Serge Heurtebise

Differentiation between populations of the Portuguese oyster, Crassostrea angulata (Lamark) and the Pacific oyster, Crassostrea gigas (Thunberg), revealed by mtDNA RFLP analysis

Restriction fragment length polymorphisms of PCR-amplified mitochondrial DNA fragments were used to examine genetic differentiation between populations of the Portuguese oyster (Crassostrea angulata) and the Pacific oyster (Crassostrea gigas). The taxonomic status of C. angulata and C. gigas has often been questioned since no morphological or genetic differences had ever been observed between the two taxa. Samples identified as C. angulata, were collected from 16 sites located in southern Portugal and Spain and samples identified as C. gigas, from European and Asian sites. Of the six haplotypes observed, one was commonly found among oysters identified as C. gigas while another one was most frequent among oysters identified as C. angulata. Analysis of haplotype diversity among sites showed that samples originating from southern Portugal and Spain cluster with the Taiwanese sample. These results implicate Taiwan as the possible origin of European C. angulata populations. The ability to differentiate between these two previously indistinguishable taxa allowed us to identify a population in Portugal as mixed. These results open new perspectives for the study of characters previously described as varying between C. gigas and C. angulata, such as growth performance and disease susceptibility.

Mitochondrial and nuclear DNA sequence variation of presumed Crassostrea gigas and Crassostrea angulata specimens: a new oyster species in Hong Kong?

Abstract In several cases, oyster taxa have been misidentified owing to their high morphological plasticity, uncertain geographical range and undocumented introductions. Recently though, molecular techniques have been efficiently applied to discriminate between oysters and to quantify genetic divergence within and among species. In the present paper, we report mitochondrial (16S and COI) and nuclear (28S) DNA sequences of presumed Crassostrea gigas and Crassostrea angulata , two taxa of aquacultural importance. Mitochondrial DNA sequences are compared with previously published sequences and PCR-RFLP haplotypes. Within C. gigas , divergence was less than 0.5% for COI, and less than 0.2% for 16S. Within C. angulata , divergence was less than 1.1% for COI and 0.2% for 16S. Our results also confirm the close genetic relationship between C. gigas and C. angulata and further document their level of divergence: 2–3% for COI and 0.5–1% for 16S. However, the initially presumed C. gigas oysters farmed in Hong Kong (Pearl River delta), presented DNA sequences strongly divergent from both C. gigas and C. angulata : 13–14% for COI, 3–4% for 16S and 1.2–1.6% for 28S. The closest related species are C. gigas and Crassostrea nippona with a divergence of 12–13% for COI, 3–4% for 16S and 1.2–1.6% for 28S. Comparisons with existing DNA sequence data available in the nucleotide sequence databases shows that this is either a new species or that it corresponds to a species for which no DNA sequence is yet available. Further studies are required to document morphological characteristics and geographical range of this putative new species.

QTL for resistance to summer mortality and OsHV-1 load in the Pacific oyster (Crassostrea gigas).

Summer mortality is a phenomenon severely affecting the aquaculture production of the Pacific oyster (Crassostrea gigas). Although its causal factors are complex, resistance to mortality has been described as a highly heritable trait, and several pathogens including the virus Ostreid Herpes virus type 1 (OsHV-1) have been associated with this phenomenon. A QTL analysis for survival of summer mortality and OsHV-1 load, estimated using real-time PCR, was performed using five F(2) full-sib families resulting from a divergent selection experiment for resistance to summer mortality. A consensus linkage map was built using 29 SNPs and 51 microsatellite markers. Five significant QTL were identified and assigned to linkage groups V, VI, VII and IX. Analysis of single full-sib families revealed differential QTL segregation between families. QTL for the two-recorded traits presented very similar locations, highlighting the interest of further study of their respective genetic controls. These QTL show substantial genetic variation in resistance to summer mortality, and present new opportunities for selection for resistance to OsHV-1.

