Male only progeny in Anastrepha suspensa by RNAi-induced sex reversion of chromosomal females Marc F. Schetelig a , Andreina Milano b , Giuseppe Saccone b , Alfred M. Handler a, * a USDA/ARS, Center for Medical, Agricultural and Veterinary Entomology,1700 SW 23rd Drive, Gainesville, FL 32608, USA b Dipartimento delle Scienze Biologiche, Sezione Genetica e Biologia Molecolare, Università degli Studi di Napoli Federico II, Via Mezzocannone 8, 80134 Naples, Italy article info Article history: Received 26 August 2011 Received in revised form 11 October 2011 Accepted 17 October 2011 Keywords: Sex-determination Transformer Transformer-2 Tephritidae Sterile insect technique Genetic-sexing abstract In Tephritidae sex determination is established by orthologs to the Drosophila melanogaster transformer and transformer-2 genes, though the primary signals for sex determination differ. The presence of the Y chromosome in the tephritid species is critical for male differentiation, while the ratio of X chromosomes to autosome ploidy is critical in drosophilids. Here the isolation, expression and function of tra and tra-2 orthologs are described for the agriculturally important tephritid, Anastrepha suspensa, and their possible use in genetically modified organisms for biologically-based pest management. The Astra and Astra-2 genes are highly conserved in structure, regulation and function with respect to those known from other tephritid species. Sex-specific transcripts for Astra were detected, one in females and three in males, whereas Astra-2 had a single common transcript found in both sexes. To test the function of these genes, Astra and Astra-2 dsRNA was injected into A. suspensa embryos from a transgenic strain having a Y-linked DsRed marker integration, allowing XY males to be distinguished from XX phenotypic males. Nearly all XX embryos developed into fully masculinized phenotypic male adults with no apparent female morphology. Upon dissection abnormal hypertrophic gonads were revealed in XX pseudomales but not in the XY males. Our findings suggest that Astra and Astra-2 are both necessary for female development, and that the potential exists for producing a male-only population when either gene alone, or both genes simultaneously, are knocked-down. Published by Elsevier Ltd. 1. Introduction In the genetic model system, Drosophila melanogaster , the Sex- lethal (Sxl) gene heads the sex-determination gene hierarchy that specifies phenotypic sex by directing the female-specific splicing of the transformer (tra) transcript, which in turn, together with transformer-2 (tra-2), directs female-specific splicing of the dou- blesex (dsx) and fruitless (fru) transcripts that are required for somatic sexual differentiation and proper sexual behavior (Salz, 2011). In the absence of tra or tra-2, the default male-specific splicing of dsx and fru transcripts results (Baker and Belote, 1983; Baker et al., 1989; Hedley and Maniatis, 1991; Wolfner, 1988). In the tephritid fruit fly, Ceratitis capitata (medfly), the orthologs of D. melanogaster tra and tra-2 are functionally conserved as sex- determining upstream regulators of the dsx and fru orthologs (Pane et al., 2002; Salvemini et al., 2009). In addition it has been found that Cctra and Cctra-2 play a novel epigenetic function in the medfly, being both required to maintain the female sexual fate established during embryogenesis (Saccone et al., 2011). Analogous epigenetic functions in Drosophila are controlled by the master ON/ OFF gene Sxl, and by its auxiliary splicing factor, fl(2)d (Granadino et al., 1990). Both D. melanogaster Sxl and C. capitata tra are ON/ OFF master regulators of sex determination that positively regulate maintenance of the female sex determination state (Graham et al., 2003). In D. melanogaster , Sxl positively regulates tra, while tra autoregulation occurs together with tra-2 in C. capitata. More recently, tra orthologs have been isolated in other tephritid species (Bactrocera oleae and Anastrepha obliqua), in a Calliphoridae (Lucilia cuprina), and in a Muscidae (Musca domestica), with all exhibiting stronger sequence conservation to C. capitata tra rather than the D. melanogaster ortholog (Concha and Scott, 2009; Hediger et al., 2010; Lagos et al., 2007; Ruiz et al., 2007). The tra orthologs isolated thus far also show higher structural and splicing regulatory similarities to those from C. capitata including conservation of positive female-specific autoregulation (Saccone et al., 2011). Transient embryonic RNAi against tra has been used in func- tional studies in C. capitata, B. oleae, and L. cuprina, (Concha and Scott, 2009; Lagos et al., 2007; Pane et al., 2002), but not in Anas- trepha species until now. This has resulted in a consistent * Corresponding author. E-mail address: ahandler@ufl.edu (A.M. Handler). Contents lists available at SciVerse ScienceDirect Insect Biochemistry and Molecular Biology journal homepage: www.elsevier.com/locate/ibmb 0965-1748/$ e see front matter Published by Elsevier Ltd. doi:10.1016/j.ibmb.2011.10.007 Insect Biochemistry and Molecular Biology 42 (2012) 51e57