Mechanisms of resistance against insecticidal dsRNA in Leptinotarsa decemlineata (Colorado potato beetle)

In the last decade, silencing of vital genes by RNA interference (RNAi) through dsRNA ingestion has been identified as a revolutionary bioinsecticide technology. As commercialization of insecticidal dsRNA technology approaches, it becomes crucial to develop resistance management tools for the sustainability of this technology. Using chronic exposure through larval development, we developed a population (CEAS) of Colorado potato beetle (CPB) that is >5,000-fold resistant to insecticidal dsRNA. In this work we share findings from research focused on the identification of candidate resistance mechanisms and cross-resistance to Cry3Aa, the most active insecticidal protein from Bacillus thuringiensis against CPB. Bioassays and comparison of dsRNA stability in digestive fluids from susceptible and resistant CPB support cross-resistance to alternative dsRNA targets and that degradation of dsRNA by nucleases is not involved in resistance. Monitoring uptake of fluorescently-labeled dsRNA by midgut cells using confocal microscopy supports reduced uptake of dsRNA in midgut cells of CEAS compared to susceptible larvae. Results from this project will guide development of Insect Resistance Management (IRM) strategies for insecticidal RNAi and its combined used with insecticidal proteins from B. thuringiensis against CPB and will allow the optimization of insecticidal RNAi technology.