Characterisation and genetic polymorphism of metallothionein gene CgMT4 in experimental families of Pacific oyster Crassostrea gigas displaying summer mortality

Summer mortality events have been observed in Pacific oyster Crassostrea gigas for several decades. This paper examines the selective pressure exerted by summer mortality on the polymorphism of a newly identified oyster metallothionein gene. CgMT4 cDNA and genomic sequences were obtained. CgMT4 was studied in two generations of oysters reared in three sites on the French Atlantic coast, using single strand conformation polymorphism analysis. Four alleles were detected. Individuals carrying genotype MT4-CD seem to have higher susceptibility to summer risk conditions. The MT4 gene could be a potential new genetic marker for susceptibility; further validation studies are recommended.     

利用抑制差减杂交技术分离受水稻抗性调控的褐飞虱基因

为分离受水稻抗性调控的褐飞虱Nilaparvata lugens基因, 以取食感虫水稻台中1号和高抗水稻B5的2叶1芯秧苗24 h的褐飞虱4龄若虫为起始材料, 采用抑制差减杂交技术构建了两个群体间的正反向差减cDNA文库。通过斑点杂交从差减文库中筛选代表受水稻抗性调控的基因的cDNA克隆, 进行测序和功能分析, 挑选具功能的基因进行Northern杂交验证。结果表明, 通过斑点杂交筛选到的98个阳性克隆代表92个互不重复的单基因, 其中25个与动物的已知蛋白基因存在较高的同源性。Northern杂交表明, 这25个基因有11个表达上调, 8个表达下调, 提示它们可能在褐飞虱适应抗性水稻过程中发挥了重要作用。本研究结果为克隆上述新基因的全长cDNA序列及进一步研究其在褐飞虱与水稻互作中的功能奠定了基础。     

水稻穗瘟防卫反应相关基因的分离和鉴定

以遗传背景相近、对叶瘟抗性相同但对穗瘟抗性不同的两个水稻株系为材料,利用抑制消减杂交（SSH）技术构建穗瘟抗／感消减cDNA文库,经差异筛选及序列分析,共获得90个独立的差异表达cDNA克隆,根据与它们刚源的基因功能推测,这些克隆可能参与了对病原菌的防卫反应、信号传导和转录等一些重要的生物学过程。利ⅢRT-PCR分析了26个所筛选到的cDNA克隆在抗／感植株接种后的表达,17个基因的表达差异得到验证。对这螳差异表达基因在抗感株系接种后不同时间点的表达谱也进行了RT-PCR的分析。文章首次报道了什关水稻对穗瘟抗性在mRNA水平进行研究,为深入研究水稻对穗瘟抗性的遗传机理打下了基础。     

Characterization of pathogenic bacteria of the cupped oyster Crassostrea gigas

The French mollusc production is mainly based on the Pacific cupped oyster, Crassostrea gigas. Since 1991, outbreaks of mass mortality of juveniles are reported during the summer period. These outbreaks are a major concern of oyster industry. Several studies have established given bacterial strains to be pathogenic for bivalve species, including oysters. Here we present a study of mortality outbreaks of C. gigas, as initiated in 1995. In a first step, bacterial strains were isolated during mass mortality outbreak and were biochemically characterised. Among the isolated strains, some strains of Vibrio splendidus biovar II were found to be pathogenic by means of experimental challenge of oyster juveniles. In the second step, a genotypical identification of the pathogenic strain was undertaken, based on 16S RNA sequences and phylogenetic analysis. It confirmed that the pathogenetic strain belonged to Vibrio splendidus biovar II.     

Sequence polymorphism in a novel noncoding region of Pacific oyster mitochondrial DNA

Nucleotide sequence polymorphism in a 641-bp novel major noncoding region of mitochondrial DNA (mtDNA-NC) of the Pacific oyster Crassostrea gigas was analysed for 29 cultured individuals within the Goseong population. A total of 30 variable sites were detected, and the relative frequency of nucleotide alteration was determined to be 4.68. Alterations were mostly single nucleotide substitutions. Transition, transversion, both transition and transversion, and both transversion and nucleotide deletion were observed at 18, 9, 2 and 1 sites, respectively. Among 29 specimens, 22 haplotypes were identified, and pairwise genetic diversity of haplotypes was calculated to be 0.988 from multiple sequence substitutions using the two-parameter model. A phylogenetic tree, obtained for haplotypes by the neighbor-joining method, showed a single cluster of linkages. The cluster comprised 11 haplotypes associating with 14 specimens, while the other 11 haplotypes associating with 15 specimens were scattered. This mtDNA-NC presenting a high nucleotide sequence polymorphism is a potential mtDNA control region. It therefore can serve as a genetic marker for intraspecies phylogenetic analysis of the Pacific oyster and is more useful than the less polymorphic mtDNA coding genes.     

Simian virus 40-permissive cell interactions: selection and characterization of spontaneously arising monkey cells that are resistant to simian virus 40 infection.

