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

## TL;DR

Scientists inserted a gene into mosquitoes that could help produce more malaria vaccines by eliminating males, but this changed egg production and hatching rates.

## Contribution

First successful insertion of a tetracycline transactivator in Anopheles stephensi mosquitoes using the vasa promoter for conditional gene expression.

## Key findings

- A driver line expressing rtTA under the vasa promoter achieved higher mRNA levels and homozygosity compared to other lines.
- The homozygous-viable line produced 18% more eggs but had a 39% lower hatching rate compared to wild-type mosquitoes.
- Insertion positions, not rtTA expression levels, determined homozygosity success in the mosquito lines.

## Abstract

Sanaria® has pioneered production of aseptic, purified, vialed cryopreserved Plasmodium falciparum (Pf) sporozoites (SPZ) as vaccines and for controlled human malaria infections. More than 3500 individuals have received more than 9700 injections of PfSPZ worldwide. The PfSPZ are manufactured in aseptically reared female Anopheles stephensi mosquitoes. Since PfSPZ vaccines are intended primarily for some of the most disadvantaged people in the world, keeping costs low is imperative. One approach to reducing the cost of goods is to eliminate male mosquitoes from the production process, thereby doubling the numbers of PfSPZ-producing mosquitoes per unit space. We intend to do this by creating An. stephensi with a male-lethal allele controlled by the tetracycline conditional gene expression system.

Herein, we report the first step in this process, the creation of a driver line that expresses the reverse tetracycline transactivator (rtTA).

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).

The online version contains supplementary material available at 10.1186/s13071-025-07003-7.