Resistance Mechanisms To Emamectin Benzoate And Diamide Insecticides In Spodoptera Exigua(H(?)bner)

The beet armyworm,S.exigua(Lepidoptera:Noctuidae),is a major pest of global importance on a wide range of cultivated crops.Because of its sporadic outbreaks in China,insecticides have been heavily used to control S.exigua.Due to serious resistance to conventional chemicals(organophosphates,carbamates and pyrethroids),emamectin benzoate and chlorantraniliprole have been heavily used against S.exigua since its commercialization in China.Due to repeated and intensive use of emamectin benzoate and chlorantraniliprole,S.exigua has developed resistance to emamectin benzoate and chlorantraniliprole in some field populations from China.However,resistance mechanisms to emamectin benzoate and chlorantraniliprole in S.exigua remains largely unknown.The resistant strain(WH-EB)is 244-fold resistant to emamectin benzoate,and the resistance is incompletely dominant.Therefore,the aim of this study was to identify the resistance gene(s)of S.exigua to emamectin benzoate by genetic mapping,so as to clarify the molecular genetic mechanism of dominant resistance to emamectin benzoate in S.exigua.Meanwhile,the relationship between RyR mutations and diamide insecticides was studied by establishing isogenic lines of S.exigua and site-specific mutation of SeRyR.These results are helpful to improve the resistance management strategy of S.exigua and the development of appropriate resistance management practices for delaying resistance evolution of S.exigua to emamectin benzoate and chlorantraniliprole.1.Effects of P-glycoprotein of Spodoptera exigua knockout on susceptibility to abamectin and emamectin benzoateP-glycoprotein(P-gp/ABCB1)is an important player in multidrug resistance(MDR)of cancer cells.It has also been found that overexpression of P-gp gene in insects is related to abamectin resistance.In this study,SeP-gp was cloned and characterized.The deduced SeP-gp protein possesses structural characteristics of a typical P-gp,and is clustered within the insect ABCB1.To analyse the potential role of SeP-gp in excreting insecticide or Bt resistance,a knockout line with a frame shift deletion of 4 nucleotides was established using CRISPR-Cas9 gene-editing technology.The results showed that loss function of SeP-gp significantly increased susceptibility of S.exigua to abamectin and emamectin benzoate(EB),but not to spinosad,chlorfenapyr,beta-cypermethrin,carbosulfan indoxacarb,chlorpyrifos,phoxim,diafenthiuron,chlorfluazuron,chlorantraniliprole,Bt toxins(Cry1C and Cry1F).Our data not only demonstrate that the CRISPR/Cas9 technique can provide a functional evidence for insecticide resistance,but also suggest that the overexpression of P-gp maybe play a role in abamectin-and EB-resistance in S.exigua.2.Genetic mapping,cloning and in vivo functional verification of resistance gene of WH-EB strain to emamectin benzoate in S.exiguaIn this study,genetic mapping of emamectin benzote resistance gene in WH-EB strain of S.eigua was carried out.According to the synteny of lepidoptera genome,single nucleotide polymorphism(SNP)markers of exon or insertion deletion(Indel)markers of intron in specific gene was used to mark 31 chromosomes of S.exigua,and the resistance gene was located on chromosome 29(Chr29).Then,through the molecular markers in the Chr29,resistance gene was mapped on 0-2.5 Mbp of Chr29.CYP9A gene cluster is within the mapped area of Chr29,therefore,we hypothesize that the dominant resistance of the WH-EB strain to emamectin benzoate is related to one or more P450 genes in the CYP9A gene cluster of WH-EB.In order to test the above hypothesis,CRISPR/Cas9 gene editing technology was used to completely knock out CYP9A gene cluster(?130 KB)of the resistant WH-EB strain of S.exigua.Knockout of the full CYP9A gene cluster made the WH-EB strain as susceptible as the WH-S strain,indicating the hypothesis is true and that the resistance gene(s)to emamectin benzoate in WH-EB is within the CYP9A cluster.By a large fragment knockout within CYP9A gene cluster and P450 gene sequence alignment,it was revealed that the F116V mutation of CYP9A58 in WH-EB strain,located in SRS1 region of substrate binding site,is related to emamectin benzoate resistance.Then,CYP9A58 gene in WH-EB resistance strain was knocked out,and susceptibily to emamectin benzoate was restored.Our results clarified that the F116V mutation of CYP9A58 gene was the genetic basis for the WH-EB strain of S.exigua to confer