Individual relationship between aneuploidy of gill cells and growth rate in the cupped oysters Crassostrea angulata, C. gigas and their reciprocal hybrids

The Portuguese oyster, Crassostrea angulata, is taxonomically close to the Pacific oyster, C. gigas, but there are clear genetic and phenotypic differences between these taxa. Among those differences, the faster growth of C. gigas compared with C. angulata has often been observed in the field. Crosses between C. angulata and C. gigas were performed to investigate the relationship between growth variation and somatic aneuploidy at the individual level in the two taxa and their reciprocal hybrids. The different progenies were reared in Ria Formosa (Portugal) under standard farming conditions. Growth rate and survival were significantly higher in C. gigas than in C. angulata, and the hybrids showed intermediate performances. Significant differences were also observed in the proportion of aneuploid cells (PAC) and of missing chromosomes (PMC) between the two taxa, C. angulata showing the highest values. Intermediate values of PAC and PMC were observed in the hybrids, supporting additive genetic bases of these parameters. Our results also confirm the negative correlation between somatic aneuploidy and growth rate at the individual level, as previously reported in C. gigas.     

Interspecific hybridization in oysters: Restriction Enzyme Digestion Chromosome Banding confirms Crassostrea angulata × Crassostrea gigas F1 hybrids

The taxonomic status of the two commercially important cupped oysters, Crassostrea angulata, the Portuguese oyster (Lamarck, 1819) and Crassostrea gigas, the Japanese oyster (Thunberg, 1793) has long been in question. The recent observation of the hybridization between C. gigas and C. angulata and the production of fertile F1s led us to search for cytogenetic evidence of both parental genomes in the interspecific hybrids. The cytogenetic characterization of the hybrids was performed by the use of restriction endonuclease treatments. This technique has recently shown the potential for individual chromosome identification by banding in oysters. Chromosomes of C. gigas, C. angulata and their hybrids were treated with two different restriction enzymes (ApaI and HaeIII), stained with Giemsa, and examined for banding patterns. These chromosome markers allowed the parental haploid sets to be identified in the hybrids. The analysis of the banded karyotypes of the interspecific hybrids showed that for each chromosome pair, one of the homologues presented a banding pattern consistent with that of C. gigas and the other homologue presented a banding pattern consistent with that of C. angulata. These cytogenetic results substantiate the reported interspecific hybridization between C. gigas and C. angulata. In view of these results and taking into account the present expansion of C. gigas aquaculture in southern Europe, the question of the need for preservation of pure C. angulata stocks should be raised as only a few populations remain in the south of Spain and Portugal. Recently, changes in the genetic composition of populations in southern Portugal have indeed been observed, showing that human activities have created contact zones between the two taxa while no natural sympatric zones exist in Europe.     

Microsatellite Analysis of 6-Hour-Old Embryos Reveals No Preferential Intraspecific Fertilization Between Cupped Oysters Crassostrea gigas and Crassostrea angulata

Experimental examination of reproductive isolation is the first step in understanding hybridization processes. Here, we studied preferential fertilization between 2 cupped oyster taxa, Crassostrea angulata and Crassostrea gigas, as a potential prezygotic reproductive isolation. Early examination of sperm competition is now possible by molecular analysis of oyster embryos. This avoids the confounding effect of differential mortality during the larval stage. Six hundred embryos were sampled from 2 crosses. Three microsatellite loci were enough to determine without ambiguity the taxa of contributing sires of embryos. No evidence of preferential fertilization between gametes from the same taxa was shown. A significantly higher contribution of the C. gigas males was revealed with the C. angulata females, but not with the C. gigas females, which might suggest early heterosis or interaction differences between gametes. In the light of these results, natural hybridization between both taxa can be expected in cases of their geographical coexistence, as in the Southern European populations in which both taxa are in contact as a result of aquaculture development. Received May 6, 2000; accepted March 6, 2001.     

