Genomic analysis of the Pacific oyster (Crassostrea gigas) reveals possible conservation of vertebrate sex determination in a mollusc

Abstract

Despite the prevalence of sex in animal kingdom, we have only limited understanding of how sex is determined and evolved in many taxa. The mollusc Pacific oyster Crassostrea gigas exhibits complex modes of sexual reproduction that consists of protandric dioecy, sex change, and occasional hermaphroditism. This complex system is controlled by both environmental and genetic factors through unknown molecular mechanisms. In this study, we investigated genes related to sex-determining pathways in C. gigas through transcriptome sequencing and analysis of female and male gonads. Our analysis identified or confirmed novel homologs in the oyster of key sex-determining genes (SoxH or Sry-like and FoxL2) that were thought to be vertebrate-specific. Their expression profile in C. gigas is consistent with conserved roles in sex determination, under a proposed model where a novel testis-determining CgSoxH may serve as a primary regulator, directly or indirectly interacting with a testis-promoting CgDsx and an ovary-promoting CgFoxL2. Our findings plus previous results suggest that key vertebrate sex-determining genes such as Sry and FoxL2 may not be inventions of vertebrates. The presence of such genes in a mollusc with expression profiles consistent with expected roles in sex determination suggest that sex determination may be deeply conserved in animals, despite rapid evolution of the regulatory pathways that in C. gigas may involve both genetic and environmental factors.

Keywords: FoxL2; Mollusca; Sry; doublesex; oyster; sex determination.

Figure 1

A DM domain gene in C. gigas (CgDsx) and its male-specific expression profile. (A) Alignment of CgDsx and other DM domain containing proteins involved in sex determination from model species. (B) Domain structure of CgDsx and selected DM domain genes from model species (purple designates low complexity regions). (C) Expression profile of CgDsx in different adult organs with standard deviation as error bars (n = 3). Species names are abbreviated as Cg for Crassostrea gigas, Dm for Drosophila melanogaster, Ce for Caenorhabditis elegans, Dr for Danio rerio, Mm for Mus musculus, and Hs for Homo sapiens. Accession numbers: CgDsx KJ489413; DmDsx NP_731197.1, CeMAB-3-a and CeMAB-3-b CAB16489.1, DrDmrt1 AAQ04555.1, MmDmrt1 AAF12826.1, and HsDmrt1 AAD40474.1.

Figure 2

SoxE and SoxH genes identified in C. gigas and their expression profile. (A) Alignment of HMG domains of CgSoxE, CgSoxH, and homologs from selected vertebrates. (B) Phylogenetic tree of protein sequences of CgSoxE, CgSoxH, and selected genes. (C) Expression profile of CgSoxE and CgSoxH in adult organs of C. gigas with standard deviation as error bars (n = 3). Species names are abbreviated as Cg for Crassostrea gigas, Dm for Drosophila melanogaster, Dr for Danio rerio, Mm for Mus musculus, and Hs for Homo sapiens. Numbers in the tree represent bootstrap values. Accession numbers: CgSoxE EKC31659.1, CgSoxH EKC38002.1, DmSox100B AAF57112.2, MmSry AAI11529.1, HsSry AFG33955.1, HsSox8 AAH31797.1, HsSox9 CAA86598.1, HsSox10 CAG30470.1, and HsSox30 BAA37146.1.

Figure 3

Analysis of selected Fox genes identified in C. gigas. (A) Phylogenetic tree of forkhead domains from selected Fox genes from C. gigas and reference species. (B) Expression profile of CgFoxL2 showing high expression in female gonad. (C) Expression of CgFoxL2, CgSoxH, and CgDsx in three females and three males showing possible interaction among the three genes in M2. (D) Male-specific expression of a novel Fox gene Cg23645. Error bars represent standard deviation (n = 3). Species names are abbreviated as Cg for Crassostrea gigas, Hs for Homo sapiens and Sp for Strongylocentrotus purpuratus. Numbers in the tree are bootstrap values. Accession numbers: CgFoxL2 ACN80999.1, Cg06159 EKC20378.1, Cg24546 EKC30312.1, Cg23645 EKC35023.1, SpFoxL1 ABB89488.1, SpFoxL2 ABB89483.1, SpFoxM, ABB89490.1, SpFoxN2/3 ABB89482.1, SpFoxQ1 ABB89489.1, SpFoxQ2 ABB89473.1, SpFoxY AF517552, HsFoxL1 AAG40312.1, HsFoxL2 AAY21823.1, HsFoxM1 NP_973731.1, HsFoxN1 NP_003584.2, HsFoxN2 AAH63305.1, HsFoxN3 AAH07506.1, HsFoxN4 AAI46826.1, HsFoxN5 AAH38969.2, HsFoxN6 AAH12934.1, and HsFoxQ1 AAH53850.1.

Figure 4

Hypothesized sex-determining pathway in C. gigas compared with that in model organisms as summarized by Gamble and Zarkower (2012). For clarity, only selected key sex-specific regulators are shown. Dashed black lines indicate temporal relationships, and dashed red lines indicate hypothetical relationships based on expression data only. [Modified based on Gamble and Zarkower (2012)]. FF genotype permits sex change (Guo et al. 1998).