Untangling the complex molecular interplay in vector-pathogen interactions using bioinformatics, gene silencing, and gene editing.

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Insect vectors transmit parasites and pathogens to humans, yet the vast majority of parasites that enter vectors are killed by components of the insects' innate immune system. This response is germ-line encoded and is neither learned nor acquired, unlike the characteristics of vertebrate immunity. Several mechanisms are used by the insects to recognize parasites and pathogens as "non-self". Recognition activates immune pathways (Toll, IMD, RNAi and Jak-STAT) to encapsulate, melanixe, or express potent antimicrobial peptides (AMPs) all of which can kill parasites. Despite the power of innate immunity, many insects still transmit parasites. The relationship between vector and parasite is very specific. Each vector species transmits one, or a very limited number of, parasites or pathogens. If the immune system functions why do vectors not kill all parasites? In Cali Colombia, approximately 30% of field collected Ae. aegypti, the major vector of Dengue and Zika viruses, actually kill the viruses while 70% continue to transmit the viruses to humans. If being refractory to viruses is a benefit, why do all female Ae. aegypti not kill the viruses? Within Ae. aegypti, Dengue viruses exploit existing mosquito proteins to establish, enter cells, and replicate, but in the refractory strains of Ae. aegypti these genes are not expressed. We will determine the role and importance of these genes by knocking down their expression and exposing these mosquitoes to Dengue virus to confirm the role of these genes. We then will use gene editing techniques to render susceptible phenotype-determining genes non-functional to determine their absence on virus development and on general mosquito fitness parameters. The Immune deficiency pathway (IMD) is a highly conserved immune pathway in insects that eliminates infections with Gram-negative bacteria. In the kissing bug, Rhodnius prolixus, however, the IMD pathway was reported to be absent, non-functional, or significantly reduced. The IMD pathway was reported to be absent in highly related insects. Despite these claims, the antimicrobial peptides normally (AMPs) regulated by the IMD pathway are expressed in these insects. We will use a series of bioinformatics and wet bench approaches to determine if the pathway is intact, but hidden in the genome, whether new proteins have replaced "missing: IMD components, or whether these AMPs are controlled by other pathways. Preliminary data indicate that the IMD pathway is present and functional R. prolixus. All of these experiments and approaches are designed to identify the regulation of innate immune responses in insects, how insects respond to parasites and pathogens, what factors determine vector-parasite specificity, and the mechanisms they use to restrict or eliminate parasite development. Ultimately, we may identify essential molecules or pathways that we might exploit to induce incompatibility between vectors and pathogens, and therefore eliminate transmission.