Female-specific flightless phenotype for mosquito control

Abstract

Dengue and dengue hemorrhagic fever are increasing public health problems with an estimated 50-100 million new infections each year. Aedes aegypti is the major vector of dengue viruses in its range and control of this mosquito would reduce significantly human morbidity and mortality. Present mosquito control methods are not sufficiently effective and new approaches are needed urgently. A "sterile-male-release" strategy based on the release of mosquitoes carrying a conditional dominant lethal gene is an attractive new control methodology. Transgenic strains of Aedes aegypti were engineered to have a repressible female-specific flightless phenotype using either two separate transgenes or a single transgene, based on the use of a female-specific indirect flight muscle promoter from the Aedes aegypti Actin-4 gene. These strains eliminate the need for sterilization by irradiation, permit male-only release ("genetic sexing"), and enable the release of eggs instead of adults. Furthermore, these strains are expected to facilitate area-wide control or elimination of dengue if adopted as part of an integrated pest management strategy.

Fig. 1.

Schematic representation of Actin4 gene and the constructs used: (A) native Actin4 gene; (B) AeAct-4-DsRed; (C) OX3545; (D) OX3604; (E) marker construct OX3576 (tRE-DsRed2) and the effector constructs OX3547 (tRE-Nipp1Dm) and OX3582 (tRE-michelob_x); (F) alternative splicing of AeAct-4 in OX3604C; RT-PCR products from females (583 bp) and males (826 bp). (A–E) Origins of coding region (CDS) and 3^′ UTR sequences are indicated [K10 indicates the Drosophila gene fs(1)K10]. Short vertical lines above and below the thick horizontal line represent potential translation start (ATG) and stop codons, respectively. The first ATG of the female splice variant of AeAct-4 is that of the AeAct-4 coding region; the male splice variant has multiple 5^′-end ATGs. (D) OX3604 has an additional engineered 5^′-end ATG, indicated with an asterisk; this leads to the alternatively spliced intron being within the tTAV open reading frame (ORF), consequently this ORF is interrupted by stop codons in the male splice variant. (E) tTA response element (tRE) comprises multiple copies of the tTA binding sequence, tetO, and a minimal promoter (hsp^min), here from Drosophila hsp70.

Fig. 2.

Phenotype of OX3604C crossed with OX3576 (tRE-DsRed2) and reared in the absence of tetracycline. (A) Functional elements of OX3604. 3^′ UTR sequences and piggyBac ends have been omitted for clarity. Protein coding regions are as follows, from left to right: (i) tTAV under the control of a promoter fragment from AeAct4. OX3604C has an engineered translation initiation codon (ATG) 5^′ to the alternatively spliced intron of AeAct4 (AeAct4; Fig. 1), so this intron is within the tTA open reading frame; (ii) VP16 under control of tRE, and hence responsive to tTAV protein; and (iii) fluorescent marker (DsRed2) under control of the Hr5-IE1 promoter. (B) late fourth-instar female larvae. White (C) and fluorescent (D) light micrographs of male and female pupae from OX3604C alone (Top) and OX3604C + tRE-DsRed (Bottom). Male and female adults (OX3604C + tRE-DsRed) under white (E) and fluorescent light (F). Strong red fluorescence is visible in the IFMs of females, but not of males. Some background red fluorescence is visible from the transformation markers, especially the punctate overall fluorescence in B, the developing IFMs are the bright pair of spots in each thorax (for one larva these are indicated by white arrows).