Pesticides and Ostreid Herpesvirus 1 Infection in the Pacific Oyster,Crassostrea gigas

Since 2008, mass mortality outbreaks have been reported in all French regions producing Pacific oysters, and in several Member States of the European Union. These mass mortality events of Pacific oysters are related to OsHV-1 infection. They occur during spring and summer periods leaving suspect the quality of the marine environment and the role of seasonal use of pesticides associated with the arrival of freshwater in oyster rearing areas. Pesticides have been also detected in French coastal waters, especially in areas of oyster production. Using PMA real-time PCR we showed that a mixture of 14 pesticides has no effect on the integrity of virus capsids from viral suspension in the conditions tested. A contact of oysters with this pesticide mixture was related to higher mortality rates among experimentally infected animals in comparison with control ones (no previous pesticide exposure before experimental infection). We therefore suggest that pesticides at realistic concentration can exert adverse effects on Pacific oysters and causes an increased susceptibility to the viral infection in experimental conditions.     

Extraction and Identification of the Pigment in the Adductor Muscle Scar of Pacific Oyster Crassostrea gigas

In this study, UV (ultraviolet) and IR (infrared radiation) spectral analysis were integrated to identify the pigment in the adductor muscle scar of the Pacific oyster Crassostrea gigas. The pigment was extracted from the adductor muscle scars of cleaned oyster shells that were pulverized, hydrolyzed in hot hydrochloric acid, purified with diethyl ether, and dissolved in 0.01 mL/L NaOH. The maximum absorption of the pigment in the UV absorption spectrum within the range of 190–500 nm was observed between 210–220 nm. The UV absorbance decreased with increasing wavelength which was consistent with the UV spectral absorption characteristics of melanin. In addition, Fourier transform infrared spectroscopy scanning revealed characteristic absorption peaks that emerged near 3440 cm^-1 and 1630 cm^-1, which was consistent with infrared scanning features of eumelanin (a type of melanin). This study has demonstrated for the first time that the pigment in the adductor muscle scar of the Pacific oyster is melanin, hinting that the adductor muscle could be another organ pigmenting the mollusc shell with melanin other than mantle.     

Settlement and Survival of the Oyster Crassostrea virginica on Created Oyster Reef Habitats in Chesapeake Bay

Efforts to restore the Eastern oyster (Crassostrea virginica) reef habitats in Chesapeake Bay typically begin with the placement of hard substrata to form three‐dimensional mounds on the seabed to serve as a base for oyster recruitment and growth. A shortage of oyster shell for creating large‐scale reefs has led to widespread use of other materials such as Surf clamshell (Spisula solidissima), as a substitute for oyster shell. Oyster recruitment, survival, and growth were monitored on intertidal reefs constructed from oyster and Surf clamshell near Fisherman’s Island, Virginia, U.S.A. and on a subtidal Surf clamshell reef in York River, Virginia, U.S.A. At the intertidal reefs, oyster larvae settlement occurred at similar levels on both substrate types throughout the monitoring period but higher levels of post‐settlement mortality occurred on clamshell reefs. The oyster shell reef supported greater oyster growth and survival and offered the highest degree of structural complexity. On the subtidal clamshell reef, the quality of the substrate varied with reef elevation. Large shell fragments and intact valves were scattered around the reef base, whereas small, tightly packed shell fragments paved the crest and flank of the reef mound. Oysters were more abundant and larger at the base of this reef and less abundant and smaller on the reef crest. The availability of interstitial space and appropriate settlement surfaces is hypothesized to account for the observed differences in oyster abundance across the reef systems. Patterns observed emphasize the importance of appropriate substrate selection for restoration activities to enhance natural recovery where an underlying habitat structure is destroyed.     

Development of Novel Microsatellite DNA Markers from the Pacific Oyster Crassostrea gigas

We document the potential of novel microsatellites as a genetic tool in furthering our understanding of the Crassostrea gigas genetic structure. From the microsatellite-enriched libraries we constructed, 123 repeat regions that had sufficient sequence information to design polymerase chain reaction primer sets were isolated. From these, 9 primer pairs were screened in a C. gigas population of 67 individuals to evaluate the genetic variability. All but 1 of the 9 loci showed allelic variation (number of alleles, 2–20; observed heterozygosity, 0.119–0.925; unbiased expected heterozygosity, 0.139–0.914). Considerable discrepancy of genotypic proportions from the Hardy-Weinberg equilibrium was observed at 1 locus with an apparent heterozygote deficiency. Several loci were successfully amplified in 3 other related species with the appropriate allele size: 6 loci in C. sikamea, 4 loci in C. ariakensis, and 5 loci in C. nippona.     

