The OX3097 transposon and induced phenotypes in olive fly. (A) Diagrammatic representation of the OX3097 transposon. OX3097 comprises a fluorescent marker (hr5-IE1-DsRed2), and the female-specific tTAV expression system (tetO-Dmhsp70 minimal promoter - Cctra:tTAV) . Sex-specific alternative splicing of the Cctra intron leads to production of tTAV and the initiation of a lethal tTAV positive-feedback loop in females only [14, 15, 34]. (b) Products of alternative splicing of Cctra:tTAV in (lane 1) male and (lane 2) female OX3097D-Bol olive flies. Three splice variants were detected, corresponding to Cctra transcripts M1, M2 and F1  (identity confirmed by sequencing). Only females produce the F1 splice variant, corresponding to the reconstitution of the tTAV open reading frame and leading to production of functional tTAV. Lane M shows DNA size standards: 200-1,000 bp in 200-bp increments (Eurogentec Smartladder). (C) Penetrance and (D) tetracycline repressibility of female lethality in five OX3097 olive fly lines. Strains OX3097A-D-Bol &F-Bol are five insertion lines of OX3097 in olive fly. Penetrance and repressibility of female-specific lethality was assessed by crossing heterozygous males of each strain to virgin wild-type (WT) females, and collecting eggs on filter paper saturated with water containing either 0 μg/ml tetracycline or 100 μg/ml tetracycline. The sex ratio of adult progeny expressing the DsRed2 fluorescent marker is shown for each strain compared with wild-type (WT) progeny. Lines A, C and D showed fully penetrant female-specific lethality when reared in the absence of tetracycline (off-tet); that is, they produced no female progeny off-tet in this assay. In lines C and D, female-specific lethality was also efficiently repressed on-tet. (E) Fluorescence microscopy allows discrimination of OX3097D-Bol from wild type at larval, pupal, and adult stages. Photomicrographs of OX3097D-Bol and wild-type olive flies under (upper panels) fluorescence and (lower panels) bright-field illumination. Each panel shows OX3097D-Bol to the left and wild-type to the right: OX3097D-Bol and wild-type (1,2) larvae, (3,4) pupae, and (5,6) adults are shown. Expression of DsRed2 is clearly visible all over the OX3097D-Bol larvae and pupae, and in areas of less opaque cuticle (for example, the labellum, upper thorax, leg joints, and anus) of OX3097D-Bol adults.