Specific Detection of Pacific Oyster (Crassostrea gigas) Larvae in Plankton Samples Using Nested Polymerase Chain Reaction

Management of sustainable Pacific oyster fisheries would be assisted by an early, rapid, and accurate means of detecting their planktonic larvae. Reported here is an approach, based on polymerase chain reaction (PCR), for the detection of Pacific oyster larvae in plankton samples. Species-specific primers were designed by comparing partial mitochondrial cytochrome oxidase subunit I (COI) sequences from Crassostrea gigas, with other members of the family Ostreidae including those of Crassostrea angulata. Assay specificity was empirically validated through screening DNA samples obtained from several species of oysters. The assay was specific as only C. gigas samples returned PCR-positive results. A nested PCR approach could consistently detect 5 or more D-hinge-stage larvae spiked into a background of about 146 mg of plankton. The assay does not require prior sorting of larvae. We conclude that the assay could be used to screen environmental and ballast water samples, although further specificity testing against local bivalve species is recommended in new locations.     

A cDNA Microarray for Crassostrea virginica and C. gigas

The eastern oyster, Crassostrea virginica, and the Pacific oyster, C. gigas, are species of global economic significance as well as important components of estuarine ecosystems and models for genetic and environmental studies. To enhance the molecular tools available for oyster research, an international group of collaborators has constructed a 27,496-feature cDNA microarray containing 4460 sequences derived from C. virginica, 2320 from C. gigas, and 16 non-oyster DNAs serving as positive and negative controls. The performance of the array was assessed by gene expression profiling using gill and digestive gland RNA derived from both C. gigas and C. virginica, and digestive gland RNA from C. ariakensis. The utility of the microarray for detection of homologous genes by cross-hybridization between species was also assessed and the correlation between hybridization intensity and sequence homology for selected genes determined. The oyster cDNA microarray is publicly available to the research community on a cost-recovery basis.     

New Resources for Marine Genomics: Bacterial Artificial Chromosome Libraries for the Eastern and Pacific Oysters (Crassostrea virginica and C. gigas)

Large-insert genomic bacterial artificial chromosome (BAC) libraries of two culturally and economically important oyster species, Crassostrea virginica and C. gigas, have been developed as part of an international effort to develop tools and reagents that will advance our ability to conduct genetic and genomic research. A total of 73,728 C. gigas clones with an average insert size of 152 kb were picked and arrayed representing an 11.8-fold genome coverage. A total of 55,296 clones with an average insert size of 150 kb were picked and arrayed for C. virginica, also representing an 11.8-fold genome coverage. The C. gigas and C. virginica libraries were screened with probes derived from selected oyster genes using high-density BAC colony filter arrays. The probes identified 4 to 25 clones per gene for C. virginica and 5 to 50 clones per gene for C. gigas. We conducted a preliminary analysis of genetic polymorphism represented in the C. gigas library. The results suggest that the degree of divergence among similar sequences is highly variable and concentrated in intronic regions. Evidence supporting allelic polymorphism is reported for two genes and allelic and/or locus specific polymorphism for several others. Classical inheritance studies are needed to confirm the nature of these polymorphisms. The oyster BAC libraries are publicly available to the research community on a cost-recovery basis at     

Perkinsus marinus in pleasure oyster Crassostrea corteziensis from Nayarit, Pacific coast of México

Culture of the pleasure oyster Crassostrea corteziensis is emerging as an alternative to the Pacific oyster (Crassostrea gigas) for oyster producers, who face severe mortalities since 1997 in Northwest México. For determining the health status of this species, we conducted a histopathological analysis of cultured populations from two estuaries in the Pacific coast of México. Macroscopical analysis revealed animals with transparent and retracted mantle. Histopathological analysis of these specimens showed tissue alterations and parasitic forms consistent with Perkinsus sp. infection. Stages of the parasite identified included tomont and trophozoites with an eccentric vacuole characteristic of Perkinsus spp. Pieces of tissues of infected oysters were incubated in Fluid Thioglycollate Medium (FTM) resulting in blue–black hypnospores after incubation. The identity of the parasite was confirmed by species specific PCR-based assay in DNA samples from oysters, tissue fractions from FTM cultures, and deparaffined samples with Perkinsus-like parasite detected by histology. Sequencing of positive amplified fragments (307 bp) showed a sequence similar to Perkinsus marinus strain TXsc NTS ribosomal RNA gene (100% coverage and 98% identity, GenBank Accession No. AF497479.1) and to P. marinus, Genomic DNA, (100% coverage and 97% identity, GenBank Accession No. S78416.1). The prevalence of P. marinus varied from 1 to 5% in Boca del Camichín and from 1 to 6% in Pozo Chino. In general, the intensity of infection was moderate. The infection was observed in oysters from 31 to 110 mm of shell length. This is the first record of P. marinus in oysters from the North America Pacific coast and the first record in C. corteziensis. The origin of this parasite in the area is unknown, but it may be associated to introductions of Crassostrea virginica from the East coast of United States of America or Gulf of México.     

