近江牡蛎HSP70基因对溶藻弧菌感染的反应

采用实时荧光定量RT-PCR方法,检测了注射溶藻弧菌(Vibiro alginolyticus)后近江牡蛎鳃,闭壳肌,消化腺,外套膜,心脏以及血细胞中HSP70基因的表达变化。结果显示近江牡蛎这五种器官组织中的HSP70基因表达量均出现显著性高表达,且在鳃、外套膜和血细胞中的HSP70基因表达变化规律表现为典型的时间依赖性。血细胞中,显著高表达的峰值出现在24h,至72h恢复到对照水平,高表达持续时间最长:鳃中表达峰值出现时间较早,在第3h,随后在第12h便恢复到对照水平;外套膜,消化腺以及心脏中的峰值分别出现在6h,6h和3h,而在闭壳肌组织中,没出现显著性高表达。由此可见,近江牡蛎HSP70s可能在机体抗菌免疫过程中起了重要作用。     

Cadmium Accumulation and Metallothionein Biosynthesis in Cadmium-Treated Freshwater Mussel Anodonta woodiana

This study investigated the distribution of cadmium (Cd) and the protein level of metallothionein (MT) and examined the relationship of Cd accumulation and the MT concentration in different tissues of freshwater mussel Anodonta woodiana following Cd treatment. The mussels were exposed to Cd (4.21, 8.43, 16.86, 33.72 and 67.45 mg L^-1) for 24, 48, 72 and 96 h, respectively. After Cd treatment, the gills, mantle, foot, visceral mass and digestive gland tissues were collected for analysis. We found that, in the controls, Cd distributed in all tissues in the concentration order of gills>mantle>foot>visceral mass>digestive gland. Upon Cd treatment, Cd concentration significantly increased in all tissues. The highest Cd accumulation was found in the digestive gland, which was 0.142 mg g^-1 (P<0.05). MT levels in the gills and mantle of the mussels increased significantly (P<0.05), which were in positive correlation with Cd accumulation in the tissues (P<0.05). In conclusion, our results demonstrated a correlation between Cd accumulation and MT up-regulation in gills and mantle of the mussels after Cd treatment. It is suggested that the protein level of MT in gills and mantle of Anodonta woodiana is a good biomarker for Cd contamination.     

Tissue distribution of a coliphage and Escherichia coli in mussels after contamination and depuration.

Experiments were undertaken to determine the tissue distribution of Escherichia coli and a coliphage after contamination of the common mussel (Mytilus edulis). Mussels were contaminated with high levels of feces-associated E. coli and a 22-nm icosahedral coliphage over a 2-day period in a flowing-seawater facility. After contamination, individual tissues were carefully dissected and assayed for E. coli and the coliphage. Contaminated mussels were also analyzed to determine the tissue distribution of the contaminants after 24- and 48-h depuration periods. The majority of each contaminant was located in the digestive tract (94 and 89% of E. coli and coliphage, respectively). Decreasing concentrations were found in the gills and labial palps, foot and muscles, mantle lobes, and hemolymph. Our results indicate that contamination above levels in water occurred only in the digestive tract. Contaminated mussels were depurated in a commercial-scale recirculating UV depuration system over a 48-h period. The percent reductions of E. coli occurred in the following order: digestive tract, hemolymph, foot and muscles, mantle lobes, and gills and labial palps. The percent reductions of the coliphage were different, occurring in the following order: hemolymph, foot and muscles, gills and labial palps, mantle lobes, and digestive tract. Our results clearly demonstrate that E. coli and the coliphage are differentially eliminated from the digestive tract. The two microorganisms are eliminated at similar rates from the remaining tissues. Our results also clearly show that the most significant coliphage retention after depuration for 48 h is in the digestive tract. Thus, conventional depuration practices are inappropriate for efficient virus elimination from mussels.     

Tulane Virus as a Potential Surrogate To Mimic Norovirus Behavior in Oysters

Oyster contamination by noroviruses is an important health and economic problem. The present study aimed to compare the behaviors of Norwalk virus (the prototype genogroup I norovirus) and two culturable viruses: Tulane virus and mengovirus. After bioaccumulation, tissue distributions were quite similar for Norwalk virus and Tulane virus, with the majority of viral particles detected in digestive tissues, while mengovirus was detected in large amounts in the gills and mantle as well as in digestive tissues. The levels of persistence of all three viruses over 8 days were comparable, but clear differences were observed over longer periods, with Norwalk and Tulane viruses displaying rather similar half-lives, unlike mengovirus, which was cleared more rapidly. These results indicate that Tulane virus may be a good surrogate for studying norovirus behavior in oysters, and they confirm the prolonged persistence of Norwalk virus in oyster tissues.     

