Competitive AChE inhibition: a novel secondary mechanism driving pyridaben's sublethal neurotoxicity

Abstract BACKGROUND The widely‐used acaricide pyridaben is classified as a mitochondrial complex I inhibitor (IRAC Group 21). However, a persistent disconnect exists, as it induces acute neuro‐excitatory symptoms including ataxia, tremors, leg paralysis and wing buzzing that are phenotypically inconsistent with a purely metabolic mode‐of‐action but hallmark cholinergic poisoning. This mechanistic ambiguity complicates resistance prediction and obscures the true sublethal hazard to nontarget arthropods. We therefore tested the hypothesis that its documented sublethal neurotoxicity is driven by direct, off‐target inhibition of acetylcholinesterase (AChE) and characterized the mode of this inhibition. RESULTS Sublethal pyridaben exposure (LC 20 = 5.2 μg mL −1 ; LC 30 = 8.2 μg mL −1 ) in Drosophila melanogaster caused significant neurobehavioral impairment, including 58% reduced locomotion and abolished olfactory attraction with response index declining from +0.50 to −0.20. In vitro , pyridaben acted as a competitive AChE inhibitor (mean inhibitory concentration IC 50 = 9.8 ± 1.2 μ m ; inhibitory K ᵢ = 7.2 ± 0.8 μ m ; n = 6 replicates). Critically, ex vivo analysis of exposed flies confirmed dose‐dependent AChE inhibition (29% reduction at LC 20 ; 46% reduction at LC 30 ), concurrent with behavioral deficits, demonstrating in vivo target engagement. This mechanism was conserved in the agriculturally significant pest Drosophila suzukii . CONCLUSION Competitive AChE inhibition is a novel secondary mechanism contributing to pyridaben's sublethal neurotoxicity. These findings resolve the toxicological paradox of its symptomology and provide a mechanistic basis for understanding behavioral impairment in exposed insects, with implications for refining diagnostic resistance monitoring approaches and informing mechanism‐based insecticide rotation strategies for sustainable pest management. © 2026 Society of Chemical Industry.