Characterization of 79 microsatellite DNA markers in the Pacific oyster Crassostrea gigas

We characterized 79 microsatellite DNA markers, which were obtained from genomic libraries enriched for CA, GA, ATG and TAGA motif repeats, in the Pacific oyster Crassostrea gigas. For eight F₁ grandparents or great‐grandparents of mapping families, the average heterozygosity, 0.705, and average number of alleles per locus, 5.7, did not vary among motif‐repeat or motif‐complexity categories. Non‐amplifying polymerase chain reaction null alleles, which were confirmed by segregation in the mapping families, were detected at 41 (51.9%) of the 79 loci. Cross‐species amplifications from C. angulata, C. sikamea, C. ariakensis and C. virginica showed a precipitous decline with distance from the focal species C. gigas.     

Introduced Pacific oysters (Crassostrea gigas) in the northern Wadden Sea: invasion accelerated by warm summers?

Among the increasing number of species introduced to coastal regions by man, only a few are able to establish themselves and spread in their new environments. We will show that the Pacific oyster (Crassostrea gigas) took 17 years before a large population of several million oysters became established on natural mussel beds in the vicinity of an oyster farm near the island of Sylt (northern Wadden Sea, eastern North Sea). The first oyster, which had dispersed as a larva and settled on a mussel bed, was discovered 5 years after oyster farming had commenced. Data on abundance and size-frequency distribution of oysters on intertidal mussel beds around the island indicate that recruitment was patchy and occurred only in 6 out of 18 years. Significant proportions of these cohorts survived for at least 5 years. The population slowly expanded its range from intertidal to subtidal locations as well as from Sylt north- and southwards along the coastline. Abundances of more than 300 oysters m^–2 on mussel beds were observed in 2003, only after two consecutive spatfalls in 2001 and 2002. Analyses of mean monthly water temperatures indicate that recruitment coincided with above-average temperatures in July and August when spawning and planktonic dispersal occurs. We conclude that the further invasion of C. gigas in the northern Wadden Sea will depend on high late-summer water temperatures.Communicated by H.D. Franke     

Assessment of genetic diversity and population structure in cultured Australian Pacific oysters

The recent development of Pacific oyster (Crassostrea gigas) SNP genotyping arrays has allowed detailed characterisation of genetic diversity and population structure within and between oyster populations. It also raises the potential of harnessing genomic selection for genetic improvement in oyster breeding programmes. The aim of this study was to characterise a breeding population of Australian oysters through genotyping and analysis of 18 027 SNPs, followed by comparison with genotypes of oyster sampled from Europe and Asia. This revealed that the Australian populations had similar population diversity (H_E) to oysters from New Zealand, the British Isles, France and Japan. Population divergence was assessed using PCA of genetic distance and revealed that Australian oysters were distinct from all other populations tested. Australian Pacific oysters originate from planned introductions sourced from three Japanese populations. Approximately 95% of these introductions were from geographically, and potentially genetically, distinct populations from the Nagasaki oysters assessed in this study. Finally, in preparation for the application of genomic selection in oyster breeding programmes, the strength of LD was evaluated and subsets of loci were tested for their ability to accurately infer relationships. Weak LD was observed on average; however, SNP subsets were shown to accurately reconstitute a genomic relationship matrix constructed using all loci. This suggests that low‐density SNP panels may have utility in the Australian population tested, and the findings represent an important first step towards the design and implementation of genomic approaches for applied breeding in Pacific oysters.     

