Identifying mechanisms of pyrethroid resistance in the navel orangeworm and novel methods of control

The navel orangeworm (Amyelois transitella) (Walker) is the most important economic pest of almonds and pistachios in California orchards. Increasing demand for these commodities has resulted in acreage expansions and substantial increases in the application of insecticides to reduce damage by A. transitella. Pyrethroid insecticides have historically been the most heavily applied insecticides registered for A. transitella control because of their efficacy against all life stages, broad-spectrum activity in orchards, and significantly reduced costs relative to other insecticides. The first incidence of pyrethroid resistance in A. transitella occurred in Kern County almond orchards in 2013, and the initial mechanism reported suggested overexpression of cytochrome P450 monooxygenases (P450s) and carboxylesterases (COEs). In the first chapter of my dissertation, I investigated the role of all P450s in the CYP3 and CYP4 clans associated with metabolism of xenobiotics in A. transitella through a comparative analysis using a susceptible population collected from Madera County almond orchards in 2016 (ALM) with a pyrethroid-resistant population (R347) collected from Kern County orchards in 2016. The objective of this research was to examine changes in gene expression in the ALM and R347 populations of A. transitella in order to identify P450s induced by bifenthrin and associated with pyrethroid resistance. I extracted RNA from midguts of fifth instar larvae that fed on artificial diets with and without bifenthrin, and I carried out quantitative real-time PCR (qRT-PCR) analyses of 65 P450s in the CYP3 and CYP4 clans of A. transitella. I identified only two P450s induced by bifenthrin, both of which occurred in the ALM population. Nine P450s were overexpressed in R347 larvae that fed on control diets, which suggested constitutive overexpression as a potential mechanism of pyrethroid resistance in this population. Among the nine P450s overexpressed in the resistant population, two were associated with the synthesis of cuticular hydrocarbons (CHCs) in the CYP4G subfamily, which has been linked to resistance in other insects by preventing or delaying passage of insecticides across the cuticle. CHCs were then extracted and quantified between the susceptible and resistant populations, and results confirmed that the resistant population produces more CHCs in eggs and adults. I carried out a series of bioassays of topical toxicity to determine if elevated CHCs in the resistant population contribute to differences in egg and larval mortality through bifenthrin sprays. R347 egg mortality was reduced at low bifenthrin concentrations, and more R347 larvae survived bifenthrin treatment when challenged with higher concentrations. Whether or not CHCs contribute to enhanced survival of R347 under field conditions remains an open question. For the second chapter of my dissertation, I annotated the carboxylesterases (COEs) in the A. transitella genome. Insect COEs are involved in developmental and neurological processes, pheromone processing and degradation, and metabolism of xenobiotics. Insect COEs are classified into subfamilies that include alpha-esterases, juvenile hormone esterases, integument esterases, beta-esterases, acetylcholinesterases, gliotactins, glutactins, neuroligins, and neurotactins through phylogenetic and functional analyses. I discovered 64 total COEs in the A. transitella genome and placed them all into their corresponding subfamilies by constructing a phylogeny with Bombyx mori, Plutella xylostella, and Trichoplusia ni, which are the only lepidopterans with fully annotated COEs. I identified an expansion in the number of alpha-esterases in A. transitella, which is consistent with all other lepidopteran insects described to date. Among the alpha-esterases, there are two clades in my phylogeny with orthologs from each species that are likely noncatalytic, with functions that are potentially unique to Lepidoptera. These findings can provide a foundation for future research on investigating COE involvement in resistance to insecticides. The third chapter of my dissertation was based upon previous research that identified a selective sweep in the A. transitella genome delimited by a point mutation kdr in the para gene that alters the conformation of the voltage-gated sodium channel and confers resistance to pyrethroids. This mutation was identified in three separate populations from the San Joaquin Valley of California, two of which came from areas without any previously described pyrethroid resistance. Although the sweep was present in the reference genome population, the mutation in para was absent. I re-sequenced the para gene to confirm that the mutation was absent in the reference genome population and also sequenced a population collected from two counties in northern California where pyrethroid applications have historically been less intense in almond orchards. After unexpectedly detecting the resistance mutation in the northern populations, I investigated the history of insecticide use in Kern County, Madera County, Colusa County, and Yolo County, where these A. transitella individuals were collected, with emphasis on the pyrethroid use. The insecticide records maintained through the California Department of Pesticide Regulations revealed a surge in bifenthrin use from 2009 to 2013 throughout the state before the first reported case of resistance arose in Kern County almond orchards in 2013. The heavy use of bifenthrin may have resulted from patent expiration and the availability of alternative product forms. The number of trade name products containing bifenthrin increased from one in 2009 to thirteen by 2013 in statewide almond orchards and one to nine products in pistachio orchards during this time. Comparisons of bifenthrin use relative to all other pyrethroids by pounds applied from 2009 to 2017 revealed that Kern County and Madera County applications were similar to statewide use at approximately 70% of all pounds of pyrethroids applied. Bifenthrin use was higher in Kern County pistachio orchards at 59.6% all of all pyrethroid pounds applied compared to 51.1% statewide and 45.8% in Madera County. Bifenthrin may have accelerated resistance acquisition in A. transitella, although this analysis of insecticide use was based on county pooled averages and did not account for site-specific applications. Site-specific pyrethroid use may have been a determining factor in the development of resistance in Kern County where as few as ten companies may control 75% of the almond acreage. Records of pyrethroid use for the northern counties of Colusa and Yolo revealed lower bifenthrin selection pressure from 2009 to 2013 and did not correlate with trends in Kern County, Madera County, and statewide use. I suggest a need to examine a broader range of populations to determine the spread of pyrethroid resistance resulting from the kdr mutation in the para gene. In the fourth and final chapter of my dissertation, I describe a series of experiments aimed at determining the potential for agricultural adjuvants to synergize the toxicity of two diamide insecticides registered at the time for A. transitella control. Despite widespread adoption of insecticide sprays for A. transitella control, the potential toxicity of adjuvants applied in combination with insecticides is unknown. In these experiments, five adjuvants currently applied by growers to manage A. transitella (Cohere®, Dyne-Amic®, FastStrike®, Induce®, Latron B-1956®) were examined alone and in combination with two diamide insecticides registered for use in almond and pistachio orchards, chlorantraniliprole (Altacor®) and flubendiamide (Belt®). Toxicity of adjuvant and adjuvant-diamide combinations was assessed against A. transitella eggs and adults through a series of laboratory experiments involving a spray tower. A series of field trials tested adjuvant-diamide combinations using orchard air-blast sprayers against the same life stages. Laboratory exposure of eggs and adults demonstrated that all adjuvants were intrinsically toxic to A. transitella and that toxicity of adjuvants and adjuvant-diamide combinations varied across life stages. Field experiments demonstrated that adjuvants affected the toxicity of insecticides sprayed for A. transitella control. This study examined an overlooked and vital component to insecticide applications in tree nuts and suggests adjuvant choice has the potential to improve insecticide performance for A. transitella control.U of I OnlyAuthor requested U of Illinois access only (OA after 2yrs) in Vireo ETD syste