Christophe Ledu

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.

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.

The mass mortality of blue mussels (Mytilus spp) from the Atlantic coast of France is associated with heavy genomic abnormalities as evidenced by flow cytometry

Since 2014, Frances blue mussel industry has been facing heavy mortality outbreaks (90-100%) affecting both juveniles and adults. This report presents evidence of heavy genomic abnormalities associated with mortality outbreaks in blue mussels, Mytilus edulis-galloprovincialis, from the Atlantic coast of France. In this study, ploidy characteristics of hemic cells were investigated using Flow CytoMetry (FCM), revealing an unusual, broad continuum of ploidy distribution from hypodiploidy to tetraploidy. FCM was additionally used to evaluate, at individual and populations levels, different thresholds of genomic abnormality (GA%) using the percentage of non-diploid nuclei. Individual mussels were considered to be abnormal when more than 10% of hemocytes in S-G2/M phase were present. At the population level, a threshold of 6% for the mean intensity of the abnormality is proposed, which means in the population, more than 6% of individual mussels have to present with more than 10% of their hemocytes in S-G2/M phase. GA% was found to be significantly predictive of the final mortality. Based on the established thresholds, only two mussel stocks analyzed in this study were considered to have good cytogenetic quality, while all other stocks appeared to be affected. FCM offers a very powerful tool to help manage current blue mussel mortality in France. We also believe that annual and extensive determination of cytogenetic quality of wild and cultivated mussel beds along with exclusive use of FCM-qualified mussel seeds should be a priority.

Autotetraploid Pacific oysters (Crassostrea gigas) obtained using normal diploid eggs: induction and impact on cytogenetic stability.

We describe two methods of producing viable and fertile autotetraploid Pacific oyster (Crassostrea gigas Thunberg) based on the use of normal-sized oocytes produced by normal diploid females. Our methods showed that the oocyte size is not a limiting factor for the success of the induction to autotetraploidy. These methods offer means of direct introgression of genetic progress from elite diploid lines to tetraploids used as broodstock, avoiding a triploid step with the risk of transferring undesirable traits from highly fecund triploids. High variability in the level of cytogenetic stability was found among the different tetraploid oysters tested, showing that induction method has an important impact on the long-term cytogenetic stability of the tetraploids. It appears that induction method based on the use of triploid females induces a greater cytogenetic instability among tetraploids so obtained, and this compared to tetraploids originating from the two methods described in our present study. As the aneuploidies and reversions observed in tetraploids can have serious consequences for the sustainability of tetraploid broodstock itself, as well as their triploid offspring, the two tetraploid induction methods described in the present work offer means to produce tetraploids with optimal cytogenetic, genetic, and zootechnical performances.

Genomic abnormalities affecting mussels (Mytilus edulis-galloprovincialis) in France are related to ongoing neoplastic processes, evidenced by dual flow cytometry and cell monolayer analyses

In the context of the abnormal mass mortality of mussels in France since 2014, Flow CytoMetry (FCM) was used in 2015 and 2016 to study the DNA content and cell cycle characteristics of hemic circulating cells collected from 2000 mussels. The mussels were sampled from 12 wild and cultivated blue mussels stocks distributed along the French Atlantic coast from the south Brittany to Pertuis Charentais areas. During these surveys, various genetic abnormalities were frequently detected, and ploidy characteristics revealed contrasting profiles that corresponded to respective contrasting sanitary status, i.e. healthy mussels with high cytogenetic quality (HCQ) versus diseased mussels with low cytogenetic quality (LCQ). In the present work, FCM and hemocytology cell monolayer techniques were combined in order to determine the putative causes of the observed genetic abnormalities that were significantly associated with mortality levels. FCM and cell monolayer approaches permitted the definition of new threshold values delimiting HCQ mussels from LCQ ones. FCM histograms of mussels from the HCQ group showed one single or a largely dominant population of diploid (2n) nuclei and a large majority of normal hemocytes. Hemolymph cell-monolayer analyses showed predominantly acidophil granulocytes characterized by nuclei of normal size and a large cytoplasm with numerous granulations. In contrast, FCM histograms for the LCQ group showed, in addition to the normal diploid (2n) nuclei, populations of nuclei that displayed aneuploidy patterns in a broad ploidy range, including diploid-triploid (2-3n), tetraploid-pentaploid (4-5n) and heptaploid-octaploid levels (7-8n). The corresponding hemolymph cell-monolayer showed cellular features characteristic of disseminated neoplasia disease with frequent abnormal anaplastic cells that exhibited noticeable numbers of mitotic figures with both normal and aberrant chromosomes segregation patterns. These neoplastic cells were a rounded shape with a reduced, granulation-free cytoplasm and large (11-12 µm) to very large (up to 21 µm) round or ovoid nuclei that correspond to the 4-5n and 7-8n nuclei previously detected by FCM analyses. These characteristics suggest that the genetic abnormalities detected by means of FCM were related to an ongoing neoplastic process that is affecting blue mussels in France, at least since the onset in 2014 of the mortality that heavily impacted French blue mussels stocks.