5S rDNA and U2 snRNA are linked in the genome of Crassostrea angulata and Crassostrea gigas oysters: does the (CT)n.(GA)n microsatellite stabilize this novel linkage of large tandem arrays?

The 5S rRNA genes from 2 species of the Ostreidae family, Crassostrea angulata and Crassostrea gigas, were molecularly characterized. The genes were amplified, cloned, and sequenced. The results revealed a 5S rDNA tandem array with a nucleotide sequence in an inverted position within the nontranscribed spacer region that corresponded to the U2 small nuclear RNA (snRNA) gene. The sequence analysis indicated that both genes could be functionally active. The presence of the microsatellite (CT)n x (GA)n at the 3' end of both genes and the possible involvement of concerted evolution are discussed.     

Restriction enzyme digestion chromosome banding in Crassostrea and Ostrea species: comparative karyological analysis within Ostreidae.

Reliable banding techniques are a major necessity for genetic research in oysters. In this study, we carried out the cytogenetic characterization of four oyster species (family Ostreidae) using restriction endonuclease treatments. Chromosomes were treated with three different restriction enzymes, stained with Giemsa, and examined for banding patterns. The following species were studied: Crassostrea gigas (2n = 20; total number of bands with ApaI, 74; HaeIII, 61; PstI, 76), Crassostrea angulata (2n = 20; ApaI, 62; HaeIII, 61; PstI, 55) (subfamily Crassostreinae), Ostrea edulis (2n = 20; ApaI, 82; HaeIII, 59; PstI, 66), and Ostrea conchaphila (2n = 20; ApaI, 68; HaeIII, 62; PstI, 69) (subfamily Ostreinae). Treatment of samples with ApaI, HaeIII, and PstI produced specific banding patterns, which demonstrates the potential of these enzymes for chromosome banding in oysters. This is of special interest, since it has been recently shown in mammalian chromosomes that restriction enzyme banding is compatible with fluorescence in situ hybridization. This study therefore provides a fundamental step in genome mapping of oysters, since chromosome banding with restriction enzymes facilitates physical gene mapping in these important aquaculture species. The analysis of the banded karyotypes revealed a greater similarity within the genera of Crassostrea and Ostrea than between them.     

Exploiting EST databases for the development and characterization of EST-SSRs in the Pacific oyster (Crassostrea gigas)

A total of 147 microsatellite-containing expressed sequence tags (ESTs) (3.63%) were detected from 4053 ESTs of the Pacific oyster (Crassostrea gigas) in GenBank. The average density of simple sequence repeats (SSRs) was 1 per 8.25 kb of EST after redundancy elimination. Dinucleotide repeat motifs appeared to be the most abundant type. Sixteen new polymorphic EST-SSRs were developed. The number of alleles per locus varied from 3 to 12, with an average of 5.9 alleles per locus. Marker transferability was tested on 2 other Crassostrea species, and 14 loci gave successful amplifications in both species. Twenty EST-SSRs were tested on 3 families of C. gigas for examination of inheritance mode of EST-SSRs. Thirty-five tests of segregation ratios revealed 5 significant departures from expected Mendelian ratios, 4 of which confirmed Mendelian expectations when accounting for the presence of null alleles. Null alleles were detected at 3 loci (15.0%) of the 20 loci, and the frequency of null alleles at EST-SSRs was lower than that in genomic SSRs in C. gigas. The results obtained in this study suggest that C. gigas EST-SSRs will complement the currently available genomic SSR markers and may be useful for comparative mapping, marker-assisted selection, and evolutionary studies.     

Demonstration of a true phenoloxidase activity and activation of a ProPO cascade in Pacific oyster, Crassostrea gigas (Thunberg) in vitro

The prophenoloxidase (ProPO) system is the origin of melanin production and is considered to be an innate defence mechanism in invertebrates. In different bivalve species, phenoloxidase (PO) is present in the haemolymph as an inactive form of ProPO. The present study focuses on the Pacific adult oyster, Crassostrea gigas, an economically important bivalve species along French coasts. The results indicate that many factors may inhibit the PO-like activity. These include: phenylthiourea (PTU), sodium diethylthiocarbamate (DETC), beta-mercaptoethanol and tropolone, which repressed the spontaneous PO activity. The activation of PO-like activity in C. gigas acellular fraction by lipopolysaccharide (LPS) involved participation of other factors, including at least one serine protease. PO was present as proPO in the acellular fraction of haemolymph and haemocytes of C. gigas and could be activated by an exogenous protease (trypsin-N-tosyl-l-phenylalanine chloromethyl ketone) when used at 1 gL(-1). Treatment of acellular fractions with other modulators/activators namely LPS (1 gL(-1)), zymosan (0.6 gL(-1)) or laminarin (0.6 gL(-1)) also increased PO-like activity but to a less important way. The study demonstrated the evidence of a true phenoloxidase activity in Pacific oyster, C. gigas (Thunberg). The activation of a proPO system by non-self molecules suggests the role played by PO in vivo in the internal defence mechanisms. Understanding the activation of the ProPO system could enable the evaluation of the health of oyster stocks.     