The Distribution of an Introduced Mollusc and its Role in the Long-term Demise of a Native Confamilial Species

The estuarine mud snail, Batillaria attramentaria, was transported to the Pacific coast of North America with the Pacific oyster, Crassostrea gigas (C. gigas), imported from Japan in the early part of this century, and has proliferated in several bays where the oyster was introduced. Since the arrival of Batillaria there have been declines in populations of its native ecological equivalent, Cerithidea californica. This study documents the distribution of the exotic Batillaria throughout its entire introduced range, concentrating on the few bays in northern California where both snails exist sympatrically. Using dates of initial importation of C. gigas and dates of first documentation of Batillaria within a bay, I established the earliest possible date for the introduction of Batillaria in a particular area. In cases where Cerithidea also was, or had been, present within a bay, I calculated a range of time for either the continued coexistence of the two species or the time until local exclusion of the native. Density measurements of Cerithidea within these bays where the species co-occurred allowed comparison of present Cerithidea numbers to historical accounts. Results indicated that Batillaria is replacing Cerithidea in the northern marshes of California. This replacement of the northernmost Cerithidea populations is not only reducing Cerithidea's overall range, but also eliminating a race recently shown to be a genetically distinct from southern Cerithidea populations. Other studies that have demonstrated superior exploitative competitive ability by Batillaria provide a potential mechanism for this displacement pattern. Regardless, the results presented here indicate that the displacement process is slow, taking on average >50 years to complete. This study illustrates a gradual, but predictable process of exotic replacement of native species, and argues strongly against complacency toward invaders that may currently seem innocuous. This revised version was published online in July 2006 with corrections to the Cover Date.     

Variability of haemocyte and haemolymph parameters in European flat oyster Ostrea edulis families obtained from brood stocks of different geographical origins and relation with infection by the protozoan Bonamia ostreae

A research project to compare productive traits (growth and mortality), disease susceptibility and immune capability between Ostrea edulis stocks was performed. This article reports the results on the immune capability and its relation with infection by the intrahaemocytic protozoan Bonamia ostreae. Four to five oyster spat families were produced from each of four European flat oyster populations (one from Ireland, one from Greece and two from Galicia, Spain) in a hatchery. The spat were transferred to a raft in the Ría de Arousa (Galicia) for on growing for 2 years. Total haemocyte count (THC) and differential haemocyte count (DHC) were estimated monthly through the second year of growing-out. Three types of haemocytes were distinguished: granulocytes (GH), large hyalinocytes (LHH) and small hyalinocytes (SHH). Significant correlations between the mean relative abundance of GH and SHH of the families and the mean prevalence of B. ostreae, the overall incidence of pathological conditions and the cumulative mortality of the families were found; these correlations supported the hypothesis that high %GH and low %SHH would enhance oyster immune ability and, consequently, would contribute to lower susceptibility to disease and longer lifespan. Infection by B. ostreae involved a significant increase of circulating haemocytes, which affected more markedly the LHH type. The higher the infection intensity the higher the %LHH. This illustrates the ability of B. ostreae to modulate the immune responses of the O. edulis to favour its own multiplication. A significant reduction of the phenoloxidase activity in the haemolymph of oysters O. edulis infected by B. ostreae was observed. Nineteen enzymatic activities in the haemolymph of O. edulis and Crassostrea gigas (used as a B. ostreae resistant reference) were measured using the kit api ZYM^®, Biomerieux. Qualitative and quantitative differences in enzyme activities in both haemocyte and plasma fractions between B. ostreae noninfected O. edulis from different origins were recorded. However, no clear positive association between enzyme activity and susceptibility to bonamiosis was found. The only enzyme detected in the resistant species C. gigas that was not found in the susceptible one O. edulis was β-glucosidase (in plasma). B. ostreae infected O. edulis showed significant increase of some enzyme activities and the occurrence of enzymes that were not detected in noninfected oysters. These changes could be due to infection-induced enzyme synthesis by the host or to enzyme synthesis by the parasite.     