Population demography and genetic characteristics of the Pacific Oyster Crassostrea gigas in Japan

The Pacific oyster Crassostrea gigas is a commercially important bivalve distributed along the northwest Pacific coast. Here C. gigas in Japan was investigated using mtDNA and microsatellite markers to elucidate its genetic structure and phylogeny. On the basis of mtDNA all populations showed high genetic diversity with limited genetic differentiation among populations. The pattern of MtDNA diversity suggested that C. gigas had experienced population expansion about 112 Kya, prior to the last glacial maximum (LGM), which accorded well with other marine organisms. For microsatellites, a Bayesian-based assignment test demonstrated that C. gigas is nearly panmictic. However, on the basis of estimates of F_ST, Kumano populations differed significantly from other populations, a recent occurrence based on low R_ST. Irrespective of geographical distance, genetic similarity was observed in the main aquaculture regions with large-scale transportation of cultured spat. Unlike in the Yellow Sea, a genetic bottleneck was not detected in Japanese populations. These results imply, contrary to the prevailing view, that C. gigas in Japan was demographically stable during the LGM. Gene flow by larval dispersal seems to be regionally restricted to localities of congenital areas by ocean currents, while genetic homogenization by cultivated oysters might have occurred in aquaculture areas.     

Reactive Oxygen Species in Unstimulated Hemocytes of the Pacific Oyster Crassostrea gigas: A Mitochondrial Involvement

The Pacific oyster Crassostrea gigas is a sessile bivalve mollusc whose homeostasis relies, at least partially, upon cells circulating in hemolymph and referred to as hemocytes. Oyster’s hemocytes have been reported to produce reactive oxygen species (ROS), even in absence of stimulation. Although ROS production in bivalve molluscs is mostly studied for its defence involvement, ROS may also be involved in cellular and tissue homeostasis. ROS sources have not yet been described in oyster hemocytes. The objective of the present work was to characterize the ROS sources in unstimulated hemocytes. We studied the effects of chemical inhibitors on the ROS production and the mitochondrial membrane potential (Δψ_m) of hemocytes. First, this work confirmed the specificity of JC-10 probe to measure Δψ_m in oyster hemocytes, without being affected by ΔpH, as reported in mammalian cells. Second, results show that ROS production in unstimulated hemocytes does not originate from cytoplasmic NADPH-oxidase, nitric oxide synthase or myeloperoxidase, but from mitochondria. In contrast to mammalian cells, incubation of hemocytes with rotenone (complex I inhibitor) had no effect on ROS production. Incubation with antimycin A (complex III inhibitor) resulted in a dose-dependent ROS production decrease while an over-production is usually reported in vertebrates. In hemocytes of C. gigas, the production of ROS seems similarly dependent on both Δψ_m and ΔpH. These findings point out differences between mammalian models and bivalve cells, which warrant further investigation about the fine characterization of the electron transfer chain and the respective involvement of mitochondrial complexes in ROS production in hemocytes of bivalve molluscs.     

牡蛎中糖蛋白成分的分离纯化及其性质研究

本文以青岛产牡蛎为原料,通过低温水提取工艺得到牡蛎糖蛋白粗提物,然后用凝胶柱层析(Sephacryls-100 HR)进行纯化,最后用高效液相色谱制备得到纯度较高的物质F22.由SDS-聚丙烯酰胺凝胶电泳证实F22是纯度较高的单一组分;分子量为34.2 kDa;等电聚焦的结果显示等电点为5.5;红外色谱呈现出典型的糖的特征吸收峰;气相色谱分析结果显示F22的中性单糖是由葡萄糖这一种单糖组成的同多糖;β-消去反应表明F22的糖肽键应为N-型糖苷键.     

Identification of Conserved and Novel MicroRNAs in the Pacific Oyster Crassostrea gigas by Deep Sequencing

MicroRNAs (miRNAs) play important roles in regulatory processes in various organisms. To date many studies have been performed in the investigation of miRNAs of numerous bilaterians, but limited numbers of miRNAs have been identified in the few species belonging to the clade Lophotrochozoa. In the current study, deep sequencing was conducted to identify the miRNAs of Crassostrea gigas (Lophotrochozoa) at a genomic scale, using 21 libraries that included different developmental stages and adult organs. A total of 100 hairpin precursor loci were predicted to encode miRNAs. Of these, 19 precursors (pre-miRNA) were novel in the oyster. As many as 53 (53%) miRNAs were distributed in clusters and 49 (49%) precursors were intragenic, which suggests two important biogenetic sources of miRNAs. Different developmental stages were characterized with specific miRNA expression patterns that highlighted regulatory variation along a temporal axis. Conserved miRNAs were expressed universally throughout different stages and organs, whereas novel miRNAs tended to be more specific and may be related to the determination of the novel body plan. Furthermore, we developed an index named the miRNA profile age index (miRPAI) to integrate the evolutionary age and expression levels of miRNAs during a particular developmental stage. We found that the swimming stages were characterized by the youngest miRPAIs. Indeed, the large-scale expression of novel miRNAs indicated the importance of these stages during development, particularly from organogenetic and evolutionary perspectives. Some potentially important miRNAs were identified for further study through significant changes between expression patterns in different developmental events, such as metamorphosis. This study broadened the knowledge of miRNAs in animals and indicated the presence of sophisticated miRNA regulatory networks related to the biological processes in lophotrochozoans.     

