Functional characterization of the Tetranychus urticae CYP392A11, a cytochrome P450 that hydroxylates the METI acaricides cyenopyrafen and fenpyroximate

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Highlights

  • Resistance against cyenopyrafen, a novel METI acaricide, has been associated with the upregulation of CYP392A11.

  • The recombinant protein CYP392A11 was shown to be capable of metabolizing cyenopyrafen and fenpyroximate.

  • Ectopic expression of tuCYP392A11 in D. melanogaster confers significant levels of fenpyroximate resistance.

  • TuCYP392A11 is overexpressed in strains, not previously exposed to cyenopyrafen, and is correlated with cyenopyrafen cross-resistance.

  • Luciferin-ME EGE is as a potential diagnostic substrate for detecting CYP392A11 – associated METI resistance.

Abstract

Cyenopyrafen is a Mitochondrial Electron Transport Inhibitor (METI) acaricide with a novel mode of action at complex II, which has been recently developed for the control of the spider mite Tetranychus urticae, a pest of eminent importance globally. However, some populations of T. urticae are cross-resistant to this molecule, and cyenopyrafen resistance can be readily selected in the lab. The cytochrome P450s genes CYP392A11 and CYP392A12 have been strongly associated with the phenotype. We expressed the CYP392A11 and the CYP392A12 genes with T. urticae cytochrome P450 reductase (CPR) in Escherichia coli. CYP392A12 was expressed predominately as an inactive form, witnessed by a peak at P420, despite optimization efforts on expression conditions. However, expression of CYP392A11 produced a functional enzyme, with high activity and preference for the substrates Luciferin-ME EGE and ethoxycoumarin. CYP392A11 catalyses the conversion of cyenopyrafen to a hydroxylated analogue (kcat = 2.37 pmol/min/pmol P450), as well as the hydroxylation of fenpyroximate (kcat = 1.85 pmol/min/pmol P450). In addition, transgenic expression of CYP392A11 in Drosophila melanogaster, in conjunction with TuCPR, confers significant levels of fenpyroximate resistance.

The overexpression of CYP392A11 in multi-resistant T. urticae strains, not previously exposed to cyenopyrafen, which had been indicated by microarray studies, was confirmed by qPCR, and it was correlated with significant levels of cyenopyrafen and fenpyroximate cross-resistance. The implications of our findings for insecticide resistance management strategies are discussed.

Introduction

The spider mite Tetranychus urticae is one of the most damaging agricultural pests world-wide. Its control has been based on the use of chemicals for many years. However, T. urticae is globally one of most resistant arthropod pests, both in terms of the total number of acaricides (93 currently) to which populations have become resistant and the levels of resistance (over 100-fold in many cases), due to its biology (arrhenotokous reproduction, short life-cycle and high fecundity) and strong insecticide selection pressure (Van Leeuwen et al., 2010; www.pesticideresistance.org).

Mitochondrial electron transport inhibitors (METI's) belong to a class of acaricides, which are known to effectively control T. urticae and other tetranychid mite species for many years, including populations resistant to other chemical classes of insecticides/acaricides. Although they are also effective against some other pest groups such as aphids and whiteflies, their main use is against spider mites. The METI acaricide family is divided into four different main groups by the Insecticide Resistance Action Committee (IRAC), i.e. complex I, II, III and IV inhibitors represented by a diverse range of chemical classes (Sparks and Nauen, 2015). Group 21 acaricides and insecticides share the same mode of action and target complex I of the respiratory chain in mitochondria, in particular the translocation of protons from NADH to ubiquinone oxidoreductase (Hollingworth and Ahammadsahib, 1995, Lümmen, 2007). Fenpyroximate is a complex I inhibitor and was discovered by Nihon Nohyaku in 1985 and commercialized in 1991 (Dekeyser, 2005). This compound is very effective against all life stages of T. urticae (Koch) and Panonychus citri (Motoba et al., 1992), while it has very low toxicity against beneficial insects, animal-parasitic mites and soil-living mites (Motoba et al., 1992). Cyenopyrafen is a relatively recently developed and commercialized acaricide (2009), which also targets mitochondrial electron transport, but at complex II (IRAC Group 25). It is a beta-ketonitrile which was developed by Nissan Chemicals in 2009 and shows strong acaricidal activity (Nakahira, 2011, Yu et al., 2012). This compound inhibits succinate-CoQ reductase in mitochondrial complex II (Nakahira, 2011) and shows very low toxicity against beneficial insects (Yu et al., 2012).

