Resistance to Pyrethroid Insecticides in Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae): Bioassay Validation, Voltage-Gated Sodium Channel Mutations and CYP6B Overexpression Analysis

Helicoverpa zea is one of the most costly insect pests of food and fiber crops throughout the Americas. Pyrethroid insecticides are widely applied for control as they are effective and relatively inexpensive; however, resistance threatens sustainability because alternative insecticides are often more expensive or less effective. Pyrethroid resistance has been identified since 1990 and monitoring has utilized cypermethrin in the adult vial test, but resistance mechanisms have not yet been elucidated at the molecular level. Here we examined field-collected H. zea males resistant to cypermethrin for target site and metabolic resistance mechanisms. We report the cDNA sequence of the H. zea sodium channel a-subunit homologous to the Drosophila para gene and identified known resistance-conferring mutations L1029H and V421M, along with two novel mutations at the V421 residue, V421A and V421G. An additional mutation, I951V, may be the first example of a pyrethroid resistance mutation caused by RNA-editing. We identified other specimens with significantly higher transcriptional expression levels of cytochrome P450 genes CYP6B8 and CYP6B9 compared to the susceptible, ranging from a factor of 3.7 to 34.9 and 5.6 to 39.6, respectively. In addition, we investigated if differences in insect growth stage and pyrethroid structure affect our ability to predict resistance in the adult vial test. Vial bioassays with cypermethrin, esfenvalerate, and bifenthrin were conducted on third instars and male moths from a susceptible laboratory colony and the F1 generation of a resistant field population. For the resistant population, vial assays using either growth stage gave similar resistance ratios for each of the three pyrethroids, respectively, proving the adult vial test accurately reflects larval resistance. However, resistance ratios varied considerably depending on the pyrethroid used, so values obtained with one pyrethroid may not be predictive of another. This dissertation is the first to identify molecular mechanisms associated with H. zea pyrethroid resistance. Our results suggest carefully chosen pyrethroid structures diagnostic for specific resistance mechanisms could improve regional monitoring programs and development of high throughput assays to detect the resistance mechanisms used in tandem with traditional monitoring may greatly improve our ability to identify and predict resistance and make better control recommendations.