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

## TL;DR

Researchers inserted a gene into mosquitoes to control reproduction, increasing egg production but reducing hatching rates, as a step toward cheaper malaria vaccine production.

## Contribution

Creation of a driver mosquito line with a tetracycline-controlled transactivator for potential male-lethal gene expression.

## Key findings

- Using the vasa promoter increased rtTA mRNA levels compared to the bZip promoter.
- One homozygous line showed 18% higher egg production and 39% lower hatching rates.
- Insertion position, not rtTA expression, likely caused failure to achieve homozygosity in other 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 3,500 individuals have received more than 9,700 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 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 A. 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 sub-optimal results using the bZip early embryonic promoter, we produced 3 mosquito driver lines that expressed rtTA from 3 different genomic loci under the early embryonic vasa promoter. Expressing the rtTA under the vasa promoter significantly increased rtTA mRNA levels compared to its level under bZip. We were unable to achieve homozygosity in two of these lines even after 26 generations. In the third line, the insertion was in an intron of a putative juvenile hormone diol kinase gene. Homozygosity was reached in this line after passage through 7 generations, indicating that the inserted vasa-rtTA nucleotides did not interfere with the function of an essential genomic locus. rtTA mRNA expression levels were higher than in the other two lines, supporting the hypothesis that failure to achieve homozygosity was not due to rtTA expression but to insertion position. The homozygous line produced ~ 18% more eggs per female and a hatching rate of larvae from eggs was 39% lower than of wild-type A. 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).