Impacts Of Ace-1 Resistance Mutations On The Growth And Reproduction Of Spodoptera Frugiperda And On The Insecticide-binding Mode

The fall armyworm（FAW）,Spodoptera frugiperda,is a globally migratory agricultural pest,and its resistance to insecticides has become one of the main obstacles to its effective control.Therefore,revealing the molecular mechanism of resistance in the FAW is crucial for its efficient management.One of the primary mechanisms of reduced sensitivity to organophosphorus insecticides（OPs）and carbamate insecticides（CXs）in the FAW is the target mutation of acetylcholinesterase-1（Ace-1）.The reported Ace-1 target mutation sites in the FAW include A201S,G227A,and F290V（numbered according to the Torpedo californica acetylcholinesterase nomenclature）,with the F290V mutation being the most common in field populations.However,there is a lack of systematic research on the structural biology mechanisms of FAW Ace-1 target mutations leading to FAW resistance to OPs and CXs,as well as the impact of the F290V mutation on the growth and reproduction of FAW.Therefore,this study investigates the impact of the Ace-1 F290V mutation on FAW growth and reproduction through field mutation detection,molecular docking,and construction of the relatively sensitive population（FAW-SS）and F290V mutation population（FAW-F290V）,and preliminarily explores the structural biology mechanism of FAW Ace-1 target mutations leading to resistance.The main research results are as follows:The genotype and mutation frequency of three Ace-1 mutation sites（A201S,G227A,and F290V）in eight field populations of the FAW in Hubei Province（Jingzhou,Xiantao,Wuhan,Shishou,Huanggang,Zigui,and Yichang）from 2021 to 2022 were investigated.Two mutation sites（A201S and F290V）were detected,with mutation frequencies ranging from 0%to 25.0%and 7.15%to 76.0%,respectively.The G227A mutation was not found.To rapidly and accurately detect the F290V mutation in the field populations,a tetra-primer ARMS-PCR primer was designed,combined with a paper-based DNA rapid extraction technique,which could identify the Ace-1 F290V mutation type within 120 min.The three mutations A201S,G227A,and F290V were introduced into the amino acid sequence of wild-type FAW Ace-1,and the protein conformations of Ace-1 wild-type（Ace1-WT）and three mutants（Ace1-A201S,Ace1-G227A,and Ace1-F290V）were constructed using Alpha Fold2 software.Molecular docking results showed that OPs and CXs mainly inhibited the degradation of acetylcholine（ACh）in the synaptic cleft by binding to the catalytic site of Ace-1,leading to the continuous excitation and death of insects.The introduction of a hydroxyl group（-OH）via the A201S mutation increased the affinity of Ace-1 for ACh,thereby enhancing the enzyme’s catalytic efficiency and reducing the sensitivity of the FAW to OPs and CXs.The G227A mutation introduced a methyl group（-CH₃）and lengthened the side chain,also potentially increasing the affinity of Ace-1 for ACh and reducing the sensitivity of the FAW to OPs and CXs.The F290V mutation,resulting from the substitution of a larger phenylalanine with a smaller valine,widened the catalytic gorge of Ace-1,increasing the likelihood of ACh entering the active site under insecticide inhibition and reducing the sensitivity of the FAW to OPs and CXs.Relatively susceptible（FAW-SS）and Ace-1 F290V mutant（FAW-F290V）strains with the same genetic background were isolated from the 2021-Wh population.The sensitivity of the two strains to various OPs and CXs was examined,and the results showed that the sensitivity of the FAW-F290V strain to multiple OPs and CXs was significantly reduced compared to the FAW-SS strain.The age-stage specific survival rate and age-stage reproduction rate of the two strains were analyzed using TWOSEX-MSChart software.The results indicated that the F290V mutation had a minimal impact on the growth and reproduction of the FAW.his study is an attempt to investigate the causes of resistance to the FAW Ace-1 locus mutation from the perspective of structural biology,which is important for predicting the trend of resistance mutation frequency in the field population of the FAW and guiding the development and scientific use of insecticides.