Physiological and gut microbial responses of <scp> <i>Apis cerana</i> </scp> to sub‐chronic exposure to binary pesticide mixtures at environmentally relevant concentrations

BACKGROUND: As an irreplaceable indigenous pollinator safeguarding biodiversity and crop productivity across mountainous Southwest China, Apis cerana is commonly exposed to pesticide mixtures. The neonicotinoid thiamethoxam (TMX), diamide chlorantraniliprole (CAP) and triazole difenoconazole (DIF) are widely detected in its beehives across Guizhou karst regions as single or binary mixtures. However, the combined toxicological effects of these pesticides on honeybees remain unclear. In this study, newly emerged A. cerana workers were sub-chronically exposed to individual pesticides and their binary mixtures (CAP-DIF, CAP-TMX, DIF-TMX) at field-relevant concentrations. The toxic impacts of these single pesticides and their binary combinations on honeybee survival, body weight, food consumption and physiological functions were investigated. In addition, their effects on honeybee gut microbiota were evaluated. RESULTS: Our results demonstrated that CAP-DIF and CAP-TMX induced oxidative damage, inhibited acetylcholinesterase activity, and elevated honeybee mortality. Although DIF-TMX did not significantly reduce survival rates, it triggered oxidative damage and decreased the relative abundances of core microbiota. Furthermore, all binary mixtures downregulated expression of the immune gene abaecin. CONCLUSION: Our study reveals that binary pesticide mixtures can disrupt physiological homeostasis in newly emerged A. cerana workers even at environmentally relevant concentrations. CAP-DIF and CAP-TMX increase mortality via synergistic toxicity, highlighting health hazards stemming from such combined pesticide exposure. Unlike reported azole-neonicotinoid synergism, DIF failed to enhance TMX toxicity in A. cerana under field-relevant concentrations. These findings advance regional pesticide risk evaluation, inform targeted pesticide management and native honeybee conservation, securing intact ecosystems and pollination-dependent food security. © 2026 Society of Chemical Industry.