Is fertility of hybrids enough to conclude that the two oysters Crassostrea gigas and Crassostrea angulata are the same species

The distinction of the two cupped oysters Crassostrea gigas(Thunberg, 1793) and Crassostrea angulata(Lamark, 1819) into two species was chiefly due to their differing geographical distributions, C. gigas being present in Asia and C. angulata in Europe. Today it is commonly accepted that C. angulata and C. gigas are a single species according to morphological, genetic and F1 hybridization data. However, the demonstration of the fertility of their hybrids and the absence of any reproductive isolation remained to be investigated. Consequently, we studied the fertility of hybrids and sperm competition by performing three different experiments and producing G1 and G2 hybrid progenies between wild populations of C. angulata and C. gigas. Progenies showed very close developmental yields, at 24 hours after fertilization, according to dam taxa suggesting a strong maternal transmission of oocyte quality, but no reproductive isolation was observed between the two taxa. Significant decreases of developmental yields were noticed in C. angulata females with sperm competition, most probably due to early larval mortality. The fertility of hybrids C. angulata × C. gigas was demonstrated, which is further evidence that they are the same species. To definitively state the precise taxonomic classification of C. angulata and C. gigas, further studies are needed to (i) identify geographical zones where these taxa are in contact and (ii) assess their level of hybridization in these zones.

Combining two-stage testing and interval mapping strategies to detect QTL for resistance to bonamiosis in the european flat oyster Ostrea edulis.

We have identified quantitative trait loci (QTL) in the flat oyster (Ostrea edulis) for resistance to Bonamia ostreae, a parasite responsible for the dramatic reduction in the aquaculture of this species. An F2 family from a cross between a wild oyster and an individual from a family selected for resistance to bonamiosis was cultured with wild oysters injected with the parasite, leading to 20% cumulative mortality. Selective genotyping of 92 out of a total of 550 F2 progeny (i.e., 46 heavily infected oysters that died and 46 parasite-free oysters that survived) was performed using 20 microsatellites and 34 amplification fragment length polymorphism primer pairs. Both a two-stage testing strategy and QTL interval mapping methods were used. The two-stage detection strategy had a high power with a low rate of false positives and identified nine and six probable markers linked to genes of resistance and susceptibility, respectively. Parent-specific genetic linkage maps were built for the family, spanning ten linkage groups (n = 10) with an observed genome coverage of 69–84%. Three QTL were identified by interval mapping in the first parental map and two in the second. Good concordance was observed between the results obtained after the two-stage testing strategy and QTL mapping.

Population genomics shed light on the demographic and adaptive histories of European invasion in the Pacific oyster, Crassostrea gigas.

Crassostrea gigas originated from the Pacific coast of Asia, but was introduced into several European countries in the early 1970s. Natural populations have now spread across the length of the western seaboard of Europe. To elucidate the demographic and selective processes at play during this rapid expansion, genome‐scan analysis was performed on different populations. High diversities and low differentiation were observed overall, but significant genetic differentiation was found among newly established populations and between the newly established northern group and a nearly panmictic group composed of southern European populations and a population from Japan. Loss of genetic diversity was also seen in the north, likely caused by founder events during colonization. The few strongly supported outlier loci revealed a genetic structure uncorrelated with the north/south differentiation, but grouping two samples from the Danish fjords (northern group) and one from the Dutch Scheldt estuary (southern group) with the one from Japan. These findings might reflect the following: (i) parallel adaptation to similar environmental pressures (fjord‐like environment) within each of the two groups or (ii) a footprint of a secondary introduction of an alternative genomic background maintained by multifarious isolation factors. Our results call for a closer examination of adaptive genetic structure in the area of origin.

