| The beet armyworm,Spodoptera exigua,is an omnivorous insects around the world and harm a variety of crops.Due to the extensive use of pesticides,the beet armyworm has high resistance to various traditional insecticides.Chlorantraniliprole is a new diamide insecticide developpedbyDupont company,which activate the ryanodine receptor and lead the insect to excessively release calcium ion,to paralyze and disable,and to make insectsdeath ultimately.In this paper,the effects of sublethal doses of chlorantraniliprole on the detoxification enzymes activities,the growth and reproduction of the beet armyworm were researched.The effects gene expression of the beet aramyworm induced by chlorantraniliprole were also analysized by transcriptome analysis.The cytochrome P450 genes of significant up-regulation expression induced by chlorantraniliprole were cloned the genes full-length and the functionswere analyzed by RNA interference technique.The above mentioned research revealed the molecular mechanism of cytochrome P450 genes in the process of adaptation of beet armyworm to chlorantraniliprole,and provided theoretical basis to elucidate the resistance molecular mechanism ofbeet armyworm on chlorantraniliprole and to manage the resistance.The full text is summarized as follows:The toxicities of chlorantraniliprole on SE-Lab and SE-Sel strains were assayed by incorporation bioassay and the SE-Sel strain was achieved with continuous selecting with LC25 sublethal doses for six generations from SE-Lab strain.According to the age-stage two-sex life tables theory,the growth,mortality,fecundity data of the tested insects were also recorded to analyze the differences of the age-stage two-sex life table parameters between the strains of SE-Lab and SE-Sel.The adult preoviposition period and total preoviposition period of SE-Sel strains became longer and spawning quantity and survival rate decreased.The intrinsic rate of increase(r),finite rate of increase(?)and net reproductive rate(Ro)of SE-Sel strain were significantly lower than those in SE-Lab strain.The values of r,? and Ro in SE-Lab and SE-Sel strains were 0.18 and 0.16 d-1,1.20 and 1.17d-1,358.42 and 203.12 d-1,respectively.The mean generation times of SE-Sel strain was not significant with those in SE-Lab.The synergistic effects of enzyme inhibitors(TPP,DEM,PBO)with chlorantraniliprole on the SE-Lab and SE-Sel strains were assayed using the dip-leaf method.The toxicities of chlorantraniliprole on the insects,which were fed the cabbage leaves soaked the solution of enzyme inhibitor or 0.1%TritonX-100 as blank control before 12 hours,were assayed by the dip-leaf method.The results indicated that the synergistic effects of PBO were the strongest amongthree enzyme inhibitors and the synergic ratios on the strains of SE-Sel and SE-Lab were 1.58-and 1.69-fold,respectively.The changes of three metabolic enzyme(CarE,GSTs,MFOs)activities in the midgut and fatbody of S.exigua treated by the sublethal doses of chlorantraniliprole were further detected and found that three detoxification enzymes were all promoted,meanwhile the MFOs activities were the most significant enhancement,which of the midgut and fatbody in SE-Sel strain were enhanced 2.07-and 2.10-folds,and the MFOs activities of SE-Sel induced by the sublethal dose of chlorantraniliprole again were also enlarged 4.02-and 3.44-fold,respectively.The activities of three detoxifying enzymes were decreased when the tested insects were treated by enzyme inhibitors and the descend range of MFOs activity among three enzymes was maximum,which was only 42.3%-44.8%compared to the treatment not treated with the enzyme inhibitor.Transcriptome sequencing of SE-Lab and SE-Sel strains were held and two biological repeats were set for each treatment.52756 Unigenes of S.exigua were achieved and 28488 genes were annotated.With significance analysis(FDR<0.05,|log2FC|>1),2935 significant differentially expressed genes were demonstrated,which of 2306 genes were significant down-regulated expression and 629 geneswere significant up-regulated expression.These difference genes were divided into 3 categories,including cellular component,molecular function and biological process.Transcriptome analysis indicated that up-regulated expression ofcytochrome P450 genes was one of the important factors of chlorantraniliprole resistance of the beet armyworm.The up-regulated expression P450 genes on the transcriptome data were verified by qRT-PCR,and ten cytochrome P450 genes which were up-regulated expression induced by chlorantraniliprole were screened out,followed by the detection of expression often genes in the resistant field populations,including SE-PZ(with 184.0 folds resistance ratio on chlorantraniliprole)and SE-ZY(with 507.4 folds resistance on chlorantraniliprole).Four cytochrome P450 genes,including Unigene0033153,Unigene0034605,Unigene0035508,Unigene0034612,which could be related to the resistance of chlorantraniliprole,were identified andbelonged to the cytochrome CYP9 family.The expression of Unigene0035508 on the strains of SE-Sel and SE-ZY were enhanced 8.5-fold.Unigene0035508 gene in SE-ZY strain was silenced by feeding dsRNA and the interference effect of 12 h after treatment reached the peak.Compared to the control group,the relative expression of Unigene0035508 in SE-ZY strain decreased by 91.7%.After RNA interference,the insects were treated by the median lethal concentration of chlorantraniliprole and found that the mortality rate was increased at 24 after treatment compared to control group.However,the mortality rates were not significant between the two groups at 48 h after treatment.In generel,the sublethal doses of chlorantraniliprole have a continuous control effect on S.exigua in a short period and the resistance is related to its long-term use.MFOs could bethe major detoxication enzyme involved in the metabolic detoxification of chlorantraniliprole in the S.exigua.The cytochrome P450 gene(Unigene0035508)associated with the detoxification could be related to generate the resistance of chlorantraniliprole on the beet armyworm. |
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