Characterization of a defensin from the oyster Crassostrea gigas. Recombinant production, folding, solution structure, antimicrobial activities, and gene expression.
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In invertebrates, defensins were found in arthropods and in the mussels. Here, we report for the first time the identification and characterization of a defensin (Cg-Def) from an oyster. Cg-def mRNA was isolated from Crassostrea gigas mantle using an expressed sequence tag approach. To gain insight into potential roles of Cg-Def in oyster immunity, we produced the recombinant peptide in Escherichia coli, characterized its antimicrobial activities, determined its solution structure by NMR spectroscopy, and quantified its gene expression in vivo following bacterial challenge of oysters. Recombinant Cg-Def was active in vitro against Gram-positive bacteria but showed no or limited activities against Gram-negative bacteria and fungi. The activity of Cg-Def was retained in vitro at a salt concentration similar to that of seawater. The Cg-Def structure shares the so-called cystine-stabilized alpha-beta motif (CS-alphabeta) with arthropod defensins but is characterized by the presence of an additional disulfide bond, as previously observed in the mussel defensin (MGD-1). Nevertheless, despite a similar global fold, the Cg-Def and MGD-1 structures mainly differ by the size of their loops and by the presence of two aspartic residues in Cg-Def. Distribution of Cg-def mRNA in various oyster tissues revealed that Cg-def is mainly expressed in mantle edge where it was detected by mass spectrometry analyses. Furthermore, we observed that the Cg-def messenger concentration was unchanged after bacterial challenge. Our results suggest that Cg-def gene is continuously expressed in the mantle and would play a key role in oyster by providing a first line of defense against pathogen colonization. (+info)
Vibrio gigantis sp. nov., isolated from the haemolymph of cultured oysters (Crassostrea gigas).
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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). (+info)
In vitro research of anti-HSV-1 activity in different extracts from Pacific oysters Crassostrea gigas.
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Mortalities related to the detection of Ostreid Herpesvirus 1 (OsHV-1) have been previously reported in France among larvae and spat of the Pacific oyster Crassostrea gigas. Adult oysters appear less sensitive to herpesvirus infections, although OsHV-1 has been detected in adults without signs of disease or mortality. This suggests that the virus is able to persist in its host and that adult oysters may be able to control OsHV-1 infection. Little is known about antiviral substances in invertebrates. The present work concerns the research of antiviral substances in adult oyster C. gigas, where putative antiviral activities were monitored using 3 strategies: (1) in metabolites with variable polarity, (2) in peptidic extracts and (3) in crude haemolymph. In vitro antiviral assays were based on inhibition of Herpes simplex virus type 1 (HSV-1) replication in Vero cell monolayers. All extracts presented no cytotoxicity. Antiviral activity was detected in the fresh filtered haemolymph (EC50:425 microg ml(-1)) and seasonal variation of the haemolymph antiviral activity was monitored. (+info)
Purification and antigenic characteristics of a rickettsia-like organism from the oyster Crassostrea ariakensis.
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A rickettsia-like organism (RLO) has been suggested to be the etiological agent responsible for heavy losses of the oyster Crassostrea ariakensis Gould in China. Because of the lack of molluscan cell lines for in vitro culture of intracellular prokaryotes, antigenic analysis of RLOs has been limited by the inherent difficulties of their purification. In this report, we describe the use of differential speed centrifugation and renografin density gradient centrifugation to purify the RLO directly from infected oyster tissues. The purity and integrity of purified prokaryotes were validated by transmission electron microscopy. Thirteen major constituent proteins, with molecular weights ranging between 17 and 99 kDa, were electrophoretically identified by silver staining, and 8 major proteins were identified with Coomassie blue R staining. Specific mouse polyclonal antiserum was prepared for serological characterization of the RLO and was used in an immunoblot assay, and 3 major antigen groups were identified. The present results advance our knowledge of RLO protein antigens, and several proteins have been identified that could potentially be useful for diagnostic assays or for production of experimental immunostimulants. (+info)
A PCR-based diagnostic assay for the detection of Roseovarius crassostreae in Crassostrea virginica affected by juvenile oyster disease (JOD).
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We have developed a PCR-assay for the diagnosis of juvenile oyster disease (JOD) based on the detection of Roseovarius crassostreae directly from affected oysters. Species-specific primers are used to amplify the 16S-23S rDNA internal transcribed spacer (ITS) of R. crassostreae, and confirmation of product identity is accomplished by restriction enzyme analysis. No false positives were obtained with either closely related bacterial species or from other DNAs present in oyster samples. The assay has the potential to detect as few as 10 cells of R. crassostreae per oyster when samples are taken from the inner valve surfaces of the animal. Inclusion of material from soft body surfaces is not necessary, and may reduce sensitivity approximately 10-fold. In a JOD-affected population, a positive PCR result was obtained from all oysters from which these bacteria were subsequently cultured. The assay also detected the presence of R. crassostreae in 2 oysters from which no R. crassostreae isolates were recovered. No R. crassostreae was detected by either PCR or bacteriology in oysters from a population that was not exhibiting JOD-signs. This assay is expected to advance regional disease management efforts and provide valuable insights into the disease process and epizootiology of JOD. (+info)
Isolation by distance in the eastern oyster, Crassostrea virginica, in Chesapeake Bay.
