Genomic organization and splicing evolution of the doublesex gene, a Drosophila regulator of sexual differentiation, in the dengue and yellow fever mosquito Aedes aegypti

Abstract

BackgroundIn the model system Drosophila melanogaster, doublesex (dsx) is the double-switch gene at the bottom of the somatic sex determination cascade that determines the differentiation of sexually dimorphic traits. Homologues of dsx are functionally conserved in various dipteran species, including the malaria vector Anopheles gambiae. They show a striking conservation of sex-specific regulation, based on alternative splicing, and of the encoded sex-specific proteins, which are transcriptional regulators of downstream terminal genes that influence sexual differentiation of cells, tissues and organs.ResultsIn this work, we report on the molecular characterization of the dsx homologue in the dengue and yellow fever vector Aedes aegypti (Aeadsx). Aeadsx produces sex-specific transcripts by alternative splicing, which encode isoforms with a high degree of identity to Anopheles gambiae and Drosophila melanogaster homologues. Interestingly, Aeadsx produces an additional novel female-specific splicing variant. Genomic comparative analyses between the Aedes and Anopheles dsx genes revealed a partial conservation of the exon organization and extensive divergence in the intron lengths. An expression analysis showed that Aeadsx transcripts were present from early stages of development and that sex-specific regulation starts at least from late larval stages. The analysis of the female-specific untranslated region (UTR) led to the identification of putative regulatory cis-elements potentially involved in the sex-specific splicing regulation. The Aedes dsx sex-specific splicing regulation seems to be more complex with the respect of other dipteran species, suggesting slightly novel evolutionary trajectories for its regulation and hence for the recruitment of upstream splicing regulators.ConclusionsThis study led to uncover the molecular evolution of Aedes aegypti dsx splicing regulation with the respect of the more closely related Culicidae Anopheles gambiae orthologue. In Aedes aegypti, the dsx gene is sex-specifically regulated and encodes two female-specific and one male-specific isoforms, all sharing a doublesex/mab-3 (DM) domain-containing N-terminus and different C-termini. The sex-specific regulation is based on a combination of exon skipping, 5' alternative splice site choice and, most likely, alternative polyadenylation. Interestingly, when the Aeadsx gene is compared to the Anopheles dsx ortholog, there are differences in the in silico predicted default and regulated sex-specific splicing events, which suggests that the upstream regulators either are different or act in a slightly different manner. Furthermore, this study is a premise for the future development of transgenic sexing strains in mosquitoes useful for sterile insect technique (SIT) programs.

Sex Determination in Insects: Variations on a Common Theme

“…It is thus likely that tra is a part of an ancestral module, which relays the instructive signals to downstream effectors. The gene doublesex (dsx) has been identified as a prime target of tra in all insect systems studied so far [Burtis and Baker, 1989;Shearman and Frommer, 1998;Kuhn et al, 2000;Ohbayashi et al, 2001;Suzuki et al, 2001;Hediger et al, 2004;Scali et al, 2005;Ruiz et al, 2007a;Oliveira et al, 2009;Concha et al, 2010;Shukla and Nagaraju, 2010;Salvemini et al, 2011;Shukla and Palli, 2012a]. In addition, there is growing evidence which suggests that the pathway below tra bifurcates employing a different target, the fruitless gene (fru) , for sex-specific differentiation of CNS-related aspects such as male courtship behavior [Bertossa et al, 2009;Salvemini et al, 2010;Clynen et al, 2011;Meier et al, 2013].…”

An Unusual Role fordoublesexin Sex Determination in the DipteranSciara

“…Strictly speaking, it is not appropriate to talk in terms of default dsx pre-mRNA splicing in Sciara because both male and female dsx mRNAs are simultaneously produced in the two sexes. Sciara could parallel the situation hypothesized in Aedes (Salvemini et al 2011); that is, putative SR-M and SR-F splicing factors, which would be functional in males and females, respectively, are present in both sexes, but in Sciara these factors would be simultaneously functional in both sexes. 3.…”

“…No putative NvdsxRE sites for TRA-TRA2 binding were found. These sequences, which have been found in the hymenopteran Nasonia dsx (Verhulst et al 2010), have been also observed in the dsx gene of Aedes (Salvemini et al 2011) and Tribolium (Shukla and Palli 2012). Finally, no BmPSI-exonic splicing suppressor (BmPSI-ESS) sequences were detected in Scdsx pre-mRNA.…”

“…No type A RBP1 sequences were found in Scdsx pre-mRNA and several type B RBP1 sequences were localized in the female-specific exons, one in exon E2 and the other in exon E3, plus numerous copies scattered along all introns ( Figure 4A). Salvemini et al (2011) reported the existence of TRA2-intronic splicing silencer (TRA2-ISS) sequences in Aedes dsx primary transcript in the region involved in its sex-specific splicing regulation. These ISS sequences (CAAGR) are bound by TRA2, causing suppression of splicing (Qi et al 2007).…”

“…Outside the drosophilids, the gene dsx has been characterized in the dipterans Megaselia scalaris (Sievert et al 1997;Kuhn et al 2000), Musca domestica (Hediger et al 2004), Lucilia cuprina (Concha et al 2010), Anopheles gambiae (Scali et al 2005), and Aedes aegypti (Salvemini et al 2011); in Bactrocera tryoni (Shearman and Frommer 1998), B. oleae (Lagos et al 2005), and Ceratitis capitata (Saccone et al 2008) and in 12 Anastrepha species (Ruiz et al , 2007; in the lepidopterans Bombyx mori Suzuki et al 2001), Antheraea assama, and A. mylitta (Shukla and Nagaraju 2010); in the hymenopterans Apis mellifera (Cho et al 2007) and Nasonia vitripennis (Oliveira et al 2009); in the coleopteran Tribolium castaneum (Shukla and Palli 2012); and in the crustacean Daphnia magna (Kato et al 2011) and other cladoceran species (Toyota et al 2013). The molecular organization of the dsx ORF varies among these organisms, yet in all cases excluding the cladoceran species dsx produces male-and female-specific mRNAs that encode putative male-and female-specific DSX proteins as in Drosophila.…”