| Lymantria dispar is a worldwide pest with wide distribution and serious damage,which often causes serious damage to agriculture and forestry in large-scale outbreak period.The traditional methods of controlling L.dispar mainly depend on chemical agents,but the 3R problems of resistance,residue and resurgence caused by chemical control have become increasingly prominent.Populus is one of the main hosts of L.dispar,which can produce secondary metabolites to resist feeding.In order to explore the effect of poplar main secondary metabolites on the growth and development of L.dispar and its sensitivity to chemical agents,the representative secondary metabolites flavonoids,quercetin and rutin were added into artificial feed according to the content of poplar leaves.Two of CYP genes(LdCYP6B53 and LdCYP6AN15v1),and four of GST genes(LdGSTe2,LdGSTs1,LdGSTs2 and LdGSTz1)were selected,and the effects of these genes on detoxification function of flavonoids and quercetin in secondary metabolites were investigated by RNAi technology.The secondary metabolites flavonoids,quercetin and rutin were added to the diet containing cyanoacetamide to observe the survival rate of L.dispar under different combined treatments,and the cytochrome P450,glutathione S-transferase(GST),carboxyesterase(CarE),acetyl cholinesterase(AChE),superoxide dismutase(SOD),Catalase(CAT)in L.dispar were measured.The expression levels of the CYP and GST genes in different treatments were detected by real-time RT-PCR to comprehensively evaluate the sensitivity of the second instar larvae of L.dispar to cyanoacetamide under secondary metabolites stress.The main results are as follows.1.RNAi was used to silence the genes of LdCYP6B53,LdCYP6AN15v1,LdGSTe2,LdGSTs1,LdGSTs2 and LdGSTz1 in the third instar larvae of L.dispar.The effective silencing efficiency of different genes showed a time effect.The expression level of LdGSTe2 at 72 h was 57.96%lower than that of the control dsGFP and the silencing efficiency was the highest,indicating a very significantly difference from other time(P<0.01).The maximum silencing efficiency of genes LdCYP6B53,LdCYP6AN15v1,LdGSTs1,LdGSTs2 and LdGSTz1 was reached at 48 h,which were lower than those of the control by 59.24%,34.21%,23.19%,43.80%and 32.89%,respectively.The silencing of CYP and GST gene in L.dispar had no significant effect on the survival rate(above 90%),but there were significant differences in body weight and nutritional utilization indicators.The body weight and relative growth rate in control group were significantly higher than that of the dsRNA injection group.The body weight at 72 h and relative growth rate(RGR)at 120 h in different groups were GFP>LdCYP6B53>LdGSTz1≈LdCYP6AN15v1>LdGSTs1≈LdGSTs2>LdGSTe2.The lowest RGR and relative food intake(RCR)were 62.01%and 184.69%in the LdGSTe2 group,respectively,while the lowest food utilization rate(ECI)and food conversion rate(ECD)were 28.89%and 31.70%in the LdGSTs1 group,respectively.Furthermore,the survival rate and body weight of L.dispar were significantly changed after different L.dispar silencers were added into the diet containing secondary metabolism flavonoids and quercetin.The survival rate of the control group fed with flavonoids was 90%,while that of the treatment group injected with dsRNA was 56.67%-76%,and there was significantly lower than that of the control group.The survival rate of the control group dsGFP fed with quercetin was 95%,which was significantly higher than that of the treatment group injected with the target gene dsRNA.The highest survival rate was 83.33%after injection of LdGSTs2,and the lowest survival rate was 62.86%after injection of LdGSTs1.At 72 h after treatment with flavonoid-containing feed,the body weight of control dsGFP was 20.16 mg,which was significantly higher than that of dsRNA injection treatment group,in which LdGSTe2 had the lowest body weight,16.30 mg;after being fed with quercetin-containing feed,the body weight of control dsGFP was 20.36 mg,which was significantly higher than those of other treatment groups.These results indicated that CYP and GST gene silencing of L.dispar could inhibit insect feeding and food nutrition utilization,and reduced the adaptability to secondary metabolism such as flavonoids and quercetin.2.Adding flavone,quercetin and rutin to the diet containing cyanoacetamide and detecting the survival rate for 48 h,it was found that the survival rate of the treatment group fed with cyanoacetamide was significantly lower than that of the control(DMSO)and the secondary metabolism treatment groups,while the survival rate of the combined treatment group was lower than that of the cyanoacetamide treatment group.The activities of protective enzymes SOD,CAT and detoxification enzymes P450,GST,CarE and AChE of L.dispar showed that different stress treatments induced SOD,CAT,P450 and GST,and the induction effect of combined treatment group was mainly higher than that of cyanoacetamide treatment;the activity of CarE and AChE were inhibited,and the inhibition effect of cyanoacetamide treatment group and combined treatment group gradually weakened with time.Furthermore,the expression levels of L.dispar genes LdCYP6B53,LdCYP6AN15vl,LdGSTe2,LdGSTs1,LdGSTs2 and LdGSTz1 were detected.Except for LdGSTs2,other genes mainly showed induction effect under different stresses,and the induction degree was 1.01～46.38 times that of the control.The above results showed that the addition of secondary metabolites could induce the up regulation of protective enzyme and detoxifying enzyme activities,CYP and GST genes of L.dispar,but compete with insecticides for detoxifying enzyme binding sites,resulting in the reduction of pesticide degradation efficiency and the enhancement of toxicity,forming a joint effect of toxicity synergy.In this paper,the LdCYP6B53,LdCYP6AN15v1,LdGSTe2,LdGSTs1,LdGSTs2 and LdGSTz1 gene of the third instar larvae of L.dispar was silenced,confirmed that the genes can affect the process of feeding,food utilization and detoxification metabolism of plant secondary metabolites.The survival rate of the combined treatment of cyanoacetamide and secondary metabolites flavonoids,quercetin and rutin was lower than that of single cyanoacetamide treatment.The results showed that the single and combined treatments of cyanoacetamide and secondary metabolism could induce the activities of protective enzymes(SOD and CAT)and detoxifying enzymes(P450 and GST)of L.dispar,and induce the expression of CYP and GST genes increased,which indicated that the participation of secondary metabolites could compete with chemical insecticides for detoxification enzyme binding sites of L.dispar,thus enhancing the toxicity of insecticides.The above results can be helpful to explore the adaptability mechanism of L.dispar to host plant secondary metabolites and chemical agents,provide theoretical basis for further research on plant-insect interaction resistance mechanism,and provide reference for the use and mixing of insecticides. |
