Stock enhancement or sea ranching? Insights from monitoring the genetic diversity, relatedness and effective population size in a seeded great scallop population (Pecten maximus).

The mass release of hatchery-propagated stocks raises numerous questions concerning its efficiency in terms of local recruitment and effect on the genetic diversity of wild populations. A seeding program, consisting of mass release of hatchery-produced juveniles in the local naturally occurring population of great scallops (Pecten maximus L.), was initiated in the early 1980s in the Bay of Brest (France). The present study aims at evaluating whether this seeding program leads to actual population enhancement, with detectable effects on genetic diversity and effective population size, or consists of sea ranching with limited genetic consequences on the wild stock. To address this question, microsatellite-based genetic monitoring of three hatchery-born and naturally recruited populations was conducted over a 5-year period. Results showed a limited reduction in allelic richness but a strong alteration of allelic frequencies in hatchery populations, while genetic diversity appeared very stable over time in the wild populations. A temporal increase in relatedness was observed in both cultured stock and wild populations. Effective population size (Ne) estimates were low and variable in the wild population. Moreover, the application of the Ryman-Laikre model suggested a high contribution of hatchery-born scallops to the reproductive output of the wild population. Overall, the data suggest that the main objective of the seeding program, which is stock enhancement, is fulfilled. Moreover, gene flow from surrounding populations and/or the reproductive input of undetected sub-populations within the bay may buffer the Ryman-Laikre effect and ensure the retention of the local genetic variability.

Pleiotropic role of the RNA chaperone protein Hfq in the human pathogen Clostridium difficile.

Clostridium difficile is an emergent human pathogen and the most common cause of nosocomial diarrhea. Our recent data strongly suggest the importance of RNA-based mechanisms for the control of gene expression in C. difficile. In an effort to understand the function of the RNA chaperone protein Hfq, we constructed and characterized an Hfq-depleted strain in C. difficile. Hfq depletion led to a growth defect, morphological changes, an increased sensitivity to stresses, and a better ability to sporulate and to form biofilms. The transcriptome analysis revealed pleiotropic effects of Hfq depletion on gene expression in C. difficile, including genes encoding proteins involved in sporulation, stress response, metabolic pathways, cell wall-associated proteins, transporters, and transcriptional regulators and genes of unknown function. Remarkably, a great number of genes of the regulon dependent on sporulation-specific sigma factor, SigK, were upregulated in the Hfq-depleted strain. The altered accumulation of several sRNAs and interaction of Hfq with selected sRNAs suggest potential involvement of Hfq in these regulatory RNA functions. Altogether, these results suggest the pleiotropic role of Hfq protein in C. difficile physiology, including processes important for the C. difficile infection cycle, and expand our knowledge of Hfq-dependent regulation in Gram-positive bacteria.

Starch supplementation modulates amylase enzymatic properties and amylase B mRNA level in the digestive gland of the Pacific oyster Crassostrea gigas.

In the oyster Crassostrea gigas consumption-related traits, amylase properties and growth were found to be linked through genotypes that differed for polymorphism in the two amylase genes AMYA and AMYB. Modulation of AMYA mRNA level had already been observed in response to food availability, whereas the functional role of AMYB was still unknown. To improve knowledge about the regulation of amylase expression in C. gigas and the respective roles of the two genes, we made an assay of amylase expression at mRNA and enzymatic levels in the digestive gland of oysters that had received dietary supplements of starch. After 18 days, a significant increase of translatable mRNA for AMYB was observed, with a correlated increase in Michaelis-Menten constant Km values and a decrease in total amylase activity. This modulation is the first evidence of observable functioning of AMYB in digestive processes. Amylase B is suggested to display a higher Km than amylase A, offering a means of adapting to high substrate concentrations. The highest starch supplement level (10 mgL(-1)) induced alteration in oyster physiology. The 1 mgL(-1) treatment should be tested as a practical food supplement that could lead to growth benefits for oysters.

