Reproduction of the pearl oyster Pinctada margaritifera : study of the female sex determinants in adult oyster

Depuis plusieurs années il est devenu essentiel de comprendre le déterminisme sexuel des espèces à fort intérêt économique afin d’optimiser leur production au sein d’écloseries émergentes.L’objectif principal de cette thèse était de mettre en évidence les mécanismes impliqués dans la détermination et la différenciation sexuelle, et notamment du sexe femelle, chez l’huître perlière P. margaritifera, espèce hermaphrodite protandre et espèce clé de la perliculture, la seconde ressource économique pour la Polynésie française. Pour atteindre cet objectif, deux approches ont été menées : une approche transcriptomique visant à étudier les mécanismes moléculaires du déterminisme et de la différenciation sexuelle, et une approche expérimentale visant à comprendre le phénomène de la sexualisation par des forçages environnementaux et hormonaux en s’intéressant plus particulièrement au déterminisme et à la différenciation sexuelle femelle.Dans l’approche transcriptomique, le transcriptome de la gonade de P. margaritifera a été séquencé à partir de plusieurs échantillons gonadiques d’huîtres de sexe mâle et femelle à différents stades de développement. Après le séquençage Illumina et l'assemblage du transcriptome, 70 147 contigs ont été obtenus. L’analyse fonctionnelle de ces 70 147contigs, a permis d’identifier des gènes d’intérêt et ainsi de constituer un catalogue de 87 ARNm codant pour 67 protéines impliquées dans la détermination, la différenciation sexuelle et/ou la gamétogenèse. Ensuite une analyse stricte des données de quantification RNAseq a révélé 1 937 contigs exprimés de manière différentielle entre les catégories histologiques des gonades. À partir de l’analyse de leurs profils d’expression au sein de chaque échantillon, un nouveau modèle de la reproduction de P. margaritifera, basé sur une double approche analytique, eg. histo-moléculaire, a été proposé. Ce modèle révèle notamment que le déterminisme sexuel de P. margaritifera chez l’adulte se produirait durant une phase de régression de la gonade. Considérant ainsi les nouveaux stades définis par ce modèle, 9 gènes biomarqueurs de la voie sexuelle femelle ont pu être identifiés révélant un modèle prédictif de la voie sexuelle basé sur 3 rapports d’expressions de gènes impliquant 2 gènes inconnus pmarg-c43476 et pmarg-c54338 et 2 gènes connus pmarg-foxl2 et pmarg-fem1-like. Ce deuxième modèle suggère fortement l'implication de pmarg-foxl2 et pmarg-fem1-like dans le déterminisme du sexe de P. margaritifera. Dans l’approche expérimentale, deux expérimentations séparées ont été réalisées pour mettre en évidence l’effet i) de plusieurs combinaisons de température et de niveau trophique, et ii) de l’œstradiol-17β administré par injection directe dans la gonade ; sur le sexe, la gamétogenèse et l’expression des neuf gènes biomarqueurs de la voie sexuelle femelle identifiés précédemment. Les résultats ont montré que la condition combinant la température de 28°C et la concentration en algues de 40 000 cellules mL-1 était la plus favorable non seulement à la maturation des gonades mâles et femelles mais aussi au maintien du sexe femelle. Ce serait dans cette condition environnementale qu’il serait possible d’induire un changement de sexe de mâle vers femelle. Dans la seconde expérimentation, il a été clairement démontré que la reproduction de P. margaritifera pouvait être régulée par les hormones œstrogènes. Les résultats montrent un effet négatif de l’œstradiol sur le développement et la différenciation mâle. Enfin les résultats du modèle prédictif de la voie sexuelle de P. margaritifera, suggèrent une programmation génétique du sexe femelle qui toutefois resterait soumise aux conditions environnementales validant ainsi l’hypothèse d’un mode de détermination mixte du sexe chez P. margaritifera.For several years it has become essential to understand sex determination of species with high economic interest to maximize their production in emerging hatcheries.The main objective of this thesis was to identify the mechanisms involved in sex determination and sex differentiation, and particularly in female sex, in the pearl oyster P. margaritifera, a protandrous hermaphrodite species and the key species of the pearl farming, the second economic resource for French Polynesia. To achieve this goal, two approaches were undertaken: a transcriptomic approach to investigate the molecular mechanisms of sex determinism and sex differentiation, and an experimental approach to understand the phenomenon of sexualization by environmental and hormonal forcing focusing especially on female sex determinism and female sex differentiation.In the transcriptomic approach, the gonad transcriptome of P. margaritifera was sequenced from several samples of male and female oyster gonads at different stages of development. After Illumina sequencing and assembly of the transcriptome, 70,147 contigs were obtained. Functional analysis of these 70,147 contigs identified genes of interest and allowed the constitution of a catalog of 87 mRNAs encoding 67 proteins involved in sex determination, sex differentiation and/or gametogenesis. Then a strict analysis of RNAseq quantification data revealed 1,937 contigs differentially expressed between the histological categories of gonad. From the analysis of their expression profiles in each sample, a new model of reproduction of P. margaritifera, based on dual analytical approach, i.e. histo-molecular, has been proposed. This model shows that sex determination of adult P. margaritifera pearl oysters occur during a regression phase of the gonad. And considering the new stages defined on this model, 9 biomarkers genes of the female sexual pathway have been identified revealing a 3-gene-pair expression ratio based model, which makes it possible to predict the sexual pathway in this hermaphrodite species. This predictive model involves two unknown genes pmarg-c43476 and pmarg-c54338 and 2 known genes pmarg-foxl2 and pmarg-fem1-like, and strongly suggests the involvement of pmarg-foxl2 and pmarg-fem1-like in sex determinism in P. margaritifera.In the experimental approach, two separated experiments were conducted to demonstrate the effect of i) various combinations of temperature and trophic level, and ii) 17β-estradiol administered by direct injection into the gonad; on sex, gametogenesis and expression of the nine biomarkers genes of the female sexual pathway previously identified. The results showed that the condition combining a temperature of 28 °C and a concentration of 40 000 cells of algae mL-1 was the most favorable not only for the maturation of the male and female gonads but also for the maintenance of the female sex. It would be in this environmental condition that it would be possible to induce a sex change from male to female. In the second experiment, it was clearly demonstrated that the reproduction of P. margaritifera could be regulated by estrogen hormones. The results show a negative effect of estradiol on male development and differentiation. Finally the results of the predictive model of the sexual pathway of P. margaritifera, suggest a genetic programming of the female sex, which however remain subject to environmental conditions, thus validating the hypothesis of a mixed sex determinism mode in P. margaritifera

