New insights in flat oyster Ostrea edulis resistance against the parasite Bonamia ostreae.

Bonamiosis due to the parasite Bonamia ostreae has been associated with massive mortality in flat oyster stocks in Europe. Control of the disease currently relies on disease management practices and transfer restriction. Previously, massal selections based on survival to challenge to infection with B. ostreae have been applied to produce flat oyster families with resistant progeny. In an attempt to understand the molecular mechanisms involved in disease resistance, differentially expressed sequence tags between resistant and wild Ostrea edulis haemocytes, both infected with the parasite, were identified using suppression subtractive hybridisation. Expression of seven ESTs has been studied using quantitative reverse-transcriptase PCR. The base-line expression of an extracellular superoxide dismutase, inhibitor of apoptosis (OeIAP), Fas ligand (OeFas-ligand) and Cathepsin B was significantly increased, whilst cyclophilin B appeared significantly decreased in resistant oysters. Considering their great interest for further studies, the open reading frames of the OeFas-ligand and OeIAP were completely sequenced.

Detection of early effects of a single herbicide (diuron) and a mix of herbicides and pharmaceuticals (diuron, isoproturon, ibuprofen) on immunological parameters of Pacific oyster (Crassostrea gigas) spat.

In the context of massive summer mortality events of the Pacific oyster Crassostrea gigas, the aim of this study was to investigate the early effects on genes, enzymes and haemocyte parameters implicated in immune defence mechanisms in C. gigas oysters exposed to a potentially hostile environment, i.e. to an herbicide alone or within a mixture. Following 2 h of exposure to the herbicide diuron at 1 µg L(-1), the repression of different genes implicated in immune defence mechanisms in the haemocytes and the inhibition of enzyme activities, such as laccase-type phenoloxidase (PO) in the plasma, were observed. The inhibition of superoxide dismutase (SOD) activity in the plasma was also observed after 6 and 24 h of exposure. In the mixture with the herbicides diuron and isoproturon, and the pharmaceutical ibuprofen, catecholase-type PO activity in the plasma and the percentage of phagocytosis in the haemocytes were reduced after 6 h of exposure. Our results showed that early effects on molecular, biochemical and cellular parameters can be detected in the presence of diuron alone or within a mixture, giving an insight of its potential effect in situations that can be found in natural environments, i.e. relatively high concentrations for short periods of time.

Suppression substractive hybridisation (SSH) and real time PCR reveal differential gene expression in the Pacific cupped oyster, Crassostrea gigas, challenged with Ostreid herpesvirus 1.

Virus-induced genes were identified using suppression subtractive hybridisation (SSH) from Pacific cupped oyster, Crassostrea gigas, haemocytes challenged by OsHV-1. A total of 304 clones from SSH forward library were sequenced. Among these sequences, some homologues corresponded to (i) immune related genes (macrophage express protein, IK cytokine, interferon-induced protein 44 or multicopper oxidase), (ii) apoptosis related genes (Bcl-2) and (iii) cell signalling and virus receptor genes (glypican). Molecular characterization and phylogenic analysis of 3 immune-related genes (macrophage expressed protein, multicopper oxidase and immunoglobulin domain cell adhesion molecule) were performed. Finally, quantitative PCR revealed significant changes in the expression of immune related genes (multicopper oxidase, macrophage expressed protein, myeloid differentiation factor 88 and interferon-induced protein 44) in oysters experimentally challenged with OsHV-1. These findings provide a first basis for studying the role of innate immunity in response to viruses in bivalves and identified genes may serve as markers of interest in breeding programs in order to obtain selected oysters presenting OsHV-1 resistance.

Experimental ostreid herpesvirus 1 infection of the Pacific oyster Crassostrea gigas: kinetics of virus DNA detection by q-PCR in seawater and in oyster samples.

Herpes- and herpes-like viruses are known to infect a wide range of bivalve mollusc species throughout the world. Abnormal summer mortalities associated to the detection of ostreid herpesvirus 1 (OsHV-1) have been currently reported in France among larvae and spat of the Pacific cupped oyster Crassostrea gigas. In the present work, we have developed an experimental protocol of horizontal transmission based on the cohabitation between healthy and experimentally infected oysters. Through a cohabitation trial, the kinetics of OsHV-1 detection in different oyster organs and seawater samples were investigated and characterized for the first time using real time quantitative PCR.

Vibrio gigantis sp. nov., isolated from the haemolymph of cultured oysters (Crassostrea gigas).

Polyphasic analysis of four new Vibrio isolates originating from the haemolymph of diseased cultured oysters is described. The new isolates were closely related to Vibrio splendidus, having 98 % 16S rRNA gene sequence similarity. Phylogenetic analysis based on DNA gyrase subunit B (gyrB), RNA polymerase sigma70 factor (rpoD), replication origin-binding protein (rctB) and transmembrane regulatory protein (toxR) genes, fluorescent amplified fragment length polymorphism and DNA-DNA hybridization experiments clearly showed that the new isolates form a tight genomic group that is different from the currently known Vibrio species. It is proposed that these new isolates should be accommodated in a novel species, Vibrio gigantis sp. nov. Phenotypic features that differentiate V. gigantis from other known Vibrio species include arginine dihydrolase, gelatinase and beta-galactosidase activities, NO(2) production, growth at 35 degrees C, and utilization of sucrose, melibiose, amygdalin, glycerol, galactose, starch and glycogen. The type strain is LGP 13T (=LMG 22741T=CIP 108656T).

Vibrio crassostreae sp. nov., isolated from the haemolymph of oysters (Crassostrea gigas).

Polyphasic analysis of five new Vibrio isolates originating from the haemolymph of diseased cultured oysters is described. The new isolates were closely related to Vibrio splendidus, having 98 % 16S rRNA gene sequence similarity. gyrB phylogenetic analysis, fluorescent amplified-fragment length polymorphism (FAFLP) fingerprinting and DNA-DNA hybridization experiments clearly showed that the new isolates form a tight genomic group that is different from the currently known Vibrio species. It is proposed to accommodate these isolates in a novel species, Vibrio crassostreae sp. nov. (type strain LGP 7(T)=LMG 22240(T)=CIP 108327(T)). Phenotypic and chemotaxonomic features that differentiate V. crassostreae from other known Vibrio species include arginine dihydrolase, utilization and fermentation of various carbon sources, beta-galactosidase activity, NO(2) production and the presence of the fatty acids 14 : 0 iso and 16 : 0 iso.