title: Molecular analysis of resistance to acaricidal spirocyclic tetronic acids in Tetranychus urticae: CYP392E10 metabolizes spirodiclofen, but not its corresponding enol.
abstract: Spirodiclofen is one of the most recently developed acaricides and belongs to the new family of spirocyclic tetronic acids (ketoenols). This new acaricidal family is an important chemical tool in resistance management strategies providing sustainable control of spider mites such as Tetranychus urticae. Spirodiclofen targets lipid biosynthesis mediated by direct inhibition of acetyl coenzyme A carboxylase (ACCase). In this study, we investigated two genetically distant spider mite strains with high resistance to spirodiclofen. Despite the strong resistance levels to spirodiclofen (up to 680-fold), only limited cross-resistance with other members of this group such as spiromesifen and spirotetramat could be detected. Amplification and sequencing of the ACCase gene from resistant and susceptible strains did not reveal common non-synonymous mutations, and expression levels of ACCase were similar in both resistant and susceptible strains, indicating the absence of target-site resistance. Furthermore, we collected genome-wide expression data of susceptible and resistant T. urticae strains using microarray technology. Analysis of differentially expressed genes revealed a broad response, but within the overlap of two resistant strains, several cytochrome P450s were prominent. Quantitative PCR confirmed the constitutive over-expression of CYP392E7 and CYP392E10 in resistant strains, and CYP392E10 expression was highly induced by spirodiclofen. Furthermore, stage specific expression profiling revealed that expression levels were not significantly different between developing stages, but very low in eggs, matching the age-dependent resistance pattern previously observed. Functional expression of CYP392E7 and CYP392E10 confirmed that CYP392E10 (but not CYP392E7) metabolizes spirodiclofen by hydroxylation as identified by LC-MS/MS, and revealed cooperative substrate binding and a Km of 43 μM spirodiclofen. CYP392E10 also metabolizes spiromesifen, but not spirotetramat. Surprisingly, no metabolism of the hydrolyzed spirodiclofen-enol metabolite could be detected. These findings are discussed in the light of a likely resistance mechanism.
authors.affiliation: Department of Crop Protection, Faculty of Bioscience Engineering, Coupure Links 653, Ghent University, B-9000 Ghent, Belgium.
authors.forename: Peter
authors.initials: P
authors.lastname: Demaeght
authors.forename: Wannes
authors.initials: W
authors.lastname: Dermauw
authors.forename: Dimitra
authors.initials: D
authors.lastname: Tsakireli
authors.forename: Jahangir
authors.initials: J
authors.lastname: Khajehali
authors.forename: Ralf
authors.initials: R
authors.lastname: Nauen
authors.forename: Luc
authors.initials: L
authors.lastname: Tirry
authors.forename: John
authors.initials: J
authors.lastname: Vontas
authors.forename: Peter
authors.initials: P
authors.lastname: Lümmen
authors.forename: Thomas
authors.initials: T
authors.lastname: Van Leeuwen
mesh_terms: D015107:4-Butyrolactone / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology; D000103:Acetyl-CoA Carboxylase / Q000037:antagonists & inhibitors / Q000096:biosynthesis*; D000818:Animals; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D005663:Furans / Q000494:pharmacology*; D007305:Insecticide Resistance / Q000187:drug effects / Q000235:genetics*; D007306:Insecticides / Q000494:pharmacology; D008055:Lipids / Q000096:biosynthesis; D013141:Spiro Compounds / Q000737:chemistry / Q000494:pharmacology*; D053719:Tandem Mass Spectrometry; D040102:Tetranychidae / Q000187:drug effects / Q000378:metabolism
chemical_list: D005663:Furans; D007306:Insecticides; D008055:Lipids; D013141:Spiro Compounds; C508135:spirodiclofen; D003577:Cytochrome P-450 Enzyme System; D000103:Acetyl-CoA Carboxylase; C003422:tetronic acid; D015107:4-Butyrolactone