Investigating the Molecular Genetic Basis of Cytoplasmic Sex Determination Caused by Wolbachia Endosymbionts in Terrestrial Isopods

Abstract

In animals, sexual differences between males and females are usually determined by sex chromosomes. Alternatively, sex may also be determined by vertically transmitted intracellular microbial endosymbionts. The best known cytoplasmic sex manipulative endosymbiont is Wolbachia which can, for instance, feminize genetic males into phenotypic females in the terrestrial isopod Armadillidium vulgare. However, the molecular genetic basis of cytoplasmic sex determination is unknown. To identify candidate genes of feminization induced by Wolbachia strain wVulC from A. vulgare, we sequenced the genome of Wolbachia strain wCon from Cylisticus convexus, the most closely related known Wolbachia strain to wVulC that does not induce feminization, and compared it to the wVulC genome. Then, we performed gene expression profiling of the 216 resulting wVulC candidate genes throughout host developmental stages in A. vulgare and the heterologous host C. convexus. We identified a set of 35 feminization candidate genes showing differential expression during host sexual development. Interestingly, 27 of the 35 genes are present in the f element, which is a piece of a feminizing Wolbachia genome horizontally transferred into the nuclear genome of A. vulgare and involved in female sex determination. Assuming that the molecular genetic basis of feminization by Wolbachia and the f element is the same, the 27 genes are candidates for acting as master sex determination genes in A. vulgare females carrying the f element.

Keywords: Wolbachia; cytoplasmic sex factor; feminization; gene expression profile; genome sequencing; sexual development; terrestrial isopods.

Figure 1

Ratios of wsp gene copy number relative to androgenic hormone (AH) copy number (A) and cytochrome oxidase I (COI) copy number (B), in ovaries (OV), nervous cord (CN) and hemolymph (HL). Proportions of base pairs based of wCon (red), mitochondrial (green) and nuclear genomes (blue) estimated by quantitative PCR (qPCR) without (C) and with (D) enrichment, and by mapping of pyrosequencing reads on assembled genomes (E). WSP: please define.

Figure 2

Candidate gene log2 expression of 35 candidate genes during both homologous and heterologous host development (A. vulgare in red and C. convexus in blue, respectively). All candidate genes are consistently over- or under-expressed during native host development (in A. vulgare). All genes present in the f element are labeled with a red “f”. The two genes showing a significant cross-correlation with exactly one-stage shift between the two hosts are boxed.