我国棉蚜田间种群对氧乐果的抗性监测及靶标抗性机制解析OA

[Objective]The objective of this study is to determine the resistance level of Aphis gossypii populations from the main cotton planting areas of China to omethoate,to elucidate the predominant mutant types in acetylcholinesterase 1(AChE1)(the target of omethoate)in these populations,and further analyze the impacts on the AChE1 structure by these major mutation types and their relationship with omethoate resistance.[Method]In 2019-2023,A.gossypii field populations were collected from the main cotton planting areas of China,and the resistance levels of these populations to omethoate were determined using the leaf-dipping method.Mutations in AChE1 were detected by single-aphid PCR amplification and sequencing,and the types of mutations carried by an individual aphid were analyzed.Monoclonal strains carrying different AChE1 mutations were established using single-female rearing protocols,and their contributions to omethoate resistance were subsequently assessed through toxicity bioassays and synergist experiments.The mechanisms of A.gossypii resistance to omethoate mediated by these mutations were revealed using homology modeling,molecular docking,and molecular dynamics simulations.[Result]In 2019-2023,38 A.gossypii field populations in China developed moderate to high levels of resistance to omethoate(17.1-207.8-fold)and remained relatively stable over the years.G221A,A302S,A332V,and S431F mutations in AChE1 were prevalent in these populations,with S431F mutation frequency reaching 100%,predominantly in homozygous form.While the average frequencies of G221A,A302S,and A332V were 37.26%,51.26%,and 74.64%,respectively.Detection of mutant types from a single aphid revealed that individuals carrying the A332V+S431F combination mutation had the highest frequency(35.24%),followed by G221A+A302S+A332V+S431F(26.80%)and A302S+A332V+S431F(12.12%).The average frequency of the S431F single mutation was 9.56%.The weighted frequencies of these major mutation types showed a certain linear correlation with omethoate resistance(R2=0.46,P＜0.001).By constructing monoclonal mutant strains,it was confirmed that A.gossypii strains carrying the combination mutations A332V+S431F,G221A+A302S+S431F,A302S+A332V+S431F,and G221A+A302S+A332V+S431F developed a moderate level of resistance to omethoate,with resistance ratios of 46.1-77.8-fold,significantly higher than that of the S431F single-mutant strain(12.5-fold).Moreover,PBO,TPP,and DEM did not exhibit significant synergistic effects on omethoate toxicity in these mutant strains.Molecular docking and dynamics simulations confirmed that the S431F single mutation and A332V+S431F,G221A+A302S+S431F,A302S+A332V+S431F,and G221A+A302S+A332V+S431F combination mutations significantly increased the geometric center distance between the AChE1 catalytic triad and omethoate by 0.074-0.185 nm compared to the wild-type.These mutations also altered the hydrophobic area and radius of gyration of the AChE1 active center,thereby affecting the active center of A.gossypii AChE1 and its binding conformation with omethoate.[Conclusion]A.gossypii populations in major cotton-planting regions of China have developed moderate to high levels of resistance to omethoate.Four mutations were identified in AChE1 across these populations,and these site mutations altered the active center of AChE1 and its binding affinity for omethoate through different combination types,thereby mediating field resistance to this insecticide.In field control practices,it is advisable to reduce the use of AChE-targeting insecticides to prevent the development of cross-resistance mediated by target mutation and further escalation of resistance levels.