Study On The Resistance Mechanisms Of Chilo Suppressalis (Walker) To Chlorantraniliprole

The rice stem borer,Chilo suppressalis(Walker)(Lepidoptera: Pyraidae),is one of the most economically important pests of rice in China.The control of C.suppressalis mainly relies on chlorantraniliprole since 2008.However,some field populations of C.suppressalis have developed high-level resistance to chlorantraniliprole and the field efficacy has declined significantly.Due to lack of effective insecticides to control the resistant C.suppressalis,the growth of rice was damaged diastrously in Zhejiang,Jiangxi and Hunan province.However,the molecular mechanism of high resistance to chlorantraniliprole against field populations is not well clear.Therefore,we carried out research on the resistance mechanism of C.suppressalis to chlorantraniliprole by detecting the mutation of RyR in C.suppressalis field populations.CRISPR/Cas9 editing was adopted to study the function of the target mutation in order to clarify the mechanism of target resistance in diamides resistance.To explore if there are other genes participate the resistance to chlorantraniliprole,transcriptome sequencing is used to find the up-expression and down-expression genes and fluorescence quantitative PCR is further used to verify the expression of differential genes in resistant populations.The GAL4/UAS expression system was used to verify the function of the candidate genes and the function of these detoxification genes on the synergism of target mutation.Another detoxification gene,FMO’s function on C.suppressalis resistance was also studied.Lastly a rapid detecting method to detect the field resistance of C.suppressalis was established.The results of the study are as follows:1.Multiple target-site mutations confer C.suppressalis resistance to diamide insecticidesIn 2016,the field population of C.suppressalis has developed high level resistance to chlorantraniliprole and the area of high resistance has expanded in recent years.To investigate the possible role of target-site mutations in resistant field populations of C.suppressalis,we detected respective RyR gene domains of thirty field populations from five provinces in 2017-2020.The results showed that the Y4667 D,Y4667C,I4758 M,G4915E mutation and one novel Y4891 F mutation were present in the field populations of C.suppressalis.Analyzing the mutation frequency of each point mutation,the data showed that I4758 M mutation was detected in 25 of the 30 field resistant populations of C.suppressalis with the frequency of 14.4-100%,of which 12 populations were only detected I4758 M mutation,and the other 13 populations detected I4758 M mutation and simultaneously detected other mutations.A novel mutation,Y4891 F,was only detected in NChe and NCha populations from Jiangxi provinces with the 11.1%-65.4% mutation frequency.Y4667 D mutation was detected Nche17(RR= 47.0)population with 45% mutation frequency(0%homozygous and 45% heterozygous).While the Y4667 D detected in Nche20(RR=1076.8)population with 100% mutation frequency(87.6% homozygous and 13.3% heterozygous).The Y4667 C mutation was detected in YY17(RR=145.4)population with 0% mutation frequency and Y4667 C was detected in YY18(RR=314.5)population with 66.7% mutation frequency(0% homozygous and 66.7% heterozygous),while the frequency of Y4667 C mutations detected in YY20(RR=1426.8)population was 70.6%(41.2% homozygous and29.4% heterozygous).This indicates that frequency of the Y4667 D and Y4667 C mutation varies with the resistance of chlorantraniliprole and the multiple mutations in the RyR gene may be involved in high level resistance to chlorantraniliprole in C.suppressalis.We generated genome modified nine fly lines possessing the RyR mutations found in C.suppressalis to elucidate the molecular mechanism of diamide resistance.The bioassay results showed that genome modified flies bearing the Y4667 D mutation exhibited high resistance to chlorantraniliprole(1542.8-fold),cyantraniliprole(487.9-fold)and tetrachlorantraniliprole(290.1-fold).The M4758 I and Y4667 D simultaneous mutations showed high resistance to chlorantraniliprole(117.2-fold)and moderate resistance to cyantraniliprole(90-fold)and tetrachlorantraniliprole(22.3-fold).The Y4667 C mutation showed high resistance to chlorantraniliprole(172.1-fold)and moderate resistance to cyantraniliprole(90-fold),tetrachlorantraniliprole(22.3-fold)and flubendiamide(19.5-fold).The M4758 I and Y4667 C simultaneous mutations and the M4758 I and Y4891 F simultaneous mutations showed low resistance to chlorantraniliprole(6.0-fold).The M4758 I and G4915 E simultaneous mutations showed high resistance to chlorantraniliprole(153.1-fold)and cyantraniliprole(323.5-fold),and moderate resistance to flubendiamide(28.9-fold)and