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Intensive efforts are underway to restore depleted stocks of Crassostrea virginica in Chesapeake Bay. However, the extent of gene flow among local populations, an important force mediating the success of these endeavors, is poorly understood. Spatial and temporal population structures were examined in C. virginica from Chesapeake Bay using eight microsatellite loci. Deficits in heterozygosity relative to Hardy-Weinberg expectations were seen at all loci and were best explained by null alleles. Permutation tests indicated that heterozygote deficiency reduced power in tests of differentiation. Nonetheless, genotypic exact tests demonstrated significant levels of geographic differentiation overall, and a subtle pattern of isolation by distance (IBD) was observed. Comparisons between age classes failed to show differences in genotype frequencies, allelic richness, gene diversity, or differentiation as measured by F(ST), contrary to predictions made by the sweepstakes hypothesis. The IBD pattern could reflect an evolutionary equilibrium established because local gene flow predominates, or be influenced in either direction by recent anthropogenic activities. An evolutionary interpretation appears justified as more parsimonious, implying that local efforts to restore oyster populations will have local demographic payoffs, perhaps at the scale of tributaries or regional subestuaries within Chesapeake Bay. (+info)
Effects of acclimation temperature and cadmium exposure on cellular energy budgets in the marine mollusk Crassostrea virginica: linking cellular and mitochondrial responses.
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In order to understand the role of metabolic regulation in environmental stress tolerance, a comprehensive analysis of demand-side effects (i.e. changes in energy demands for basal maintenance) and supply-side effects (i.e. metabolic capacity to provide ATP to cover the energy demand) of environmental stressors is required. We have studied the effects of temperature (12, 20 and 28 degrees C) and exposure to a trace metal, cadmium (50 microg l(-1)), on the cellular energy budget of a model marine poikilotherm, Crassostrea virginica (eastern oysters), using oxygen demand for ATP turnover, protein synthesis, mitochondrial proton leak and non-mitochondrial respiration in isolated gill and hepatopancreas cells as demand-side endpoints and mitochondrial oxidation capacity, abundance and fractional volume as supply-side endpoints. Cadmium exposure and high acclimation temperatures resulted in a strong increase of oxygen demand in gill and hepatopancreas cells of oysters. Cd-induced increases in cellular energy demand were significant at 12 and 20 degrees C but not at 28 degrees C, possibly indicating a metabolic capacity limitation at the highest temperature. Elevated cellular demand in cells from Cd-exposed oysters was associated with a 2-6-fold increase in protein synthesis and, at cold acclimation temperatures, with a 1.5-fold elevated mitochondrial proton leak. Cellular aerobic capacity, as indicated by mitochondrial oxidation capacity, abundance and volume, did not increase in parallel to compensate for the elevated energy demand. Mitochondrial oxidation capacity was reduced in 28 degrees C-acclimated oysters, and mitochondrial abundance decreased in Cd-exposed oysters, with a stronger decrease (by 20-24%) in warm-acclimated oysters compared with cold-acclimated ones (by 8-13%). These data provide a mechanistic basis for synergism between temperature and cadmium stress on metabolism of marine poikilotherms. Exposure to combined temperature and cadmium stress may result in a strong energy deficiency due to the elevated energy demand on one hand and a reduced mitochondrial capacity to cover this demand on the other hand, which may have important implications for surviving seasonally and/or globally elevated temperatures in polluted estuaries. (+info)
Accumulation of paralytic shellfish poison (PSP) and biotransformation of its components in oysters, Crassostrea gigas, fed with the toxic dinoflagellate Alexandrium tamarense.
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As a part of our studies on the mechanism of uptake of paralytic shellfish poison (PSP) and the kinetics of its accumulation in bivalves, oysters Crassostrea gigas were experimentally contaminated with PSP by being fed with the toxic dinoflagellate Alexandrium tamarense for 2, 4, 6, 8 and 10 days. Temporal variations in the PSP contents and their profiles in oysters during the feeding experiment were monitored by high-performance liquid chromatography (HPLC) and the toxin profile of the oysters was compared with that of A. tamarense. Toxins excreted from the infested oysters into the seawater for 2 and 10 days were recovered and analyzed by HPLC. PSP toxicity rapidly appeared in the tissues of oysters and their toxicity levels reached 0.6 (0.3), 2.2 (1.1), 1.0 (0.5), 3.4 (1.6) and 1.1 (0.5) MU/g (nmol/g) shucked meat at 2, 4, 6, 8 and 10 days, respectively. The accumulation rates of toxin, calculated from the total amount (nmol) of toxins expressed by the total cell number fed during the exposure period and the toxicity of the oysters, were 14.1, 18.7, 5.1, 14.9 and 3.2% for 2, 4, 6, 8 and 10 days. During feeding experiments, the toxin profile of oysters changed substantially, showing marked differences from the proportions found in the toxigenic dinoflagellate used as food. The toxin components in this strain existed almost exclusively as beta-epimers, which accounted for 66.3 mol% of the total. This contrasts with the case of the oysters, where the beta-epimers represented 24.8, 29.8, 25.1, 27.3 and 25.2 mol% of the total at 2, 4, 6, 8 and 10 days, respectively. The amount of gonyautoxin-1 (GTX1) accumulated in oysters increased linearly and slowly for 8 days and the maximum content of GTX1 reached 51.3 mol%. The composition of GTX group compounds recovered from the seawater in which the oysters had been reared was a little different from that within the oyster tissues. (+info)