Novel Insecticide Resistance-Monitoring Bioassays for Lepidopteran and Hemipteran Cotton Pests and 454 Pyrosequencing To Identify Potential Gene Targets for RNAi Silencing in Hemipteran Cotton Pests

Feeding disruption test (FDT) bioassays for resistance-monitoring were developed to circumvent the limitations of traditional vial and diet bioassays. In FDT assays, insects feed on insecticide in artificial diet containing a blue dye. The assay endpoint is the dose-dependent amount of blue feces produced, usually within 24 hours.;FDT assays were developed for monitoring larval Lepidoptera (caterpillars) for resistance to three Bt proteins: Cry1Ac, Cry1F, and Cry1Ab. The assays rely on a diagnostic dose of Bt toxin in 100-mul rehydratable meal pads of artificial diet containing blue indicator dye. The assay was formatted as a portable (palm-sized), plastic plate containing an array of 16 insect test wells with each containing a single hydratable meal pad. The diagnostic dose was the concentration of Bt in meal pad rehydration solution that reduced 24-hour dyed fecal production of Bt-susceptible neonates to ≤ 2 fecal pellets. Bt-resistant neonates were able to consume the diagnostic dose of the insecticidal protein and produce >2 blue feces which were distinctly visible on the FDT plate's white background. Diagnostic doses were determined with lab-strain Bt-susceptible Heliothis virescens and Helicoverpa zea. The assays were validated against lab-strain Bt-resistant H. virescens and with susceptible larval H. virescens collected as eggs from field-grown tobacco in North Carolina. The difference in 24-hour dyed fecal production for Bt-susceptible H. virescens vs. Bt- resistant was ≥ 341-fold.;An FDT for lepidopteran adults (moths) was developed. Lab-strain H. virescens adults were fed different concentrations of permethrin and spinosad in 10% sucrose containing 0.08 mg/ml trypan blue dye to obtain dose-dependant nectar-ingestion, fecal production and mortality. A permethrin diagnostic dose of 20 mug/ml distinguished lab-strain pyrethroid-resistant H. virescens from lab-strain susceptible moths on the basis of both mortality and relative amounts of dyed feces. The assay produced no false-positives or false-negatives (error rate = 0%).;A consequence of the success of Bt cotton in controlling lepidopteran pests has been the emergence of several species of plant bugs (Miridae) and stink bugs (Pentatomidae) to become major pests of cotton. The feasibility of developing FDT assays for plant bugs and stink bugs was examined. Lab-strain adult Lygus lineolaris fed different concentrations of beta-cyfluthrin and thiamethoxam in rehydratable meal pads of dyed artificial diet in FDT plates showed a dose-response in production of dyed feces for both insecticides. Lab-strain adult Euschistus servus fed different concentrations of thiamethoxam in sucrose solution showed a mortality dose response. E. servus fed dyed artificial diet produced dyed feces; thus, FDT assays for stink bugs, as well as plant bugs, should be feasible.;A potential transgenic alternative or complement to Bt transgenes for control of plant bugs and stink bugs was explored. A transcriptome was constructed by 454 pyrosequencing of combined nymph and adult whole-body homogenate of green stink bugs, Acrosternum hilare. The whole-body cDNA library produced 162,000 reads which were assembled into 1,598 contigs with the assembly cutoff set at 400 bp. BLASTp was used to compare the contigs to the Uniprot database to determine putative sequence identity. Of these contigs, 974 had an E (expect value) of ≤ 1e-05 and 876 of these had an assigned Uniprot GO (Gene Ontology) ID and were mapped and annotated. Wego software was used to translate the Uniprot GO ID for these contigs into GO Level 2 functional categories. Analysis of this transcriptome is aimed at the discovery of new insecticide targets for applications in plant genetic pest management.