An integrative perspective on fish health: Environmental and anthropogenic pathways affecting fish stress.

Multifactorial studies assessing the cumulative effects of natural and anthropogenic stressors on individual stress response are crucial to understand how organisms and populations cope with environmental change. We tested direct and indirect causal pathways through which environmental stressors affect the stress response of wild gilthead seabream in Mediterranean costal lagoons using an integrative PLS-PM approach. We integrated information on 10 environmental variables and 36 physiological variables into seven latent variables reflecting lagoons features and fish health. These variables concerned fish lipid reserves, somatic structure, inorganic contaminant loads, and individual trophic and stress response levels. This modelling approach allowed explaining 30 % of the variance within these 46 variables considered. More importantly, 54 % of fish stress response was explained by the dependent lagoon features, fish age, fish diet, fish reserve, fish structure and fish contaminant load latent variables included in our model. This integrative study sheds light on how individuals deal with contrasting environments and multiple ecological pressures.

Harsh intertidal environment enhances metabolism and immunity in oyster (Crassostrea gigas) spat.

The Pacific oyster Crassostrea gigas is established in the marine intertidal zone, experiencing rapid and highly dynamic environmental changes throughout the tidal cycle. Depending on the bathymetry, oysters face oxygen deprivation, lack of nutrients, and high changes in temperature during alternation of the cycles of emersion/immersion. Here we showed that intertidal oysters at a bathymetry level of 3 and 5 m delayed by ten days the onset of mortality associated with Pacific Oyster Mortality Syndrome (POMS) as compared to subtidal oysters. Intertidal oysters presented a lower growth but similar energetic reserves to subtidal oysters but induced proteomic changes indicative of a boost in metabolism, inflammation, and innate immunity that may have improved their resistance during infection with the Ostreid herpes virus. Our work highlights that intertidal harsh environmental conditions modify host-pathogen interaction and improve oyster health. This study opens new perspectives on oyster farming for mitigation strategies based on tidal height.

Environmental optima for an ecosystem engineer: a multidisciplinary trait-based approach.

A complex interplay of biotic and abiotic factors underpins the distribution of species and operates across different levels of biological organization and life history stages. Understanding ecosystem engineer reproductive traits is critical for comprehending and managing the biodiversity-rich habitats they create. Little is known about how the reproduction of the reef-forming worm, Sabellaria alveolata, varies across environmental gradients. By integrating broad-scale environmental data with in-situ physiological data in the form of biochemical traits, we identified and ranked the drivers of intraspecific reproductive trait variability (ITV). ITV was highest in locations with variable environmental conditions, subjected to fluctuating temperature and hydrodynamic conditions. Our trait selection pointed to poleward sites being the most physiologically stressful, with low numbers of irregularly shaped eggs suggesting potentially reduced reproductive success. Centre-range individuals allocated the most energy to reproduction, with the highest number of intermediate-sized eggs, whilst equatorward sites were the least physiologically stressful, thus confirming the warm-adapted nature of our model organism. Variation in total egg diameter and relative fecundity were influenced by a combination of environmental conditions, which changed depending on the trait and sampling period. An integrated approach involving biochemical and reproductive traits is essential for understanding macro-scale patterns in the face of anthropogenic-induced climate change across environmental and latitudinal gradients.

[The oyster Crassostrea gigas, a new model against cancer]. / Crassostrea gigas, une huître au service de la recherche sur le cancer.

The Warburg effect is one of the hallmarks of cancer cells in humans. It is a true metabolic reprogramming to aerobic glycolysis, allowing cancer cells to meet their particular energy needs for growth, proliferation, and resistance to apoptosis, depending on the microenvironment they encounter within the tumor. We have recently discovered that the Crassostrea gigas oyster can naturally reprogram its metabolism to the Warburg effect. Thus, the oyster becomes a new invertebrate model useful for cancer research. Due to its lifestyle, the oyster C. gigas has special abilities to adapt its metabolism to the extreme changes in the environment in which it is located. The oyster C. gigas is therefore a model of interest to study how the environment can control the Warburg effect under conditions that could not be explored in vertebrate model species.

The Voltage-Dependent Anion Channel (VDAC) of Pacific Oysters Crassostrea gigas Is Upaccumulated During Infection by the Ostreid Herpesvirus-1 (OsHV-1): an Indicator of the Warburg Effect.

Voltage-dependent anion channel (VDAC) is a key mitochondrial protein. VDAC drives cellular energy metabolism by controlling the influx and efflux of metabolites and ions through the mitochondrial membrane, playing a role in its permeabilization. This protein exerts a pivotal role during the white spot syndrome virus (WSSV) infection in shrimp, through its involvement in a particular metabolism that plays in favor of the virus, the Warburg effect. The Warburg effect corresponds to an atypical metabolic shift toward an aerobic glycolysis that provides energy for rapid cell division and resistance to apoptosis. In the Pacific oyster Crassostrea gigas, the Warburg effect occurs during infection by Ostreid herpesvirus (OsHV-1). At present, the role of VDAC in the Warburg effect, OsHV-1 infection and apoptosis is unknown. Here, we developed a specific antibody directed against C. gigas VDAC. This tool allowed us to quantify the tissue-specific expression of VDAC, to detect VDAC oligomers, and to follow the amount of VDAC in oysters deployed in the field. We showed that oysters sensitive to a mortality event in the field presented an accumulation of VDAC. Finally, we propose to use VDAC quantification as a tool to measure the oyster susceptibility to OsHV-1 depending on its environment.

