De Novo Assembly of the Manila Clam Ruditapes philippinarum Transcriptome Provides New Insights into Expression Bias, Mitochondrial Doubly Uniparental Inheritance and Sex Determination

Abstract

Males and females share the same genome, thus, phenotypic divergence requires differential gene expression and sex-specific regulation. Accordingly, the analysis of expression patterns is pivotal to the understanding of sex determination mechanisms. Many bivalves are stable gonochoric species, but the mechanism of gonad sexualization and the genes involved are still unknown. Moreover, during the period of sexual rest, a gonad is not present and sex cannot be determined. A mechanism associated with germ line differentiation in some bivalves, including the Manila clam Ruditapes philippinarum, is the doubly uniparental inheritance (DUI) of mitochondria, a variation of strict maternal inheritance. Two mitochondrial lineages are present, one transmitted through eggs and the other through sperm, as well as a mother-dependent sex bias of the progeny. We produced a de novo annotation of 17,186 transcripts from R. philippinarum and compared the transcriptomes of males and females and identified 1,575 genes with strong sex-specific expression and 166 sex-specific single nucleotide polymorphisms, obtaining preliminary information about genes that could be involved in sex determination. Then we compared the transcriptomes between a family producing predominantly females and a family producing predominantly males to identify candidate genes involved in regulation of sex-specific aspects of DUI system, finding a relationship between sex bias and differential expression of several ubiquitination genes. In mammalian embryos, sperm mitochondria are degraded by ubiquitination. A modification of this mechanism is hypothesized to be responsible for the retention of sperm mitochondria in male embryos of DUI species. Ubiquitination can additionally regulate gene expression, playing a role in sex determination of several animals. These data enable us to develop a model that incorporates both the DUI literature and our new findings.

Proteomic Analysis of Eggs from Mytilus edulis Females Differing in Mitochondrial DNA Transmission Mode

“…It is also in agreement with a substantial transcriptomics study on the clam Ruditapes phippinarum in which 17,186 transcripts were analyzed using body tissue from clams (46). Two families were compared, one with a sex ratio biased toward males and the other with a female bias.…”

“…Ubiquitinization of sperm mitochondria may play an important role in the tagging of sperm mitochondria for destruction in mammals that exhibit standard maternal mtDNA inheritance (43,44) resulting in degradation in the proteasome pathway (45). The possibility that this ubiquitin-proteasome system is involved in DUI has been previously referred to in studies considering a model for DUI (12,15,17,34,46). An earlier proteomics study on Mytilus (47) and a recent transcriptomics study of the clam Ruditapes phippinarum, which also has DUI (46), have identified proteasome proteins as showing expression differences between a male sex ratio biased family and female sex ratio biased family.…”

“…The possibility that this ubiquitin-proteasome system is involved in DUI has been previously referred to in studies considering a model for DUI (12,15,17,34,46). An earlier proteomics study on Mytilus (47) and a recent transcriptomics study of the clam Ruditapes phippinarum, which also has DUI (46), have identified proteasome proteins as showing expression differences between a male sex ratio biased family and female sex ratio biased family. These studies taken together provide an a priori hypothesis, which we test here.…”

Sex Determining Mechanisms in Bivalves

“…By comparing transcriptomes of males and females in the Manila clam Ruditapes philippinarum, they identified 1,575 genes with strong sex-specific expression including the malebiased gene Sry (sex determining region-y)-box 30 (Sox30), a transcription factor involved in the differentiation of developing male germ cells in mammals (Wallis et al 2008;Ghiselli et al 2012). Because Sox30 was not only highly expressed in testis but also in females producing male-biased progenies, the authors suggested that this gene was expressed in eggs of male-biased mothers to induce the development of future embryos towards maleness (Ghiselli et al 2012).…”

Gene expression profile analysis of Manila clam (Ruditapes philippinarum) hemocytes after a Vibrio alginolyticus challenge using an immune-enriched oligo-microarray

“…Our two groups released 457,717 and 975,190 raw reads from adult/larval tissues and hemocytes, respectively [20,21]. Concurrently, studies by Ghiselli et al on gonad tissue yielded approximately 90 million raw sequences [22].…”