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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.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.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.

The fat body was stained was stained for the wild-type control (Myo-GAL4; Top row) along with Mdr65KO (2nd row down), Mdr49KO (3rd row down), Mdr50KO (bottom). Blue indicates nuclei while red indicates the C219 P-gp signal.

The central nervous system was stained was stained for the wild-type control (Myo-GAL4; Top row) along with Mdr65KO (2nd row down), Mdr49KO (3rd row down), Mdr50KO (bottom). Blue indicates nuclei while red indicates the C219 P-gp signal.

Additional file 1.

Bioassay data is shown for both spinosad and fipronil against genotypes selectively overexpressing a single P-glycoprotein orthologue (Mdr49; C-D, Mdr50; E-F, or Mdr65; A-B) in the specific tissue corresponding to their proposed localization. Grey bars indicate genetically matched controls in all samples while colors correspond to specified overexpression genotypes.

Additional file 3.

The Malpighian tubule was stained was stained for the wild-type control (Myo-GAL4; Top row) along with Mdr65KO (2nd row down), Mdr49KO (3rd row down), Mdr50KO (bottom). Blue indicates nuclei while red indicates the C219 P-gp signal. Green indicates actin.

Additional file 6.

Additional file 5.

Additional file 4.

Additional file 2.

Additional file 2: Figure S2. The predicted numbers of ABC transporters in the ABC_scan pipeline were benchmarked against previously published ABC transporter datasets. The % difference in total number of ABC transporters (y-axis) was plotted for each species (x-axis). For all species tested, the difference was less than 10% (dotted line).

Additional file 4: Figure S4. A CAFE analysis for ABC-C proteins was performed with a focus on Coleoptera. Higher numbers of ABC-C proteins were observed in Cucujiformia beetles (e.g. T. castaneum, L. decemlineata) compared to other beetles and non-beetle arthropods. The numbers present next to each tip correspond to the number of predicted ABC-C transporters, while the node numbers correspond to CAFE predictions for ABC-B full transporter family sizes. Color coding of the nodes signifies bootstrap support with >90 % percent = Green; 70-90% =Yellow

Additional file 5: Figure S5. qPCR was performed for all ABC-H genes knocked down in N. viridula. For each gene at least 3 biological replicates were performed, and all values were calculated using the ΔΔCt method and normalized to 1.

Additional file 1: Figure S1. The total number of ABC transporters in each species (x-axis) was calculated for each species included in the analysis.

Additional file 3: Figure S3. A comparison was made among the non-variable ABC families (ABC-BH, ABC-D, ABC-E, and ABC-F) with family size (y-axis) broken down by both taxonomic order (x-axis) and family (panels). Orders are color coded, and boxplots include a horizontal black bar for median, boxes for upper quartiles, dots for outliers. Lower case letters above the boxes signify statistical groups generated by the Kruskal-Wallis test.