New Insight for the Genetic Evaluation of Resistance to Ostreid Herpesvirus Infection,a Worldwide Disease,in Crassostrea gigas

The Pacific oyster, Crassostrea gigas, is the most important commercial oyster species cultivated in the world. Meanwhile, the ostreid herpesvirus 1 (OsHV-1) is one of the major pathogens affecting the Pacific oyster, and numerous mortality outbreaks related to this pathogen are now reported worldwide. To assess the genetic basis of resistance to OsHV-1 infection in spat C. gigas and to facilitate breeding programs for such a trait, if any exist, we compared the mortality of half- and full-sib families using three field methods and a controlled challenge by OsHV-1 in the laboratory. In the field, three methods were tested: (A) one family per bag; (B) one family per small soft mesh bag and all families inside one bag; (C) same as the previous methods but the oysters were individually labelled and then mixed. The mean mortality ranged from 80 to 82% and was related to OsHV-1 based on viral DNA detection. The narrow-sense heritability for mortality, and thus OsHV-1 resistance, ranged from 0.49 to 0.60. The high positive genetic correlations across the field methods suggested no genotype by environment interaction. Ideally, selective breeding could use method B, which is less time- and space-consuming. The narrow sense heritability for mortality under OsHV-1 challenge was 0.61, and genetic correlation between the field and the laboratory was ranged from 0.68 to 0.75, suggesting a weak genotype by environment interaction. Thus, most of families showing the highest survival performed well in field and laboratory conditions, and a similar trend was also observed for families with the lowest survival. In conclusion, this is the first study demonstrating a large additive genetic variation for resistance to OsHV-1 infection in C. gigas, regardless of the methods used, which should help in selective breeding to improve resistance to viral infection in C. gigas.     

Allograft Inflammatory Factor 1 Functions as a Pro-Inflammatory Cytokine in the Oyster,Crassostrea ariakensis

The oyster Crassostrea ariakensis is an economically important bivalve species in China, unfortunately it has suffered severe mortalities in recent years caused by rickettsia-like organism (RLO) infection. Prevention and control of this disease is a priority for the development of oyster aquaculture. Allograft inflammatory factor-1 (AIF-1) was identified as a modulator of the immune response during macrophage activation and a key gene in host immune defense reaction and inflammatory response. Therefore we investigated the functions of C. ariakensis AIF-1 (Ca-AIF1) and its antibody (anti-CaAIF1) in oyster RLO/LPS-induced disease and inflammation. Ca-AIF1 encodes a 149 amino acid protein containing two typical Ca²⁺ binding EF-hand motifs and shares a 48–95% amino acid sequence identity with other animal AIF-1s. Tissue-specific expression analysis indicates that Ca-AIF1 is highly expressed in hemocytes. Significant and continuous up-regulation of Ca-AIF1 is detected when hemocytes are stimulated with RLO/LPS (RLO or LPS). Treatment with recombinant Ca-AIF1 protein significantly up-regulates the expression levels of LITAF, MyD88 and TGFβ. When anti-CaAIF1 antibody is added to RLO/LPS-challenged hemocyte monolayers, a significant reduction of RLO/LPS-induced LITAF is observed at 1.5–12 h after treatment, suggesting that interference with Ca-AIF1 can suppress the inflammatory response. Furthermore, flow cytometric analysis indicated that anti-CaAIF1 administration reduces RLO/LPS-induced apoptosis and necrosis rates of hemocytes. Collectively these findings suggest that Ca-AIF1 functions as a pro-inflammatory cytokine in the oyster immune response and is a potential target for controlling RLO infection and LPS-induced inflammation.     

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.     

A Centromeric Satellite Sequence in the Pacific Oyster (Crassostrea gigas Thunberg) Identified by Fluorescence In Situ Hybridization

A highly repetitive satellite sequence was previously identified in the Pacific oyster Crassostrea gigas Thunberg. The sequence has 168 bp per unit, present in tandem repeats, and accounts for 1% to 4% of the genome. We studied the chromosomal location of this satellite sequence by fluorescence in situ hybridization (FISH). A probe was made by polymerase chain reaction and incorporation of digoxigenin-11-dUTP. Hybridization was detected with fluorescein-labeled antidigoxigenin antibodies. FISH signals were located at centromeric regions of 7 pairs of the Pacific oyster chromosomes. No interstitial site was found. Signals were strong and consistent on chromosomes 1, 2, 4, and 7, but weak or variable on chromosomes 5, 8, and 10. No signal was observed on chromosomes 3, 6, and 9. Our results showed that this sequence is clearly a centromeric satellite, disputing its previous assignment to the telomeric and submetacentric regions of 2 chromosomes. No signal was detected in the American oyster (Crassostrea virginica Gmelin). Received February 2, 2001; accepted May 24, 2001     

