Fabrice Pernet

Temperature adaptation in two bivalve species from different thermal habitats: energetics and remodelling of membrane lipids

SUMMARY We compared lipid dynamics and the physiological responses of blue mussels Mytilus edulis, a cold-adapted species, and oysters Crassostrea virginica, a warmer-water species, during simulated overwintering and passage to spring conditions. To simulate overwintering, animals were held at 0°C, 4°C and 9°C for 3 months and then gradually brought to and maintained at 20°C for 5 weeks to simulate spring–summer conditions. Changes in lipid class and fatty acid composition were related to clearance rate and oxygen consumption. We found major differences between species in triglyceride (TAG) metabolism during overwintering. Mussels used digestive gland TAG stores for energy metabolism or reproductive processes during the winter, whereas oysters did not accumulate large TAG stores prior to overwintering. Mussel TAG contained high levels of 20:5n-3 compared to levels in oysters and in the diet. This may help to counteract the effect of low temperature by reducing the melting point of TAG and thus increasing the availability of storage fats at low temperature. Mussels seemed better able to mobilise 20:5n-3 and 18:4n-3 than other fatty acids. We also found that both bivalves underwent a major remodelling of membrane phospholipids. The unsaturation index decreased in the gills and digestive glands of both species during the early stages of warming, principally due to decreases in 22:6n-3 and 20:5n-3. In digestive glands, the unsaturation index did not increase with decreasing temperature beyond a threshold attained at 9°C whereas a perfect negative relationship was observed in gills, as predicted by homeoviscous adaptation. The presence of digestive enzymes and acids in the digestive gland microenvironment may lead to specific requirements for membrane stability. That oysters had lower metabolic rates than mussels coincides with a lower unsaturation index of their lipids, as predicted by Hulberts theory of membranes as metabolic pacemakers. Both species showed increased 20:4n-6 levels in their tissues as temperature rose, suggesting an increasing availability of this fatty acid for eicosanoid biosynthesis during stress responses. The contrast between the species in TAG dynamics and the similarity of their phospholipid remodelling emphasises the essential functional role of membrane phospholipid structure and the contrasting use of TAG by oysters and mussels during overwintering.

Temperature influence on pathogen transmission and subsequent mortalities in juvenile Pacific oysters Crassostrea gigas

Since 2008, mass mortalities of 1-yr-old Crassostrea gigas associated with the ostreid herpesvirus OsHV-1 μVar have occurred along all the coasts of France when seawater temperature reaches 16 to 17°C. The present study aimed to characterize the effect of temperature on oyster survival in combination with OsHV-1 DNA quantification by standard real-time PCR and total vibrio population levels in oyster tissues. To examine the effect of seawater temperature on disease transmission and related mortality of oysters, cohabitation experiments were conducted between healthy naive oysters and oysters previously exposed to field conditions in areas where mortalities were occurring. Oysters initially maintained in controlled conditions (free of mortality and negative for OsHV-1), and then transferred to an area where high mortalities were occurring among farmed stocks, became infected with OsHV-1 and exhibited high loads of vibrios followed by significant mortalities. When previously exposed oysters were maintained indoors at 13.0°C for 40 d and then at 20.6°C, they exhibited no mortality, were negative for OsHV-1 detection, and did not transmit the disease to healthy oysters. Survival of previously exposed oysters maintained indoors at 8 temperatures ranging from 13.4 to 29.0°C varied from 25 to 48% and was negatively correlated with holding temperature. Concomitantly, survival of naive cohabiting animals (62 to 98%) decreased with increasing seawater temperature until a plateau was reached between 16.2 and 21.9°C, and increased at higher temperatures. Therefore, the optimal temperature range for disease transmission from field-exposed to naive animals was between 16.2 and 21.9°C. Our results suggest that a long-term period (40 d) at low temperature (13°C) may offer a method of mitigating mortalities in oysters that have been exposed to an infective environment.

