Andre Gerard

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.

Bivalve hatchery technology: The current situation for the Pacific oyster Crassostrea gigas and the scallop Pecten maximus in France

Abstract The French oyster farming industry relies almost exclusively on juveniles collected from the natural environment; the supply of spat produced by hatcheries is low, about 10 % of the industrys requirements. Development through selective breeding of oyster stocks which are better suited for aquaculture purposes, is likely to reverse this tendency since only hatcheries will be able to supply such animals. Scallop farming, which at present is poorly developed in France, relies exclusively on hatchery produced spat. Although hatchery technology is constantly being improved, significant production problems remain which must be solved before hatcheries become a major supplier of juveniles for the industry. This paper describes the present state of hatchery technology in France based on experimental results obtained with the great scallop Pecten maximus and the Pacific oyster Crassostrea gigas , over the past ten years. Compared to the great scallop, the Pacific oyster has higher D larva yields (60 % for C. gigas vs. 30 % for P. maximus ) and a faster larval growth rate (10 μm·d −1 for C. gigas vs. 5 μm·d −1 for P. maximus ). However, Pacific oysters have a greater heterogeneity during larval development and higher mortality rates (40 % for C. gigas vs. 25 % for P. maximus ) which produce lower overall yields of pediveliger larvae ready-to-set for Pacific oysters (15 %) compared to the great scallop (30 %). Development of continuous larval and post-larval culture methods along with development of continuous phytoplankton technology offers one of the most promising methods to improve molluscan hatchery techniques.

Detection of oyster herpesvirus DNA and proteins in asymptomatic Crassostrea gigas adults

Since 1972, several herpes-like virus infections have been reported among different bivalve species around the world. Most of these reports involved larvae or juveniles presenting high mortalities. Two case reports of herpes-like viruses concerned adult oysters, Crassostrea virginica in USA and Ostrea angasi in Australia. Molecular techniques including PCR and in situ hybridization (ISH) have been recently developed to detect the oyster herpesvirus genome. In the present study, 30 Pacific oyster, Crassostrea gigas, adults have been analyzed using three different techniques: PCR, ISH and immunochemistry, in order to detect herpesviruses in asymptomatic individuals. PCR and ISH allowed detection of oyster herpesvirus DNA in 93.3 and 86.6%, respectively, of analyzed oysters while polyclonal antibodies allowed detection of viral proteins in 76.6% of analyzed adult oysters. These results suggest that oyster herpesvirus infects adult oysters with high prevalence and that the virus may persist in its host after primary infection. The detection of viral DNA and viral proteins in the gonad of several individuals supports the hypothesis of a possible vertical transmission of the infection. Lastly, concordance among the three techniques used in this study is discussed.

ReviewBivalve hatchery technology: The current situation for the Pacific oyster Crassostrea gigas and the scallop Pecten maximus in FranceÉcloserie de mollusques en France: situation actuelle de l'huître creuse Crassostrea gigas et de la coquille Saint-Jacques Pecten maximus

Abstract The French oyster farming industry relies almost exclusively on juveniles collected from the natural environment; the supply of spat produced by hatcheries is low, about 10 % of the industrys requirements. Development through selective breeding of oyster stocks which are better suited for aquaculture purposes, is likely to reverse this tendency since only hatcheries will be able to supply such animals. Scallop farming, which at present is poorly developed in France, relies exclusively on hatchery produced spat. Although hatchery technology is constantly being improved, significant production problems remain which must be solved before hatcheries become a major supplier of juveniles for the industry. This paper describes the present state of hatchery technology in France based on experimental results obtained with the great scallop Pecten maximus and the Pacific oyster Crassostrea gigas , over the past ten years. Compared to the great scallop, the Pacific oyster has higher D larva yields (60 % for C. gigas vs. 30 % for P. maximus ) and a faster larval growth rate (10 μm·d −1 for C. gigas vs. 5 μm·d −1 for P. maximus ). However, Pacific oysters have a greater heterogeneity during larval development and higher mortality rates (40 % for C. gigas vs. 25 % for P. maximus ) which produce lower overall yields of pediveliger larvae ready-to-set for Pacific oysters (15 %) compared to the great scallop (30 %). Development of continuous larval and post-larval culture methods along with development of continuous phytoplankton technology offers one of the most promising methods to improve molluscan hatchery techniques.

