Philippe Miner

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.

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.

Mortality control of scallop larvae in the hatchery

Larval mortalities occurring in molluscan hatcheries have often been associated with bacterial contamination, and more specifically with vibrios. Although batches of oyster and clam larvae have been routinely reared in the hatchery of Argenton (North Brittany, France) without antibiotics, high larval mortalities have been recorded with the great scallop, Pecten maximus, under similar conditions. For this species, an addition of antibiotics was found necessary and chloramphenicol was used at a concentration of 8 mg l−1. However, this chemical has now been banned in Europe, making either substitution products or an improvement in the rearing procedures essential. Studies carried out have shown that neither a decrease in larval density (to 1 larva ml−1) nor an increase in seawater change frequency (to one per day) had any positive effects. Furthermore, elective substances such as sugars were not suitable and the use of another antibiotic, erythromycin, led to inconsistent results. The only positive effects were obtained with lower levels of chloramphenicol, which does not resolve the problem. Because no alternative solutions have as yet been found, further research needs to be undertaken.

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.

Effects of bioactive extracellular compounds and paralytic shellfish toxins produced by Alexandrium minutum on growth and behaviour of juvenile great scallops Pecten maximus

Dinoflagellates of the genus Alexandrium are a major cause of harmful algal blooms (HABs) that have increasingly disrupted coastal ecosystems for the last several decades. Microalgae from the genus Alexandrium are known to produce paralytic shellfish toxins (PST) but also bioactive extracellular compounds (BEC) that can display cytotoxic, allelopathic, ichtyotoxic or haemolytic effects upon marine organisms. The objective of this experimental study was to assess the effects of PST and BEC produced by A. minutum upon juvenile great scallops Pecten maximus. Scallops were exposed for one week to two different strains of A. minutum, the first producing both PST and BEC and the second producing only BEC. Escape response to starfish, daily shell growth, histological effects, and accumulation of PST were recorded after one week of exposure, and after two subsequent weeks of recovery. Daily shell growth was delayed three days in scallops exposed to the BEC-producing A. minutum strain, probably during the three first days of exposure. An increase of reaction time to predators was observed in scallops exposed to the BEC condition, suggesting that BEC may have altered sensing processes. Scallops exposed to PST displayed a less-efficient escape response and muscular damage which could reflect the effects of paralytic toxins upon the nervous system of scallops. This study demonstrates contrasting effects of the distinct toxic compounds produced by A. minutum upon marine bivalves, thus highlighting the importance to better characterize these extracellular, bioactive compounds to better understand responses of other marine organisms.

Non-additive effects of ocean acidification in combination with warming on the larval proteome of the Pacific oyster, Crassostrea gigas

UNLABELLED Increasing atmospheric carbon dioxide results in ocean acidification and warming, significantly impacting marine invertebrate larvae development. We investigated how ocean acidification in combination with warming affected D-veliger larvae of the Pacific oyster Crassostrea gigas. Larvae were reared for 40h under either control (pH8.1, 20 °C), acidified (pH7.9, 20 °C), warm (pH8.1, 22 °C) or warm acidified (pH7.9, 22 °C) conditions. Larvae in acidified conditions were significantly smaller than in the control, but warm acidified conditions mitigated negative effects on size, and increased calcification. A proteomic approach employing two-dimensional electrophoresis (2-DE) was used to quantify proteins and relate their abundance to phenotypic traits. In total 12 differentially abundant spots were identified by nano-liquid chromatography-tandem mass spectrometry. These proteins had roles in metabolism, intra- and extra-cellular matrix formations, stress response, and as molecular chaperones. Seven spots responded to reduced pH, four to increased temperature, and six to acidification and warming. Reduced abundance of proteins such as ATP synthase and GAPDH, and increased abundance of superoxide dismutase, occurred when both pH and temperature changes were imposed, suggesting altered metabolism and enhanced oxidative stress. These results identify key proteins that may be involved in the acclimation of C. gigas larvae to ocean acidification and warming. SIGNIFICANCE Increasing atmospheric CO2 raises sea surface temperatures and results in ocean acidification, two climatic variables known to impact marine organisms. Larvae of calcifying species may be particularly at risk to such changing environmental conditions. The Pacific oyster Crassostrea gigas is ecologically and commercially important, and understanding its ability to acclimate to climate change will help to predict how aquaculture of this species is likely to be impacted. Modest, yet realistic changes in pH and/or temperature may be more informative of how populations will respond to contemporary climate change. We showed that concurrent acidification and warming mitigates the negative effects of pH alone on size of larvae, but proteomic analysis reveals altered patterns of metabolism and an increase in oxidative stress suggesting non-additive effects of the interaction between pH and temperature on protein abundance. Thus, even small changes in climate may influence development, with potential consequences later in life.