Variation of lipid class and fatty acid composition of Chaetoceros muelleri and Isochrysis sp. grown in a semicontinuous system

The lipid class and the fatty acid composition of microalgae are of primary importance in feeding filter-feeding animals properly and variations of those parameters have never been investigated in a cultivation system applied to hatcheries. The objective of this study was to document the lipid class and fatty acid composition of the diatom Chaetoceros muelleri (CHGRA) and the flagellate Isochrysis sp. (Clone TISO) during the entire process of hatchery cultivation in relation to the period of the year (summer vs. winter). At the beginning of the cultivation process of CHGRA, in a small volume of culture, triacylglycerol (TAG), sterol, saturated, monounsaturated, and polyunsaturated fatty acids (particularly 20:5n3) were high, sharply dropping when volume was increased. An inverse relationship has been observed for acetone mobile polar lipids (AMPLs; pigment) and phospholipids (PLs), thus leading to no significant effect of the volume of culture on the total lipid (TL) content of CHGRA. The total lipid content of TISO increased with the volume of culture due to AMPL and PL classes and saturated and polyunsaturated fatty acids. The short- and long-term variability of lipid class composition has been observed for both species. Such results highlight the high variability of marine microalgae lipid content during the culture process and season as well. Hatchery implication is discussed. D 2003 Elsevier Science B.V. All rights reserved.

Dietary BMAA Exposure in an Amyotrophic Lateral Sclerosis Cluster from Southern France

Background Dietary exposure to the cyanotoxin BMAA is suspected to be the cause of amyotrophic lateral sclerosis in the Western Pacific Islands. In Europe and North America, this toxin has been identified in the marine environment of amyotrophic lateral sclerosis clusters but, to date, only few dietary exposures have been described. Objectives We aimed at identifying cluster(s) of amyotrophic lateral sclerosis in the Hérault district, a coastal district from Southern France, and to search, in the identified area(s), for the existence of a potential dietary source of BMAA. Methods A spatio-temporal cluster analysis was performed in the district, considering all incident amyotrophic lateral sclerosis cases identified from 1994 to 2009 by our expert center. We investigated the cluster area with serial collections of oysters and mussels that were subsequently analyzed blind for BMAA concentrations. Results We found one significant amyotrophic lateral sclerosis cluster (p = 0.0024), surrounding the Thau lagoon, the most important area of shellfish production and consumption along the French Mediterranean coast. BMAA was identified in mussels (1.8 µg/g to 6.0 µg/g) and oysters (0.6 µg/g to 1.6 µg/g). The highest concentrations of BMAA were measured during summer when the highest picocyanobacteria abundances were recorded. Conclusions While it is not possible to ascertain a direct link between shellfish consumption and the existence of this ALS cluster, these results add new data to the potential association of BMAA with sporadic amyotrophic lateral sclerosis, one of the most severe neurodegenerative disorder.

Effect of ultrasonication and grinding on the determination of lipid class content of microalgae harvested on filters

To ensure complete lipid extraction of algal samples collected on glass fiber filters, one must facilitate the access of extracting solvent to the lipids by using ultrasonication, grinding, or a combination of these two methods. This study examines the effect of these three treatments, in combination with storage time and sampling volumes, on the determination of lipid class composition of the diatom Chaetoceros gracilis. The TAG level was significantly influenced by treatment in relation to either storage time or sampling volume. FFA and other degradation products increased markedly with storage time at the expense of TAG and phospholipids (PL). Finally, sampling volume and storage time interacted in their effects on TAG and PL contents in an inverse manner.

Integrative Study of Physiological Changes Associated with Bacterial Infection in Pacific Oyster Larvae

Background Bacterial infections are common in bivalve larvae and can lead to significant mortality, notably in hatcheries. Numerous studies have identified the pathogenic bacteria involved in such mortalities, but physiological changes associated with pathogen exposure at larval stage are still poorly understood. In the present study, we used an integrative approach including physiological, enzymatic, biochemical, and molecular analyses to investigate changes in energy metabolism, lipid remodelling, cellular stress, and immune status of Crassostrea gigas larvae subjected to experimental infection with the pathogenic bacteria Vibrio coralliilyticus. Findings Our results showed that V. coralliilyticus exposure induced (1) limited but significant increase of larvae mortality compared with controls, (2) declined feeding activity, which resulted in energy status changes (i.e. reserve consumption, β-oxidation, decline of metabolic rate), (3) fatty acid remodeling of polar lipids (changes in phosphatidylinositol and lysophosphatidylcholine composition`, non-methylene–interrupted fatty acids accumulation, lower content of major C20 polyunsaturated fatty acids as well as activation of desaturases, phospholipase and lipoxygenase), (4) activation of antioxidant defenses (catalase, superoxide dismutase, peroxiredoxin) and cytoprotective processes (heat shock protein 70, pernin), and (5) activation of the immune response (non-self recognition, NF-κκ signaling pathway, haematopoiesis, eiconosoids and lysophosphatidyl acid synthesis, inhibitor of metalloproteinase and antimicrobial peptides). Conclusion Overall, our results allowed us to propose an integrative view of changes induced by a bacterial infection in Pacific oyster larvae, opening new perspectives on the response of marine bivalve larvae to infections.