Recovery capabilities of Xenopus laevis after exposure to Cadmium and Zinc

International audienceThe present investigation evaluates the recovery capabilities of Xenopus laevis following 12days of exposure to 30μg CdL(-1) and 1000μg ZnL(-1) alone or mixed, followed by a depuration phase in laboratory conditions. Focused endpoints, which were investigated at different times of depuration, are bioaccumulation of Cd and Zn, micronucleus induction, quantification of metallothioneins (MTs), and expression of genes involved in metal toxicity mechanisms. The results show that at the end of the contamination phase, there was higher metal bioaccumulation capability and MT synthesis in remaining tissues than in the liver. An increased expression of genes involved in detoxification and oxidative stress mechanisms was observed, suggesting an additive effect of both metals and a higher Zn regulation in the liver. During the depuration phase, the results show the recovery capability of Xenopus from 7days of depuration related to metamorphosis processes, which were observed at the end of the experiment. The results confirm the relevance of the amphibian model and the complementarities between a marker of genotoxicity, MT production, bioaccumulation and transcriptional analysis in the evaluation of the ecotoxicological impact. The results also highlight the reversible effects of Cd and Zn toxicity

Molecular Signatures Discriminating the Male and the Female Sexual Pathways in the Pearl Oyster Pinctada margaritifera

The genomics of economically important marine bivalves is studied to provide better understanding of the molecular mechanisms underlying their different reproductive strategies. The recently available gonad transcriptome of the black-lip pearl oyster Pinctada margaritifera is a novel and powerful resource to study these mechanisms in marine mollusks displaying hermaphroditic features. In this study, RNAseq quantification data of the P. margaritifera gonad transcriptome were analyzed to identify candidate genes in histologically-characterized gonad samples to provide molecular signatures of the female and male sexual pathway in this pearl oyster. Based on the RNAseq data set, stringent expression analysis identified 1,937 contigs that were differentially expressed between the gonad histological categories. From the hierarchical clustering analysis, a new reproduction model is proposed, based on a dual histo-molecular analytical approach. Nine candidate genes were identified as markers of the sexual pathway: 7 for the female pathway and 2 for the male one. Their mRNA levels were assayed by real-time PCR on a new set of gonadic samples. A clustering method revealed four principal expression patterns based on the relative gene expression ratio. A multivariate regression tree realized on these new samples and validated on the previously analyzed RNAseq samples showed that the sexual pathway of P. margaritifera can be predicted by a 3-gene-pair expression ratio model of 4 different genes: pmarg-43476, pmarg-foxl2, pmarg-54338 and pmarg-fem1-like. This 3-gene-pair expression ratio model strongly suggests only the implication of pmarg-foxl2 and pmarg-fem1-like in the sex inversion of P. margaritifera. This work provides the first histo-molecular model of P. margaritifera reproduction and a gene expression signature of its sexual pathway discriminating the male and female pathways. These represent useful tools for understanding and studying sex inversion, sex differentiation and sex determinism in this species and other related species for aquaculture purposes such as genetic selection programs

Gonad transcriptome analysis of pearl oyster Pinctada margaritifera: identification of potential sex differentiation and sex determining genes