Responses of diploid and triploid Pacific oysters Crassostrea gigas to Vibrio infection in relation to their reproductive status.

Several Vibrio species are known to be pathogenic to the Pacific oyster Crassostrea gigas. Survival varies according to pathogen exposure and high mortality events usually occur in summer during gametogenesis. In order to study the effects of gametogenetic status and ploidy (a factor known to affect reproduction allocation in oysters) on vibriosis survival, we conducted two successive experiments. Our results demonstrate that a common bath challenge with pathogenic Vibrio splendidus and Vibrio aestuarianus on a mixture of mature, spawning and non-mature oysters can lead to significant mortality. Previous bath challenges, which were done using only non-mature oysters, had not produced mortality. Immunohistochemical analyses showed the affinity of Vibrio for gonadic tissues, highlighting the importance of sexual maturity for vibriosis infection processes in oysters. Mortality rate results showed poor repeatability between tanks, however, in this bath challenge. We then tested a standardized and repeatable injection protocol using two different doses of the same combination of two Vibrio species on related diploid and triploid oysters at four different times over a year. Statistical analyses of mortality kinetics over a 6-day period after injection revealed that active gametogenesis periods correspond to higher susceptibility to vibriosis and that there is a significant interaction of this seasonal effect with ploidy. However, no significant advantage of triploidy was observed. Triploid oysters even showed lower survival than diploid counterparts in winter. Results are discussed in relation to differing energy allocation patterns between diploid and triploid Pacific oysters.

Variance in the reproductive success of flat oyster Ostrea edulis L. assessed by parentage analyses in natural and experimental conditions.

In order to document further the phenomena of variance in reproductive success in natural populations of the European flat oyster Ostrea edulis, two complementary studies based on natural and experimental populations were conducted. The first part of this work was focused on paternity analyses using a set of four microsatellite markers for larvae collected from 13 brooding females sampled in Quiberon Bay (Brittany, France). The number of individuals contributing as the male parent to each progeny assay was highly variable, ranging from 2 to more than 40. Moreover, paternal contributions showed a much skewed distribution, with some males contributing to 50-100% of the progeny assay. The second part of this work consisted of the analysis of six successive cohorts experimentally produced from an acclimated broodstock (62 wild oysters sampled in the Quiberon Bay). Allelic richness was significantly higher in the adult population than in the temporal cohorts collected. Genetic differentiation (F(st) estimates) was computed for each pair of samples and all significant values ranged from 0.7 to 11.9%. A limited effective number of breeders (generally below 25) was estimated in the six temporal cohorts. The study gives first indications of the high variance in reproductive success as well as a reduced effective size, not only under experimental conditions but also in the wild. Surprisingly, the pool of the successive cohorts, based on the low number of loci used, appeared to depict a random and representative set of alleles of the progenitor population, indicating that the detection of patterns of temporal genetic differentiation at a local scale most likely depends on the sampling window.

Regulation of FADS2 expression and activity in European sea bass (Dicentrarchus labrax, L.) fed a vegetable diet.

Supplies of marine fish oils are limited, and continued growth in aquaculture production dictates that lipid substitutes in fish diets must be used without compromising fish health and product quality. In this study, the total substitution of a fish meal and fish oil by a blend of vegetable meals (corn, soybean, wheat and lupin) and linseed oil in the diet of European sea bass (Dicentrachus labrax) was investigated. Two groups of European sea bass were fed with fish diet (FD) or vegetable diet (VD) for 9months. VD, totally deprived of eicosapentaenoate (EPA; 20:5n-3) and docosahexaenoate (DHA; 22:6n-3), revealed a nutritional deficiency and affected growth performance. Whilst VD induced a significant increase in fatty acid desaturase 2 (FADS2) and sterol binding regulatory element-binding protein 1 (SREBP-1) mRNA levels, the desaturation rate of [1-(14)C]18:3n-3 into [1-(14)C]18:4n-3, analysed in microsomal preparations using HPLC method, did not show an upregulation of FADS2 activities in liver and intestine of fish fed VD. Moreover Western-blot analysis did not revealed any significant difference of FADS2 protein amount between the two dietary groups. These data demonstrate that sea bass exhibits a desaturase (FADS2) activity whatever their diet, but a post-transcriptional regulation of fads2 RNA prevents an increase of enzyme in fish fed a HUFA-free diet. This led to a lower fish growth and poor muscle HUFA content.

