Pierre Boudry

High variance in reproductive success of the Pacific oyster (Crassostrea gigas, Thunberg) revealed by microsatellite-based parentage analysis of multifactorial crosses

Abstract Marine bivalves are known to have small N e / N ratios, both in wild and hatchery-propagated populations. This has been interpreted as the consequence of a high variance in reproductive success. However, how much of this variance could be attributed to pre-zygotic reproductive factors or to post-zygotic selection was not yet estimated. To estimate the relative effect of these factors, we have conducted a parentage analysis on two 5×5 outbred crosses of Pacific oyster, Crassostrea gigas , using a single microsatellite marker. This was made possible by the high polymorphism encountered at this locus and careful selection of progenitors according to their genotypes. Results show large variance in parental contributions at various developmental stages, leading to a strong reduction of experiment-wide effective population sizes. Segregation distortions fluctuating with time were also observed. Our results show that the observed variance in reproductive success can be attributed to three main factors: gamete quality, sperm–egg interaction and differential viability among genotypes. The comparison of two kinds of crosses, where sperm competition was allowed or not, allowed us to estimate a 20% decrease of the effective population size under sperm competition. A further decrease was observed from larval to juvenile stages. Taken together, the observed gametic, zygotic and genetic effects warn against using too limited a number of progenitors in breeding programmes.

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.

Mytilin B and MGD2, two antimicrobial peptides of marine mussels: gene structure and expression analysis

Previous research has shown that mytilins and MGDs are two types of 4-kDa, cysteine-rich, cationic antimicrobial peptides, which are abundant in hemocytes of the mussels, Mytilus galloprovincialis and M. edulis. The expression of the genes encoding these peptides has been analyzed in the hemocytes of animals subjected to various stress factors, as well as during larval development. Variations in gene expression in adult mussels have been tested under conditions of physical stress, bacterial challenge and heat shock. The results suggest that in adult mussels, the MGD2 gene may be over-expressed with physical and temperature stress, but that reduced expression occurs with bacterial challenge. Gene expression during development has been analyzed using different larval and post-larval stages, ranging from 4-day-old veliger larvae to 32-day-old post-larvae. The results show that the expression of both mytilin B and MGD2 is developmentally regulated, but neither gene is expressed in mussels until after larval settlement and metamorphosis. Finally, the genes encoding two isoforms of these peptides have been cloned and sequenced, revealing that both genes contain four exons and three introns.

Ostreid herpes virus 1 infection in families of the Pacific oyster, Crassostrea gigas, during a summer mortality outbreak: differences in viral DNA detection and quantification using real-time PCR.

Ostreid herpes virus 1 (OsHV-1) infections, notably reported in Europe and the USA, are closely associated with significant mortalities of the Pacific oyster, Crassostrea gigas, especially during its early stages of life. In summer 2006, we monitored mortality by strict daily verification of three full-sib families of oysters reared under common conditions. We quantified OsHV-1 using real-time PCR in dead and living individuals during and after a mortality event. Mortality events were severe and brief, but significantly different between tested families (cumulative mortality ranging from 1.2 to 49%). Real-time PCR assays revealed different viral DNA loads in dead individuals from different families (P<0.001). Moreover, the mean level of infection among families was correlated with mortality (P<0.05). Living oysters showed a significantly lower amount of viral DNA compared with dead ones. This is the first experiment showing the daily changes of individual OsHV-1 DNA load during a mortality outbreak. Our results also support the previously reported high genetic basis underlying the variance of resistance of Pacific oyster to summer mortality, suggesting that there might be a possibility to improve resistance to OsHV-1 by selective breeding.

Plasticity in resource allocation based life history traits in the Pacific oyster, Crassostrea gigas. I. Spatial variation in food abundance.

We investigated the quantitative genetics of plasticity in resource allocation between survival, growth and reproductive effort in Crassostrea gigas when food abundance varies spatially. Resource allocation shifted from survival to growth and reproductive effort as food abundance increased. An optimality model suggests that this plastic shift may be adaptive. Reproductive effort plasticity and mean survival were highly heritable, whereas for growth, both mean and plasticity had low heritability. The genetic correlations between reproductive effort and both survival and growth were negative in poor treatments, suggesting trade‐offs, but positive in rich ones. These sign reversals may reflect genetic variability in resource acquisition, which would only be expressed when food is abundant. Finally, we found positive genetic correlations between reproductive effort plasticity and both growth and survival means. The latter may reflect adaptation of C. gigas to differential sensitivity of fitness to survival, such that genetic variability in survival mean might support genetic variability in reproductive effort plasticity.