Long dsRNAs promote an anti-viral response in Pacific oyster hampering ostreid herpesvirus 1 replication

ABSTRACT Double-stranded RNA (dsRNA)-mediated genetic interference (RNAi) is a widely used reverse genetic tool for determining the loss-of-function phenotype of a gene. Here, the possible induction of an immune response by long dsRNA was tested in a marine bivalve (Crassostrea gigas), as well as the specific role of the subunit 2 of the nuclear factor κB inhibitor (IκB2). This gene is a candidate of particular interest for functional investigations in the context of oyster mass mortality events, as Cg-IκB2 mRNA levels exhibited significant variation depending on the amount of ostreid herpesvirus 1 (OsHV-1) DNA detected. In the present study, dsRNAs targeting Cg-IκB2 and green fluorescent protein genes were injected in vivo into oysters before being challenged by OsHV-1. Survival appeared close to 100% in both dsRNA-injected conditions associated with a low detection of viral DNA and a low expression of a panel of 39 OsHV-1 genes as compared with infected control. Long dsRNA molecules, both Cg-IκB2- and GFP-dsRNA, may have induced an anti-viral state controlling the OsHV-1 replication and precluding the understanding of the specific role of Cg-IκB2. Immune-related genes including Cg-IκB1, Cg-Rel1, Cg-IFI44, Cg-PKR and Cg-IAP appeared activated in the dsRNA-injected condition, potentially hampering viral replication and thus conferring a better resistance to OsHV-1 infection. We revealed that long dsRNA-mediated genetic interference triggered an anti-viral state in the oyster, emphasizing the need for new reverse genetics tools for assessing immune gene function and avoiding off-target effects in bivalves. Summary: Double-stranded ribonucleic acid (dsRNA) injection in the Pacific oyster induced an anti-viral state controlling ostreid herpesvirus 1 replication and precluding the understanding of the role of the inhibitor of nuclear factor-κB, Cg-IκB2.

Bioactive extracellular compounds produced by the dinoflagellate Alexandrium minutum are highly detrimental for oysters

Blooms of the dinoflagellate Alexandrium spp., known as producers of paralytic shellfish toxins (PSTs), are regularly detected on the French coastline. PSTs accumulate into harvested shellfish species, such as the Pacific oyster Crassostrea gigas, and can cause strong disorders to consumers at high doses. The impacts of Alexandrium minutum on C. gigas have often been attributed to its production of PSTs without testing separately the effects of the bioactive extracellular compounds (BECs) with allelopathic, hemolytic, cytotoxic or ichthyotoxic properties, which can also be produced by these algae. The BECs, still uncharacterized, are excreted within the environment thereby impacting not only phytoplankton, zooplankton but also marine invertebrates and fishes, without implicating any PST. The aim of this work was to compare the effects of three strains of A. minutum producing either only PSTs, only BECs, or both PSTs and BECs, on the oyster C. gigas. Behavioral and physiological responses of oysters exposed during 4 days were monitored and showed contrasted behavioral and physiological responses in oysters supposedly depending on produced bioactive substances. The non-PST extracellular-compound-producing strain primarily strongly modified valve-activity behavior of C. gigas and induced hemocyte mobilization within the gills, whereas the PST-producing strain caused inflammatory responses within the digestive gland and disrupted the daily biological rhythm of valve activity behavior. BECs may therefore have a significant harmful effect on the gills, which is one of the first organ in contact with the extracellular substances released in the water by A. minutum. Conversely, the PSTs impact the digestive gland, where they are released and mainly accumulated, after degradation of algal cells during digestion process of bivalves. This study provides a better understanding of the toxicity of A. minutum on oyster and highlights the significant role of BECs in this toxicity calling for further chemical characterization of these substances.