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

Abstract

Background: 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.

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

Results: 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).

Keywords: Anopheles; Driver line; Tetracycline; rtTA.

Fig. 1

Schematic representation of the driver line construct. The rtTA open reading frame (ORF) was designed to be expressed under the vasa or bZip promoters. The mBanana is expressed under the 3xP3 promoter. The SV40 3′ untranslated region (UTR) is used as transcription termination for both genes. The cassette was placed between two piggyBac transposon elements (yellow arrows)

Fig. 2

rtTA mRNA abundance in embryos under bZip and vasa early embryonic promoters. Quantitative RT-PCR performed on cDNA from pools of eggs laid by ten females from each group. The results represent the mean mRNA abundances, relative to egg pool from bZip-rtTA M1-2 line ± standard deviation (SD), n = 3. The data were analyzed using one-way analysis of variance (ANOVA), ^*P ≤ 0.05. The ribosomal S7 protein gene was used as the housekeeping gene

Fig. 3

Diagnostic PCR to identify the zygosity status of the insertion in M1f (a), M2i (b), and F2c (c) driver lines. PCR was performed on genomic DNA from three individual mosquitoes (a–c) from all three lines, collected at G⁷. At G²⁴, mosquitoes (a–c) were collected for M1f and F2c, and six were collected for M2i (a–f). Genomic DNA from WT An. stephensi female mosquitoes was used as a negative control, and water was used as a no template control (NTC). The expected PCR products for the M1F insertion and WT alleles were 7560 and 354 base pairs (bp), respectively. For M2i, the products of the insertion and WT alleles were 7534 and 328, respectively. The expected PCR products for the F2c insertion and WT alleles were 7636 and 430 bp, respectively. Black arrows indicate the position of the PCR products on the gel. Homozygous mosquitoes were found only in M2i

Fig. 4

Fecundity in WT and driver line mosquitoes. a The number of eggs laid per individual female in Drosophila tubes in experiment no. 1. b The percentage of eggs hatching in experiment no. 1 was determined by counting the number of larvae in the Drosophila tubes 1 day after egg laying. In both panels the results are reported as the geometric means, n = 34 for F2c and n = 35 for WT, M1f, and M2i lines. The data in both a and b were analyzed using non-parametric Kruskal Wallis test (^** P ≤ 0.01, ^*** P ≤ 0.001, and ^**** P ≤ 0.0001). In b, some values were 0. Whenever a value was 0, it was arbitrarily assigned a value of 1 to allow calculation of the geometric means. c The number of eggs laid per individual female in Drosophila tubes in experiment no. 2. d The percentage of eggs hatching in experiment no. 2. The data in both c and d were analyzed using non-parametric Mann–Whitney test (^* P ≤ 0.05, ^**** P ≤ 0.0001)