Selecting the flat oyster Ostrea edulis (L.) for survival when infected with the parasite Bonamia ostreae

Abstract In the early 1970s, the appearance of the protozoan parasite Marteilia refringens restricted the production of the European flat oyster Ostrea edulis to subtidal areas. Ten years later a second protozoan parasite, Bonamia ostreae, caused a further serious decline in flat oyster farming in subtidal areas. Prophylactic measures were taken to sustain oyster farming, but the continuing presence of the two parasites led to the initiation of a programme to select for resistant oysters. Efforts were concentrated on B. ostreae and two selected strains were obtained in 1985 (S85) and 1989 (S89). The programme was based on mass selection and parasite inoculation or natural infections. This paper focuses on the survival and weights of the third generation of S85 (S85-G3), the second generation of S89 (S89-G2) and a cross between S85-G2 and S89-G1. The % survival of S85-G3 oysters was more than 4-times higher than the control group after a 20 month experiment in the wild, and significant differences in parasite prevalence were recorded. No significant differences between S89-G2 and the control group were observed for either survival or parasite prevalence. This was attributed either to a less powerful experiment or to the effect of genetic load clearing as a result of inbreeding. The % survival of the cross between the two strains was more than twice as high as the control group and significant differences were recorded for parasite prevalence. Good evidence for additivity of the resistance was provided by both the intermediate behaviour of a cross between S85-G2 and the controls and the rapid improvement that was obtained from the first generations. The selected strains showed a tendency toward higher weights and higher weight variances when compared to controls. These populations are suspected to have undergone bottlenecks, which would explain the increase in phenotypic variance. No measurable natural resistance has arisen in the wild. This could be due first to the relatively short time that has elapsed since the first infections, secondly to the early sale of resistant oysters as soon as they reach a marketable size, and thirdly to the slow kinetics of B. ostreae infection that allows both susceptible and potentially resistant oysters to participate in recruitment. Improved resistance seems to be related to the delayed mortality of selected oysters. This last result is discussed in the light of a previous study conducted in the laboratory which showed that resistant and susceptible oysters have different haemograms.

Population bottleneck and effective size in Bonamia ostreae-resistant populations of Ostrea edulis as inferred by microsatellite markers

Genetic variability at five microsatellite loci was analysed in three hatchery-propagated populations of the flat oyster, Ostrea edulis. These populations were part of a selection programme for resistance to the protozoan parasite Bonamia ostreae and were produced by mass spawns, without control of the genealogy. Evidence for population bottlenecks and inbreeding was sought. A reduction in the number of alleles, mainly due to the loss of rare alleles, was observed in all selected populations, relative to the natural population from which they were derived. Heterozygote excesses were observed in two populations, and were attributed to substructuring of the population into a small number of families. Pedigree reconstruction showed that these two populations were produced by at most two spawning events involving a limited number of parents. Most individuals within these populations are half or full-sib, as shown by relatedness coefficients. The occurrence of population bottlenecks was supported by estimates of effective number of breeders derived by three methods: temporal variance in allelic frequencies, heterozygote excess, and a new method based on reduction in the number of alleles. The estimates from the different methods were consistent. The evidence for bottleneck and small effective number of breeders are expected to lead to increasing inbreeding, and have important consequences for the future management of the three O. edulis selected populations.

A genetic and metabolic basis for faster growth among triploids induced by blocking meiosis I but not meiosis II in the larviparous European flat oyster, Ostrea edulis L.

