| Since 2009,the outbreak of Galeruca daurica Joannis in the grasslands of Xilingol,Inner Mongolia,has caused the destruction of natural pastures and seriously affected the development of animal husbandry.Because of the black color of its body,herders call it "black worm".Sand onion leaf beetle is mainly distributed in Siberia,the Mongolian plateau,Korea and many places in northern China.In recent years,the insects are mostly found in desert grasslands and degraded grasslands,which makes the fragile grassland ecosystem environment face a more serious situation.Chlorantraniliprole belongs to the class of o-formylaminobenzamide.In practice,some pests are killed directly by the insecticide,while others are exposed to concentrations of the insecticide that are not sufficient to kill them,or the insects are exposed to sublethal concentrations due to the degradation of the insecticide,and the insects experience sublethal effects.Insects are bound to develop target resistance to insecticides when exposed to this growth environment for a long time.However,the level of G.daurica susceptibility to chlorantraniliprole remains unclear.The aim of this study was to determine the effects of sublethal concentrations of chlorobenamide on the growth and detoxification enzyme activity of G.daurica.To detect the mutation frequency of the ryanodine receptor(Gd Ry R)gene associated with diamide insecticide resistance in the field,it was found that there were resistance site mutations in the local population;The changes of binding patterns of diamide insecticides and Gd Ry R before and after mutation were further analyzed.This study explored the mechanism of resistance of G.daurica to diamide insecticides from the perspectives of molecular biology and structural biology,so as to provide reference for the reasonable selection of insecticides,and to prevent the rapid development of resistance to chlorantraniliprole by scientific and effective means.The main results are as follows:1.Sublethal effect of chlorantraniliprole on biological traits of G.daurica parent generationThe toxicity of chlorantraniliprole to the 3rd instar larvae of leaf beetle was determined by Leaf drug film method.The developmental effects of chlorantraniliprole at LC15,LC30 and LC50 on the 3rd instar larvae were compared: the developmental time of 3rd instar larvae,pupal stage and adult stage of the 3rd instar larvae was significantly longer compared with the control group.The larval survival rate,pupation rate,fledging rate and adult stagnation rate decreased under different sublethal concentrations of chlorantraniliprole,and were negatively correlated with the sublethal concentrations;the larval body weight,pupal weight and adult body weight of the 3rd instar larvae of the sand onion leaf beetle decreased significantly compared with the control group.The above results indicated that the sublethal concentration of chlorantraniliprole could cause significant inhibition of insect growth and development.2.Sublethal effects of chlorantraniliprole on the activity and RNA relative expression levels of detoxifying enzymes in G.daurica 3rd larvaIn this study,the activities of detoxification enzymes(one metabolic enzyme carboxylesterase Car E and cytochrome P450s;two metabolic enzymes glutathione-S-transferase GST and uridine diphosphate glycosyltransferase UGT)were measured in the 3rd instar larvae of G.daurica after 24 h treatment with LC15,LC30 and LC50 concentrations of chlorantraniliprole,respectively.The expression levels of detoxification enzyme genes in the G.daurica were detected by q PCR after 24 h treatment with LC15,LC30 and LC50 concentrations.Car E,GST and UGT viability were induced by different sublethal concentrations of chlorfenvinphos;CYP450 activity was not significantly different after chlorfenvinphos treatment compared with the control.The expression profiles of Car E(17),GST(2),UGT(13)and 54CYP450 genes(26 genes of CYP4 family,22 genes of CYP6 family,3 genes of CYP9 family and 3 genes of other CYP families)screened by transcriptome data were analyzed by different sublethal concentrations of chlorantraniliprole treatment,which revealed that 6 Car E,2 GST,11 UGT and 32 CYP450 genes were found to be up-regulated in expression by different sublethal concentrations of chlorantraniliprole.3.Sublethal effects of chlorantraniliprole on the RNA relative expression levels of calcium-binding protein in G.daurica 3rd larvaIn order to understand the role of calc-binding protein in methylol resistance,the expression levels of Ca M,CAPSL,Tn Cl and CRT in the third instar larvae of G.daurica treated with chlorfenamide at concentrations of LC15,LC30 and LC50 for 24 h were determined.The expressions of Ca M and Tn Cl were positively correlated with the sublethal concentration of chloranisamide.The expression of Gd CAPS decreased gradually with the increase of the sublethal concentration.The expression of Gd CRT in LC15 treatment group was the highest,and significantly higher than that in control group(P<0.05).Compared with the control group,the expression of Gd CRT in LC30 and LC50 treatment groups was significantly down-regulated(P<0.05).4.Gene cloning of Gd Ry R gene and analysis of the G4911 E and I4754 M allelic mutation frequenciesThe c DNA sequence(Gen Bank number: OP828593)was obtained by piecework cloning of the ryanodine receptor(Gd Ry R)gene.The length of the gene was 15,399 bp,encoding 5,133 amino acids.Sequence analysis showed that the N-terminal of the nidin receptor had 11 typical conserved structures,and the C-terminal had 6 transmembrane helical structures.The homology analysis showed that Ry R of G.daurica and Leptinotarsa decemlineata(Gen Bank number: QZZ63290.1)had the highest homology.Both were Coleoptera,and the amino acid sequence consistency reached 91.33%.Two potential allelic mutation sites and frequency of Gly4911 Glu and Ile4754 Met were detected by single head RNA extraction using reverse transcriptional c DNA as template.The results showed that only heterozygous genotypes of G4911 E existed in the tested population,accounting for 12% of the population.The heterozygous and homozygous genotypes of I4754 M were 32% and 2%,respectively.5.Homology modeling of Gd Ry R and analysis of its binding mode with diamide insecticidesIn this study,3D structural models of Gd Ry R,Gd Ry R-E4911 and Gd Ry R-M4754 mutants were successfully constructed using computer simulation technology,and molecular docking models of Gd Ry R,Gd Ry R-E4911 and Gd Ry R-M4754 with two diamide insecticides were constructed.And its combination mode is analyzed.It was found that the affinity of Gd Ry R’s G4911 E mutation model with chloranisamide and cyancyanamide decreased,and the forming force mode decreased,which affected the binding stability of fish nitin receptor and diamide insecticides. |
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