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.

Infestation of the cupped oysters Crassostrea angulata, C. gigas and their first-generation hybrids by the copepod Myicola ostreae: differences in susceptibility and host response.

SUMMARY: We studied the prevalence and intensity of the parasitic copepod Myicola ostreae in 2 closely related oysters Crassostrea angulata and C. gigas and their F1 hybrids. The effects on host and host reaction were also analysed to better understand host-parasite relationships between copepods and bivalve molluscs. Full reciprocal crosses were carried out between C. angulata and C. gigas and the progenies were reared in the wild in Ria Formosa Lagoon (Portugal), allowing natural infestation by M. ostreae. Prevalence and intensity were significantly higher in C. angulata than in C. gigas. The parasite level of F1 hybrids was similar to C. angulata and significantly higher than in C. gigas. The results of our study support a hypothesis of dominantly inherited susceptibility to M. ostreae infestation. Moreover, copepods were observed on the gill surface of C. gigas engulfed by a capsule-like structure. Histological analyses revealed that the copepods were surrounded by a massive agglomerate of haemocyte-like cells encircled by a thin layer of fibroblast-like cells. This encapsulation response was not observed in C. angulata or in F1 hybrids. These results suggest that the differential susceptibility to M. ostreae between C. angulata and C. gigas may be ascribed to host defence factors.

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 F(2) 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 F(2) 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.

Identification and characterization of 18 novel polymorphic microsatellite makers derived from expressed sequence tags in the Pacific oyster Crassostrea gigas.

We report the development of 18 new polymorphic microsatellite DNA markers derived from Crassostrea gigas expressed sequences tags. Genotyping of 48 wild adult oysters sampled from Marennes-Oléron bay (France) revealed 12 to 48 alleles per locus. Observed and expected heterozygosity levels ranged from 0.64 to 1 and from 0.77 to 0.97, respectively. The development of these new markers creates a useful complementary tool for population genetics studies, parentage analysis and mapping in Pacific oyster, a species of major aquacultural and ecological importance.

Association among growth, food consumption-related traits and amylase gene polymorphism in the Pacific oyster Crassostrea gigas.

To examine further a previously reported association between amylase gene polymorphism and growth in the Pacific oyster Crassostrea gigas, ecophysiological parameters and biochemical and molecular expression levels of alpha-amylase were studied in Pacific oysters of different amylase genotypes. Genotypes that previously displayed significantly different growth were found to be significantly different for ingestion and absorption efficiency. These estimated parameters, used in a dynamic energy budget model, showed that observed ingestion rates (unlike absorption efficiencies) allowed an accurate prediction of growth potential in these genotypes. The observed association between growth and amylase gene polymorphism is therefore more likely to be related to ingestion than to absorption efficiency. Additionally, relative mRNA levels of the two amylase cDNAs were also strongly associated with amylase gene polymorphism, possibly reflecting variation in an undefined regulatory region, although no corresponding variation was observed in specific amylase activity. Amylase gene sequences were determined for each genotype, showing the existence of only synonymous or functionally equivalent non-synonymous polymorphisms. The observed associations among growth, food consumption-related traits and amylase gene polymorphism are therefore more likely to be related to variation in the level of amylase gene expression than to functional enzymatic variants.

A first-generation genetic linkage map of the European flat oyster Ostrea edulis (L.) based on AFLP and microsatellite markers.