Metabolism of the Oyster, Crassostrea virginica

SYNOPSIS. The American oyster, Crassostrea virginica, has somemetabolic traits typical of marine bivalve molluscs, andsomethat are apparently unique. Cvconsumption is rapid when thevalves are open, but may become and remain zero for days onclosure. Rates of nitrogen-excretion are similar to those ofother marine bivalves; the chief end-products are ammonia (65–70%) and amino acids (5–21 %), but small amountsof urea and uric acid are regularly found. Deoxyribonucleicacid (DNA) of marine molluscs has a low content of the base-pair,guanine-cytosine (GC); oyster DN'A has thelowest GC (about 28%) of nine species studied. There are six sterols, the majorone cholesterol, and there is evidence of changes in steroidmetabolism with the gonadal cycle. Aminotransferase activitiesof marine bivalves are proportional to rates of loss of aminoacids, suggesting that the enzymes function to assure continuousreplenishment. The oyster isunique in its extraordinarily lowrate of conversion of alanine to pyruvate. Most of the intermediatesand enzymes requiredfor complete oxidation of glucose have beenfound in some species of Crassostrea, but anaerobic glycolysisproduces more succinic than lactic acid. A partial explanationlies in ready reversibility of succinate and laclate oxidoreductases.     

AFLP-Based Genetic Linkage Maps of the Pacific Oyster <Emphasis Type="Italic">Crassostrea gigas</Emphasis> Thunberg

Amplified fragment length polymorphisms (AFLPs) were used for genome mapping in the Pacific oyster Crassostrea gigas Thunberg. Seventeen selected primer combinations produced 1106 peaks, of which 384 (34.7%) were polymorphic in a backcross family. Among the polymorphic markers, 349 were segregating through either the female or the male parent. Chi-square analysis indicated that 255 (73.1%) of the markers segregated in a Mendelian ratio, and 94 (26.9%) showed significant (P < 0.05) segregation distortion. Separate genetic linkage maps were constructed for the female and male parents. The female framework map consisted of 119 markers in 11 linkage groups, spanning 1030.7 cM, with an average interval of 9.5 cM per marker. The male map contained 96 markers in 10 linkage groups, covering 758.4 cM, with 8.8 cM per marker. The estimated genome length of the Pacific oyster was 1258 cM for the female and 933 cM for the male, and the observed coverage was 82.0% for the female map and 81.3% for the male map. Most distorted markers were deficient for homozygotes and closely linked to each other on the genetic map, suggesting the presence of major recessive deleterious genes in the Pacific oyster.     

Expression of Overdominance for Specific Activity at the Phosphoglucomutase-2 Locus in the Pacific Oyster, Crassostrea Gigas

Environmental and genetic components of specific activity variation at the phosphoglucomutase-2 locus in the Pacific oyster, Crassostrea gigas, were examined to assess the direct role played by this polymorphism in a heterozygosity/growth relationship. Both environmental variables studied, season and intertidal position, exerted highly significant effects on phosphoglucomutase specific activity but no interactions occurred between these factors and Pgm-2 genotype. Highly significant differences were also detected between Pgm-2 genotypes. The three most common heterozygotes (Pgm-2(92/100), Pgm-2(96/100) and Pgm-2(100/104) consistently expressed greater specific activities than the Pgm-2(92/92), Pgm-2(96/96), Pgm-2(100/100) and Pgm-2(104/104) homozygotes. Overall, the specific activities of heterozygotes for the Pgm-2(100) allele exceeded heterozygotes by 24% and 20% in the mantle and adductor muscle tissues, respectively. Heterozygotes formed between the three less frequent Pgm-2(92), Pgm-2(96) and Pgm-2(104) alleles differed sharply from those possessing the Pgm-2(100) allele in being indistinguishable from homozygotes. The possibility of these patterns arising from the undetected presence of an inactive Pgm-2 allele was examined and found to be inconsistent with all of its predicted effects on the specific activity data. Genuine overdominance was shown to be capable of explaining the specific activities of ten structural locus genotypes, allelic frequency distributions in natural populations, and the maintenance of the enzyme polymorphism in a balanced state. The results provide evidence favoring the overdominance explanation for one locus involved in a heterozygosity/growth relationship and suggest that the reported effects of this locus on adult body weight may have been caused by the greater flux capacities of heterozygotes for the Pgm-2(100) allele.