Polymorphism of metallothionein genes in the Pacific oyster Crassostrea gigas as a biomarker of response to metal exposure.

Quantification of metallothioneins (MTs) is classically associated with a cellular response to heavy metal contamination and is used in the monitoring of disturbed ecosystems. Despite the characterization of several MT genes in marine bivalves, only a few genetic studies have used MT genes as potential biomarkers of pollution. The aim of this study was to assess whether MT gene polymorphism could be used to monitor exposure of the Pacific oyster Crassostrea gigas to heavy metals and to develop specific genetic markers for population genetic studies in relation to environmental stress. The polymorphism of two exons of the C. gigas MT gene CgMT1 were studied using polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) in both field populations exposed to various metals concentrations and in experimentally exposed populations. High frequencies of two SSCP types in exons 2 and 3 of the CgMT1 gene have found to be significantly associated with tolerance to metals in experimental and field oyster populations. The use of MT1 gene polymorphism in C. gigas as in the present study should therefore be of high ecological relevance. In conclusion, the analysis of the types in these two CgMT1 gene exons, which can confer a greater tolerance to heavy metals, can constitute a good biomarker of effect of the presence of heavy metals in ecosystems.     

Response of haemocyte lysosomes to bacterial inoculation in the oysters Ostrea edulis L. and Crassostrea gigas (Thunberg) and the scallop Pecten maximus (L)

Data are presented that demonstrate the application of the neutral red retention assay (NRR) to monitor the effects of a bacterial inoculation on the haemocyte lysosomes of the European flat oyster Ostrea edulis, Pacific oyster Crassostrea gigas and scallop Pecten maximus. Bivalves were acclimated to three temperature regimes (5, 15 and 25 degrees C), at constant salinity for 7 days in the laboratory. Once baseline responses to acclimation temperature had been established, the effects of an in vivo inoculation on haemocyte lysosomal stability were assessed using the NRR assay. Lysosomal membrane stability was reduced in the presence of bacteria for all three species of bivalve, but destabilisation of C. gigas haemocyte lysosomes appeared to be most sensitive to the presence of the bacterium Listonella anguillarum. For all three bivalve species, the reduction in lysosomal stability appeared to be proportional to the growth of the bacterial inoculate. Using appropriate controls, the NRR assay was demonstrated to have great potential as a tool with which to make rapid initial assessments of the immune status of bivalve molluscs.     

Survival of Vibrio vulnificus in shellstock and shucked oysters (Crassostrea gigas and Crassostrea virginica) and effects of isolation medium on recovery

When two species of shellstock oysters were artificially contaminated with Vibrio vulnificus, the bacterium survived when the oysters were stored at 10 degrees C and below. Large numbers of endogenous V. vulnificus cells were found after 7 days at both 0.5 and 10 degrees C in uninoculated control oysters (Crassostrea virginica). Oysters allowed to take up V. vulnificus from seawater retained the bacterium for 14 days at 2 degrees C. The presence of V. vulnificus in the drip exuded from the shellstock presented a possibility of contamination of other shellstock in storage. V. vulnificus injected into shucked Pacific (Crassostrea gigas) and Eastern (C. virginica) oysters survived at 4 degrees C for at least 6 days. An 18-h most-probable-number enrichment step in alkaline peptone water gave higher recovery levels of V. vulnificus than did direct plating to selective agars. The survival of this pathogen in both shellstock and shucked oysters suggests a potential for human illness, even though the product is refrigerated.     

EST‐SSR markers from the Pacific oyster,Crassostrea gigas

Ten polymorphic microsatellite repeat markers were identified from Crassostrea gigas, expressed sequence tags (EST) deposited in public sequence database. Number of alleles per locus ranged from three to 18, expected and observed heterozygosities ranged from 0.071 to 0.738 and from 0.306 to 0.913, respectively. Marker transferability was tested on other two Crassostrea species and polymorphic products were detected at nine loci. EST‐derived simple sequence repeats provide robust, informative and potentially transferable polymorphic markers suitable for population genetic, parentage, and mapping studies of C. gigas.     

Comparative Methylome Analysis Reveals Epigenetic Signatures Associated with Growth and Shell Color in the Pacific Oyster,Crassostrea gigas

Marine Biotechnology - Fast growth is one of the most important breeding goals for all economic species such as the Pacific oyster (Crassostrea gigas), an aquaculture mollusk with top global...     

Survival of Vibrio vulnificus in shellstock and shucked oysters (Crassostrea gigas and Crassostrea virginica) and effects of isolation medium on recovery.

