The Molecular Mechanism Of Glutathione Stransferase And Carboxylesterase Mediated Chlorantraniliprole Stress Response In Hyphantria Cunea

Hyphantria cunea is a global pest in agriculture and forestry.At present,chemical pesticide control is still the effective measure for H.cunea.Chlorantraniliprole,a new generation of highly effective insecticide,acts on insect ryanodine receptors and releases Ca²⁺from cells,causing muscle contraction,twitching and death in insects.Glutathione S-transferase（GST）and carboxylesterase（CarE）play an important role in the detoxification metabolism of insects to a variety of chemical insecticides.In this study,the toxicity and detoxification enzyme activity of common insecticide chlorantraniliprole to H.cunea were investigated.The differentially expressed GST and CarE genes of H.cunea treated with chlorantraniliprole were screened by transcriptome sequencing technique.The differences of GST and CarE gene expression levels of H.cunea treated with sublethal concentration of chlorantraniliprole were detected by real-time fluorescence quantitative technique.In addition,RNA interference（RNAi）technology was used to verify the function of GST and CarE genes,which laid a foundation for further investigation of the detoxification metabolic process of chlorantraniliprole.The main results were as follows:1.The bioassay showed that the median lethal concentration(LC₅₀)and sublethal concentration(LC₃₀)of chlorantraniliprole in the 3^rd instar larvae of H.cunea were 21.40μg/L and 11.13μg/L,respectively.Chlorantraniliprole had high biological activity and good insecticidal activity against H.cunea at lower concentration.The 3^rd instar larvae of H.cunea were treated with LC₃₀ concentration of chlorantraniliprole and acetone artificial diet,and the transcriptome library was constructed by Illumina technique.A total of 20 GST differential genes were screened,including 7 genes in Sigma subfamily,2 genes in Omega subfamily,6genes in Delta subfamily,4 genes in Epsilon subfamily,1 gene in Theta subfamily,of which 6GST genes were up-regulated and 14 GST genes were down-regulated.A total of 39differentially expressed CarE genes were screened,including 5 in acetylcholinesterase subfamily,14 inα-esterase subfamily,16 in juvenile hormone esterase subfamily,3 in neuroligin subfamily and 1 in neurotactin subfamily,of which 19 CarE genes were up-regulated,and 20 genes were down-regulated.2.Effects of 72 h chlorantraniliprole LC₃₀ concentration treatment on the activities of GST and CarE enzymes in the 3^rd instar larvae of H.cunea were investigated.The results showed that the activities of GST enzyme in the 3^rd instar larvae of H.cunea were inhibited by LC₃₀ chlorantraniliprole treatment for 24 h and 48 h,which were 57.83%and 91.33%of that of the control group,respectively.The GST enzyme activity of the treatment group was 15.45μmol/min/g fresh weight after 48 h of treatment,which was 1.17 times significantly higher than that of the control group.After treatment for 24 h and 72 h,CarE enzyme activity were14.41 U/g and 18.54 U/g,which were 22.51%and 60.80%of that of the control group,respectively.The CarE enzyme activity of the 48 h treatment group was 38.86 U/g,which was1.36 times higher than that of the control group.3.The expression patterns of GST and CarE genes in different developmental stages and tissues showed that the relative expression of HcGSTs2,Hc CarE4 and Hc CarE37 reached the peak at the 4^th instar,the relative expression of HcGSTs3 and HcGSTe1 were the highest at the7^th instar,and HcGSTd3 was highly expressed in the male pupae.HcGSTe1 was highly expressed in fat body,the relative expression of Hc CarE4 and HcGSTs2 were the highest in the midgut.HcGSTs3 was highly expressed in foregut.And in the malpighian tubes,the relative expression of HcGSTd3 and Hc CarE37 were the highest.It is speculated that the specific expression patterns of GST and CarE genes in different developmental stages and tissues are related to the detoxification metabolism of H.cunea larvae.4.11 GST family genes and 12 CarE family genes were screened from transcriptome data.Real-time fluorescence quantitative RT-PCR was used to detect the differences of gene expression in the 3^rd instar larvae of H.cunea treated with chlorantraniliprole LC₃₀ for 24 h,48h and 72 h.The most significant increase of GST and CarE gene expression in H.cunea exposed to chlorantraniliprole stress were as follows:The expression of HcGSTd1 in the treatment group was 2.29 times higher than that in the control group treated with chlorantraniliprole for 48 h.After 72 h of treatment,the expression level of HcGSTs2 was 6.47times higher than that of the control group.At 24 h,48 h and 72 h,Hc CarE38 gene expression was 3.16,9.66 and 9.45 times higher than that of the control group,respectively.5.The molecular mechanism of chlorantraniliprole metabolism mediated by HcGSTs2,HcGSTs3,HcGSTe1,HcGSTd3,Hc CarE4,and Hc CarE37 in H.cunea were analyzed by RNAi technique.The silencing efficiency was significant at 48 h and 72 h of treatment.After 48 h of treatment,the most significant silencing effect was HcGSTe1,which was 89.07%.The lowest silencing efficiency was HcGSTs2,which was 33.74%.The most significant interference effect in 72 h was Hc CarE4,and the silencing efficiency was 85.33%.The lowest silencing efficiency was Hc CarE37,which was 21.38%after treatment for 72 h.Silencing Hc CarE4,Hc CarE37,HcGSTe1,HcGSTd3,HcGSTs2 and HcGSTs3 genes respectively,the susceptibility of H.cunea to chlorantraniliprole significantly increased,and the mortality was 83.33%,76.67%,70.00%,66.67%,83.33%and 90.00%,respectively,while that of the control group was 43.33%.The above results show that these genes are the key genes of detoxification metabolism of chlorantraniliprole.