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Proc. Arthropod. Embryol. Soc. Jpn. 48, 55–56 (2017)Ⓒ 2017 Arthropodan Embryological Society of Japan
ISSN 1341–1527

Identification of Sex-specific Transcripts of the doublesex Gene in theSawfly, Athalia rosae ruficornis (Hymenoptera)*1,*2
Megumi SUMITANI1), Kazuki SEKINÉ2), Hideki SEZUTSU1)
and Masatsugu HATAKEYAMA2)
Genetically Modified Organism Research Center, National Institute of Agrobiological Sciences, Owashi, Tsukuba, Ibaraki 305–8634,
Japan2)Division of Insect Sciences, National Institute of Agrobiological Sciences, Owashi, Tsukuba, Ibaraki 305–8634, JapanE-mail: sumikasashima@affrc.go.jp (MS)1)

Homologs of the doublesex (dsx) gene of Drosophilamelanogaster are highly conserved elements that regulate
genetic sex determination in a wide range of organisms (Kopp,2012). The dsx pre-mRNA is alternatively spliced by transacting proteins, resulting in sex-specific variants. Distinctive
dsx isoforms in males and females are known to induce
somatic sexual differentiation in a variety of insects (Oliveiraet al., 2009). However, functional assays of dsx gene arerestricted to Diptera (Baker and Wolfner, 1988; Raymond etal., 1998) and Lepidoptera (Suzuki et al., 2005). Hymenoptera
is a unique order in which arrhenotokous parthenogenesis(development of haploid males from unfertilized eggs) is thegeneral mode of reproduction. The sawfly, Athalia rosae, is a
new model species in this order that has been used to conduct
studies in genetics and developmental biology with tools suchas germline transformation by the piggyBac transposonderived vector and gene knockdown by RNAi (Sumitani et al.,
2003; Yoshiyama et al., 2013). In addition, its genomic sequenceis publicly available (http://www.ncbi.nlm.nih.gov/nuccore/
AOFN00000000.1/). To investigate the regulatory mechanismsof sex determination in A. rosae, we identified its dsx homolog(Ardsx) using whole transcriptome shotgun sequencing(RNA-Seq) (Hatakeyama et al., 2017). In this report, we
describe the sex-specific transcripts of A. rosae.
The complete Ardsx coding sequence was confirmed
using reverse transcription-polymerase chain reaction (RTPCR) with cDNA constructed from adult females and males astemplates. The Ardsx gene corresponded to a region spanningapproximately 12 kb of the genome. The deduced amino acidsequence showed the typical Dsx structure, with a doublesex
and mab-3 (DM) DNA-binding domain (which is characteristicof the zinc-finger motif in the N-terminal region) and a Dsx
dimerization domain in the C-terminal region. In males, exons
1, 2, and 3 joined to form a transcript encoding 234 aminoacids. In females, specific splicing of 119 nucleotides withinexon 3 resulted in the female-specific variant. The resultant

female-specific transcript encoded 338 amino acids and had alonger C-terminal region than the male variant. In addition,
we identified the transformer 2 (tra2) homolog, which is
known to regulate sex-specific alternative splicing of dsx premRNA in D. melanogaster (Ryner and Baker 1991). In contrast,
in silkworm (Bombyx mori), the tra2 homolog is not required
for sex-specific splicing of B. mori dsx pre-mRNA (Suzuki etal., 2012). It would be interesting to determine whether tra2 isinvolved in dsx splicing in A. rosae.
References
Baker, B. S. and M. F. Wolfner (1988) A molecular analysis of doublesex, abifunctional gene that controls both male and female sexualdifferentiation in Drosophila malanogaster. Genes and Development,2, 477–489.
Hatakeyama, M., M. Sumitani and K. Sekiné (2017) Identification ofgene transcripts using whole transcriptome shotgun sequencing(RNA-Seq) in the sawfly Athalia rosae ruficornis (Hymenoptera).
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Kopp, A. (2012) Dmrt genes in the development and evolution ofsexual dimorphism. Trends in Genetics, 28, 175–184.
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*1 Abstract of paper read at the 49th Annual Meeting of the Arthropodan Embryological Society of Japan, June 7–8, 2013 (Tsukuba-san, Ibaraki).
*2 This article, which was accepted in 2013 and should have been published in 2014, was printed in 2017 being much delayed due to various circumstances.

56Suzuki, M. G., S. Funaguma, T. Kanda, T. Tamura and T. Shimada

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