Contrasting roles of cytochrome P450s in amitraz and chlorfenapyr resistance in the crop pest Tetranychus urticae.

The molecular mechanisms of amitraz and chlorfenapyr resistance remain only poorly understood for major agricultural pests and vectors of human diseases. This study focusses on a multi-resistant field strain of the crop pest Tetranychus urticae, which could be readily selected in the laboratory to high levels of amitraz and chlorfenapyr resistance. Toxicity experiments using tralopyril, the active toxophore of chlorfenapyr, suggested decreased activation as a likely mechanism underlying resistance. Starting from the same parental strain, transcriptome profiling revealed that a cluster of detoxifying genes was upregulated after amitraz selection, but unexpectedly downregulated after chlorfenapyr selection. Further functional validation associated the upregulation of CYP392A16 with amitraz metabolism and the downregulation of CYP392D8 with reduced activation of chlorfenapyr to tralopyril. Genetic mapping (QTL analysis by BSA) was conducted in an attempt to unravel the genetic mechanisms of expression variation and resistance. This revealed that chlorfenapyr resistance was associated with a single QTL, while 3 QTLs were uncovered for amitraz resistance. Together with the observed contrasting gene expression patterns, we argue that transcriptional regulators most likely underly the distinct expression profiles associated with resistance, but these await further functional validation.

A glutamate-gated chloride channel as the mite-specific target-site of dicofol and other diphenylcarbinol acaricides.

Dicofol has been widely used to control phytophagous mites. Although dicofol is chemically related to DDT, its mode of action has remained elusive. Here, we mapped dicofol resistance in the spider mite Tetranychus urticae to two genomic regions. Each region harbored a glutamate-gated chloride channel (GluCl) gene that contained a mutation-G314D or G326E-known to confer resistance against the unrelated acaricide abamectin. Using electrophysiology assays we showed that dicofol and other diphenylcarbinol acaricides-bromopropylate and chlorobenzilate-induce persistent currents in Xenopus oocytes expressing wild-type T. urticae GluCl3 receptors and potentiate glutamate responses. In contrast, the G326E substitution abolished the agonistic activity of all three compounds. Assays with the wild-type Drosophila GluClα revealed that this receptor was unresponsive to dicofol. Homology modeling combined with ligand-docking confirmed the specificity of electrophysiology assays. Altogether, this work elucidates the mode of action of diphenylcarbinols as mite-specific agonists of GluCl.

Activity, selection response and molecular mode of action of the isoxazoline afoxolaner in Tetranychus urticae.

BACKGROUND: Afoxolaner is a novel representative of the isoxazolines, a class of ectoparasiticides which has been commercialized for the control of tick and flea infestations in dogs. In this study, the biological efficacy of afoxolaner against the two-spotted spider mite Tetranychus urticae was evaluated. Furthermore, as isoxazolines are known inhibitors of γ-aminobutyric acid-gated chloride channels (GABACls), the molecular mode of action of afoxolaner on T. urticae GABACls (TuRdls) was studied using functional expression in Xenopus oocytes followed by two-electrode voltage-clamp (TEVC) electrophysiology, and results were compared with inhibition by fluralaner, fipronil and endosulfan. To examine the influence of known GABACl resistance mutations, H301A, I305T and A350T substitutions in TuRdl1 and a S301A substitution in TuRdl2 were introduced. RESULTS: Bioasassays revealed excellent efficacy of afoxolaner against all developmental stages and no cross-resistance was found in a panel of strains resistant to most currently used acaricides. Laboratory selection over a period of 3 years did not result in resistance. TEVC revealed clear antagonistic activity of afoxolaner and fluralaner for all homomeric TuRdl1/2/3 channels. The introduction of single, double or triple mutations to TuRdl1 and TuRdl2 did not lower channel sensitivity. By contrast, both endosulfan and fipronil had minimal antagonistic activities against TuRdl1/2/3, and channels carrying single mutations, whereas the sensitivity of double and triple TuRdl1 mutants was significantly increased. CONCLUSIONS: Our results demonstrate that afoxolaner is a potent antagonist of GABACls of T. urticae and has a powerful mode of action to control spider mites. © 2022 Society of Chemical Industry.

Untangling a Gordian knot: the role of a GluCl3 I321T mutation in abamectin resistance in Tetranychus urticae.

BACKGROUND: The cys-loop ligand-gated ion channels, including the glutamate-gated chloride channel (GluCl) and GABA-gated chloride channel (Rdl) are important targets for drugs and pesticides. The macrocyclic lactone abamectin primarily targets GluCl and is commonly used to control the spider mite Tetranychus urticae, an economically important crop pest. However, abamectin resistance has been reported for multiple T. urticae populations worldwide, and in several cases was associated with the mutations G314D in GluCl1 and G326E in GluCl3. Recently, an additional I321T mutation in GluCl3 was identified in several abamectin resistant T. urticae field populations. Here, we aim to functionally validate this mutation and determine its phenotypic strength. RESULTS: The GluCl3 I321T mutation was introgressed into a T. urticae susceptible background by marker-assisted backcrossing, revealing contrasting results in phenotypic strength, ranging from almost none to 50-fold. Next, we used CRISPR-Cas9 to introduce I321T, G314D and G326E in the orthologous Drosophila GluCl. Genome modified flies expressing GluCl I321T were threefold less susceptible to abamectin, while CRISPRed GluCl G314D and G326E flies were lethal. Last, functional analysis in Xenopus oocytes revealed that the I321T mutation might reduce GluCl3 sensitivity to abamectin, but also suggested that all three T. urticae Rdls are affected by abamectin. CONCLUSION: Three different techniques were used to characterize the role of I321T in GluCl3 in abamectin resistance and, combining all results, our analysis suggests that the I321T mutation has a complex role in abamectin resistance. Given the reported subtle effect, additional synergistic factors in resistance warrant more investigation. © 2020 Society of Chemical Industry.

A G326E substitution in the glutamate-gated chloride channel 3 (GluCl3) of the two-spotted spider mite Tetranychus urticae abolishes the agonistic activity of macrocyclic lactones.

BACKGROUND: The macrocyclic lactones abamectin and milbemectin are frequently used to control phytophagous mites such as Tetranychus urticae. Consequently, resistance has developed and was genetically linked with substitutions in the glutamate-gated chloride channel (GluCl) subunits TuGluCl1 and TuGluCl3. Here, we functionally validated a G326E substitution in TuGluCl3 by functional expression in Xenopus laevis oocytes followed by two-electrode voltage-clamp electrophysiology. RESULTS: Homomeric wild-type and mutated GluCl3 were successfully expressed. l-glutamic-acid-induced currents exhibited a rapid onset equal in both channels and EC50 for l-glutamic-acid was in the micromolar range (384.2 µm and 292.7 µm, respectively). Abamectin and milbemycin A4 elicited sustained currents in wild-type GluCl3, but the G326E substitution completely abolished the agonistic activity of macrocyclic lactones. CONCLUSION: A target-site mutation in Tu GluCl3 contributes to avermectin resistance in T. urticae. However, given the multitude of channel genes and the potential additive or synergistic effects of mutations, to what extent mutations determine the often extremely strong resistance phenotype in the field deserves further study. © 2017 Society of Chemical Industry.