Tissue distribution of a coliphage and Escherichia coli in mussels after contamination and depuration

Experiments were undertaken to determine the tissue distribution of Escherichia coli and a coliphage after contamination of the common mussel (Mytilus edulis). Mussels were contaminated with high levels of feces-associated E. coli and a 22-nm icosahedral coliphage over a 2-day period in a flowing-seawater facility. After contamination, individual tissues were carefully dissected and assayed for E. coli and the coliphage. Contaminated mussels were also analyzed to determine the tissue distribution of the contaminants after 24- and 48-h depuration periods. The majority of each contaminant was located in the digestive tract (94 and 89% of E. coli and coliphage, respectively). Decreasing concentrations were found in the gills and labial palps, foot and muscles, mantle lobes, and hemolymph. Our results indicate that contamination above levels in water occurred only in the digestive tract. Contaminated mussels were depurated in a commercial-scale recirculating UV depuration system over a 48-h period. The percent reductions of E. coli occurred in the following order: digestive tract, hemolymph, foot and muscles, mantle lobes, and gills and labial palps. The percent reductions of the coliphage were different, occurring in the following order: hemolymph, foot and muscles, gills and labial palps, mantle lobes, and digestive tract. Our results clearly demonstrate that E. coli and the coliphage are differentially eliminated from the digestive tract. The two microorganisms are eliminated at similar rates from the remaining tissues. Our results also clearly show that the most significant coliphage retention after depuration for 48 h is in the digestive tract. Thus, conventional depuration practices are inappropriate for efficient virus elimination from mussels.     

Differential tissue distribution and specificity of phenoloxidases from the Pacific oyster Crassostrea gigas

Phenoloxidases (POs) play a key role in melanin production, are involved in invertebrate immune mechanisms, and have been detected in different bivalves. Recently, we identified catecholase- and laccase-like PO activities in plasma and haemocyte lysate supernatant (HLS) of the Pacific oyster Crassostrea gigas. To go further in our investigations, the aims of this study were (i) to determine the tissue distribution of PO activities in C. gigas, and (ii) to identify and characterise the different sub-classes of POs (i.e. tyrosinase, catecholase and/or laccase) involved in these oxido-reductase activities. With dopamine and p-phenylenediamine (PPD) but not with l-tyrosine used as substrates, PO-activities were detected by spectrophotometry in the gills, digestive gland, mantle, and muscle. These results suggest the presence of catecholase and laccase but not of tyrosinase activities in oyster tissues. The highest activity was recovered in the digestive gland. PO-like activities were all inhibited by 1-phenyl-2-thiourea (PTU) and by the specific laccase inhibitor, cethyltrimethylammonium bromide (CTAB). With dopamine as substrate, the catecholase inhibitor 4-hexylresorcinol (4-HR) only inhibited PO in the muscle. SDS-PAGE zymographic assays with dopamine and PPD elicited a unique ~40kDa protein band in the muscle. In the other tissues, laccase-like activities could be related to ~10kDa and/or ~200kDa protein bands. The ~10kDa protein band was also detected in plasma and HLS, confirming the presence of a laccase in the later compartments, and probably in most of the tissues of C. gigas. This is the first time to our knowledge that a ~10kDa protein band is associated to a laccase-like activity in a mollusc species, contributing to the characterisation of phenoloxidase activities in marine bivalves.     

