RNA interference-based sterile insect technique in mosquitoes: Overcoming barriers to implementation

The yellow fever mosquito (Aedes aegypti) is an important pathogen vector and model organism for mosquito researchers. Gene silencing by RNA interference (RNAi) has been used to determine genes’ functions in the vinegar fly, Drosophila melanogaster, but comparable, genome-wide loss-of-function data are not available for mosquitoes. This is due to the difficulty of achieving transcript depletion by dsRNA feeding and to the complexity and size of the Ae. aegypti genome. To respond to these challenges, I set out to understand dsRNA degradation in the mosquito gut, analyze the transcriptome of male and female mosquito larvae and improve dsRNA delivery methods. Several enzymes predicted to degrade dsRNA are found in the mosquito genome. I suspected that some of these enzymes could reduce RNAi efficiency of ingested dsRNAs in the larvae. I identified ten putative nuclease genes in the insect’s genome, two of which are expressed in the gut of fourth instar larvae. By delivering dsRNA against these nuclease genes, RNAi efficiency against other transcripts was improved. Sex determination in mosquitoes is controlled by a cascade of differentially-spliced transcripts that encode different transcription factors in the two sexes. To explore whether genes other than the few sex-determination genes are also spliced in a sex-specific manner, I used nanopore sequencing to sequence full-length transcripts from single reads in sex-sorted fourth instar larvae. This is the stage where development of sex organs begins, and I targeted some of these sex-biased transcripts using RNAi, inducing female-specific mortality. I also described several male-biased genes which may provide effective targets to produce sterile males. To enable low-cost RNAi assays, I developed and optimized dsRNA expression systems in bacteria and yeast. An understanding of dsRNA degradation, together with identification of new sex-specific gene targets and improved dsRNA delivery methods will allow researchers to exp