Characterization Of The Nicotinic Acetylcholine Receptor Gene Family Of Helicoverpa Armigera And Functional Validation Of NAChR α6 Subunit As The Target Of Spinosad

Cotton bollworm(Helicoverpa armigera)is one of the most destructive crop pests in the world.This species is highly polyphagous and can feed on more than 200 host plants.H.armigera has developed resistance to many traditional insecticides,including pyrethroids and carbamates.Therefore,the control of H.armigera depends on newer selective insecticides,such as spinosad,emamectin benzoate,indoxacarb and chloantraniliprole.Spinosad is a macrolide insecticide,which has a good control effect on lepidopteran pests.Its target is the insect nicotinic acetylcholine receptors(nAChRs).However,mutations in the nAChR α6 subunit(nAChRα6)detected in several agricultural pests have been found to be associated with high levels of resistance to spinosad in field and laboratory-selected strains.Although resistance to spinosad has not been reported in H.armigera,there is still a need to understand its resistance mechanism in order to develop new resistance monitoring methods and improve resistance management strategies.In this study,the nAChR gene family in the H.armigera genome was identified and analyzed.The role of the two nAChR subunits of H.armigera(Haα6 and Haα7)in spinosad resistance was also assessed using CRISPR/Cas9 gene editing technology.A total of 12 nAChR subunits were identified in the H.armigera genome.Gene knockout experiments confirmed that Haα6 is a functional receptor for spinosad in H.armigera,while Haα7 is not associated with the toxicity of spinosad.This study lays a foundation for the development of molecular detection techniques for the target resistance of H.armigera to spinosad,and provides a basis for developing resistance management strategies based on the rotation of insecticides with different modes of action.1.Analysis of the nAChR gene family of H.armigeraUsing the BLAST sequence analysis tool,12 nAChR subunit genes were identified from the reference genome of the Australian H.armigera.According to the synteny between lepidopteran insects,the 12 nAChR subunit genes of H.armigera were located on 6 chromosomes.Alignment of amino acid sequences indicates that the nAChR subunits of H.armigera have the common structural features of the superfamily of Cys-loop ligand-gated ion channels(LGICs).Among the 12 nAChR subunits of H.armigera,the amino acid similarity between Haα6 and Haα7 is the highest(66.02%).Phylogenetic relationship analysis indicates that the nAChR subunits of insects are conserved among different lepidopteran species.Transcriptome data analysis shows that most of the nAChR subunits of H.armigera have high expression levels in the embryo stage and larval ventral nerve cords.Identification and analysis of the nAChR gene family of H.armigera laid a foundation for future investigation on the function and evolution of H.armigera nAChR subunits.2.Construction of two knockout strains of H.armigera nAChR α6 and α7 subunitsCRISPR/Cas9-mediated NHEJ was employed to construct two knockout strains of H.armigera nAChR α6 and α7 subunits based on the susceptible SCD strain of H.armigera.The two knockout strains were named Haα6-KO and Haα7-KO respectively.Sequencing results showed that the 5bp insertion at exon 6 of Haα6 and 5bp deletion at exon 8 of Haα7 are homozygous in Haα6-KO and Haα7-KO strains,respectively.It is predicted that these two types of indels will result in a pre-mature stop codon,lead to premature truncation of the translated proteins,and thus loss of function for the two nAChR subunits of H.armigera.3.Effects of the two knockouts of Haα6 and Haα7 on insecticide susceptibilityIn order to clarify the effects of the Haα6 and Haα7 knockouts of H.armigera on insecticide sensitivity,the two knockout strains and SCD strains were bioassayed against 6 insecticides.Haα6-KO strain exhibited high levels of resistance to spinosad(531)and spinetoram(1105),while the sensitivity of Haα7-KO strain to both spinosyns did not change significantly.No significant difference in sensitivity to another four insecticides(emamectin benzoate,chlorantraniliprole,beta-cypermethrin,and indoxacarb)was observed among the two knockout strains and the SCD strain.Genetic analyses demonstrated that the resistance to spinosad conferred by knockout of Haα6 was autosomal,incomplete recessive and tightly linked to the mutation of Haα6.These results indicate that Haα6 is a functional receptor for spinosad in H.armigera,while Haα7 is not associated with the toxicity of spinosad.