Evaluating the combined toxicity of broflanilide and myclobutanil on honeybees (Apis mellifera L.): Molecular mechanisms and protective effects of curcumin

Pesticide toxicity to honeybees has become a pressing ecological issue, yet the effects of pesticide co-exposure are still not fully understood. This research investigates the toxicological implications of concurrent exposure to broflanilide (BFL), a novel diamide insecticide, and myclobutanil (MYC), a commonly used triazole fungicide, on honeybees (Apis mellifera L.), while exploring potential preventive strategies. Acute toxicity tests revealed a significantly lower 96-hour lethal concentration 50 (LC₅₀) for BFL (0.34 mg a.i. L⁻¹) compared to MYC (82.3 mg a.i. L⁻¹), and their co-exposure resulted in pronounced synergistic toxicity. Worker bees were exposed to environmentally relevant doses of BFL and MYC for 7 days, and midgut toxicity was assessed. The co-exposure caused severe midgut damage, including G-layer deterioration, loss of columnar epithelium integrity, and downregulation of the tight junction protein ZO-2. Additionally, oxidative stress-related genes (Sod1, Catalase, SelK, GstD1) were upregulated, accompanied by higher MDA levels and increased CAT and SOD activities. Furthermore, a greater number of TUNEL-positive cells were detected, along with elevated expression of apoptosis-related genes (Caspase-3-like, Caspase-8-like, Caspase-9-like) and higher caspase enzyme activities. Curcumin (Cur) was tested for its protective effects, and it significantly alleviated midgut damage, oxidative stress, and apoptosis. This study reveals the synergistic ecotoxicological effects of pesticide combinations and suggests Cur as a potential prevention strategy for mitigating their harmful impact on honeybees.