Study On The Molecular Targets Of N Neonicotinoids And Related Insecticides

Nicotinic acetylcholine receptors（nAChRs）,the principal excitatory receptor of the insect central nervous system,are also the molecular targets of several important insecticides,such as neonicotinoids.The model animals Drosophila melanogaster have ten genes（Dα1-7 and Dβ1-3）encoding nAChR subunits.The insect nAChR is a pentameric assembly by these subunits,therefore,many combinations of these subunits could exist produced a series of candidate nAChR with different physiological properties.An important question is these insecticides act on which nAChR subunits against agricultural pests?In this study,we collected 8 subunit mutant flies and constructed a point mutation nAChRβ1^R81T by CRISPR/Cas9technology.We systematically tested the sensitivity of 9 mutants to eleven insecticides,including seven neonicotinoids,sulfoxaflor,flupyradifurone,triflumezopyrim and spinetoram.We also analysed the differential expression of nAChR subunit genes in nAChR mutants.Construction of nAChRβ1^R81T mutant and nAChRα1KORFP,nAChRβ1^R81Tdouble mutants.We found that homozygous mutations of nAChRα4KORFP and nAChRβ1KORFP were lethal,which proved that Dα4 and Dβ1 play a vital role in normal development of flies.Therefore,we used CRISPR/Cas9 technology to construct Dβ1^R81T mutant.Previous studies have been reported that mutation of nAChRβ1 subunit,arginine mutated to threonine substitution（R81T）,is associated with resistance to neonicotinoids in the aphid Myzus persicae.In this study,we successfully constructed nAChRβ1^R81T mutant.and we also constructed two double mutants nAChRα1KORFP,nAChRβ1^R81T by Genetic recombination.The sensitivity of each nAChR subunit mutant on nAChR modulatorsWe systematically tested the sensitivity of 9 mutants to 11 insecticides target on nAChRs by bioassays.We found that nAChRβ1^R81T showed high resistance to all chemicals but spinetoram.We also found that neonicotinoids acted on multiple nAChR subunits,spinetoram,however,showed specific resistance to nAChRα6KORFP mutants.Mutation of a specific nAChR subunit showed significantly different resistance to neonicotinoids and other nAChR competitive modulators--sulfoxaflor,flupyradifurone and triflumezopyrim,demonstrating that the nAChR subtypes they target do not completely overlap.Differential expression levels of nAChR subunit genes in nAChR mutants.We detected the expression levels of subunit genes by qPCR in the adult flies’head,and no significant genetic compensation effect was foundOur study initially explores the molecular targets of neonicotinoids and related nAChR modulators,and promotes the study of the composition and function of insect nAChRs.