Molecular Mechanisms Underlying The Involvement Of GST And NAChR Genes In The Response Of Monochamus Alternatus To Thiacloprid Stress

Monochamus alternatus Hope belongs to the Coleopteran Cerbycidae family.It is an important stem borer of pine trees and also serves as the main vector of the devastating forest disease known as pine wilt disease.The neonicotinoid insecticide thiacloprid possesses several advantages,such as a broad spectrum of activity,high efficiency,low toxicity and minial residue,it could cause stomach poisoning and endotoxic activity against M.alternatus.However,insects can develop resistance to insecticides by modulating their physiology,biochemistry and behavior.In insect resistance research,Glutathione-S-transferase（GST）and nicotinic acetylcholine receptors（nAChRs）have been shown to be involved in the development of resistance in a variety of insects in insect resistance research.Nevertheless,there is limited research available on the mechanisms of detoxification and resistance to insecticides in M.alternatus.In this study,the virulence of thiacloprid on the larvae was determined through feeding experiments.Transcriptome sequencing technology was employed to screen the expressed nAChR genes and GST genes in M.alternatus under LC₃₀ thiacloprid treatment.Subsequently,the spatial-temporal expression responses of GST and nAChR genes were detected by real-time PCR under LC₃₀ thiacloprid treatment.Finally,the RNAi technology was used to verify the function of the Ma GSTs1 and ManAChRα2 genes,which lays the foundation for further exploration of detoxification metabolism and resistance risk assessment of thiacloprid against M.alternatus.The main results are shown below:（1）Virulence determination of thiacloprid against M.alternatus larvaeThe virulence of the larvae was determined by feeding experiment,and the results showed that the LC₃₀ of thiacloprid was 33.290 mg/L for 72 h,and the 95%confidence interval(CI₉₅)was 24.454 mg/L to 46.255 mg/L.（2）Identification of GST and nAChR genesA total of 93,646 transcripts were obtained through Illumina high-throughput sequencing of RNA samples from larvae treated with LC₃₀ thiacloprid.After removing redundancy,a total of 74,675 Unigenes were obtained.Based on the sequence similarity search,12candidate GST genes and 3 candidate nAChR genes were identified.（3）Spatial-temporal expression responses of GST and nAChR genes under thiacloprid stressReal-time PCR was used to verify the differential expression of genes in different tissues（hemolymph,gut,fat body）of M.alternatus at 24 h,48 h and 72 h under LC₃₀ thiaclolrid treatment.Ma GSTs1 exhibited a significantly upregulation in the hemolymph,with a 6.7-fold increase compared to the control group.In the gut,it displayed a notable upregulation of10.9-fold,while in the fat body,the upregulation was observed to be 3.8-fold after 48 hours of treatment.On the other hand,ManAChRα2 demonstrated a significant upregulation in the fat body,showing a 2.8-fold increase relative to the control group at 48 h post-treatment.Conversely,in the intestines,ManAChRα2 exhibited significant downregulation at both 48hours and 72 hours after treatment,with a decrease of 0.45-fold and 0.16-fold,respectively,in comparison to the control group.（4）RNAi validation of Ma GSTs1 and ManAChRα2 genesThe expression of Ma GSTs1 and ManAChRα2 genes were silenced using oral-induced RNAi technology.The results showed that the mortality rate of M.alternatus larvae treated with LC₃₀ thiaclolrid for 72h reached 17.78%after interfering with ManAChRα2 gene.This downregulation was significant,showing an 11.11%decrease compared to the control group.It indicates that the sensitivity of M.alternatus larvae to thiacloprid was reduced upon silencing the ManAChRα2 gene.Furthermore after silencing the Ma GSTs1 gene,the mortality rate of the M.alternatus larvae increased to 46.67%,showing a significant upregulation of 17.78%compared to the control group.This finding suggested that the Ma GSTs1 gene played an important role in the detoxification metabolism of thiacloprid in M.alternatus.However,when both genes were silenced simultaneously,the mortality rate of M.alternatus larvae was 31.11%.This further indicated that the ManAChRα2 and Ma GSTs1 genes played important roles in the response to thiacloprid stress in M.alternatus.The study suggested that M.alternatus enhance its resistance against thiacloprid through modulating metabolic resistance and resistance mechanisms.