This study establishes a genetic and metabolic basis to faster triploid growth in the oyster Ostrea edulis. Triploidy was induced using cytochalasin B, and image analysis of biopsied tissue employed to ensure similar ploidy of all animals within each class. Results indicate that lifetime growth in total dry tissue weight over 15 months was more than 60% faster (p<0.001) in meiosis I triploids than in diploid siblings or meiosis II triploids, with no difference between meiosis II triploids and their diploid siblings. For six polymorphic enzyme loci, single-locus heterozygosity was consistently greatest in meiosis I triploids (p<0.001), so that average multiple-locus heterozygosity in meiosis I triploids was 49% higher than in normal diploids, and 55% higher than in meiosis II triploids (p<0.001). This suggests that faster growth resulted from increased allelic diversity, rather than the increased allelic quantity that results from the addition of one entire set of chromosomes among triploids generally. Results also confirm that the faster growth of meiosis I triploids resulted from reduced energy expenditure, associated with lower concentrations of RNA per unit total tissue protein, which infer reduced rates of whole-body protein turnover. Statistical analyses confirmed that differences in oxygen consumption and growth were associated with both ploidy class and average multiple-locus heterozygosity, indicating that performance in meiosis I triploids is not only improved as a result of reduced reproductive output, but also through the metabolic consequences associated with increased heterozygosity.

Influences of triploidy, parentage and genetic diversity on growth of the Pacific oyster Crassostrea gigas reared in contrasting natural environments.

An increasing number of hypotheses are being proposed to explain the faster growth potential of triploids in molluscs, including their partial sterility or their higher heterozygosity compared to diploids. Triploid advantage however, remains controversial for poorer sites, because of a potential trade‐off with survival. These questions were addressed in Crassostrea gigas by deploying meiosis II triploids and their diploid siblings from a single mass spawning of three males and seven females, in two contrasting locations for their trophic resources. One hundred and fifty individuals were sampled at each site after nine months, measured for weight and biochemical composition, and genotyped using three microsatellite and seven allozyme loci. Higher performance was observed at the fast‐growing site for all traits except shell weight, and triploids had greater weights and biochemical contents than diploids at harvest. Triploids also grew faster at the poorer site, and showed similar survival rates to diploids at both sites. Triploids had significantly higher average allozyme and microsatellite diversity. However, they performed better for a wide range of individual heterozygosity values, arguing for an advantage of the triploid state per se, that could be due to positive effects on growth of both sterility of triploids with subsequent resource re‐allocation and possible faster transcription with three copies of each gene. Despite evidence of very low or no inbreeding in the diploid sample, positive associations between individual allozyme diversity and growth were detected, which explained little but significant amounts of phenotypic variation. These associations were interpreted as direct effects of allozymes, either alone or including epistatic interactions with other loci. In addition, measures of individual distance (mean‐d2) specific to microsatellites, were negatively correlated with growth in diploids, indicating possible effects of outbreeding depression between more distant genomes of parents from distinct populations.

A novel method to produce triploids in bivalve molluscs by the use of 6-dimethylaminopurine.

Abstract To date, pressure shock, heat shock, and chemical treatment with cytochalasin B have been the major methods used to induce triploid bivalves. In this study, triploid bivalves were induced by a new chemical treatment using 6-dimethylaminopurine (6-DMAP). The capacity of 6-DMAP to produce triploid eggs and larvae was investigated in the Pacific oyster, Crassostrea gigas , the giant sea scallop, Placopecten magellanicus , and the blue mussel, Mytilus edulis . The triploid yields from the 6-DMAP treatments were compared with those of cytochalasin treatments. The highest percentage of triploid production was 90% in the Pacific oysters when the fertilized eggs were treated for 20 min with 300 μM 6-DMAP at 15 min after fertilization and 95% in the giant scallops when treated for 15 min with 400 μM 6-DMAP at 70 min after fertilization. Increasing durations of 6-DMAP treatments improved the efficiency of triploid induction. However, long incubations with 6-DMAP, which overlapped the period of first mitotic cleavage, led to the development of abnormal larvae. These included low percentages of normal veliger larvae in the Pacific oysters and developmental arrest at the trochophore stage in the blue mussels. The percentage of abnormalities increased with increased treatment duration. Triploid larvae of Pacific oysters produced by 6-DMAP or cytochalasin treatments had equivalent growth rates and were similar to those of control diploid larvae. However, triploid larvae showed high mortalities following these two chemical treatments. Overall, the results clearly demonstrate that 6-DMAP was an efficient and practical inducer of triploidy in bivalve molluscs. Moreover, the described procedure is the most simple and reproducible method ever reported. In addition, 6-DMAP is safer to handle than cytochalasin which is classified as a carcinogen.

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.