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

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).

Keywords: Anopheles; Tetracycline; driver line; rtTA.

Figures 1. Schematic representation of the driver line construct.

The rtTA ORF was designed to be expressed under the vasa or bZip promoters. The mBanana is expressed under the 3Xp3 promoter. The SV40 3’UTR is used as transcription termination for both genes. The cassette was placed between 2 piggyBac transposon elements (yellow)

Figure 2. rtTA mRNA abundance under bZip and vasa early embryonic promoters.

(a) Quantitative RT-PCR done on cDNAs produced from RNAs, extracted from individual female adult mosquitoes expressing rtTA using the bZip or vasa promoters, following egg laying. The abundances are relative to a female mosquito from bZip-rtTA M1–2 line. (b) qRT-PCR done on cDNA from pools of eggs laid by 10 females from each group. The results represent the mean mRNA abundances, relative to bZip-rtTA M1–2 line ±SD, n=3. The data were analyzed by one-way ANOVA, * p≤0.05. In both qRT-PCR reactions the ribosomal S7 protein gene was used as the house keeping gene.

Figure 3. Diagnostic PCR to identify the zygosity status of the insertion in M1f (a), M2i (b) and F2c (c) driver lines.

PCR was done on genomic DNA from 3 individual mosquitoes (a-c) from all 3 lines, collected at G7. At G24, mosquitoes (a-c) were collected for M1f and F2c and 6 were collected for M2i (a-f). Genomic DNA from wt. A. 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 bp, respectively. For M2i the products of the insertion and WT alleles were 7534 and 328, respectively. The expected PCR product 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’ labels are bolded. Homozygous mosquitoes were found only in M2i.

Figure 4. Fecundity in WT and driver line mosquitoes.

(a) The number of eggs laid per individual female in Drosophila tubes in experiment #1. (b) The percentage of eggs hatching in experiment #1 was determined by counting the number of larvae in the Drosophila tubes one 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. In panel B, some values were zero. 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 #2. (d) The percentage of eggs hatching in experiment #2.