at the beginning of April. The management of the energetic resources (storage, consumption) was estimated with respect to biochemical changes of proteins, lipids and carbohydrates. In order to evaluate the effect of the sediment nearness at cellular level, the metabolic rate was evaluated by assessing pyruvate kinase (PK) and phosphoenolpyruvate carboxykinase (PEPCK) enzyme activities and electron transport system (ETS) activity. The metabolic pathways were assessed by measuring ATP and intermediary metabolites such as alanine, succinate and aspartate. Despite similar survival and growth at the two rearing levels, the protein, lipid and carbohydrate content were significantly lower in the oysters close to the bottom. In the oysters reared at 10 cm, PK activity was also significantly reduced and a significant negative correlation between alanine content and PK activity was revealed. The sediment nearness decreased significantly the ATP content in the oyster. There was a significant relationship between ATP and aspartate in these oysters. The use of aspartate contributed to complete the metabolic pathways along with carbohydrate allowing to maintain the same biological performance as the oyster reared far from the bottom. The energy state (proteins, lipids, carbohydrates and ATP contents) could suggest that the oysters close to the sediment were fed less but the metabolic enzyme activities allow to suggest an oxygen deficiency. However, the reduced PK activity, the absence of PEPCK activity stimulation and the utilization of aspartate suggest a transition stage to anaerobiosis