Identification and functional characterization of the sex-determining gene doublesex in the sawfly, Athalia rosae\xa0(Hymenoptera: Tenthredinidae)

Shotaro Mine,Megumi Sumitani,Masataka G Suzuki,Fugaku Aoki,Masatsugu Hatakeyama

doi:10.1007/s13355-017-0502-3

Shotaro Mine, Megumi Sumitani + Show 3 more

Open Access

https://doi.org/10.1007/s13355-017-0502-3

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Abstract

Sexual fate of the sawfly, Athalia rosae (Hymenoptera: Tenthredinidae) is determined by the complementary sex determination (CSD) mechanism as is the case in honeybees. However, to date, genes involved in sex determination have not been identified in this species. In this study, we attempted to identify orthologs of complementary sex-determiner (csd), feminizer (fem), and doublesex (dsx) from the A. rosae genome, all of which are crucial components of the sex determination cascade in the honeybee. As a result, we identified a sawfly ortholog of dsx (designated as Ardsx). Rapid amplification of cDNA ends (RACE) using total RNA extracted from male and female larvae identified three male-specific variants and three female-specific variants. Comparison between the full-length Ardsx cDNAs and the genomic sequence revealed that exon 5 was differentially spliced between the male- and female-specific variants. RT-PCR analysis demonstrated that Ardsx pre-mRNA was spliced alternatively in a sex-dependent manner at almost all the developmental stages. RNAi-mediated knockdown of Ardsx in males caused severe defects in the reproductive organs and, notably, induced development of the ovipository apparatus containing the dorsal pair of blades and the sheath. These males also showed abnormalities in testes and seminal vesicles and lacked mature sperm. The present study provides the first direct evidence that dsx is essential for sexual development in hymenopteran species.

Highlights

Sexual fate is determined by the complementary sex determination (CSD) mechanism, in which heterozygosity at a single locus determines femaleness in diploid individuals, while haploid individuals are hemizygous for the CSD locus and develop into males (Whiting 1933)
In order to analyze the function of Ardsx, we investigated the effects of Ardsx knockdown on sexual developments
The genitalia observed in the Ardsx KD males contained several imcomplete parts of the ovipository apparatus including the dorsal pair of blades and the sheath, both of which are derived from the second valvifers (Fig. 5e)

Summary

Introduction

Sexual fate is determined by the complementary sex determination (CSD) mechanism, in which heterozygosity at a single locus (the CSD locus) determines femaleness in diploid individuals, while haploid individuals are hemizygous for the CSD locus and develop into males (Whiting 1933). The CSD locus was first molecularly identified in the honeybee Apis mellifera and found to be a homolog of transformer (tra) (Beye et al 2003). The tra gene is known to be a conserved upstream component of the insect sex determination cascade and induces female development by regulating sexspecific alternative splicing of downstream targets such as doublesex (dsx) (Gempe and Beye 2011; Hoshijima et al 1991). A. mellifera has two copies of tra homologs (Hasselmann et al 2008). One copy is named complementary sex-determiner (csd) that is the primary signal for femaleness. It activates the other copy, named feminizer (fem), which is more conserved and retains the ancestral function of regulating sex-specific alternative splicing dsx. The csd was considered to have arisen from duplication of the fem gene (Schmieder et al 2012)

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