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.

Physiological and biochemical traits correlate with differences in growth rate and temperature adaptation among groups of the eastern oyster Crassostrea virginica.

SUMMARY We tested two hypotheses in this study: first, that intraspecific growth variations in a marine bivalve are correlated with physiological (basal metabolic rate and scope for growth) and biochemical (membrane lipids) characteristics, and, second, that this bivalve shows intraspecific variations in physiological and biochemical adaptations to temperature. To test these hypotheses, five genetically distinct groups of juvenile oysters Crassostrea virginica that showed differences in their growth rates were maintained in the laboratory (1) for further measurements of growth and standard metabolic rates and (2) subjected to acclimation at 4°C, 12°C and 20°C and further examined for scope for growth and determination of membrane lipid composition. Our results show that a lower basal metabolic rate and lower unsaturation index of membrane lipids coincides with higher growth rates and a higher scope for growth in oysters. We provide evidence that intraspecific differences in basal metabolic rate in oysters are related to membrane unsaturation as predicted by Hulberts theory of membranes as metabolic pacemakers. Furthermore, our results suggest that the theory of membranes as metabolic pacemakers is related to intraspecific differences in growth. A perfect negative relationship was observed between the acclimation temperature and the unsaturation index of membrane lipids in oysters, as predicted by the homeoviscous adaptation theory. However, changes in the unsaturation index in response to temperature were mainly due to variations in the eicosapentaenoic (20:5n-3) fatty acid in fast-growing oysters, whereas slow-growing animals changed both docosahexaenoic acid (22:6n-3) and 20:5n-3. Thus, the pattern of biochemical compensation in response to temperature in this species shows intraspecific variation.

Lipid remodeling in wild and selectively bred hard clams at low temperatures in relation to genetic and physiological parameters

SUMMARY A temperature decrease usually induces an ordering effect in membrane phospholipids, which can lead to membrane dysfunction. Poikilotherms inhabiting eurythermal environments typically counteract this temperature effect by remodeling membrane lipids as stipulated in the homeoviscous adaptation theory (HVA). Hard clams, Mercenaria mercenaria, can suffer high overwintering mortalities in the Gulf of St Lawrence, Canada. The selectively bred M. mercenaria var. notata can have higher overwintering mortalities than the wild species, thus suggesting that the two varieties have different degrees of adaptation to low temperatures. The objective of this study was to investigate the changes in lipid composition of soft tissues in wild and selected hard clams in relation to their metabolic and genetic characteristics. Clams were placed at the northern limit of their distribution from August 2003 to May 2004; they were exposed to a gradual temperature decrease and then maintained at <0°C for 3.5 months. This study is the first to report a major remodeling of lipids in this species as predicted by HVA; this remodeling involved a sequential response of the phospholipid to sterol ratio as well as in levels of 22:6n-3 and non-methylene interrupted dienoic fatty acids. Hard clams showed an increase in 20:5n-3 as temperature decreased, but this was not maintained during overwintering, which suggests that 20:5n-3 may have been used for eicosanoid biosynthesis as a stress response to environmental conditions. Selectively bred hard clams were characterized by a higher metabolic demand and a deviation from Hardy-Weinberg equilibrium at several genetic loci due to a deficit in heterozygote frequency compared with wild clams, which is believed to impose additional stress and render these animals more vulnerable to overwintering mortality. Finally, an intriguing finding is that the lower metabolic requirements of wild animals coincide with a lower unsaturation index of their lipids, as predicted by Hulberts theory of membranes as pacemakers of metabolism.

Infectious diseases in oyster aquaculture require a new integrated approach

Emerging diseases pose a recurrent threat to bivalve aquaculture. Recently, massive mortality events in the Pacific oyster Crassostrea gigas associated with the detection of a microvariant of the ostreid herpesvirus 1 (OsHV-1µVar) have been reported in Europe, Australia and New Zealand. Although the spread of disease is often viewed as a governance failure, we suggest that the development of protective measures for bivalve farming is presently held back by the lack of key scientific knowledge. In this paper, we explore the case for an integrated approach to study the management of bivalve disease, using OsHV-1 as a case study. Reconsidering the key issues by incorporating multidisciplinary science could provide a holistic understanding of OsHV-1 and increase the benefit of research to policymakers.