Although METI-acaricides have been used successfully for several years against T. urticae, there have been numerous reports of resistance development (reviewed in Van Leeuwen et al., 2010). Cross-resistance between METI's has also been observed in several cases (Stumpf and Nauen, 2001, Van Pottelberge et al., 2009). For example, laboratory selections with fenpyroximate and pyridaben conferred cross-resistance between METI's, while field selection with tebufenpyrad led to high cross-resistance to pyridaben, fenazaquin and fenpyroximate. Biochemical and synergism studies indicated that METI-resistance is, at least partially, associated with elevated cytochrome P450 monooxygenase (P450) activity in many different strains (Stumpf and Nauen, 2001, Tirrelo et al., 2012, Van Pottelberge et al., 2009). The highly METI resistant strain MR-VP characterized by Van Pottelberge et al. (2009), and cross-resistant to pyridaben, fenpyroximate and tebufenpyrad, exhibited a more than 20-fold increased P450 activity as measured by model substrates and synergistic ratios of almost 100-fold in combination with piperonylbutoxide (PBO), a P450 inhibitor, were strongly suggests oxidative detoxification.

Cyenopyrafen resistance mechanisms have been only recently studied in T. urticae. Sugimoto and Osakabe (2014) studied the possibility of cross–resistance between cyenopyrafen and the complex I inhibitor pyridaben. Experiments with synergists revealed that resistance to cyenopyrafen and pyridaben is at least partially driven by P450s and esterases. A more recent microarray-based study pointed towards two P450s, CYP392A11 and CYP392A12 as the most likely candidates conferring cyenopyrafen resistance (Khalighi et al., in press).

Cytochrome P450s (CYPs) belong to a protein superfamily of Heme-containing enzymes that catalyze the mono-oxygenation of many xenobiotics and endogenous compounds. CYPs have been implicated in the insecticide resistance of many pests (Feyereisen, 2005). Eighty-six P450 genes were found in the T. urticae genome, and among them several were shown to be associated with the multi-resistant phenotype, by a genome-wide microarray based expression analysis (Dermauw et al., 2013). Recently, some of these P450s have been functionally characterized and were shown to metabolize the acaricides spirodiclofen and spiromesifen (CYP392E10), and abamectin (CYP392A16) (Demaeght et al., 2013, Riga et al., 2014).

Here, we cloned and expressed CYP392A11 and CYP392A12 in Escherichia coli to examine their catalytic properties and their potential to metabolize cyenopyrafen and other acaricides. We also performed heterologous expression of CYP392A11 and TuCPR in Drosophila and conducted relevant bioassays examining acaricide toxicity of transgenic flies. We finally examined the cross resistance of multi-resistant of T. urticae strains which overexpress these genes but have never been exposed to cyenopyrafen.

Section snippets

Extraction of RNA, cDNA synthesis and RT-PCR

Total RNA was extracted from about 100 adult females or pools of 300 deutonymphs of each T. urticae strain (i.e. Marathonas and London, Dermauw et al., 2013), or 20 Drosophila flies using Tri Reagent (Sigma Aldrich). Extracted RNA samples were treated with Turbo DNase (Ambion) and were used to make first strand cDNA using oligo-dT primers with Superscript III reverse transcriptase (Invitrogen). The levels of P450 transcripts were measured by quantitative PCR (qPCR) amplification on a

Functional expression of CYP392A11/A12 with TuCPR in E. coli

The complex CYP392A11 and TuCPR was expressed functionally in E. coli and the proteins were directed to the inner bacterial membrane using the leading sequences ompA and pelB, respectively. The reduced CO-difference spectrum indicated that CYP392A11 was expressed predominately in its P450 form, indicative of a good-quality functional enzyme (Omura and Sato, 1964). However, CYP392A12 was expressed predominately as P420, despite several efforts to optimize expression conditions (data not shown).

Discussion

We show that the P450 CYP392A11, a gene that has been strongly implicated in cyenopyragen resistance (Khalighi et al., in press) and is also upregulated in multi-resistance T. urticae strains (Dermauw et al., 2013), encodes an enzyme that is capable of metabolizing cyenopyrafen and fenpyroximate. This is the first enzyme from an arthropod pest that is shown to metabolize at least two members of the important acaricide/insecticide family of METIs. Our study is in line with previous reports that

Acknowledgments

We thank Yannis Livadaras, Maria Monastirioti and Linda Grigoraki (Foundation for Research and Technology, Institute Molecular Biology and Biotechnology, FORTH-IMBB, Greece), for their help with the Drosophila work, and Mousaalreza Khalighi (University of Gent) for his help with the mite bioassays. We thank Philip Daborn (The University of Melbourne, Australia) for kindly sharing HR-GAL4 constructs and Drosophila lines used in our study. This research has been co-financed by the European Union

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