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

Abstract

BackgroundBlack 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.ResultsThe 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.ConclusionsThe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-491) contains supplementary material, which is available to authorized users.

Alternative Splicing Profile and Sex-Preferential Gene Expression in the Female and Male Pacific Abalone Haliotis discus hannai

“…In order to test sex-preferential expression from each transcriptome, quantitative real-time RT-PCR (qPCR) was performed using six sex-preferential genes: vitellogenin ( VTG ), forkhead box protein L2 ( FOXL2 ), condensin-2 , sperm-associated antigen 6 ( SPAG6 ), protein fem-1 homolog C-like ( FEM1-like ), and tektin-1 , which are relatively well-characterized in marine invertebrates [9,21,22,23,24]. Based on the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines [25], transcriptional levels of each sex-preferential gene were validated using qPCR.…”

Foxl2 and Its Relatives Are Evolutionary Conserved Players in Gonadal Sex Differentiation

“…2 ). In mollusks, analysis of foxl2/3 expression in the Pacific oyster Crassostrea gigas , Hong Kong oyster Crassostrea hongkongensis , and Black-lip pearl oyster Pinctada margaritifera revealed predominant expression of foxl2/3 in female gonads [Teaniniuraitemoana et al, 2014[Teaniniuraitemoana et al, , 2015Zhang et al, 2014;Tong et al, 2015]. In C. gigas , in situ hybridization specifically localized expression of foxl2/3 transcripts in the oogonia and earlydeveloped oocytes of the female ovary as well as in male germ cells from spermatogonia until spermatids of the testis [Naimi et al, 2009].…”

“…1 , table 1 ), including sponges (Suberites domuncula) [Adell and Müller, 2004], mollusks [Naimi et al, 2009;Teaniniuraitemoana et al, 2014;Zhang et al, 2014;Tong et al, 2015], arthropods including insects and crustaceans [De Loof et al, 2010;Ma et al, 2012;Farlora et al, 2014;Li et al, 2015], hemichordates [Fritzenwanker et al, . See text for details.…”

Sex Determining Mechanisms in Bivalves

“…Owing to their importance in aquaculture and fisheries, the interest in bivalve genomics and transcriptomics to identify the genes involved in reproduction, sex differentiation and sex determination processes has significantly increased in recent years (e. Teaniniuraitemoana et al 2014Teaniniuraitemoana et al , 2016. However, because this is beyond the scope and goals of the present review, studies relating genes involved in reproduction or in later steps during sexual differentiation are not discussed.…”

“…Among the genes that are known to act in sex determination in other animal species and/or during early gonadal differentiation, and thus potentially in sex determination in oysters, there are: Oyvlg (Crassostrea gigas; Fabioux et al 2004Fabioux et al , 2009), a homologue of vasa, which is a gene involved in primordial germ cell development and early sex differentiation in eukaryotes; Cg-DMI (C. gigas Dmrt-like; Naimi et al 2009), a homologue of Dmrt1 (other homologues of Dmrt1 have also been reported in the oysters Pinctada martensii and P. margaritifera; Yu et al 2011;Teaniniuraitemoana et al 2014); Cg-SoxE, a homologue of Sox9, and β-catenin, which are respectively expressed when sex is still not distinguishable, or in mature females and vitellogenic oocytes in C. gigas (Santerre et al 2014); and CgFoxL2, CgSoxH (or Sry-like) and CgDsx (C. gigas; Zhang et al 2014), the two latter genes being linked to the triggering of male development. Vasa, FoxL2 and β-catenin have also been reported in the species Crassostrea hongkongensis (Tsuda et al 2003;Tong et al 2015).…”