A fraction of permissive cells survive simian virus 40 (SV40) infection. The frequency of such surviving cells depends only upon the concentration of infecting virus, both parental and progeny, to which the cells are exposed during the course of selection. Surviving clones, which can be freed of virus by cloning in the presence of SV40 antiserum, are indistinguishable from parental cells in their growth of characteristics and display no SV40 antigen; thus they are not transformed. Most surviving clones are less than 10% as susceptible as parental cells to SV40 infection; 5 to 10% are less than 1% as susceptible. None of these SV40-resistant clones is absolutely resistant to SV40 infection. Analysis of 16 independently arising resistant clones indicates that they all block SV40 infection at an early stage after adsorption and eclipse but before full uncoating. Viral mutants have been isolated that partially overcome the block to infection in these cells; these host range viruses plaque on resistant lines fivefold more efficiently than wild-type SV40 and have a characteristic plaque morphology. Fluctuation analysis indicates that resistant cells arise spontaneously during the growth of normally susceptible permissive cells. Thus, SV40-resistant cells are selected for, not induced by, SV40 infection.     

Microarray Analysis Highlights Immune Response of Pacific Oysters as a Determinant of Resistance to Summer Mortality

Summer mortality of Crassostrea gigas is the result of a complex interaction between oysters, their environment, and pathogens. A high heritability was estimated for resistance to summer mortality, which provided an opportunity to develop lines of oysters that were resistant (R) or susceptible (S) to summer mortality. Previous genome-wide expression profiling study of R and S oyster gonads highlighted reproduction and antioxidant defense as constitutive pathways that operate differentially between these two lines. Here, we show that signaling in innate immunity also operates differentially between these lines, and we hypothesize that this is at the main determinant of their difference in survival in the field. A reanalysis of our published microarray data using separate ANOVAs at each sampling date revealed a specific “immune” profile at the date preceding the mortality. In addition, we conducted additional microarray profiling of two other tissues, gills, and muscle, and both showed an overrepresentation of immune genes (46%) among those that are differentially expressed between the two lines. Eleven genes were pinpointed to be simultaneously differentially expressed between R and S lines in the three tissues. Among them, ten are related to “Immune Response.” For these genes, the kinetics of R mRNA levels between sampling dates appeared different just before the morality peak and suggests that under field conditions, R oysters had the capacity to modulate signaling in innate immunity whereas S oysters did not. This study enhances our understanding of the complex summer mortality syndrome and provides candidates of interest for further functional and genetics studies.     

Herpes virus in juvenile Pacific oysters Crassostrea gigas from Tomales Bay, California, coincides with summer mortality episodes

Pacific Crassostrea gigas and eastern C. virginica oysters were examined between June 2002 and April 2003 from 8 locations along the east, west and south USA coasts for oyster herpes virus (OsHV) infections using the A primer set in a previously developed PCR test. Only surviving Pacific oysters from a mortality event in Tomales Bay, California, USA, where annual losses of oysters have occurred each summer since 1993, were infected with a herpes-like virus in 2002. PCR examination using template amounts of both 50 and 500 ng were essential for OsHV detection. Sequence analysis indicated that the Tomales Bay OsHV was similar to that identified in France with the exception of a single base pair substitution in a 917 bp fragment of the viral genome. However, unlike the French OsHV-1, the Tomales Bay OsHV did not amplify with the primer pair of a second OsHV-1 PCR assay, suggesting that further characterization of these viruses is warranted. No evidence of Cowdry type A viral infections characteristic of herpes virus infections or other pathogens were observed in OsHV-infected oysters. Hemocytosis, diapedesis and hemocyte degeneration characterized by nuclear pycnosis and fragmentation were observed in infected oysters, which is consistent with previous observations of OsHV infections in France. Together these data suggest that OsHV may be associated with the annual summer Pacific oyster seed mortality observed in Tomales Bay, but establishment of a causal relationship warrants further investigation.     

Synergistic impacts of heat shock and spawning on the physiology and immune health of Crassostrea gigas: an explanation for summer mortality in Pacific oysters

Mass mortality is often observed in cultured oysters during the period following spawning in the summer season. To examine the underlying causes leading to this phenomenon, thermotolerance of the Pacific oyster Crassostrea gigas was assessed using pre- and postspawning oysters that were sequentially treated with sublethal (37 degrees C) and lethal heat shocks (44 degrees C). The effects were examined on a range of immune and metabolic parameters in addition to mortality rate. A preventative 37 degrees C significantly reduced oyster mortality after exposure to a second heat shock of 44 degrees C, but in postspawning oysters mortality remained at 80%, compared with < 10% in prespawning oysters. Levels of the 72 kDa and 69 kDa heat shock proteins were low in the gill tissue from postspawning oysters stimulated by heat shock, indicating spawning reduced heat shock protein synthesis. The postspawning oysters had depleted glycogen stores in the mantle tissue and reduced adenylate energy charge after heat shock, indicative of lower energy for metabolic activity. A cumulative effect of spawning and heat shock was observed on the immunocompetence of oysters, demonstrated by reduced hemocyte phagocytosis and hemolymph antimicrobial activity. These results support the hypothesis that the energy expended during reproduction compromises the thermotolerance and immune status of oysters, leaving them easily subject to mortality if heat stress occurs in postspawning stage. This study improves our understanding of oyster summer mortality and has implications for the long-term persistence of mollusks under the influence of global warming.