Temperature influence on pathogen transmission and subsequent mortalities in juvenile Pacific oysters Crassostrea gigas

Since 2008, mass mortalities of 1-yr-old Crassostrea gigas associated with the ostreid herpesvirus OsHV-1 μVar have occurred along all the coasts of France when seawater temperature reaches 16 to 17°C. The present study aimed to characterize the effect of temperature on oyster survival in combination with OsHV-1 DNA quantification by standard real-time PCR and total vibrio population levels in oyster tissues. To examine the effect of seawater temperature on disease transmission and related mortality of oysters, cohabitation experiments were conducted between healthy naive oysters and oysters previously exposed to field conditions in areas where mortalities were occurring. Oysters initially maintained in controlled conditions (free of mortality and negative for OsHV-1), and then transferred to an area where high mortalities were occurring among farmed stocks, became infected with OsHV-1 and exhibited high loads of vibrios followed by significant mortalities. When previously exposed oysters were maintained indoors at 13.0°C for 40 d and then at 20.6°C, they exhibited no mortality, were negative for OsHV-1 detection, and did not transmit the disease to healthy oysters. Survival of previously exposed oysters maintained indoors at 8 temperatures ranging from 13.4 to 29.0°C varied from 25 to 48% and was negatively correlated with holding temperature. Concomitantly, survival of naive cohabiting animals (62 to 98%) decreased with increasing seawater temperature until a plateau was reached between 16.2 and 21.9°C, and increased at higher temperatures. Therefore, the optimal temperature range for disease transmission from field-exposed to naive animals was between 16.2 and 21.9°C. Our results suggest that a long-term period (40 d) at low temperature (13°C) may offer a method of mitigating mortalities in oysters that have been exposed to an infective environment.

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.

Trans-atlantic distribution of a mangrove oyster species revealed by 16S mtDNA and karyological analyses.

Three species of mangrove oysters, Crassostrea rhizophorae, C. brasiliana, and C. gasar, have been described along the Atlantic shores of South America and Africa. Because the distribution of these molluscs is of great biological and commercial interest, their taxonomy and distribution deserve further clarification. Therefore, 15 populations were sampled from both continents. Their 16S mitochondrial polymorphism was studied by sequencing and PCR-RFLP analysis. Two haplotypes were identified. Haplotype a was the only one observed in Africa, but it was also observed in South America together with haplotype b. Because C. gasar is the only mangrove oyster identified on the west coast of Africa, haplotype a was attributed to this species, which has thus been shown to occur in South America. Haplotype b is attributed to C. rhizophorae. The karyotypes of specimens of C. gasar, from Africa and from South America, were very similar, and both species were observed at the same location in Brazil. The occurrence of C. gasar in South America adds a third species—in addition to C. rhizophorae and C. brasiliana—to the list of species present along these coasts. The predominant surface circulation patterns in this part of the Atlantic Ocean favor the hypothesis that C. gasar was transported from Africa to America. Finally, a phylogenetic tree built with seven 16S sequences from Crassostrea and Saccostrea species showed that C. gasar is intermediate between the American Crassostrea species (C. virginica and C. rhizophorae) and the Asian species (C. gigas and C. ariakensis).

Mitochondrial and nuclear DNA phylogeography of Crassostrea angulata, the Portuguese oyster endangered in Europe

The respective status of the Portuguese oyster, Crassostrea angulata, and the Pacific oyster, Crassostrea gigas, has long been a matter of controversy. Morphological and physiological similarities, homogeneity of allozyme allelic frequencies between populations of the two taxa and the demonstration of hybridization lead most authors to suggest that they should be regrouped within the same species. The risk of introgression and the present expansion of C. gigas aquaculture in Europe raises the question of the need for preservation of C. angulata in Europe, as only a few populations remain. We studied European and Asian populations of C. gigas and C. angulata using microsatellite and mitochondrial DNA markers to estimate their genetic diversity and differentiation. The analysis of genetic distances and the distribution of allelic and haplotype frequencies revealed significant genetic differences between taxa, showing two clusters: (1) C. gigas French and Japanese populations and (2) C. angulata Portuguese and Taiwanese populations. The Asian origin of the Crassostrea angulata taxa is therefore confirmed. Unlike previous studies based on allozymes, significant nuclear genome differences were noted between C. angulata and C. gigas. Despite the presumed history of the introduction of C. angulata into Southern Europe, these populations did not show any significant reduction of variability compared to Taiwanese populations. Any conservation plans for European C. angulata populations should take its non-native origin into account. They represent a valuable genetic resources for European breeding program.

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.