Identification of expressed genes in cDNA library of hemocytes from the RLO-challenged oyster, Crassostrea ariakensis Gould with special functional implication of three complement-related fragments (CaC1q1, CaC1q2 and CaC3)

A SMARTer? cDNA library of hemocyte from Rickettsia-like organism (RLO) challenged oyster, Crassostrea ariakensis Gould was constructed. Random clones (400) were selected and single-pass sequenced, resulted in 200 unique sequences containing 96 known genes and 104 unknown genes. The 96 known genes were categorized into 11 groups based on their biological process. Furthermore, we identified and characterized three complement-related fragments (CaC1q1, CaC1q2 and CaC3). Tissue distribution analysis revealed that all of three fragments were ubiquitously expressed in all tissues studied including hemocyte, gills, mantle, digestive glands, gonads and adductor muscle, while the highest level was seen in the hemocyte. Temporal expression profile in the hemocyte monolayers reveled that the mRNA expression levels of three fragments presented huge increase after the RLO incubation at 3 h and 6 h in post-challenge, respectively. And the maximal expression levels at 3 h in post-challenge are about 256, 104 and 64 times higher than the values detected in the control of CaC1q1, CaC1q2 and CaC3, respectively.     

Histological Characterization and Glucose Incorporation into Glycogen of the Pacific Oyster Crassostrea gigas Storage Cells

In order to investigate glycogen metabolism in the oyster Crassostrea gigas, the distribution of storage cells in the whole animal was studied before histological and biochemical characterization. These cells were found mainly in the labial palps, the mantle, and gonadal area and also in gills and the digestive area. Storage cells from palps, mantle, and gonad presented the same morphological features and the same seasonal glycogen variations. Storage cells were isolated from the labial palps and the mantle plus gonadal area of the oyster by enzymatic dispersion and centrifugation through discontinuous Percoll gradient. These cells have a modal density of 1.043 g/ml. An ultrastructural study confirmed that glycogen is present in the cytoplasm either as fine particles or sequestered within vesicles. Glucose incorporation into glycogen was evaluated in vitro using [U-¹⁴C]glucose: the incorporation in isolated cells increased linearly for at least 8 hours, was proportional to the cell concentration, and showed saturation kinetics with respect to the exogenous glucose concentration. Received March 18, 1999; accepted September 27, 1999.     

受孔雀石绿胁迫的近江牡蛎HSP90基因的表达变化

HSP90是重要抗逆分子,为了探讨近江牡蛎Crassostrea hongkongensis HSP90分子在抗逆中的作用,本研究建立实时荧光定量PCR方法,研究了在孔雀石绿胁迫下近江牡蛎HSP90基因在外套膜、消化腺、鳃、闭壳肌4种器官组织中的表达变化规律.结果显示,在浓度1μg/L的孔雀石绿处理下,4种器官组织中的...     

Persistence of Vibrio vulnificus in tissues of Gulf Coast oysters, Crassostrea virginica, exposed to seawater disinfected with UV light.

Vibrio vulnificus is an estuarine bacterium which can cause opportunistic infections in humans consuming raw Gulf Coast oysters, Crassostrea virginica. Although V. vulnificus is known as a ubiquitous organism in the Gulf of Mexico, its ecological relationship with C. virginica has not been adequately defined. The objective of the present study was to test the hypothesis that V. vulnificus is a persistent microbial flora of oysters and unamenable to traditional methods of controlled purification, such as UV light depuration. Experimental depuration systems consisted of aquaria containing temperature-controlled seawater treated with UV light and 0.2-microns-pore-size filtration. V. vulnificus was enumerated in seawater, oyster shell biofilms, homogenates of whole oyster meats, and tissues including the hemolymph, digestive region, gills, mantle, and adductor muscle. Results showed that depuration systems conducted at temperatures greater than 23 degrees C caused V. vulnificus counts to increase in oysters, especially in the hemolymph, adductor muscle, and mantle. Throughout the process, depuration water contained high concentrations of V. vulnificus, indicating that the disinfection properties of UV radiation and 0.2-microns-pore-size filtration were less than the rate at which V. vulnificus was released into seawater. Approximately 10(5) to 10(6) V. vulnificus organisms were released from each oyster per hour, with 0.05 to 35% originating from shell surfaces. These surfaces contained greater than 10(3) V. vulnificus organisms per cm2. In contrast, when depuration seawater was maintained at 15 degrees C, V. vulnificus was not detected in seawater and multiplication in oyster tissues was inhibited.     

Persistence of Vibrio vulnificus in tissues of Gulf Coast oysters, Crassostrea virginica, exposed to seawater disinfected with UV light.

