Analysis Of Fipronil Resistance Of Plutella Xylostella(L.) Based On Metabolomic Data

Metabolomics is an important part of systemic biology that includes qualitative and quantitative analysis for all metabolites of a particular organism or cell during a given physiological period or condition and the identification of differential target metabolites.At present,the metabolomics focuses on disease diagnosis,drug screening or pathology and other medical fields,but there are few studies on the detoxification of pesticides in insects.Plutella xylostella is an important pest of cruciferous vegetables and has become a hotspot in resistant research.P.xylostella has a rapid development in resistance to pesticides and is extremely difficult of control.Previous studies on the resistance of P.xylostella were mainly focused on detoxification enzymes,environmental adaptation and so on,but metabolic changes or signal pathways as well as other aspects have not been reported.In this study,metabolomics was used to compare the metabolomic changes in fipronil-resistant and susceptible strains(FRS and SS)from which the differential metabolites were excavated and mapped to their corresponding metabolic pathways.Key enzymes of the metabolites between the two strains were identified using the KEGG pathway.At the same time,transcriptome data and RNAi were used to verify the important role of the key genes in the resistance of P.xylostella.The main results were as follows:1.Changes in metabolites of P.xylostellaThrough NMR,LC-MS and GC-MS analysis,82 different metabolites were qualitatively and quantitatively identified in P.xylostella samples.In FRS,the concentrations of 37 metabolites were increased,whereas those of 45 other metabolites decreased.The results showed that the metabolite profiles of the SS and FRS were significantly different regarding some fatty acids and amino acids,which involved in multiple pathways including glutamate metabolism,fat metabolism and et al.The physiological significance and potential detoxification mechanism of metabolite were changed to P.xylostella from the aspects of amino acid metabolism,energy metabolism,fatty acid metabolism and detoxification.2.Metabolic pathway changes in P.xylostellaBy matching the existing metabolic pathways of P.xylostella,the differential metabolites were mapped to metabolic pathways to identify the significantly different pathways.We found that a total of six pathways showed different expression between the SS and FRS.By metabolic and KEGG mapping,we found a metabolic network of differential metabolites,the key enzymes of which were directly related to the differential metabolite positions.Combined with the transcriptome data,we found that two key enzymes were significantly upregulated in the FRS in glutamate metabolism,namely,L-glutamate synthase and glutamate decarboxylase,which suggested that they may be associated with fipronil detoxification.3.RNAi and bioassay to verify the roles of glutamate decarboxylase(PxGAD)in P.xylostellaThe primers were designed using the sequence of the glutamate decarboxylase gene(Px004382)from the genome database of P.xylostella.Sequence analysis showed that PxGAD had an aspartate transaminase conserved domain.The expression patterns of PxGAD was detected by real-time fluorescence quantitative PCR,and it was found to be Significantly higher expression in 1st instar larvae and fat body.The primers were designed to synthesize dsRNA according to the sequencing results.After micro-injection of FRS larvae,the mRNA expression of P.xylostella larvae was significantly decreased at 12 h and 24 h,which indicated that the dsPxGAD had a significant interference effect.Twelve hours after the injection of dsPxGAD and dsGFP and administration of a slight drop of LD20 fipronil,we found that the mortality of larvae following dsPxGAD injection was higher than that of the larvae injected with dsGFP,indicating that the resistance to fipronil of P.xylostella larvae was lower than the control after dsPxGAD injection.The RNAi and bioassay results suggested that PxGAD may play an important role in the detoxification of fipronil in P.xylostella.This study was completed from a metabolomics perspective and detected the metabolite changes in P.xylostella,which were screened for pesticide resistance for a long time by metabolomics methods.The discovery of possible metabolic pathways and key genes involved in the detoxification of insecticides has opened up a new approach for the study of resistance to P.xylostella and provided a feasible basis for comprehensive control of P.xylostella and other important agricultural pests.