Effects Of Poplar Secondary Metabolites On Performance And Key Detoxifying Enzymatic Activity Of Lymantria Dispar

Lymantria dispar,as one of the important forest pests,can eat more than 500 kinds of plants,causing serious damage to the forest.Poplar is one of the most favored host plants of L dispar.In present study,six poplar secondary metabolites(i.e.,caffeic acid,salicin,rutin,quercetin,flavone,and catechol)and three mixtures containing characteristic secondary metabolites in poplar were selected to identify the effects of poplar secondary metabolites on L.dispar.Mixture 1 contained flavone and salicin;mixture 2 contained salicin,caffeic acid,and catechol;and mixture 3 contained flavone,catechol,and caffeic acid.The different secondary metabolites adding into artificial diets were used to feed on 2nd instar L.dispar larvae.And then we studied the effects of different secondary metabolites on larval growth and development,antifeedant activity,nutrient utilization,and activities of cytochrome P450(P450),glutathione S-transferase(GST),carboxylesterase(CarE)and acetyl cholinesterase(AChE)during 15 days.Based on the changes of detoxifying enzyme activity of L.dispar induced by poplar secondary metabolites,and combined with published research reports,the 25 CYP and 13 GST genes were selected as candidate genes.Real-time PCR was used to study the expression pattern of CYP and GST genes at different developmental stages and after treatment with secondary metabolites.These genes were induced by secondary metabolites including LdCYP4L23,LdCYP6AB37,LdCYP9A54v1,LdCYP332A6,LdGSTel,and LdGSTo1.RNAi technology was further used to explore the detoxifying function of these genes on secondary metabolites.The main results are as follows:1.The effects of different poplar secondary metabolites on weight and survival rate of L.dispar larvae are different.All L.dispar larvae were died under flavone,mixture 1 and mixture 3 treatments after 7 days.After 15 days of feeding,the weight of larvae descreased in the treatment groups of caffeic acid,salicin,rutin,quercetin and mixture 2.Moreover,the survival rate and body weight of larvae treated by mixture 2 were lower than those of caffeic acid,salicin and catechol.The L.dispar larvae treated with mixture 2 had a significantly longer development time than that of the control treatment.The results of food consumption and antifeeding rate showed that the artificial diets containing secondary letabolites significantly reduced the food consumption of the 2nd instar L.dispar larvae.Among them,the flavone treatment group had the highest antifeedant rate,which was 87.58%.Food selection results showed that the number of larvae on the artificial diets with flavone-treated group was significantly lower than that in the control group.The nutrition utilization results showed that the relative growth rate(RGR),relative efficiency of consumption rate(RCR),efficiency of the conversion of ingested food(ECI)and efficiency of the conversion of digested food(ECD)of the larvae in the flavone,mixture 1 and mixure 3 treatment groups were significantly reduced,but the approximate digestibility(AD)was higher than that of the other treatment groups.2.Poplar secondary metabolites significantly affected detoxifying enzyme activities in L.dispar larvae.P450 and GST activities were significantly induced by caffeic acid,salicin,rutin,quercetin,flavone,catechol,mixture 1,mixture 2,and mixture 3 treatments from 12h to 72h.The mixture 3 treatment had the highest P450 activity,which is 6.83-fold of the control when the L.dispar larvae were fed with artificial diets containing secondary metabolites for 12 hours.The GST activity was highest in the catechol treatment,which is 3.40-fold of the control.The CarE activity was inhibited from 12h to 24h,and the inhibitory rate of mixture 1 treatment was the highest with 50.78%of the control after 12h of treatment.The AChE activity was inhibited with the prolongation of time,and the AChE activity of caffeic acid,salicin,rutin,quercetin,flavone,catechol,mixture 1,mixture 2,and mixture 3 treatments decreased after 72h.3.The 25 CYP and 13 GST genes of L.dispar are expressed at all developmental stages but its expression level is different.There are differences in gene expression level