Tsakireli, D. | bioRxiv Science

bioRxiv2022

Functional characterization of cytochrome P450s associated with pyrethroid resistance in the olive fruit fly Bactrocera oleae

Resistance to pyrethroid insecticides has evolved in Bactrocera oleae populations in Greece, threatening the efficacy of control interventions based on this insecticide class. Here we report the collection of populations from Crete with resistance levels reaching up to 331-folds, compared to susceptible laboratory strains and show that pyrethroid resistance is substantially suppressed by the PBO synergist, suggesting the involvement of detoxification enzymes. To identify specific candidate genes implicated in resistance, we performed comparative transcriptomic analysis, between the pyrethroid resistant populations from Crete and the susceptible laboratory strains, using both whole bodies and Malpighian tubules. Several genes were found differentially transcribed between resistant and susceptible flies in each comparison, with P450s being among the most highly over-expressed detoxification genes in pyrethroid resistant populations. Four of the over-expressed P450s (Cyp6A61, Cyp6G6, Cyp4P6 and Cyp6G28) were recombinantly expressed in Escherichia coli and in vitro metabolism assays revealed that CYP6A61 is capable of metabolizing alpha-cypermethrin, while CYP6G6, CYP4P6 and CYP6G28 are capable of metabolizing deltamethrin. No metabolism of neonicotinoid insecticides was recorded. We further silenced CYP6G6 in vivo, via RNAi, which led to a small, but significant increase in deltamethrin toxicity. The study provides valuable information towards the development of molecular diagnostics and evidence-based insecticide resistance management strategies.

molecular biology↗

bioRxiv2021

High-resolution genetic mapping reveals cis-regulatory and copy number variation in loci associated with cytochrome P450-mediated detoxification in a generalist arthropod pest

Chemical control strategies are driving the evolution of pesticide resistance in pest populations. Understanding the genetic mechanisms of these evolutionary processes is of crucial importance to develop sustainable resistance management strategies. The acaricide pyflubumide is one of the most recently developed mitochondrial complex II inhibitors with a new mode of action that specifically targets spider mite pests. In this study, we characterize the molecular basis of pyflubumide resistance in a highly resistant population of the spider mite Tetranychus urticae. Classical genetic crosses indicated that pyflubumide resistance was incompletely recessive and controlled by more than one gene. To identify resistance loci, we crossed the resistant population to a highly susceptible T. urticae inbred strain and propagated resulting populations with and without pyflubumide exposure for multiple generations in an experimental evolution set-up. High-resolution genetic mapping by a bulked segregant analysis approach led to the identification of three quantitative trait loci (QTL) linked to pyflubumide resistance. Two QTLs were found on the first chromosome and centered on the cytochrome P450 CYP392A16 and a cluster of CYP392E6-8 genes. Comparative transcriptomics revealed a consistent overexpression of CYP392A16 and CYP392E8 in the experimental populations that were selected for pyflubumide resistance. We further corroborated the involvement of CYP392A16 in resistance by in vitro functional expression and metabolism studies. Collectively, these experiments uncovered that CYP392A16 N-demethylates the toxic carboxamide form of pyflubumide to a non-toxic compound. A third QTL coincided with cytochrome P450 reductase (CPR), a vital component of cytochrome P450 metabolism. We show here that the resistant population harbors three gene copies of CPR and that this copy number variation is associated with higher mRNA abundance. Together, we provide evidence for detoxification of pyflubumide by cytochrome P450s that is likely synergized by gene amplification of CPR. Author summaryOur understanding of the causal genetic variants that drive the evolution of quantitative traits, such as polygenic pesticide resistance, remains very limited. Here, we followed a high-resolution genetic mapping approach to localize the genetic variants that cause pyflubumide resistance in the two-spotted spider mite Tetranychus urticae. Three well-supported QTL were uncovered and pointed towards a major role for cytochrome P450-mediated detoxification. Cis-regulatory variation for cytochrome P450s was observed, and in vitro cytochrome P450 experiments showed that pyflubumide was metabolized into a non-toxic derivate. A third QTL centered on cytochrome P450 reductase (CPR), which is required for cytochrome P450 activity, and is amplified in pyflubumide resistant populations. Our results indicate that pyflubumide resistance is mediated by cytochrome P450 detoxification that is enhanced by gene amplification at the CPR locus. Data availabilitySequence data has been deposited at the Sequence Read Archive (PRJNA596790). Phenotypic quantification data is available at Supplementary Data S1.

genetics↗