Insect nicotinic receptor interactions in vivo with neonicotinoid,organophosphorus, and methylcarbamate insecticides and a synergist

The nicotinic acetylcholine (ACh) receptor (nAChR) is the principal insecticide target. Nearly half of the insecticides by number and world market value are neonicotinoids acting as nAChR agonists or organophosphorus (OP) and methylcarbamate (MC) acetylcholinesterase (AChE) inhibitors. There was no previous evidence for in vivo interactions of the nAChR agonists and AChE inhibitors. The nitromethyleneimidazole (NMI) analog of imidacloprid, a highly potent neonicotinoid, was used here as a radioligand, uniquely allowing for direct measurements of house fly (Musca domestica) head nAChR in vivo interactions with various nicotinic agents. Nine neonicotinoids inhibited house fly brain nAChR ³H]NMI binding in vivo, corresponding to their in vitro potency and the poisoning signs or toxicity they produced in intrathoracically treated house flies. Interestingly, nine topically applied OP or MC insecticides or analogs also gave similar results relative to in vivo nAChR binding inhibition and toxicity, but now also correlating with in vivo brain AChE inhibition, indicating that ACh is the ultimate OP- or MC-induced nAChR active agent. These findings on ³H]NMI binding in house fly brain membranes validate the nAChR in vivo target for the neonicotinoids, OPs and MCs. As an exception, the remarkably potent OP neonicotinoid synergist, O-propyl O-(2-propynyl) phenylphosphonate, inhibited nAChR in vivo without the corresponding AChE inhibition, possibly via a reactive ketene metabolite reacting with a critical nucleophile in the cytochrome P450 active site and the nAChR NMI binding site.The nicotinic nervous system has two principal sites of insecticide action, the nicotinic receptor (nAChR) activated by acetylcholine (ACh) and neonicotinoid agonists (1–6), and acetylcholinesterase (AChE) inhibited by organophosphorus (OP) and methylcarbamate (MC) compounds to generate and maintain localized toxic ACh levels (Fig. 1) (7). The nAChR and AChE targets have been identified in insects by multiple techniques but not by direct assays of the ACh binding site in the brain of poisoned insects. Here we use the outstanding insecticidal potency of the nitromethyleneimidazole (NMI) analog of imidacloprid (IMI) (8) as a radioligand (9), designated ³H]NMI, to directly measure the house fly (Musca domestica) nAChR not only in vitro but also in vivo, allowing us to validate by a previously undescribed method the neonicotinoid direct and OP/MC indirect nAChR targets (Fig. 2). This approach also helped solve the intriguing mechanism by which an O-(2-propynyl) phosphorus compound strongly synergizes neonicotinoid insecticidal activity (10) by dual inhibition of cytochrome P450 (CYP) (11–13) and the nAChR agonist site (described herein). Insecticide disruption at the insect nAChR can now be readily studied in vitro and in vivo with a single radioligand allowing better understanding of the action of several principal insecticide chemotypes (Fig. 3).Open in a separate windowFig. 1.The insect nicotinic receptor is the direct or indirect target for neonicotinoids, organophosphorus compounds and methylcarbamates, which make up about 45% of the insecticides by number and world market value (2, 7).Open in a separate windowFig. 2.In this study, Musca nicotinic receptor in vivo interactions with major insecticide chemotypes are revealed by a ³H]NMI radioligand reporter assay. *Position of tritium label.Open in a separate windowFig. 3.Two neonicotinoid nicotinic agonists and two anticholinesterase insecticides.