Successful insertion and expression of a tetracycline transactivator in Anopheles stephensi associated with increased egg production and decreased hatching rate

After suboptimal results using the bZip early embryonic promoter, we produced three mosquito driver lines that expressed rtTA from three different genomic loci under the early embryonic vasa promoter. Expressing the rtTA under the vasa promoter significantly increased rtTA mRNA levels compared with under bZip. We were unable to achieve homozygosity in two of these lines even after 26 generations. In a third line we observed seven distinct insertions, six of which, including one located in an intron of a protein-coding gene, were homozygous. This line achieved homozygosity after being passed through seven generations, suggesting that the insertions did not disrupt the function of any crucial genomic locus. The levels of rtTA mRNA expression in the homozygous-viable line were higher than those in the other two lines, reinforcing the idea that the inability to reach homozygosity was not due to rtTA expression levels but rather the position of the insertion. The homozygous-viable line produced ~18% more eggs per female, and a hatching rate of larvae from eggs was 39% lower than that of wild-type An. stephensi. The next step will be to cross the driver line with an effector line containing a male-linked lethal gene regulated by the tetracycline responsive element (TRE).