| Sitobion miscanthi is one of the important wheat pests in our country.The insect sucks the sap of wheat with its piercing mouthparts,harms the leaves of wheat,spreads Barley Yellow Dwarf Virus（BYDV）,and seriously affects the wheat production in our country.The neonicotinoid insecticide imidacloprid has the advantages of broad spectrum,high efficiency,low toxicity,low residue,etc.Meanwhile,imidacloprid has a strong toxicity,stomach poisoning and high active absorption on insect pests,especially shows high efficiency in control of sucking pests including aphids,planthoppers,leafhoppers and whiteflies.It is widely used in the control of wheat crop aphids in our country.With the widespread use of imidacloprid,many pests have developed resistance to it.The existing resistance monitoring results found that the field population of wheat aphid in some areas of our country has developed a certain level of resistance to imidacloprid,while the research report on the resistance to imidacloprid in S.miscanthi is relatively few.Therefore,the mechanism of resistance to imidacloprid in S.miscanthi was studied in depth.The imidacloprid-resistant strains were screened,the resistance level of the resistant strains was determined,and the cross-resistance of the imidacloprid-resistant strains to other insecticides was analyzed.Screening of differentially expressed nAChR genes by transcriptome sequencing.The functions of SMnAChRβ1,SMnAChRα3 and SMnAChRα4 genes in the resistance to imidacloprid were verified by RNA interference（RNAi）.According to literature reports,mutation of nAChRβ1 gene in homologous species Aphis gossypii and Myzus persicae is the cause of high resistance to imidacloprid.Therefore,mutation point detection was performed on the SMnAChRβ1gene of S.miscanthi.The main results are as follows:1.Cross-resistance of imidacloprid-resistant strain of S.miscanthiIn this study,imidacloprid was used for continuous screening of S.miscanthi,and laboratory resistant strains were obtained.Toxicity to imidacloprid was determined by the leaf-dipping with apids method.The LC50of the sensitive strain was 0.957 mg/L,the LC50of the resistant strain was 48.120 mg/L,and the resistance multiple was 50.28.In this study,the virulence of S.miscanthi to different insecticides was determined before and after imidacloprid resistance screening.The results showed that the resistance multiples of resistant strains to sulfoxaflor and abamectin were 16.50 and 14.08,respectively,indicating that imidacloprid,sulfoxaflor and abamectin had a moderate level of cross-resistance.However,there was no cross-resistance to the five insecticides lambda-cyhalothrin,thiamethoxam,acetamiprid,chlorpyrifos and omethoate.2.Screening of nicotinic acetylcholine receptor genes（nAChR）Based on the transcriptome sequencing data,16 nicotinic acetylcholine receptor genes were screened out from S.miscanthi.Among them,there are 6 nAChR genes with significant differences.The expressions of SMnAChRα1A,SMnAChRα1B,and SMnAChRα6 were all significantly up-regulated,which were significantly up-regulated by 1.982 times,1.5 times and1.121 times,respectively,compared with the control group.The expressions of SMnAChRβ1,SMnAChRα3 and SMnAChRα4 were significantly down-regulated by 1.149 times,1.628 times and1.184 times respectively compared with the control group.SMnAChRα1-1,SMnAChRα1C,SMnAChRα1D,SMnAChRα1E,SMnAChRα7-1,SMnAChRα3A,SMnAChRα3B,SMnAChRα3C,SMnAChRα3D and SMnAChRα3E were not significantly different to control.3.Relative expression of nAChR genes in imidacloprid resistant and susceptible strains of S.miscanthiIn this study,qRT-PCR was used to analyze the relative expression levels of six nicotinic acetylcholine receptor genes in the imidacloprid-resistant and sensitive strains of S.miscanthi.The results showed that the expression levels of three genes,SMnAChRβ1,SMnAChRα3 and SMnAChRα4,were significantly decreased compared with the sensitive strains,which were 51%,34%and 50%of the sensitive strain,respectively.Whether the decrease in gene expression is directly related to imidacloprid resistance needs to be verified in the next step.4.Functional verification of the nicotinic acetylcholine receptor genes in S.miscanthiAccording to the results of the previous analysis.After silencing the three genes SMnAChRβ1（Gene Bank:MW771289）,SMnAChRα3（Gene Bank:MW771290）,and SMnAChRα4（Gene Bank:MW771291）by RNAi technology,when the S.miscanthi was treated with LC50of0.957 mg/L imidacloprid,the sensitivity to imidacloprid was decreased,and the mortality was significantly decreased by 25.6%,20.1%and 20.9%,respectively.It indicated that the decreased expression of these three genes might be related to the resistance of S.miscanthi to imidacloprid.5.Analysis of SMnAChRβ1 in S.miscanthiThe SMnAChRβ1 has a complete open reading frame with a total of 509 amino acids translated.After online software analysis,it has a typical nicotinic acetylcholine receptor protein structure:（1）Cys-Loop with 13 amino acids in the middle;（2）The extracellular region has Loop D,Loop E and Loop F loops;（3）There are four complete transmembrane fragments TM1,TM2,TM3and TM4;（5）Amino acids 1-24 are signal peptide fragments.6.Detection of SMnAChRβ1 mutation points in S.miscanthiFour mutation sites E206K,T224A,S283P and R357K were detected in the SMnAChRβ1 receptor gene of S.miscanthi.The imidacloprid resistance-related mutation sites V62I,R81T and V101I of the homologous species cotton aphid and M.persicae that have been reported have not been detected.The mutation sites E206K and T224A are located in the N-terminal extracellular region.S283P is located on the transmembrane fragment TM2.This transmembrane fragment TM2 is arranged in a ring to form a cation entry and exit channel.Amino acid mutations may lead to altered conductance properties.This in turn affects the selectivity of the channel for cations.The mutation site R357K is located on the intracellular macrocyclic fragment.Whether these four gene mutation sites lead to resistance to imidacloprid in S.miscanthi,it needs further analysis and verification. |
