Knockin of the G275E mutation of the nicotinic acetylcholine receptor (nAChR) α6 confers high levels of resistance to spinosyns in Spodoptera exigua

Spinosyns,including spinosad and spinetoram,act on the insect central nervous system,gradually paralyzing or destroying the target insect.Spinosad resistance is associated with loss-of-function mutations in the nicotinic acetylcholine receptor(nAChR)α6 subunit in a number of agricultural pests.Using gene editing,nAChRα6 has been verified as a target for spinosyns in five insect species.Recently,a point mutation(G275E)in exon 9 of nAChRα6 was identified in spinosad-resistant strains of Thrips palmi and Tuta absoluta.To date,no in vivo functional evidence has been obtained to support that this mutation is involved in spinosyn resistance in lepidopteran pests.In this study,the G275E mutation was introduced into the nAChR of Spodoptera exigua using clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)gene-editing technology.Reverse transcriptase-polymerase chain reaction and sequencing confirmed that this mutation was present in exon 9 of the nAChR transcripts in the edited 275E strain.The results of bioassays showed that the 275E strain was highly resistant to spinosad(230-fold)and spinetoram(792-fold)compared to the unedited background strain,directly confirming that the G275E mutation of the nAChRα6 subunit confers high levels of spinosyn resistance in S.exigua.Inheritance analysis showed that the resistance trait is autosomal and incompletely recessive.This study employs a reverse genetics approach to validate the functional role played by the G275E mutation in nAChRα6 of S.exigua in spinosyns resistance and provides another example of the use of CRISPR/Cas9 gene-editing technology to confirm the role played by candidate target site mutations in insecticide resistance.