Rnai-mediated silencing methods for xenopsylla cheopis, a primary vector forplague transmission

Washington State University received a $153,000 Project Grant award from the National Institute of Allergy and Infectious Diseases (NIAID) under the Allergy and Infectious Diseases Research program (CFDA 93.855) beginning August 1, 2025, and concluding July 31, 2027. The award funds the development of RNA interference (RNAi)-mediated gene silencing methods targeting Xenopsylla cheopis, a primary flea vector responsible for plague transmission. The research aims to address the critical public health challenge posed by plague caused by Yersinia pestis, which remains endemic in wild rodent populations worldwide and has developed insecticide resistance, compromising traditional vector control strategies.

The primary deliverable under this award is the establishment of novel genetic tools and optimized RNAi delivery methodologies specific to flea biology, with particular focus on genes implicated in the immune deficiency (IMD) signaling pathway that governs the flea's response to Y. pestis infection. By developing and validating RNAi-mediated silencing techniques, this research will enable future mechanistic investigations into flea-borne plague transmission biology and support the development of vector-based bacterial pathogen control strategies that overcome current limitations of insecticide-dependent disease management approaches.

Abstract

Fleas are obligate blood-feeding arthropods that are associated with several notable bacterial-derived human diseases, i.e., rickettsioses, bartonelloses, and plague. Plague caused by the gram negative bacterium, yersinia pestis, is difficult to eradicate because flea-borne transmission is endemic in natural foci of wild rodents and their associated fleas world-wide. Insecticide control is the primary strategy for disease management, but like many vector-borne diseases this is compromised by development of insecticide resistance in fleas. Development of novel vector-based strategies for bacterial pathogen control are therefore a priority. However, to accomplish this, we must overcome lack of knowledge regarding flea biology, particularly the detailed processes of the flea host response to infection. But genetic tools enabling such research into this knowledge GAP are absent. Towards this goal and to enable previously inaccessible, yet essential, mechanistic research into understanding the transmission biology of flea borne plague in future studies, this proposal aims to develop rnai-mediated gene silencing methods for xenopsylla cheopis, a primary flea vector of plague. The biggest hurdle to effective rnai-mediated silencing in any arthropod is achieving delivery of exogenous double-stranded (DS) RNA into the cell cytoplasm to induce specific gene silencing by targeting homologous mrnas. No one effective delivery method is currently broadly applicable to all arthropods, requiring that methods be tested and optimized. Having identified x. Cheopis gene target/s in the IMD immune signaling response are specifically implicated in response to y. Pestis infection, appropriate gene targets are now available to which rnai strategies can be developed and tested. Our preliminary experiments demonstrate significant silencing of the IMD gene by electroporation and microinjection methods of dsrna delivery. Therefore, in aim 1 we will determine the most feasible dsrna delivery method that enables a high degree of and sustained gene suppression in flea GUT- specific tissue (the site of a y. Pestis infection), as well as low mortality in fleas. In aim 2 we will use the most effective method identified to determine if IMD pathway silencing modulates y. Pestis infection rates and burdens in fleas. The proposed development of a genetic tool to expand research into flea borne plague lies within a part of the NIH's mission to develop fundamental knowledge that will assist in reducing the burden of infectious diseases on human health.