Insecticide resistance is a global problem. Insecticide resistance management is very important, considering the time, effort, and cost of discovering and developing a new insecticide. There are diverse resistance mechanisms, but enhanced detoxification through overexpression of cytochrome P450s and target site insensitivity through mutation in insecticide binding site are the two most common mechanisms. The xenobiotic detoxification is divided into three successive phases (I, II and III), which ensures the metabolism and excretion of the detrimental toxins. Each phase comprises of a specific group of metabolizing enzymes such as P450s (phase I), GSTs (phase II) and ABC transporters (phase III). The major goal of my research was to understand the molecular mechanism of insecticide resistance in two economically important coleopteran pests, Leptinotarsa decemlineata and Tribolium castaneum. The transcriptional regulation of the P450 genes mediating insecticide resistance in L. decemlineata (imidacloprid-resistant) and T. castaneum (deltamethrin-resistant) were studied and the xenobiotic trans and cis-elements identified. RNA interference (RNAi), and reporter assays revealed that the cytochrome P450 genes involved in insecticide resistance are regulated by transcription factor Cap n Collar ‘CncC’ and muscle aponeurosis fibromatosis ‘Maf’ belonging to the b-ZIP transcription factor family. Site-directed mutagenesis was employed to identify the binding site for CncC and Maf. Sequencing of RNA isolated from CncC knockdown T. castaneum identified genes regulated by CncC and involved in insecticide detoxification. RNAi and insecticide bioassays confirmed the function of select phase II (glutathione-S-transferases) and phase III (ABC transporters) identified by RNA sequencing. Overall, these data revealed that the xenobiotic transcription factor CncC is the master regulator of multiple genes that are involved in insecticide resistance.

RNA interference (RNAi) is a useful reverse genetics tool for investigation of gene function as well as for practical applications in many fields including medicine and agriculture. Due to the variability in RNAi efficiency, RNAi-based methods are currently being developed for controlling only coleopteran insects which are known to be amenable to RNAi. The first chapter of my thesis includes findings from research to investigate what are the factors that make coleopteran insects relatively more efficient in RNAi. I used Colorado potato beetle (CPB), Leptinotarsa decemlineata and its cell line (Lepd-SL1) as study models to identify genes that play key roles in RNAi pathway. Five genes including Argonaute-1 (microRNA Argonaute) and Aubergine (PiwiRNA Argonaute) were identified as those required for siRNA (short interfering RNA) RNAi pathway. I also found that RNAi is completely blocked in StaufenC knockdown cells. StaufenC belongs to dsRNA binding protein family and binds to dsRNA as shown by gel mobility shift and the pull-down assays. Interestingly, I also found that StaufenC is downregulated in RNAi resistant cells and StaufenC homologous sequences are present in only coleopteran insects where RNAi works efficiently. These data suggest that StaufenC is a major contributor to efficient RNAi in coleopteran insects and is a potential target for RNAi resistance. The second part of my research is to understand the mechanisms of RNAi in those insects refractory to RNAi. The barriers for successful RNAi include the presence of double-stranded ribonucleases (dsRNase) in the lumen and hemolymph that could potentially digest double-stranded RNA (dsRNA) and the variability in the transport of dsRNA into and within the cells. Recent work in our laboratory showed that the dsRNAs are transported into lepidopteran cells, but they are not processed into siRNAs because they are trapped in acidic bodies. I focused on identification of these acidic bodies in which dsRNAs accumulate in Spodoptera frugiperda Sf9 cells. These studies showed that entrapment of internalized dsRNA in endosomes is one of the major factors contributing to inefficient RNAi. Overall, my research revealed important players involved in successful and unsuccessful RNAi in insects.