Vibrio vulnificus is an estuarine bacterium which can cause opportunistic infections in humans consuming raw Gulf Coast oysters, Crassostrea virginica. Although V. vulnificus is known as a ubiquitous organism in the Gulf of Mexico, its ecological relationship with C. virginica has not been adequately defined. The objective of the present study was to test the hypothesis that V. vulnificus is a persistent microbial flora of oysters and unamenable to traditional methods of controlled purification, such as UV light depuration. Experimental depuration systems consisted of aquaria containing temperature-controlled seawater treated with UV light and 0.2-microns-pore-size filtration. V. vulnificus was enumerated in seawater, oyster shell biofilms, homogenates of whole oyster meats, and tissues including the hemolymph, digestive region, gills, mantle, and adductor muscle. Results showed that depuration systems conducted at temperatures greater than 23 degrees C caused V. vulnificus counts to increase in oysters, especially in the hemolymph, adductor muscle, and mantle. Throughout the process, depuration water contained high concentrations of V. vulnificus, indicating that the disinfection properties of UV radiation and 0.2-microns-pore-size filtration were less than the rate at which V. vulnificus was released into seawater. Approximately 10(5) to 10(6) V. vulnificus organisms were released from each oyster per hour, with 0.05 to 35% originating from shell surfaces. These surfaces contained greater than 10(3) V. vulnificus organisms per cm2. In contrast, when depuration seawater was maintained at 15 degrees C, V. vulnificus was not detected in seawater and multiplication in oyster tissues was inhibited.     

Sublethal effects of experimental exposure to mercury in European flat oyster Ostrea edulis: Cell alterations and quantitative analysis of metal

Oysters display a diversity of uptake mechanisms for metallic elements and distribution in the target organs, namely gills and the digestive gland. Various tissues of the flat oyster, Ostrea edulis, were studied following experimental exposure to 0.025 m (5 g l) of mercury, for up to 34 days. All animals survived the treatment. Data indicate Hg accumulation in gill tissue with a maximum concentration of 38.76 g g dry weight after 25 days of exposure. Hg levels were lower in remaining tissues, in which the maximum concentration (18.47 mg g-1 dry weight) was reached after 18 days of exposure. After these times, concentration in both tissues decreased. Results show that oysters can accumulate Hg from the environment, without their survival being affected during the experimental period. Structural alteration of epithelial tissues of gill and digestive gland of flat oyster was comparable with effects described for other metallic elements in bivalve molluscs. Interstitial tissue was disorganized in the digestive gland, and ultrastructural changes in intracellular endomembranes were detected in epithelial cells of the digestive gland after 18 days of treatment. After 25 days, absorptive epithelial cells of gills showed highly dilated, swollen microvilli. These intracellular alterations are parameters of the incipient response to the accumulation of mercury.     

Identification of bacterial diversity in the oyster Crassostrea gigas by fluorescent in situ hybridization and polymerase chain reaction

AIMS: To carry out a rapid and reliable identification of bacterial diversity in the oyster Crassostrea gigas from Todos Santos Bay, México, in the current study we applied the molecular techniques of fluorescent in situ hybridization (FISH) and polymerase chain reaction (PCR). In order to reach this goal, genus and group-specific oligonucleotides targeted to 16S rDNA/rRNA were used. METHODS AND RESULTS: Oysters were collected and different tissues were analysed by means of culture-independent methodologies. In the digestive glands and gonads gamma-Proteobacteria and Gram-positive bacteria with a low G+C content, were identified as metabolically active by FISH. In the oyster gills a higher active diversity was observed, including Gram-positive bacteria with a low and high G+C content, members of the Cytophaga/Flavobacterium cluster and gamma-Proteobacteria. Consistent with FISH analysis, the amplification of 16S rDNA genes fragments with genus and group-specific oligonucleotides confirmed the presence of the same groups, as well as members of the alpha- and beta-Proteobacterias, Pseudomonas spp. and Bacillus spp. CONCLUSIONS: The combination of accurate and very easy-to-apply molecular methods allowed us to carry out a rapid screening of high bacterial diversity in oysters. SIGNIFICANCE AND IMPACT OF THE STUDY: This work is the first report about bacterial diversity in oyster tissues analysed by FISH and PCR, without using culture-dependent methods and allowed us to determine the phylogenetic diversity of the bacterial communities present in oyster cultures, including bacteria with and without metabolic activity, as well as uncultivable cells, which are generally underestimated by traditional identification.     