This study presents the first genetic linkage map for the European flat oyster Ostrea edulis. Two hundred and forty-six AFLP and 20 microsatellite markers were genotyped in a three-generation pedigree comprising two grandparents, two parents and 92 progeny. Chi-square goodness-of-fit tests revealed high segregation distortion, which was significant for 32.8% of markers. Sixteen microsatellites and 235 AFLPs (170 type 1:1 AFLPs and 65 type 3:1 AFLPs) were used to build sex-specific linkage maps using crimap software. The first parental map (P(1)) consisted of 104 markers grouped in nine linkage groups, and spanned 471.2 cM with an average spacing of 4.86 cM. The second parental map (P(2)) consisted of 117 markers grouped in 10 linkage groups (which equals the haploid chromosome number), and covered 450.0 cM with an average spacing of 4.21 cM. The estimated coverage of the genome was 82.4% for the P(1) map and 84.2% for the P(2) map. Eight linkage groups that were probably homologous between the two parents contained the same microsatellites and 3:1 AFLPs (segregating through both parents). Distorted markers were not randomly distributed across the genome and tended to cluster in a few linkage groups. Sex-specific differences in recombination rates were evident. This first-generation genetic linkage map for O. edulis represents a major step towards the mapping of QTL such as resistance to bonamiasis, a parasitosis that has drastically decreased populations of flat oysters since the 1960s.

Single Nucleotide polymorphisms and their relationship to codon usage bias in the Pacific oyster Crassostrea gigas.

DNA sequence polymorphism and codon usage bias were investigated in a set of 41 nuclear loci in the Pacific oyster Crassostrea gigas. Our results revealed a very high level of DNA polymorphism in oysters, in the order of magnitude of the highest levels reported in animals to date. A total of 290 single nucleotide polymorphisms (SNPs) were detected, 76 of which being localised in exons and 214 in non-coding regions. Average density of SNPs was estimated to be one SNP every 60 bp in coding regions and one every 40 bp in non-coding regions. Non-synonymous substitutions contributed substantially to the polymorphism observed in coding regions. The non-synonymous to silent diversity ratio was 0.16 on average, which is fairly higher to the ratio reported in other invertebrate species recognised to display large population sizes. Therefore, purifying selection does not appear to be as strong as it could have been expected for a species with a large effective population size. The level of non-synonymous diversity varied greatly from one gene to another, in accordance with varying selective constraints. We examined codon usage bias and its relationship with DNA polymorphism. The table of optimal codons was deduced from the analysis of an EST dataset, using EST counts as a rough assessment of gene expression. As recently observed in some other taxa, we found a strong and significant negative relationship between codon bias and non-synonymous diversity suggesting correlated selective constraints on synonymous and non-synonymous substitutions. Codon bias as measured by the frequency of optimal codons for expression might therefore provide a useful indicator of the level of constraint upon proteins in the oyster genome.

AFLP-based genetic linkage maps of the blue mussel (Mytilus edulis).

We report the construction of the first genetic linkage map in the blue mussel, Mytilus edulis. AFLP markers were used in 86 full-sib progeny from a controlled pair mating, applying a double pseudo-test cross strategy. Thirty-six primer pairs generated 2354 peaks, of which 791 (33.6%) were polymorphic in the mapping family. Among those, 341 segregated through the female parent, 296 through the male parent (type 1:1) and 154 through both parents (type 3:1). Chi-square goodness-of-fit tests revealed that 71% and 73% of type 1:1 and 3:1 markers respectively segregated according to Mendelian inheritance. Sex-specific linkage maps were built with mapmaker 3.0 software. The female framework map consisted of 121 markers ordered into 14 linkage groups, spanning 862.8 cM, with an average marker spacing of 8.0 cM. The male framework map consisted of 116 markers ordered into 14 linkage groups, spanning 825.2 cM, with an average marker spacing of 8.09 cM. Genome coverage was estimated to be 76.7% and 75.9% for the female and male framework maps respectively, rising to 85.8% (female) and 86.2% (male) when associated markers were included. Twelve probable homologous linkage group pairs were identified and a consensus map was built for nine of these homologous pairs based on multiple and parallel linkages of 3:1 markers, spanning 816 cM, with joinmap 4.0 software.

