Functional characterization of the Tetranychus urticae CYP392A11, a cytochrome P450 that hydroxylates the METI acaricides cyenopyrafen and fenpyroximate
Graphical abstract
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
References (32)
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding
Anal. Biochem.
(1976)- et al.
Evaluating the insecticide resistance potential of eight Drosophila melanogaster cytochrome P450 genes by transgenic over-expression
Insect Biochem. Mol. Biol.
(2007) - et al.
Molecular analysis of resistance to acaricidal spirocyclic tetronic acids in Tetranychus urticae: CYP392E10 metabolizes spirodiclofen
Insect Biochem. Mol. Biol.
(2013) Insect cytochrome P450
- et al.
Structural model and functional characterization of the Bemisia tabaci CYP6CM1vQ, a cytochrome P450 associated with high levels of neonicotinoid resistance
Insect Biochem. Mol. Biol.
(2009) - et al.
Effect of a new acaricide, Fenpyroximate, on energy metabolism and mitocondrial morphology in adult female Tetranychus urticae (two-spotted spider mite)
Pest. Biochem. Physiol.
(1992) - et al.
Species – specific detoxification metabolism of fenpyroximate, a potent acaricide
Pest. Biochem. Physiol.
(2000) - et al.
Development of a lateral flow test to detect metabolic resistance in Bemisia tabaci mediated by CYP6CM1, a cytochrome P450 with broad spectrum catalytic efficiency
Pestic. Biochem. Physiol.
(2015) - et al.
The carbon monoxide-binding pigment of liver microsomes I. evidence for its hemoprotein nature
J. Biol. Chem.
(1964) - et al.
Transgenic expression of the Aedes aegypti CYP9J28 confers pyrethroid resistance in Drosophila melanogaster
Pest. Biochem. Physiol.
(2012)
Abamectin is metabolized by CYP392A16, a cytochrome P450 associated with high levels of acaricide resistance in Tetranychus urticae
Insect Biochem. Mol. Biol.
Purification and properties of NADPH cytochrome P-450 reductase
Methods Enzymol.
Acaricide resistance mechanisms in the two-spotted spider mite Tetranychus urticae and other important Acari: a review
Insect Biochem. Mol. Biol.
Cis-regulatory elements in the Accord retrotransposon result in tissue specific expression of the Drosophila melanogaster insecticide resistance gene Cyp6g1
Genetics
Acaricide mode of action
Pest. Manag. Sci.
Link between host plant adaptation and pesticide resistance in the polyphagous spider mite Tetranychus urticae
Proc. Natl. Acad. Sci. U. S. A.
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