tetrachlorantraniliprole(25.2-fold).These results suggested that multiple point mutations in RyR confered diamide resistance against C.suppressalis.Additionally,an allele-specific polymerase chain reaction(AS-PCR)method was developed to detect the target resistance in C.suppressalis populations.2.Transcriptome analysis of field chlorantraniliprole resistant populations of C.suppressalisAlthough it is clear that multiple point mutations in RyR confer diamide resistance of C.suppressalis,only one I4758 M mutation was detected in some field high-resistant populations.The contribution of I4758 M mutation showed 20-fold resistance to chlorantraniliprole in genome modified fruitfly indicate that there must be other resistance mechanisms exsited.In order to further study the resistance mechanism of C.suppressalis to chlorantraniliprole,we have performed high-throughput sequencing on C.suppressalis susceptible strain and three field high-resistant populations.Compared with the susceptible strain,three field high-resistant populations had 114 genes up-regulated and the 107 genes downregulated during the differential expression gene screening process.KEGG functional enrichment analysis showed that these differential genes were mainly enriched in metabolic pathways.Subsequently,q-PCR technology was used to confirm that four P450 genes(CYP6SN2,CYP324A12,CYP6AB45 and CYP6CV5),one glycosyltransferase 2,one cytochrome b5-related protein gene,one juvenile hormone binding protein gene,one juvenile hormone epoxide hydrolase precursor gene,one lysozyme 1B gene,one immulectin-2 gene,CYP9A68 and CYP321F3 gene were overexpressed in three field high-resistant populations.These results indicate that cytochrome P450 and other differential expression genes may be involved in the resistance of C.suppressalis to chlorantraniliprole,which provide clues for finding the potential genes involved in chlorantraniliprole resistance.3.The synergistic effect of the overexpression of the P450 gene on the target-site mutations confers the high-level resistance of C.suppressalis to chlorantraniliproleThe results of transcriptome analysis had indicated that cytochrome P450 genes may be involved in the resistance of C.suppressalis to chlorantraniliprole.In order to clarify the role of P450 gene in chlorantraniliprole resistance,we generated transgenic Drosophila lines expressing C.suppressalis P450 gene by GAL4/UAS expression system.Toxicity assays showed that the transgenic fruit fly overexpressing CYP321F3,CYP9A68,CYP6CV5,CYP6AB45 and CYP6SN2 gene showed no significantly sensitivity to chlorantraniliprole compared to the control flies.These results indicated that the overexpression of CYP321F3,CYP9A68,CYP6CV5,CYP6AB45 and CYP6SN2 genes does not directly participate in the resistance of C.suppressalis to chlorantraniliprole.To further study whether metabolic resistance and target resistance synergistically mediate the high-level resistance of C.suppressalis to chlorantraniliprole.we generated strain enabled CYP transgenic respectively overexpression in genetic background RyR mutations which confer target-site resistance.the bioassay results showed that the strain Y4667D;da-Gal4>UAS-CYP321F3 and the strain Y4667D;da-Gal4>UAS-CYP6CV5 with a 1.8-fold and 1.9-fold resistance increasing to chlorantraniliprole compared to the control flies which bearing Y4667 D mutation alone.These results showed that metabolic gene had an synergism on target mutation might confer the high resistance of C.suppressalis to chlorantraniliprole.4.The function of FMO in resistance of C.suppressalis to chlorantraniliproleOverexpression of a flavin-dependent monooxgenase(FMO)confers resistance to the diamide chlorantraniliprole in Plutella xylostella.In order to explore the role of FMO in resistance of C.suppressalis to chlorantraniliprole,the full-length sequences of four FMO genes(CsFMO)of C.suppressalis were obtained by PCR amplification.The open reading frames was 1374 bp,1323 bp,1299 bp,and 1578 bp,encoding 458,441,433,and 526 amino acids,respectively.q PCR analysis revealed that CsFMO1 was mainly expressed in the cuticle of larvae,CsFMO2 was highly expressed in fat body and midgut,CsFMO3-1 was highly expressed in malpighian tubule,and CsFMO3-2 gene is highly expressed in malpighian tubule and midgut.Compared with the expression of susceptible strain,the CsFMO3-1 and CsFMO3-2 genes were 2.2-7.4 times and 3.1-4.7 times higher in the three field resistant populations of C.suppressalis,while the expression levels of CsFMO1 and CsFMO2 genes were no significant difference between three field resistant populations and susceptible strain.These results indicated that CsFMO3-1 and CsFMO3-2 genes might involve in the resistance of C.suppressalis to chlorantraniliprole.