The quality of great scallop (Pecten maximus) sperm after thawing.

Most publications devoted to the cryopreservation of mollusc sperm have focused on the definition of technical protocols, avoiding the description of sperm quality after thawing. The present study investigated the effects of cryopreservation on sperm quality in the great scallop. Wild scallop were fished during the natural spawning period and conditioned in the hatchery before use. Sperm samples were obtained after intragonadal injection of serotonin and cryopreserved using a previously published protocol. Sperm quality was assessed using a panel of four parameters: sperm motility characteristics, using a computer assisted sperm analysis plugin with Image J, intracellular ATP content using an ATP-Lite kit, sperm integrity, using flow cytometry and sperm morphology, using transmission electron microscopy. For each parameter, fresh (control) and thawed spermatozoa were compared. A significant decrease of both the percentage of motile spermatozoa (reduction: 75%) and sperm swimming speed (86%) were observed for thawed sperm compared with fresh sperm. The percentage of living spermatozoa, as assessed using flow cytometry, was significantly lower for thawed sperm (72.4±2.5%) compared with fresh sperm (86.4±1.1). However, no significant difference of intracellular sperm ATP content was observed between fresh and thawed sperm. Post thawing, while some spermatozoa showed little or no morphological differences compared with fresh sperm, others had undergone drastic changes, including swelling of the plasma membrane, structural alterations of the chromatin and damage to mitochondria. In conclusion, the descriptive parameters studied in the present work showed that the quality of thawed great scallop sperm was lower than that of fresh cells but was still sufficient for use in aquaculture programs and sperm cryobanking for this species.

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

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.

Energy and antioxidant responses of pacific oyster exposed to trace levels of pesticides.

Here, we assess the physiological effects induced by environmental concentrations of pesticides in Pacific oyster Crassostrea gigas. Oysters were exposed for 14 d to trace levels of metconazole (0.2 and 2 µg/L), isoproturon (0.1 and 1 µg/L), or both in a mixture (0.2 and 0.1 µg/L, respectively). Exposure to trace levels of pesticides had no effect on the filtration rate, growth, and energy reserves of oysters. However, oysters exposed to metconazole and isoproturon showed an overactivation of the sensing-kinase AMP-activated protein kinase α (AMPKα), a key enzyme involved in energy metabolism and more particularly glycolysis. In the meantime, these exposed oysters showed a decrease in hexokinase and pyruvate kinase activities, whereas 2-DE proteomic revealed that fructose-1,6-bisphosphatase (F-1,6-BP), a key enzyme of gluconeogenesis, was up-regulated. Activities of antioxidant enzymes were higher in oysters exposed to the highest pesticide concentrations. Both pesticides enhanced the superoxide dismutase activity of oysters. Isoproturon enhanced catalase activity, and metconazole enhanced peroxiredoxin activity. Overall, our results show that environmental concentrations of metconazole or isoproturon induced subtle changes in the energy and antioxidant metabolisms of oysters.

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.

Proteomic signatures of the oyster metabolic response to herpesvirus OsHV-1 µVar infection.

Pacific oyster Crassostrea gigas were inoculated with OsHV-1 at low load (control) or high load (challenged) to better understand the pathogenesis of ostreid herpesvirus 1 (OsHV-1 µVar) and to determine which metabolic pathways might be affected during infection. Animals were sampled for proteomic analysis two days post-injection, at the same time as OsHV-1 initiated an intense replication phase in challenged oysters. Twenty-five abundant protein spots that showed a marked change in accumulated levels were identified using a two-dimensional electrophoresis (2-DE) proteomic approach. Overall, these proteins are involved in cytoskeleton organization, protein turnover, induction of stress signals, signalling pathways and energy metabolism. Challenged oysters exhibited an increased glycolysis and VDAC accumulation, which reflect a "Warburg effect" as initially reported in cancer cells and more recently in shrimp infected with virus. The results presented here should be useful for identifying potential biomarkers of disease resistance and developing antiviral measures. BIOLOGICAL SIGNIFICANCE: This study is the first 2-DE proteomic analysis dedicated to the pathogenesis of ostreid herpesvirus 1 (OsHV-1 µVar) in oyster Crassostrea gigas, the most important bivalve produced in the world. OsHV-1 has affected oysters every year since 2008. All the proteins identified in this paper are key targets involved in OsHV-1 infection processes. We presented evidence that the metabolic changes during infection in oyster somehow resemble the Warburg effect occurring in cancer cells. This work constitutes a real advance in the comprehension of the host metabolic pathways affected during OsHV-1 disease. Overall, this work contributes to a better understanding of disease mortalities in aquatic ecosystems which could guide management actions to mitigate their impacts.

