Differential regulation of cytochrome P450 genes associated with biosynthesis and detoxification in bifenthrin-resistant populations of navel orangeworm (Amyelois transitella)

Authors: DEMKOVICH, MARK - Sygenta Seeds; CALLA, BERNARDA - University Of Illinois; NGUMBI, ESTHER - University Of Illinois; HIGBEE, BRADLEY - Trece, Inc; Siegel, Joel; BERENBAUM, MAY - University Of Illinois

Submitted to: PLOS ONE
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/7/2021
Publication Date: 1/22/2021
Citation: Demkovich, M.R., Calla, B., Ngumbi, E., Higbee, B.S., Siegel, J.P., Berenbaum, M.R. 2021. Differential regulation of cytochrome P450 genes associated with biosynthesis and detoxification in bifenthrin-resistant populations of navel orangeworm (Amyelois transitella). PLoS ONE. 16(1). Article e0245803. https://doi.org/10.1371/journal.pone.0245803.
DOI: https://doi.org/10.1371/journal.pone.0245803

Interpretive Summary: The navel orangeworm, Amyelois transitella, is the most destructive pest of California almonds and pistachios and is also a pest of walnuts and pomegranates. Currently, this moth is controlled by a combination of sanitation, insecticides and mating disruption, but control has become increasingly challenging as the number of acres planted with these four commodities has increased because of their value. The insecticides most commonly used to control this pest belong to three families (pyrethroid, diamide and diacyl hydrazine) and the pyrethroid family is the cheapest and most widely used. Resistance to the pyrethroid insecticide bifenthrin was first reported in Kern County in 2013 and resistant populations have been reported statewide. At this point in time pyrethroids insecticides can still be successfully used to control navel orangeworm, and there is considerable interest in both prolonging their use, as well as determining if pyrethroid resistant individuals are also resistant to other insecticide families with differing modes of action (multiple resistance). In this study, we examine the role played in pyrethroid breakdown by a major class of detoxification enzymes, the cytochrome P450 monooxygenases, by comparing differences in P450 activity between a resistant (R347) and a susceptible strain (ALM). This study is unprecedented in scale because we use a molecular technique known as high-throughput quantitative real-time PCR (qRT-PCR) to cheaply analyze the behavior of 65 P450 monooxygenases belonging to the CYP3 and CYP4 clans and identify specific enzymes that increase when the insect is exposed to pyrethroids. Nine P450s were constitutively overexpressed in resistant R347 in comparison to susceptible ALM. Among those overexpressed (= 10-fold) was CYP6AE54, belonging to a subfamily associated with metabolic pesticide detoxification in Lepidoptera. Also overexpressed in resistant individuals was CYP4G89, belonging to a subfamily associated with cuticular hydrocarbon (CHC) synthesis and resistance via reduced pesticide penetrance. We confirmed this cuticular hydrocarbon change for strain R347 using chemical analysis. This finding is troubling because changes in the cuticle, which in turn affect the rate of insecticide penetration, can affect the efficacy of other insecticide families. We paired these molecular studies with bioassays challenging eggs, larvae and adult navel orangeworm with bifenthrin. Even the eggs of strain R347 were more tolerant to low levels of bifenthrin than the eggs of strain ALM. Our laboratory bioassays support the hypothesis that enzymatic changes in R347 provide protection against pyrethroid insecticides by a combination of increased rate of breakdown and reduced penetrance through the cuticle. Future studies will determine if the cuticular changes of strain R347 provide resistance to other families of insecticide and if these changes can be reversed using adjuvants (other chemicals combined with insecticide to enhance efficacy).

Technical Abstract: Pyrethroid resistance was first reported in 2013 for the navel orangeworm Amyelois transitella, the most destructive pest of California almonds and pistachios, but the genetic underpinnings of pyrethroid resistance in this insect are unknown. We investigated the possible role of cyctochome P450 monooxygenases (P450s)belonging to the CYP3 and CYP4 clans in pyrethroid resistance, using a comparative analysis of two strains originating from a susceptible population (ALM) collected from almond orchards in Madera County in 2016 and a pyrethroid-resistant population (R347) collected in Kern County in 2014. We carried out high-throughput quantitative real-time PCR (qRT-PCR) analyses of 65 P450s belonging to the CYP3 and CYP4 clans of A. transitella, to identify the P450s induced by bifenthrin and associated with pyrethroid resistance. Nine P450s were constitutively overexpressed (= ten-fold) in resistant R347, and among the enzymes overexpressed was CYP6AE54, which belongs to a subfamily associated with metabolic pesticide detoxification in Lepidoptera. Resistant individuals also overexpressed CYP4G89, which belongs to a subfamily associated with cuticular hydrocarbon (CHC) synthesis and resistance, via reduced pesticide penetrance. Cuticular hydrocarbons were extracted, quantified, and then compared between the two strains, revealing that the resistant strain produced more CHCs in the egg and adult life stages. Topical toxicity bioassays using bifenthrin associated elevated CHCs in the resistant population with differential egg and larval mortality; R347 egg mortality was reduced at low bifenthrin concentrations and R347 larval mortality was reduced at high concentrations of bifenthrin. These findings, consistent with the proposed mechanism of reduced insecticide penetrance, suggest that the penetration rate of insecticides belonging to other chemical families may also be reduced, which in turn would make them less effective for managing resistant navel orangeworm. The potential of R347 to develop multiple resistance warrants further investigation of management strategies that will facilitate insecticide passage across the cuticle. Our study is unique because we screened the entire inventory of 65 CYP3 Clan and CYP4 Clan P450s in A. transitella across 24 total treatments (with a total of 2,304 assays per run), circumventing the limitation of single-gene qPCR, which becomes inefficient as the number of samples and genes increases. This method also proved more effective than RNA-seq, which, due to cost, limits the number of samples and treatments that can be simultaneously tested. The results from this study demonstrate the efficacy of applying this high-throughput method directed to a specific gene family.