A conserved role of the duplicated Masculinizer gene in sex determination of the Mediterranean flour moth, Ephestia kuehniella.

Sander Visser,Petr Nguyen,František Marec,Eveline C. Verhulst,Anna Voleníková,Claude Desplan

doi:10.1371/journal.pgen.1009420

Abstract

Sex determination in the silkworm, Bombyx mori, is based on Feminizer (Fem), a W-linked Fem piRNA that triggers female development in WZ individuals, and the Z-linked Masculinizer (Masc), which initiates male development and dosage compensation in ZZ individuals. While Fem piRNA is missing in a close relative of B. mori, Masc determines sex in several representatives of distant lepidopteran lineages. We studied the molecular mechanisms of sex determination in the Mediterranean flour moth, Ephestia kuehniella (Pyralidae). We identified an E. kuehniella Masc ortholog, EkMasc, and its paralog resulting from a recent duplication, EkMascB. Both genes are located on the Z chromosome and encode a similar Masc protein that contains two conserved domains but has lost the conserved double zinc finger domain. We developed PCR-based genetic sexing and demonstrated a peak in the expression of EkMasc and EkMascB genes only in early male embryos. Simultaneous knock-down experiments of both EkMasc and EkMascB using RNAi during early embryogenesis led to a shift from male- to female-specific splicing of the E. kuehniella doublesex gene (Ekdsx), their downstream effector, in ZZ embryos and resulted in a strong female-biased sex-ratio. Our results thus confirmed the conserved role of EkMasc and/or EkMascB in masculinization. We suggest that the C-terminal proline-rich domain, we have identified in all functionally confirmed Masc proteins, in conjunction with the masculinizing domain, is important for transcriptional regulation of sex determination in Lepidoptera. The function of the Masc double zinc finger domain is still unknown, but appears to have been lost in E. kuehniella.

Highlights

Sex determination in insects is under the control of a cascade of genes, each affecting the expression or splicing of the gene in the pathway [1]
We suggest that the C-terminal proline-rich domain, we have identified in all functionally confirmed Masc proteins, in conjunction with the masculinizing domain, is important for transcriptional regulation of sex determination in Lepidoptera
We provide two lines of evidence that the EkMasc and/or EkMascB genes play an essential role in masculinization: (i) they show a peak of expression during early embryogenesis in ZZ but not in WZ embryos and (ii) their simultaneous silencing by RNAi results in female-specific splicing of the E. kuehniella doublesex gene (Ekdsx) in ZZ embryos and in a female-biased sex ratio

Summary

Introduction

Sex determination in insects is under the control of a cascade of genes, each affecting the expression or splicing of the gene in the pathway [1]. This cascade evolved from the bottom up with the most conserved gene, doublesex (dsx), at the bottom of the cascade [2] present in all insects studied to date [3,4,5]. The gene initiating sexual differentiation in the honeybee Apis mellifera, complementary sex determiner (csd), originated via a duplication of the feminizer (fem) gene, an ortholog of tra [9,10]. In the housefly Musca domestica (Diptera), the masculinizing gene male determiner (Mdmd) originated through a gene duplication and is located in a locus containing multiple pseudocopies of the gene [13]

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Conclusion