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.

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.

Mitochondrial activity, hemocyte parameters and lipid composition modulation by dietary conditioning in the Pacific oyster Crassostrea gigas.

Several parameters can affect membrane lipid composition in bivalves, including diet. Although two fatty acids (FA) 22:6n-3 and 20:5n-3 are essential membrane components, they are sparingly synthesized by bivalves and must be obtained from their diet. Here, effects of dietary modifications of membrane lipid composition were studied at both cellular and subcellular levels in the oyster Crassostrea gigas. To this end, we compared oysters fed two monoalgal diets that differed markedly in their FA composition and a mix of both. As expected, algae impacted phospholipids, in particular 22:6n-3 and 20:5n-3, reflecting differences of dietary microalgae FA composition. Meantime, total saturated FA, total monounsaturated FA, total polyunsaturated FA and total non-methylene-interrupted FA varied little and phospholipid class composition was only slightly affected by diets. Measures made in hemocytes indicated that only mitochondrial membrane potential was affected by diets. Total ROS production as well as mitochondrial superoxide production did not differ with diet. There was no difference in phosphorylating (state 3) and non-phosphorylating (state 4) rates of oxygen consumption rates or in cytochrome c oxidase activity of mitochondria isolated from gills between the three diets. Similarly, neither cytochromes a, b, c or c1 content nor citrate synthase activities were changed, suggesting that number and morphology of mitochondria were not affected by dietary treatment. These results suggest that oysters could possess high homeostatic capabilities, at both cellular and subcellular levels, to minimize the effect of dietary FA and related membrane lipid FA modifications on mitochondrial functions. These capabilities could be a means to face variations in diet composition in their natural environment and to preserve important oyster physiological functions such as growth and reproduction.

Sex-specific regulation of AMP-activated protein kinase (AMPK) in the Pacific oyster Crassostrea gigas.

The hermaphrodite Pacific oyster Crassostrea gigas displays a high energy allocation to reproduction. We studied the expression of AMP-activated protein kinase (AMPK) during gametogenesis in the gonad and characterized the mRNA sequences of the AMPK subunits: the AMPK alpha mRNA sequence was previously characterized; we identified AMPK beta, AMPK gamma, and mRNAs of putative AMPK-related targets following bioinformatics mining on existing genomic resources. We analyzed the mRNA expression of the AMPK alpha, beta, and gamma subunits in the gonads of male and female oysters through a reproductive cycle, and we quantified the mRNA expression of genes belonging to fatty acid and glucose metabolism. AMPK alpha mRNA levels were more abundant in males at the first stage of gametogenesis, when mitotic activity and the differentiation of germinal cells occur, and were always more abundant in males than in females. Some targets of fatty acid and glucose metabolism appeared to be correlated with the expression of AMPK subunits at the mRNA level. We then analyzed the sex-specific AMPK activity by measuring the phosphorylation of the catalytic AMPK alpha protein and its expression at the protein level. Both the amount of AMPK alpha protein and threonine 172 phosphorylation appeared to be almost totally inhibited in mature female gonads at stage 3, at the time when accumulation of reserves in oocytes was promoted, while it remained at a high level in mature spermatozoa. Its activation might play a sex-dependent role in the management of energy during gametogenesis in oyster.

Regulation of a truncated isoform of AMP-activated protein kinase α (AMPKα) in response to hypoxia in the muscle of Pacific oyster Crassostrea gigas.

AMP-activated protein kinase α (AMPKα) is a key regulator of energy balance in many model species during hypoxia. In a marine bivalve, the Pacific oyster Crassostrea gigas, we analyzed the protein content of adductor muscle in response to hypoxia during 6 h. In both smooth and striated muscles, the amount of full-length AMP-activated protein kinase α (AMPKα) remained unchanged during hypoxia. However, hypoxia induced a rapid and muscle-specific response concerning truncated isoforms of AMPKα. In the smooth muscle, a truncated isoform of AMPKα was increased from 1 to 6 h of hypoxia, and was linked with accumulation of AKT kinase, a key enzyme of the insulin signaling pathway which controls intracellular glucose metabolism. In this muscle, aerobic metabolism was maintained over the 6 h of hypoxia, as mitochondrial citrate synthase activity remained constant. In contrast, in striated muscle, hypoxia did not induce any significant modification of neither truncated AMPKα nor AKT protein content, and citrate synthase activity was altered after 6 h of hypoxia. Together, our results demonstrate that hypoxia response is specific to muscle type in Pacific oyster, and that truncated AMPKα and AKT proteins might be involved in maintaining aerobic metabolism in smooth muscle. Such regulation might occur in vivo during tidal intervals that cause up to 6 h of hypoxia.

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.

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

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.