among the egg,larva,pupae and adult stages.Compared with the egg stage,LdCYP4L23,LdCYP4G81,LdCYP9A56,LdCYP332A6,LdGSTt1,LdGSTd2,LdGSTe1,LdGSTe2,LdGSTe3,LdGSTo2,and LdGSTs2 genes were all up-regulated from the 1st to 3rd instar larval development stage.This result may be associated with the ingestion of secondary metabolites during the larval stage.After different secondary metabolites treatments,there were differences in the expression levels of 25 CYP genes and 13 GST genes in L.dispar.The LdCYP9A54v1 gene was induced by caffeic acid,salicin,rutin,flavone,catechol,mixture 1,mixture 2,and mixture 3,and the highest expression level after mixture 2 teratment is 46.12-fold of the control.The LdCYP4L23 and LdCYP6AB37 genes can be induced by caffeic acid,quercetin,flavone,and mixture 3.The induction range is 2.07-fold to 8.79-fold of the control.The expression of LdCYP332A6 gene can be induced by caffeic acid,salicin,rutin,and mixture 3.Its induction range is 1.50-fold to 2.63-fold of the control.Among the GST family genes,salicin can induce the expression level of LdGSTd1,LdGSTo1,and LdGSTz1 genes,which are 3.80-fold,3.15-fold,and 29.24-fold of the control,respectively.The LdGSTe1 gene can be induced up-regulated by salicin,rutin,mixture 1,mixture 2 and mixture 3,which induction range is from 1.83-fold to 8.66-fold of the control.4.The RNAi technology was used to silence LdCYP4L23,LdCYP6AB37,LdCYP9A54v1,LdCYP332A6,LdGSTel and LdGSTol genes in the 3rd instar L.dispar larvae.Different genes were silenced at different time points.The relative expression levels of LdCYP332A6,LdGSTel,and LdGSTo1 were the lowest after 48 hours with redution of 84.00%,46.18%and 79.87%compared with the control dsGFP group,respectively.While the LdCYP4L23 and LdCYP9A54v1 genes had the highest silencing efficiency at 72h,which were 66.00%and 49.06%,respectively.The LdCYP6AB37had a maximum silencing efficiency of 66.57%at 120h.Compared with the dsGFP-injected control,the larvae injected with LdCYP4L23,LdCYP6AB37,LdCYP9A54v1,LdCYP332A6,LdGSTe1,and LdGSTo1 genes have no significant differences in body weight,survival rate and nutrient utilization.The silenced L.dispar larvae were further fed with artificial diets containing secondary metabolites to study on the resistance of CYP and GST genes to secondary metabolites.These results showed that L.dispar larvae silenced with LdCYP332A6 and LdGSTe1 gene showed 14.75%and 27.81%descrease in weight body after being fed with rutin for 48 hours.Compared with the control,the weights of L.dispar larvae with LdGSTo1,LdCYP9A54v1 and LdCYP6AB37 gene silencing were reduced from 9.00%to 30.73%after treatment with salicin for 48h,72h and 96h.Moreover,it was found that the L.dispar larvae with LdCYP6AB37 gene silencing significantly prolonged the developmental period of 3rd instars compared with the control after feeding the mixture 2 diets;In the diet supplemented with rutin,the developmental period of LdGSTe1 gene silencing was also significantly prolonged.Analysis of nutritional utilization showed that the ECI and ECD of L.dispar larvae with the LdCYP6AB37 gene silencing decreased by 2.12%and 2.27%,respectively,after feeding salicin.The ECI and ECD of L.dispar larvae silenced by LdGSTe1 gene decreased 1.49%and 2.66%,respectively,after feeding rutin,which indicates that LdCYP6AB37 and LdGSTe1 are involved in the metabolic detoxification of salicin or rutin by the L.dispar.In this thesis,secondary metabolites were selected from poplars to feed L.dispar larvae.Through the performence and feeding consumption tests,flavone was found to be highly antifeedant.The P450 and GST detoxifying enzymes were significantly induced by salicin,caffeic acid,catechol,rutin,quercetin,and flavone.Meanwhile,salicin and rutin could significantly induce the expressions of LdCYP4L23,LdCYP6AB37,LdCYP9A54v1,LdCYP332A6,LdGSTe1 and LdGSTo1 genes.Under secondary metabolites stress,the LdCYP6AB37 and LdGSTe1 gene silencing affected the adaptability of L.dispar larvae to salicin and rutin.These results will contribute to explore the adaptive mechanism of L.dispar to the host plant,and provide the basic theory for the research of host resistance mechanism in the future,and provide targeted gene information for breeding resistant transgenic poplars.