Background Black pearl farming is based on culture of the blacklip pearl oyster Pinctada margaritifera (Mollusca, lophotrochozoa), a protandrous hermaphrodite species. At first maturation, all individuals are males. The female sex appears progressively from two years old, which represents a limitation for broodstock conditioning for aquaculture production. In marine mollusks displaying hermaphroditic features, data on sexual determinism and differentiation, including the molecular sex determining cascade, are scarce. To increase genomic resources and identify the molecular mechanisms whereby gene expression may act in the sexual dimorphism of P. margaritifera, we performed gonad transcriptome analysis. Results The gonad transcriptome of P. margaritifera was sequenced from several gonadic samples of males and females at different development stages, using a Next-Generation-Sequencing method and RNAseq technology. After Illumina sequencing, assembly and annotation, we obtained 70,147 contigs of which 62.2% shared homologies with existing protein sequences, and 9% showed functional annotation with Gene Ontology terms. Differential expression analysis identified 1,993 differentially expressed contigs between the different categories of gonads. Clustering methods of samples revealed that the sex explained most of the variation in gonad gene expression. K-means clustering of differentially expressed contigs showed 815 and 574 contigs were more expressed in male and female gonads, respectively. The analysis of these contigs revealed the presence of known specific genes coding for proteins involved in sex determinism and/or differentiation, such as dmrt and fem-1 like for males, or foxl2 and vitellogenin for females. The specific gene expression profiles of pmarg-fem1-like, pmarg-dmrt and pmarg-foxl2 in different reproductive stages (undetermined, sexual inversion and regression) suggest that these three genes are potentially involved in the sperm-oocyte switch in P. margaritifera. Conclusions The study provides a new transcriptomic tool to study reproduction in hermaphroditic marine mollusks. It identifies sex differentiation and potential sex determining genes in P. margaritifera, a protandrous hermaphrodite species

Temperature and Food Influence Shell Growth and Mantle Gene Expression of Shell Matrix Proteins in the Pearl Oyster Pinctada margaritifera

In this study, we analyzed the combined effect of microalgal concentration and temperature on the shell growth of the bivalve Pinctada margaritifera and the molecular mechanisms underlying this biomineralization process. Shell growth was measured after two months of rearing in experimental conditions, using calcein staining of the calcified structures. Molecular mechanisms were studied though the expression of 11 genes encoding proteins implicated in the biomineralization process, which was assessed in the mantle. We showed that shell growth is influenced by both microalgal concentration and temperature, and that these environmental factors also regulate the expression of most of the genes studied. Gene expression measurement of shell matrix protein thereby appears to be an appropriate indicator for the evaluation of the biomineralization activity in the pearl oyster P. margaritifera under varying environmental conditions. This study provides valuable information on the molecular mechanisms of mollusk shell growth and its environmental control

Relative expression of the nine selected marker genes of the female pathway of <i>P</i>. <i>margaritifera</i>.

<p>Relative expression profiles (real-time PCR) of <i>pmarg-foxl2</i> (A), <i>pmarg-c43476</i> (B), <i>pmarg-c45042</i> (C), <i>pmarg-c19309</i> (D), <i>pmarg-c54338</i> (E), <i>pmarg-vit-6</i> (F), <i>pmarg</i>-<i>zglp1</i> (G), <i>pmarg-dmrt</i> (H), and <i>pmarg-fem1-like</i> (I) in gonad categories (new set of samples). Different letters indicate statistically significant differences determined by ANOVA. M or F: male or female in gametogenesis; (M/F)R: male/female at stage of regression; Und: Undetermined sex.</p

Hierarchical clustering using Spearman’s correlation on gonad samples.

<p>Samples are divided into two main clusters based on their gene expression ratio pattern (36 variables), discriminating male and female pathways. Each cluster is divided into two sub-clusters discriminating oysters in early male or early female pathway (eMP or eFP) and oysters in the course of male or female pathway (MP or FP). M or F: male or female in gametogenesis; (M/F)R: male/female at regression stage; Und: gonad sex undetermined.</p

The histo-molecular model of the reproduction of <i>P</i>. <i>margaritifera</i>.

<p>M or F: male or female in gametogenesis; (M/F)E: male/female at early stage; (M/F)I: male/female at intermediate stage; (M/F)M: male/female at mature stage; (M/F)R: male/female at regression stage; Und(M/F): gonad undifferentiated at the cellular level, beginning a male or a female differentiation at the molecular level; Inv(M/F): oyster in sexual inversion female to male or male to female; MRF: male in regression stage at the cellular level beginning a female differentiation at the molecular level; and FRM: female in regression stage at cellular level beginning a male differentiation at the molecular level.</p

Histological features of gonads in sexual inversion in <i>P</i>. <i>margaritifera</i>.

<p>Two types of sexual inversion are distinguished: sexual inversion from male to female (InvF, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122819#pone.0122819.t001" target="_blank">Table 1</a>) (A), and sexual inversion from female to male (InvM, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122819#pone.0122819.t002" target="_blank">Table 2</a>) (B). Og: ovogonia; O: oocytes; Dg: degenerating oocytes; Spg: spermatogonia; T: connective tissue; Spc: spermatocytes; Spz: spermatozoids.</p

Results of the predictive MRT model on RNAseq sample (data from [29]).

<p>Results of the predictive MRT model on RNAseq sample (data from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122819#pone.0122819.ref029" target="_blank">29</a>]).</p