Protoplast isolation in the marine brown alga Dictyopteris prolifera (Dictyotales)

Protoplasts were isolated from thalli of Dictyopteris prolifera using a mixture of crude enzymes from vicera of live oysters (Crassostrea gigas) and the following commercial enzymes: an abalone enzyme, cellulase, polygalacturonase and hemicellulase. The enzyme mixtures produced up to 3.3 × 10⁷ cells per l g of tissue fresh weight. The conversion to protoplasts of the cells was about 100% using the oyster enzyme or the abalone enzyme alone. The optimum pH for protoplast isolation was 6.0 and 20 hours were required for conversion to protoplasts.     

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.     

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.     

Development of SNP‐genotyping arrays in two shellfish species

Use of SNPs has been favoured due to their abundance in plant and animal genomes, accompanied by the falling cost and rising throughput capacity for detection and genotyping. Here, we present in vitro (obtained from targeted sequencing) and in silico discovery of SNPs, and the design of medium‐throughput genotyping arrays for two oyster species, the Pacific oyster, Crassostrea gigas, and European flat oyster, Ostrea edulis. Two sets of 384 SNP markers were designed for two Illumina GoldenGate arrays and genotyped on more than 1000 samples for each species. In each case, oyster samples were obtained from wild and selected populations and from three‐generation families segregating for traits of interest in aquaculture. The rate of successfully genotyped polymorphic SNPs was about 60% for each species. Effects of SNP origin and quality on genotyping success (Illumina functionality Score) were analysed and compared with other model and nonmodel species. Furthermore, a simulation was made based on a subset of the C. gigas SNP array with a minor allele frequency of 0.3 and typical crosses used in shellfish hatcheries. This simulation indicated that at least 150 markers were needed to perform an accurate parental assignment. Such panels might provide valuable tools to improve our understanding of the connectivity between wild (and selected) populations and could contribute to future selective breeding programmes.     

The density and spatial arrangement of the invasive oyster Crassostrea gigas determines its impact on settlement of native oyster larvae

Understanding how the density and spatial arrangement of invaders is critical to developing management strategies of pest species. The Pacific oyster, Crassostrea gigas, has been translocated around the world for aquaculture and in many instances has established wild populations. Relative to other species of bivalve, it displays rapid suspension feeding, which may cause mortality of pelagic invertebrate larvae. We compared the effect on settlement of Sydney rock oyster, Saccostrea glomerata, larvae of manipulating the spatial arrangement and density of native S. glomerata, and non‐native C. gigas. We hypothesized that while manipulations of dead oysters would reveal the same positive relationship between attachment surface area and S. glomerata settlement between the two species, manipulations of live oysters would reveal differing density‐dependent effects between the native and non‐native oyster. In the field, whether oysters were live or dead, more larvae settled on C. gigas than S. glomerata when substrate was arranged in monospecific clumps. When, however, the two species were interspersed, there were no differences in larval settlement between them. By contrast, in aquaria simulating a higher effective oyster density, more larvae settled on live S. glomerata than C. gigas. When C. gigas was prevented from suspension feeding, settlement of larvae on C. gigas was enhanced. By contrast, settlement was similar between the two species when dead. While the presently low densities of the invasive oyster C. gigas may enhance S. glomerata larval settlement in east Australian estuaries, future increases in densities could produce negative impacts on native oyster settlement. Synthesis and applications: Our study has shown that both the spatial arrangement and density of invaders can influence their impact. Hence, management strategies aimed at preventing invasive populations reaching damaging sizes should not only consider the threshold density at which impacts exceed some acceptable limit, but also how patch formation modifies this.     

Beta-naphthoflavone inhibits the induction of hepatic oestrogen-dependent proteins by 17alpha-ethynylestradiol in mosquitofish (Gambusia holbrooki)

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.