Maternal Germline-Specific Genes in the Asian Malaria Mosquito Anopheles stephensi: Characterization and Application for Disease Control

Anopheles stephensi is a principal vector of urban malaria on the Indian subcontinent and an emerging model for molecular and genetic studies of mosquito biology. To enhance our understanding of female mosquito reproduction, and to develop new tools for basic research and for genetic strategies to control mosquito-borne infectious diseases, we identified 79 genes that displayed previtellogenic germline-specific expression based on RNA-Seq data generated from 11 life stage-specific and sex-specific samples. Analysis of this gene set provided insights into the biology and evolution of female reproduction. Promoters from two of these candidates, vitellogenin receptor and nanos, were used in independent transgenic cassettes for the expression of artificial microRNAs against suspected mosquito maternal-effect genes, discontinuous actin hexagon and myd88. We show these promoters have early germline-specific expression and demonstrate 73% and 42% knockdown of myd88 and discontinuous actin hexagon mRNA in ovaries 48 hr after blood meal, respectively. Additionally, we demonstrate maternal-specific delivery of mRNA and protein to progeny embryos. We discuss the application of this system of maternal delivery of mRNA/miRNA/protein in research on mosquito reproduction and embryonic development, and for the development of a gene drive system based on maternal-effect dominant embryonic arrest.