An amylase gene polymorphism is associated with growth differences in the Pacific cupped oyster Crassostrea gigas.

This study investigated the non-neutrality of genetic polymorphism in two alpha-amylase genes (AMYA and AMYB) in the oyster Crassostrea gigas. Bi-parental oyster families, bred to be polymorphic for markers in these genes, were monitored for growth and survival for 1 year under standard culture conditions in two French production sites. Within-family genotype frequencies indicated that the two amylase genes were closely linked (c. 1.7 cM). Within two of three families, significant differences in growth were observed between genotypes at one of the two production sites, suggesting that this polymorphism is not neutral and might be under selection because of its role in digestive function. Estimated daily yields were different between amylase genotypes, indicating the potential value of amylase markers in selective breeding programmes to improve oyster growth.

Ostreid herpesvirus 1 (OsHV-1) detection among three successive generations of Pacific oysters (Crassostrea gigas).

Ostreid Herpesvirus 1 (OsHV-1) was likely detected in Pacific oysters, Crassostrea gigas, at different stages of development. Viral infections were associated with high mortality rates in the spat and larvae. Furthermore, the persistance of OsHV-1 in asymptomatic adults was demonstrated by detection of viral DNA and proteins. In the present study, three successive generations of C. gigas (G0 and G1 parental oysters, G1 and G2 larvae) were screened for OsHV-1 by PCR. Viral DNA was detected in 2-day-old larvae, indicating that infection may take place at very early stages. Although results strengthen the hypothesis of a vertical transmission, it was not possible to predict the issue of a particular type of cross. Indeed, the detection of viral DNA in parental oysters did not systematically correspond to a productive infection or result in a successful transmission to the progeny. However, the infective status of the parents appeared to have an influence on both the infection and the survival rates of the progeny. Crosses involving an OsHV-1 infected male and a non-infected female resulted in hatching and larval survival rates statistically lower than those observed in the other types of cross. These results suggest that OsHV-1-infected females may transmit to their offspring some kind of protection or resistance against viral infection.

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.

Reduced female gene flow in the European flat oyster Ostrea edulis.

The geographical structure of 15 natural populations of the flat oyster (Ostrea edulis L.) was assessed by single-strand conformation polymorphism (SSCP) of a 313-base-pair (bp) fragment of the mitochondrial 12S-rRNA gene. Fourteen haplotypes were observed, with one being dominant in the Mediterranean samples and another one in the Atlantic populations. The geographically extreme populations sampled in Norway and the Black Sea appeared differentiated by exhibiting the dominance of a third group of haplotypes. The results were compared to available microsatellite data at five loci. The Atlantic/Mediterranean differentiation pattern was qualitatively the same with both types of markers, confirming an isolation-by-distance pattern. The average mitochondrial haplotypic diversity displayed a high among populations variance, reflecting small effective population size in some locations. Additionally, a 10-fold quantitative difference was observed in Fst between the mitochondrial and the nuclear genomes, which could be due to an unbalanced sex ratio or sex-biased differential reproductive success between males and females (or both).

Spatio-temporal variation in the genetic composition of wild populations of pearl oyster (Pinctada margaritifera cumingii) in French Polynesia following 10 years of juvenile translocation.

Abstract The genetic impact of the cultural practice of spat collection and translocation between genetically distinct stocks of black-lipped pearl oyster, Pinctada margaritifera cumingii, was studied by comparing samples collected in the 1980s and 2000s from seven atolls in French Polynesia. An amova revealed homogenization of the previously genetically distinct wild stocks of Tuamotu-Gambier and Society archipelagos (the indices of genetic differentiation among archipelagos and among populations within archipelagos, respectively, Phi(CT) and Phi(ST), decreased from 0.032* and 0.025*, respectively, to 0.006(NS) and 0.007(NS)). These results suggest high success of spontaneous reproduction in farms, probably due to the very high density of cultivated pearl oysters, and underline the importance of genetic monitoring of future hatchery produced stocks.

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.