high level resistance to emamectin benzoate.3.Functional verification of the F116V mutation of CYP9A58 and detection of the mutation frequency of CYP9A58 gene in field populations of S.exiguaIn order to ascertain whether the F116V mutation of CYP9A58 affects the metabolic capability to emamectin benzoate and abamectin,the wild type allele(116F)and mutant allele(116V)of CYP9A58 of S.exigua were expressed in vitro by the baculovirus expression system of insect cells,and their metabolic capability were determined and compared.Results showed that the mutant CYP9A58(116V)from the resistant strain had a strong metabolic activity to emamectin benzoate and abamectin,while the wild type CYP9A58 from the susceptible strain had no metabolic capability to emamectin benzoate and abamectin.The metabolites identified with mass spectrometry included both hydroxyl-and O-deoxymethylated metabolites of emamectin benzoate and abamectin.The results of in vitro functional verification demonstrated that the CYP9A58 of S.exigua can obtain the detoxification ability to emamectin benzoate and abamectin through the F116V point mutation,and this mutation can thus confer resistance to emamectin benzoate and abamectin.Biological assay and mutation frequency detection showed that five S.exigua field populations had developed high levels of resistance to emamectin benzoate(212-388-fold).The mutation frequencies of F116V in the field populations were ranged from 80.36%to 96.67%,among which the mutation frequency of QP population from Shanghai was the highest(96.67%)and that of LY population from Luoyang of Henan was the lowest(80.36%).There was a significant correlation between the resistance levels and the allele frequencies(R2=0.9794).Therefore,the mutation frequency of CYP9A58 F116V can be used to predict the resistance levels of emamectin benzoate in field populations of S.exigua,and providing scientific basis for the implementation of reasonable insecticide rotation programs.4.Contribution of amino acid point mutations of S.exigua ryanodine receptor(SeRyR)to diamide insecticides resistanceDiamide insecticides represent a new class of chemistry that target insect ryanodine receptors(RyRs).Two point mutations G4946E and 14790M(numbering as in the RyR of Plutella xylostella,PxRyR)in the transmembrane domain of the insect RyRs associated with diamide resistance have been identified in three lepidopteran pests,P.xylostella,Tuta absoluta and Chilo suppressalis.In the present study,sequencing the conserved transmembrane domain of S.exigua RyR(SeRyR)revealed that a 14743M mutation(corresponding to I4790M of PxRyR)of SeRyR was found in WF field resistance strains,but no G4900E allele(corresponding to G4946E of PxRyR)mutation.To confirm the contribution of I4743M mutation of SeRyR to diamide insecticides resistance in S.exigua,the 4743M allele in the WF strain was introgressed into the susceptible WH-S strain by crossing WF with WH-S,followed by 3 rounds of backcrossing with WH-S and molecular marker assisted selection.The introgressed strain(named as 4743M)was homozygous for the mutant 4743M allele and shared about 94%of its genetic background with that of the recipient WH-S strain.In comparison with the background WH-S strain,the near-isogenic 4743M strain showed moderate levels of resistance to chlorantraniliprole(21-fold),cyantraniliprole(25-fold)and flubendiamide(22-fold),indicating the I4743M mutation by itself confers around 20-fold resistance to the three diamides.To confirm the contribution of G4900E mutation(corresponding to G4946E of PxRyR)of SeRyR to diamide insecticides resistance in S.exigua,we introduced the PxRyR G4946E mutation with CRISPR/Cas9 technology into WH-S strain of S.exigua.The genome-edited strain(named 4946E)homozygous for the SeRyR G4900E mutation exhibited 223-,336-and>1000-fold resistance to chlorantraniliprole,cyantraniliprole and flubendiamide,respectively when compared to the wild type strain(WH-S)of S.exigua.Genetic analysis showed diamide resistance in the 4743M and 4946E strain was inherited as an autosomal recessive trait.This study has confirmed that I4743M mutation(already evolved in field populations,conferring moderate-level resistance)and G4900E mutation(to be evolved,conferring high-level resistance)of SeRyR result in different levels of resistance to diamide insecticides.Those results have important implications for developing DNA-based detection methods for resistance mutations and formulating adaptive resistance management strategy.