Schematic representations of the transformation constructs OX3545, AsOX3545 and OX3547, and the mechanism of action of the double transgene-based female-specific RIDL. OX3545 and OX3547 were previously described in detail . AsOX3545 is a similar to OX3545, with the Anopheles stephensi Actin-4 regulatory sequences replacing the Aedes aegypti Actin-4 sequence. Actin 4 promoters (Act4P) drive the expression of the tetracycline-repressible transactivator protein (tTa) in the flight muscles of adult female mosquitoes. tTA binds tetO in the absence of tetracycline, driving expression of the effector molecule, Nuclear Inhibitor of PP1 (Nipp1Dm) [22, 23], leading to disruption of normal cell functions. Tetracycline prevents tTa binding to tetO abolishing expression of Nipp1Dm. The positions of the Spe I recognition and cleavage sites in OX3545, AsOX3545 and OX3547 are indicated by vertically oriented arrows. Horizontally oriented arrows indicate the direction of transcription for each component of the constructs. Abbreviations: pBac R and L, piggyBac right and left inverted repeats, respectively; attB, φC31 recombination site; DsRed2, red fluorescent protein open reading frame; IE1, baculovirus immediate-early gene promoter driving DsRed2 expressed throughout the body; 3×P3, artificial promoter consisting of a multimer of the binding site (P3), driving high levels of expression in the eye.