Environmental risk of an insecticide: High toxicity of broflanilide and its metabolite to honeybees.

Broflanilide, a widely used meta-diamide insecticide, poses severe environmental risks to honeybees (Apis mellifera L.), yet its toxic mechanisms remain unclear. Here we demonstrate that broflanilide functions as a pro-insecticide in honeybees, requiring metabolic activation to exert its potent toxicity. Its N-demethylated metabolite (II-03) exhibits 24,000-fold stronger inhibition of the honeybee GABA receptor RDL and 10-fold higher acute toxicity than the parent compound, suggesting that metabolic activation is a critical step underlying honeybee poisoning. Further mechanistic studies reveal that this potent toxicity arises from the synergistic interaction between the meta-diamide scaffold and the polyfluorinated moiety. The polyfluorinated moiety forms a stable halogen-bond network with two critical residues in the RDL binding pocket-Gln258 and Gly320-thereby enhancing the binding affinity and inhibitory potency of II-03 toward the receptor. Multi-omics analyses revealed molecular changes associated with broflanilide exposure, including a dual-hit process of enhanced membrane degradation and blocked synthesis, along with membrane disruption, mitochondrial dysfunction, and neurotransmitter signaling disorder. These findings suggest a possible cascade that may lead to immunosuppression. Field-relevant exposure experiments confirm that due to this rapid and efficient metabolic activation, the environmental hazard of broflanilide to honeybees has been underestimated. Based on these mechanistic insights, we propose a pollinator-safety design strategy: replace the halogen-rich moiety and avoid molecular interactions with the Gln258 and Gly320 residues of the RDL receptor. This study elucidates the toxic mechanism and environmental risk of broflanilide to honeybees, providing guidance for developing safer next-generation insecticides to ensure sustainable pollinator protection.