Short-term bioaccumulation, circulation and metabolism of estradiol-17β in the oyster Crassostrea gigas

Steroids are active signal transmitters in Vertebrates. These roles have also been hypothesized in other Phyla and endocrine disrupting effects have been reported for different estrogen-like compounds in fishes and some marine invertebrates. As estradiol-17β has shown some physiological activities in the oyster and as estrogens or estrogen-like molecules can be present in water, we have investigated the bioaccumulation and metabolism of this estrogen in vivo in the oyster Crassostrea gigas. When dissolved in seawater, in less than 48 h estradiol-17β concentrated up to 31 times in the soft tissues of the suspension-feeder mollusc. Injected in the adductor muscle, estradiol-17β circulated from muscle to the gonad, the gills, the mantle, the labial palps, and to a lesser extent to the digestive gland. After 2 h, estradiol flow increased specifically towards this gland. Different hypotheses were raised concerning the circulation paths. However, in all cases estradiol metabolism primarily evidenced an in vivo transformation into estrone in the whole oyster and in its digestive gland. This strong 17β-hydroxysteroid-dehydrogenase activity confirms our previous in vitro results. In conclusion, it is proposed that oyster is able to take in charge estradiol as a potential contaminant in seawater. Therefore, its bioaccumulation and transformation into estrone could be studied as potential biomarkers of endocrine disruption. Furthermore, the experimental approach with dissolved steroids in the seawater combined to an anatomical screening appears as an interesting tool to investigate the bivalve endocrinology.     

Strain-dependent norovirus bioaccumulation in oysters

Noroviruses (NoVs) are the main agents of gastroenteritis in humans and the primary pathogens of shellfish-related outbreaks. Some NoV strains bind to shellfish tissues by using carbohydrate structures similar to their human ligands, leading to the hypothesis that such ligands may influence bioaccumulation. This study compares the bioaccumulation efficiencies and tissue distributions in oysters (Crassostrea gigas) of three strains from the two principal human norovirus genogroups. Clear differences between strains were observed. The GI.1 strain was the most efficiently concentrated strain. Bioaccumulation specifically occurred in digestive tissues in a dose-dependent manner, and its efficiency paralleled ligand expression, which was highest during the cold months. In comparison, the GII.4 strain was very poorly bioaccumulated and was recovered in almost all tissues without seasonal influence. The GII.3 strain presented an intermediate behavior, without seasonal effect and with less bioaccumulation efficiency than that of the GI.1 strain during the cold months. In addition, the GII.3 strain was transiently concentrated in gills and mantle before being almost specifically accumulated in digestive tissues. Carbohydrate ligand specificities of the strains at least partly explain the strain-dependent bioaccumulation characteristics. In particular, binding to the digestive-tube-specific ligand should contribute to bioaccumulation, whereas we hypothesize that binding to the sialic acid-containing ligand present in all tissues would contribute to retain virus particles in the gills or mantle and lead to rapid destruction.     

Modulations in antioxidant enzymes in different tissues of marine bivalvePerna viridis during heavy metal exposure

Lipid peroxidation induced by metals at sub-lethal levels, alter physiological and biochemical characteristics of biological systems. To counter the detrimental effects of the prooxidant activity of metals, a group of antioxidant enzyme systems function in the organisms. The present study was performed to investigate into the lipid peroxidation product formation due to the exposure to effects of the metals namely aluminium, lead and cadmium at sub-lethal concentrations and the biological response through protective antioxidant enzyme activity in the marine mussels,Perna viridis Lin. This organism is a known bioindicator and bioconcentrator of metals in the environment.The results of the present study were: (a) accumulation of lead showed a definite linear increase during the period of exposure whereas aluminium and cadmium showed fluctuations. Mantle and gill tissues showed greater accumulation of metals when compared to digestive gland; (b) lead and aluminium induced lipid peroxidation was greater in tissues than the peroxidation induced by cadmium. Cadmium induced peroxidation was observed only after the day 7 of the exposure; (c) anti-oxidant enzymes activity levels were significantly higher in digestive gland and mantle than gills; (d) mantle was observed to significantly contribute to the organismal response to lipid peroxidation as indicated by high activity levels of anti-oxidant enzymes.