Resistance to double-stranded RNA (dsRNA) in Colorado potato beetle (Leptinotarsa decemlineata Say): Genetic and molecular insights

Double-stranded RNA (dsRNA) insecticides delivered through transgenic and non-transgenic means have been approved for commercialization in the US. As with other insecticides development of insect resistance is expected, necessitating the implementation of an Insecticide Resistance Management (IRM) framework for their long-term use. Laboratory-selected insect resistance strains can provide useful insights for an effective IRM plan. The Colorado potato beetle (CPB) is the target pest for the first commercial dsRNA foliar spray, making it an ideal model to study dsRNA resistance. In this dissertation, we studied the previously described CEAS 300 strain of CPB that exhibits >11,100-fold resistance against V-ATPase subunit A dsRNA. We demonstrate that resistance in CEAS 300 is inherited as a recessive autosomal trait supporting the potential of the refuge strategy in delaying resistance. Resistance in CEAS 300 is s polygenic trait which suggests the presence of multiple resistance mechanisms. Bioassays with chemical pesticides indicate a lack of cross-resistance, supporting their combined use with dsRNA insecticides in reducing selection pressure. While no differences in dsRNA stability in digestive fluids were observed, uptake of dsRNA in CEAS 300 midgut cells was reduced compared to susceptible insects in fluorescence microscopy and small RNA sequencing. Transcriptomic analyses indicate suppression of clathrin-mediated endocytosis (CME), a key dsRNA uptake pathway, in CEAS 300. At the same time, several immune and stress-related pathways are activated in CEAS 300, suggesting their potential role in dsRNA resistance. Candidate resistance-associated genes in CEAS 300 were also identified using bulked segregant analysis (BSA). Following linkage analysis, these can be used to monitor resistance alleles in field populations and estimate the risk of resistance. Taken together, this dissertation provides critical information regarding resistance characteristics and mechanisms in a strain of CPB. This information can guide strategies to potentially overcome resistance as well as create a resistance management framework to delay resistance.