Study On Resistance Mechanism Of Spodoptera Exigua To Spinosad Insecticides And Analysis Of Field Population Resistance

Spinosad,one of the main control agents of Spodoptera exigua,acts on the nicotinic acetylcholine receptor(n ACh R)of insects,causing conformation change of the receptor,opening ligand gated ion channel,causing tremor and paralysis of insect pests,and ultimately leading to the death of insect pests.Although spinosad has a unique mode of action,it is easy for Spodoptera exigua to develop resistance to spinosad after long-term and large-scale field application.Low to moderate resistance to spinosad has been reported in field populations of Spodoptera exigua,but the gene frequency and molecular mechanism of resistance in field populations remain unclear.Using gene editing techniques,n ACh R α6 has been identified as a target site for spinosad insecticides in insects.Spinosad resistance is associated with n ACh Rα6 subunit functional loss mutations in many agricultural pests.In recent years,a point mutation(G275E)in exon 9 of n ACh R α6 has been found to lead to high levels of spinosad resistance in spinosad resistant strains of Thrips Palmi and Tuta Absoluta.However,no in vivo functional evidence has been obtained to support the role of this mutation in resistance to spinosad in lepidopteran pests.Therefore,in this study,α6-G275 E and α6-KO(knockout)gene editing strains were constructed to clarify the mechanism of n ACh R α6 functional loss mutation and G275 E point mutation against spinosad resistance.The target alleles of n ACh Rα6 were detected by α6-KO strain,and the resistance alleles of n ACh R α6 were expected to be found.The main research results of this paper are as follows:1.The Seα6-KO strain was successfully constructed using CRISPR/Cas9 genome editing system.The Seα6 strain was deleted about 1760 bp in the Beet Armyworm,leading to premature transcription termination of Seα6.The resistance of Seα6-KO to spinosad was 373-fold higher than that of the susceptible strain WH-S with the same genetic background,and the resistance to ethylspinosad was 850-fold higher than that of Seα6-KO.However,the sensitivity of Seα6-KO to methylabamectin and chlorantrantranamide was not significantly changed.Genetic analysis showed that Seα6-KO was an incomplete recessive trait.These results not only revealed the important role of Seα6 in resistance to spinosad insecticides in Spodoptera exigua,but also provided an example for the identification of resistance related target functional loss mutations using genome editing techniques.2.Using CRISPR/Cas9 gene editing technique,the G275 E mutation was introduced into n ACh R α6 of WH-S,a sensitive strain of Spodoptera exigua.Reverse transcriptional PCR and sequencing confirmed that the mutation was present in exon 9 of n ACh R transcript of the 275 E strain.Bioassay results showed that compared with the unedited sensitive strain WH-S,the275 E strain had a high level of resistance to spinosad(230.40-fold)and ethyl spinosad(791.67-fold),which directly confirmed that the mutation of n ACh R α6 subunit G275 E could lead to a high level of resistance to spinosad.Genetic analysis showed that the resistance traits of this strain were autosomal incomplete recessive.In this study,we verified the functional role of the G275 E mutation of n ACh R α6 in spinosad resistance using reverse genetics,and provided an example for identifying the role of candidate target site mutation in insecticide resistance using CRISPR/Cas9 gene editing techniques.(3)Bioassay of Spodoptera exigua from Shanghai Fengxian(FX),Shandong Jinan(JN),Xinjiang Shihezi(SHZ),Shaanxi Xing Ping(XP),found that the 4-25 fold resistance have been produced to spinetoram.F1 detection results of cross between Seα6-KO strain and field strain showed that each field strain of XP,FX and SHZ had a pair of test insects with 100% survival rate,and there may be target resistance alleles in field individuals.In the subsequent backcross with WH-S,the survival rate of offspring was about 50%,which may be due to the main role of metabolic resistance in the early stage of resistance generation.Therefore,the expression levels of CYP9 A series genes in the FX strain were analyzed,and it was found that the expression levels of CYP9 A series genes were all up-regulated,among which the expression levels of CYP9A9,CYP9A186 and CYP9A40 genes were most obviously up-regulated,which were 7.93,5.53 and 6.82.These results suggest that CYP9A9,CYP9A186 and CYP9A40 may be related to the metabolic resistance of the FX strain to spinetoram.In conclusion,it is clear that the n ACh R α6 subunit and its G275 E mutation play an important role in resistance to spinosad.In China,the sensitivity of Spodoptera exigua in field to spinetoram is in a declining stage,in which metabolic resistance may play a major role in resistance development.