Honey bees are more vulnerable to amitraz under organophosphate insecticide exposure

Abstract Honey bees ( Apis mellifera ) play a critical role in pollination and agricultural productivity, contributing significantly to global food security and ecosystem stability. However, their populations are increasingly threatened by multiple stressors, and pesticide exposure, especially from agricultural insecticides and in-hive acaricides, has become a major concern due to its potentially low lethal and synergistic effects on bee health. This study investigates the potential synergistic effects of two organophosphate insecticides, dimethoate and chlorpyrifos, when combined with amitraz, a widely used acaricide for Varroa destructor control. Acute oral toxicity assays revealed that dimethoate exhibited the highest toxicity (LC₅ 0 = 2.6 ppm), followed by chlorpyrifos (56.3 ppm) and amitraz (432.3 ppm). Co-exposure to organophosphates and amitraz significantly reduced survival compared to single-compound treatments, with the most pronounced effect observed in the A 10 D 30 treatment group. To understand the underlying physiological effects, we analyzed the expression of key detoxification ( Am-CYP1 , Am-CYP2 , Am-CYP3 , Am-GST ) and oxidative stress-related genes ( Am-Cat , Am-SOD ), as well as Am-Vg . Vitellogenin expression was significantly suppressed at 24 h post-exposure ( F = 19.271, p < 0.001), particularly under mixture treatments, while stress-response genes ( Am-Cat and Am-SOD ) were consistently downregulated. In contrast, co-exposure induced significant upregulation of detoxification-related genes, including Am-CYP2 , Am-CYP3 , and Am-GST ( p < 0.01 in several treatments). These findings highlight the compounded risk posed by pesticide mixtures and emphasize the importance of conducting integrative risk assessments that consider realistic, multi-chemical exposure scenarios encountered by foraging bees.