Relationship between pre- and post-metamorphic growth in the Pacific oyster Crassostrea gigas (Thunberg)

Twenty male and 20 female parental oysters, originating from four sites located along the French Atlantic coast, were crossed together. The 400 crosses were performed separately and then pooled to give a batch of larvae with a large genetic base. Successive sieving after 17 days at 23°C enabled the separation of the largest larvae from the batch. These larvae (i.e., sieving groups) were left to metamorphose and fix onto flat PVC collectors changed daily. Four groups of larvae representing distinctly different growth rates were successively separated during the attachment and metamorphosis of the whole population. This lasted 12 days, from the 17th day to the 29th after fertilisation. A part of each sieving group was settled separately as replicates on cultch. The post- metamorphic height was recorded weekly on 100 oysters per sieving group generating 400 growth curves. The oysters were removed from the collectors and weighed. The effect of the date of settlement (i.e., developmental rate) and sieving group (i.e., larval growth rate) affected (P<0.0001) the spat growth rate significantly. The correlation between sieving groups (i.e., larval growth) and spat growth rate was positive. After 11 months of growth under intensive conditions, the sieving group still had a significant effect on the total weight of juveniles settled on cultch (P<0.0001). These results justify the size-grading of larvae in bivalve hatcheries and show the great importance of early growth on growth in later stages in Crassostrea gigas.

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.

Complete mitochondrial DNA sequence of the European flat oyster Ostrea edulis confirms Ostreidae classification

BackgroundBecause of its typical architecture, inheritance and small size, mitochondrial (mt) DNA is widely used for phylogenetic studies. Gene order is generally conserved in most taxa although some groups show considerable variation. This is particularly true in the phylum Mollusca, especially in the Bivalvia. During the last few years, there have been significant increases in the number of complete mitochondrial sequences available. For bivalves, 35 complete mitochondrial genomes are now available in GenBank, a number that has more than doubled in the last three years, representing 6 families and 23 genera. In the current study, we determined the complete mtDNA sequence of O. edulis, the European flat oyster. We present an analysis of features of its gene content and genome organization in comparison with other Ostrea, Saccostrea and Crassostrea species.ResultsThe Ostrea edulis mt genome is 16 320 bp in length and codes for 37 genes (12 protein-coding genes, 2 rRNAs and 23 tRNAs) on the same strand. As in other Ostreidae, O. edulis mt genome contains a split of the rrnL gene and a duplication of trnM. The tRNA gene set of O. edulis, Ostrea denselamellosa and Crassostrea virginica are identical in having 23 tRNA genes, in contrast to Asian oysters, which have 25 tRNA genes (except for C. ariakensis with 24). O. edulis and O. denselamellosa share the same gene order, but differ from other Ostreidae and are closer to Crassostrea than to Saccostrea. Phylogenetic analyses reinforce the taxonomic classification of the 3 families Ostreidae, Mytilidae and Pectinidae. Within the Ostreidae family the results also reveal a closer relationship between Ostrea and Saccostrea than between Ostrea and Crassostrea.ConclusionsOstrea edulis mitogenomic analyses show a high level of conservation within the genus Ostrea, whereas they show a high level of variation within the Ostreidae family. These features provide useful information for further evolutionary analysis of oyster mitogenomes.

Individual growth variation and its relationship with survival in juvenile Pacific oysters, Crassostrea gigas (Thunberg)

In order to study individual growth variability and its relationship with survival in juvenile Crassostrea gigas, parental oysters were sampled at four sites located along the French Atlantic coast and bred under controlled hatchery conditions. Four groups of larvae were obtained by crossing five males and five females from each of the four sites, and a fifth group by crossing these 20 males and 20 females together in a pool. Larvae were reared under conditions allowing the maintenance of a maximum variability of size and gave five experimental groups. Oysters were individually monitored for growth and survival from 3 to 10 months after fertilization. The individual growth performances were relatively stable over time and no noticeable compensation for growth occurred. Early growth rate was a very good predictor of size later in life: 66% of variation in the live weight at 10 months could be explained by variation in the initial growth rate calculated between 3 and 4 months. A significant group effect was observed on survival and on growth rate. Mortality mostly occurred between 3 and 5 months and appeared to affect the slow-growing animals more. However, two groups can be distinguished among those which died during the experimental period, one which showed a decrease in weight and the other whose growth was similar to surviving oysters. These results are discussed in the light of usual oyster farming practices and selective breeding.