Isolation and characterization of Doublesex homologues in the Bactrocera species: B. dorsalis (Hendel) and B. correcta (Bezzi) and their putative promoter regulatory regions

Abstract

Doublesex (dsx) is a double-switch gene at the bottom of the somatic sex-determination hierarchy which regulates sexual dimorphism in many insects. Here, Drosophila melanogaster homologues of dsx were isolated in two Bactrocera species, the oriental fruit fly, B. dorsalis, and the guava fruit fly, B. correcta. Results of RT-PCR analysis suggests that both the B. dorsalis dsx (Bd1dsx) and B. correcta dsx (Bcdsx) genes are transcribed and sex-specifically spliced in accordance with the Drosophila sex-specific splicing mechanism. The cDNA sequences shared a high degree of similarity at the nucleotide level among the Bactrocera species. Structurally conserved domains for DNA-binding and oligomerization were observed in all transcripts suggesting that their proteins function as transcriptional factors for downstream sex-specific gene expression. A purine-rich element (PRE) and four repeat elements (dsxRE) for TRA/TRA-2 binding sites were also found in the 3' untranslated regions (UTR) of both the female Bd1dsx and Bcdsx mRNAs. Notably, a putative core promoter was revealed in Bd1dsx, being probably the first dsx promoter discovered in the tephritid flies.

The Am-tra2 Gene Is an Essential Regulator of Female Splice Regulation at Two Levels of the Sex Determination Hierarchy of the Honeybee

“…Similar motifs have also been detected in the dsx gene sequences of other dipteran insects, but are lacking in the Am-dsx pre-mRNAs of the honeybee (Crampton et al 1998; Hediger et al 2004; Lagos et al 2005; Ruiz et al 2005, 2007; Cho et al 2007; Saccone et al 2008; Concha et al 2010; Permpoon et al 2011). We next studied the sequence similarities within the RBDs of Tra2 proteins of different holometabolous insects (Figure 2).…”

“…In D. melanogaster , Tra2, together with the Tra proteins, binds to six repeats of a 13-nucleotide motif [TC(T/A)(A/T)C(A/G)ATCAACA] on the dsx pre-mRNA and promotes the activation of the weak female splice acceptor that directs the production of the female dsx transcripts. In other dipteran species ( M. domestica , C. capitata , Bactrocera oleae , Bactrocera dorsalis , Bactrocera correcta , Bactrocera tyroni , and different Anastrepha species), the canonical Tra/Tra2-binding motifs are consistently present in the dsx genes (Crampton et al 1998; Hediger et al 2004; Lagos et al 2005; Ruiz et al 2005, 2007; Saccone et al 2008; Concha et al 2010; Permpoon et al 2011) and are proposed to be utilized in promoting female splicing (Burghardt et al 2005; Salvemini et al 2009; Sarno et al 2010). We propose that the Am -Tra2 protein, like its ortholog in D. melanogaster , is an essential, constitutively expressed cofactor that, together with the female-specific Fem protein, directs the female processing of the Am-dsx transcript.…”

Identification and characterization of doublesex from the pumpkin fruit fly, Bactrocera tau (Diptera: Tephritidae)

“…dsx genes have been identifi ed and characterized in many tephritid insects such as Bactrocera tryoni (Shearman & Frommer, 1998), Anastrepha oblique (Ruiz et al, 2005), B. oleae (Lagos et al, 2005), Ceratitis capitata (Saccone et al, 2008), B. dorsalis (Chen et al, 2008;Permpoon et al, 2011), B. jarvisi (Morrow et al, 2014), andB. correcta (Permpoon et al, 2011).…”

“…In contrast to most dipteran dsx genes, Btau-dsx was transcribed into three (one female and two male) instead of two (one male and one female) transcript variants (An et al, 1996;Shearman & Frommer, 1998;Hediger et al, 2004;Lagos et al, 2005;Chen et al, 2008;Saccone et al, 2008;Permpoon et al, 2011;Morrow et al, 2014). The splicing of the dsx pre-mRNAs to produce more than two transcript variants had been reported in many insect spe-cies, however, almost all additional dsx transcripts are female-specifi c transcripts, such as two in Aedes aegypti (Salvemini et al, 2011), Apis mellifera (Cho et al, 2007), Bombyx mori (Ohbayashi et al, 2001), Antheraea mylitta (Shukla & Nagaraju, 2010), three in Tribolium castaneum (Shukla & Palli, 2012), and six in A. assama (Shukla & Nagaraju, 2010).…”

“…This suggests that the Btau-dsx female-specifi c splicing may also be regulated by the combined protein products of the two upstream genes, Btau-tra and Btau-tra2, which bind to the dsxREs and the PRE. Moreover, the copy number of the dsxRE domain seems to vary to as much as eight in Lucilia capurina (Concha et al, 2010); six in D. melanogaster (Inoue et al, 1992), Megaselia scalaris (Kuhn et al, 2000) and Anopheles gambiae (Scali et al, 2005); fi ve in Aedes aegypti (Salvemini et al, 2011); four in M. domestica (Hediger et al, 2004), C. ceratitis (Saccone et al, 2008) and Bactrocera species (Shearman & Frommer, 1998;Chen et al, 2008;Permpoon et al, 2011), and three in Anastrepha obligua (Ruiz et al, 2005). However, all Bactrocera dsx female-specifi c transcripts including Btau-dsx F contain four dsxRE copies and one PRE domain.…”