When two species of shellstock oysters were artificially contaminated with Vibrio vulnificus, the bacterium survived when the oysters were stored at 10 degrees C and below. Large numbers of endogenous V. vulnificus cells were found after 7 days at both 0.5 and 10 degrees C in uninoculated control oysters (Crassostrea virginica). Oysters allowed to take up V. vulnificus from seawater retained the bacterium for 14 days at 2 degrees C. The presence of V. vulnificus in the drip exuded from the shellstock presented a possibility of contamination of other shellstock in storage. V. vulnificus injected into shucked Pacific (Crassostrea gigas) and Eastern (C. virginica) oysters survived at 4 degrees C for at least 6 days. An 18-h most-probable-number enrichment step in alkaline peptone water gave higher recovery levels of V. vulnificus than did direct plating to selective agars. The survival of this pathogen in both shellstock and shucked oysters suggests a potential for human illness, even though the product is refrigerated.     

Genome Structural Variation Landscape and Its Selection Signatures in the Fast-growing Strains of the Pacific Oyster,Crassostrea gigas

Marine Biotechnology - The Pacific oyster (Crassostrea gigas) genome is highly polymorphic and affluent in structural variations (SVs), a significant source of genetic variation underlying...     

Crassostrea gigas oysters as a shrimp farm bioindicator of white spot syndrome virus

This study explored whether Crassostrea gigas oysters can be used as a bioindicator of white spot syndrome virus (WSSV) in shrimp farm water canals. Bioassays showed that C. gigas can accumulate WSSV in their gills and digestive glands but do not become infected, either by exposure to seawater containing WSSV or by cohabitation with infected shrimp. The use of a WSSV nested PCR to screen oysters placed in water canals at the entry of a shrimp farm allowed WSSV to be detected 16 d prior to the disease occurring. The finding that C. gigas can concentrate small amounts of WSSV present in seawater without being harmed makes it an ideal sentinel species at shrimp farms.     

Discriminatory predation by three invertebrates on eastern oysters (Crassostrea virginica) compared with non-native Suminoe oysters (C. ariakensis)

Abstract. Diminished populations of eastern oysters Crassostrea virginica in Chesapeake Bay have stimulated proposals to introduce Crassostrea ariakensis from Asia to restore oyster stocks. As part of a program evaluating possible ramifications of such an introduction, we studied how invertebrate predators responded to this non-native oyster. We compared predation activity under laboratory conditions by oyster drills ( Urosalpinx cinerea; Eupleura caudata ) that bore through an oyster's shell and by the seastar Asterias forbesi that pulls shell valves apart. These three predators preyed significantly (p<0.05) more on the familiar C. virginica than on the novel C. ariakensis . We previously reported that five crab species preyed significantly more on C. ariakensis than on C. virginica , with predation by polyclad flatworms similar between oyster species. Thus, the drills and the seastar differed from the crabs and the flatworms in their response to novel prey. When Urosalpinx cinerea was placed in a Y-maze after being held for 40 d with oysters of one species or the other, the drills moved toward C. virginica effluent more than toward C. ariakensis effluent. This response did not depend on the species of oyster the drills had been held with, suggesting that the drills were responding to more familiar infochemicals from eastern oysters than from the non-native oysters.     

Identification of candidate AFLP markers for shell color of the Pacific oyster (Crassostrea gigas) under artificial selection

The shell color of the Pacific oyster (Crassostrea gigas) is a desirable trait, but only a few genetic studies on shell color have been documented. Through successive selective breeding, four shell color variants of white (W), gold (G), black (B) and purple (P) C. gigas have been developed. The amplified fragment length polymorphism (AFLP) technique was used to scan the genomes of the four variants with different shell colors and one wild population (C) to identify candidate markers for shell polymorphism. Fifteen AFLP primer combinations were used, 1079 loci were scored as polymorphic loci, and the percentage of polymorphic bands was 95.5%. In the gold, white, black, purple and wild populations, the percentages of polymorphic loci were estimated to be 90.5% (G), 90.0% (W), 91.1% (B), 95.3% (P) and 93.2% (C); the expected heterozygosity values were 0.3115 (G), 0.3044 (W), 0.3102 (B), 0.3285 (P) and 0.3105 (C). The white shell variant was observed to have slightly lower genetic diversity than others, with a F_ST value of 0.1483. These results indicated that the four different shell color variants had high genetic diversity and that the genetic differentiation of populations mostly results from genetic diversity of individuals within populations. Furthermore, 11 outlier loci were considered candidate markers for shell color. This work provides new insights on relationships among color variants of C. gigas.     

Evidence of differential chromosome loss in aneuploid karyotypes of the Pacific oyster, Crassostrea gigas.

The G-banding technique was performed on aneuploid karyotypes from gill tissue of the Pacific oyster, Crassostrea gigas, to assess whether chromosome losses could be explained by differential chromosomal susceptibility and to clarify the negative correlation between aneuploidy and growth rate previously reported in different populations of this oyster. The study of 95 G-banded aneuploid karyotypes showed that only 4 of the 10 chromosome pairs (viz. 1, 5, 9, and 10) of C. gigas were affected by the loss of one homologous chromosome. Pairs 1, 9, and 10, which were lost in 56, 33, and 44% of cases, respectively, may be considered to be differentially affected. Hypotheses on this differential chromosomal susceptibility are discussed.