Contribution of <i>Nilaparvata lugens</i> Nicotinic Acetylcholine Receptor Subunits Toward Triflumezopyrim and Neonicotinoid Susceptibility

Nicotinic acetylcholine receptors (nAChRs) are the molecular targets of some important insecticides including triflumezopyrim and neonicotinoids. However, our understanding of insect nAChR pharmacology and the specific nAChR subunits targeted by these insecticides remains limited. Here, we cloned 11 nAChR subunit genes, comprising <i>Nlα1</i> to <i>Nlα8</i>, <i>Nlα10</i>, <i>Nlβ1</i> and <i>Nlβ3</i>, from <i>Nilaparvata lugens</i>, a highly damaging insect pest of rice crops worldwide. Analysis of the expression of these genes in different tissues of <i>N. lugens</i> by qPCR analysis identified the brain as the primary site of expression. Knock down of the expression of <i>Nlα1</i>, <i>Nlα2</i>, <i>Nlα8</i> and <i>Nlβ1</i> using RNAi reduced <i>N. lugens</i> sensitivity to triflumezopyrim, suggesting these genes encode potential target subunits for triflumezopyrim. Knock out of <i>Nlα2</i> and <i>Nlα8</i> nAChR subunits by CRISPR/Cas9 genome editing showed that their deletion significantly reduced the toxicity of triflumezopyrim toward <i>N. lugens</i>. Furthermore, the deletion of <i>Nlα2</i> also increased <i>N. lugens</i> resistance to imidacloprid and dinotefuran. However, numerous attempts revealed that the <i>Nlβ1</i> knockout was nonviable. <i>In vitro</i> expression of receptors composed of <i>Drosophila</i> homologous subunits showed that this all-insect nAChR was inhibited by nanomolar concentrations of triflumezopyrim. The present findings identify specific nAChR subunits that are important both as targets for monitoring resistance-associated mutations and as subjects for molecular studies aimed at developing novel insecticides targeting these essential ion channels.