Synergistic disruption of detoxification, immunity, and neural pathways in honeybees following co-exposure to cyantraniliprole and difenoconazole

The diamide insecticide cyantraniliprole (CYA) and the triazole fungicide difenoconazole (DIF) are frequently co-detected in bee-related matrices. However, the interactive effects of these compounds on honey bee (Apis mellifera L.) physiology remain insufficiently elucidated. Our results revealed that co-exposure to CYA and DIF elicited a pronounced acute synergistic toxicity. Biochemical assays demonstrated significant elevations in malondialdehyde (MDA) level, superoxide dismutase (SOD), and caspase-3 (CASP-3) activities across all treatments, with the most marked alterations occurring under co-exposure conditions. These data pointed to exacerbated oxidative stress and mitochondrial impairment when both pesticides were present concurrently. At the transcriptional level, notable dysregulation was observed in genes associated with apoptosis (caspase-1), detoxification (CYP4G11), immune modulation (dorsal-2), and lifespan regulation [vitellogenin (vtg)]. Notably, co-exposure intensified gene expression changes beyond those induced by single-pesticide treatments, underscoring a compound interaction that amplified cellular stress responses. These findings demonstrated that both CYA and DIF, especially in combination, disrupted critical physiological pathways in honey bees, compromising their detoxification capacity, immune integrity, and longevity. These insights not only unraveled key mechanistic underpinnings of pesticide mixture toxicity but also emphasized the urgent need for regulatory frameworks that address the mixture risks posed by agrochemical co-exposures in pollinator populations.