High-resolution genetic mapping revealscis-regulatory and copy number variation in loci associated with cytochrome P450-mediated detoxification in a generalist arthropod pest
Authors: Fotoukkiaii Seyedeh Masoumeh , Wybouw Nicky , Kurlovs Andre H. , Tsakireli Dimitra , Pergantis Spiros A. , Clark Richard M. , Vontas John , Van Leeuwen Thomas 
Published: February 2021

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 miteTetranychus 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 susceptibleT. urticaeinbred 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 P450CYP392A16and a cluster ofCYP392E6-8genes. Comparative transcriptomics revealed a consistent overexpression ofCYP392A16andCYP392E8in the experimental populations that were selected for pyflubumide resistance. We further corroborated the involvement of CYP392A16 in resistance byin vitrofunctional 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 ofCPRand 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 ofCPR.

Author summary

Our 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 miteTetranychus 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, andin vitrocytochrome 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 theCPRlocus.

Data availability

Sequence data has been deposited at the Sequence Read Archive (PRJNA596790). Phenotypic quantification data is available at Supplementary Data S1.