Sodium channels are excellent targets of both natural and synthetic insecticides with high insect selectivity. Indoxacarb, its active metabolite DCJW, and metaflumizone (MFZ) belong to a relatively new class of sodium channel blocker insecticides (SCBIs) with a mode of action distinct from all other sodium channel-targeting insecticides, including pyrethroids. Electroneutral SCBIs preferably bind to and trap sodium channels in the inactivated state, a mechanism similar to that of cationic local anesthetics. Previous studies identified several SCBI-sensing residues that face the inner pore of sodium channels. However, the receptor site of SCBIs, their atomic mechanisms, and the cause of selective toxicity of MFZ remain elusive. Here, we have built a homology model of the open-state cockroach sodium channel BgNa_v1-1a. Our computations predicted that SCBIs bind in the inner pore, interact with a sodium ion at the focus of P1 helices, and extend their aromatic moiety into the III/IV domain interface (fenestration). Using model-driven mutagenesis and electrophysiology, we identified five new SCBI-sensing residues, including insect-specific residues. Our study proposes the first three-dimensional models of channel-bound SCBIs, sheds light on the molecular basis of MFZ selective toxicity, and suggests that a sodium ion located in the inner pore contributes to the receptor site for electroneutral SCBIs.

Olfaction is crucial in mediating various behaviors of social insects such as red imported fire ants, Solenopsis invicta Buren. Olfactory receptor (OR) complexes consist of odor-specific ORs and OR co-receptors (Orco). Orcos are highly conserved across insect taxa and are widely co-expressed with ORs. Odorant binding proteins (OBPs) can transport semiochemicals to ORs as protein carriers and thus constitute the first molecular recognition step in insect olfaction. In this study, three OBP genes highly expressed in S. invicta antenna, OBP1, OBP5, OBP6, and Orco were partially silenced using RNA interference (RNAi). RNAi SiOBP5- and Orco-injected ants showed significantly lower EAG (electroantennography) responses to fire ant alarm pheromones and the alkaloid, 2,4,6-trimethylpyridine than water- or GFP-injected ants 72 h post injection. Subsequent qRT-PCR analysis demonstrated that the transcript level of the OBP1, OBP5, OBP6, and Orco significantly decreased 72 h after ants were injected with dsRNAs; however, there were no transcript level or EAG changes in ants fed dsRNAs. Our results suggest that S. invicta Orco and SiOBP5 are crucial to fire ants for their responses to alarm pheromones. RNAi knocking down SiOBP5 can significantly disrupt alarm pheromone communication, suggesting that disrupting SiOBP5 and Orcos could be potentially useful in the management of red imported fire ants.

The toxicity of synthetic pesticides to non-target organisms has prompted a shift towards more environmentally friendly agricultural pest control methods, including the use of essential oils as possible biopesticides. Before these natural chemicals can be widely adopted for protecting food supplies and human health, it is crucial to evaluate their impacts on pollinators, such as honey bees. In this study, we examined the effects of one commercially available essential oil mixture (EcoTec+) and four essential oil components (β-bisabolene, cinnamaldehyde, 1,8-cineole, and eugenol) on honey bee workers using feeding or spray treatment. We then assessed the responses of esterase (EST), glutathione-S-transferase (GST), acetylcholine esterase (AChE), and P450. EcoTec+ increased the P450 transcript, while bisabolene inhibited EST and AChE, increased GST, and caused a mixed P450 response without being lethal. Cinnamaldehyde exhibited toxicity when ingested, suppressing P450 and eliciting a mixed response in AChE. Cineole inhibited EST but caused a mixed P450 response. Eugenol suppressed EST and AChE and was toxic on contact. We also assayed combinations of each compound with four synthetic formulations representative of the major pesticide categories, though no significant interactions were found. Overall, the essential oils tested did not cause acute lethal toxicity to honey bees; however, their biochemical effects varied, mostly remaining sublethal. These findings suggest that these essential oils could be considered safe for use around honey bees.