Resistance Monitoring Of Epacromius Coerulipes Ivanov And Research Of Metabolic Resistance Mechanism To By Butene-fipronil

Epacromius coerulipes Ivanov is one of the locust widely distributed around China’s grasslands and farming-pastoral zone. It can cause serious economic damage and migration harm to agricultural and animal husbandry with its capability of flying short distances. Locust management based on chemical pesticide leads to the increasing resistance and declining control efficiency, thus contributing to the increasingly frequent outbreak of locust plague. Using butane-fipronil as an emergency measure for locust plague has been widely applied. It is necessary to carry out resistance monitoring and to research into the biochemistry and molecular changes of resistance for better understanding of the development of Epacromius coerulipes’resistance for various pesticide, thus contributing to the sustainable pest management.This paper uses Epacromius coerulipes as the research object and presents resistance selection and realized heritability of butane-fipronil by carrying out resistance monitoring and synergistic agent experiments. We also analyzed the risk of resistance to butane-fipronil and the resistance development of several common pesticide. Detoxifying enzyme activity was measured using both resistant and susceptible strains to analyze the biochemistry mechanism. Transcriptome data was obtained by transcriptome sequencing technology to evaluate the differential expression gene related to butane-fipronil resistance. Quantitative verification of P450, GSTs and CarE overexpression was carried out using RT-PCR technic. The results are as follows.1. Susceptible baseline for Epacromius coerulipes & field resistance monitoringEpacromius coerulipes was collected from natural grassland and subcultured in laboratory. Susceptible baselines of 10 common pesticide was established by toxicity test using topical application. Resistance monitoring was carried out using these baselines at 3 different grasslands near Daqing in 2010 and 2013 separately. The results showed a low resistance to N-nitrosodimethylamine (NDMA) and a declining susceptibility to phoxim, as well as susceptibility to all other pesticides. Resistance monitoring also showed that the pests restore their susceptibility in a short time upon discontinuing dusting at a low resistance level. Mixed use of PBO and NDMA or TPP and Phoxim pesticide can enhance the insecticidal effect and reduce pesticide use, thus benefiting the prevention and postponement of resistance development. In a word, this paper presents practical advice on locust resistance governance and pesticide selection.2. Breeding of butane-fipronil-resistant Epacromius coerulipes & Resistance risk evaluationEpacromius coerulipes was fed and screened for 7 generations while resistance increased to 11.74 times. The first 4 generations showed a relatively slow resistance growing process until the fifth generation. Epacromius coerulipes’s realized heritability was carried out(h2=0.3191). It was estimated that it would take 12-25 generations for the resistance to increase to 10 times in the field. The resistance risk evaluation was relatively small given the only 1 generation of Epacromius coerulipes in the north.3. Biochemistry mechanism of Epacromius coerulipes’resistance to butane-fipronilResearch on Epacromius coerulipes’resistance to butane-fipronil was carried out using in-vivo synergistic test and detoxifying enzyme activity determination method. The detoxifying enzyme activity determination showed that all three detoxifying enzyme increased in resistant strains(4.04 times for multi-function oxidase,1.94 times for esterase and 1.34 times for glutathions-transferase). The results also showed a significant induction of multi-function oxidase and esterase in both resistant and susceptible strains, while only the resistant strain was inducted by the glutathions-transferase after different dose of butane-fipronil induction. The synergistic test showed that PBO has the most significant synergism effect to both resistant and susceptible strains, followed by TPP, while DEM only has synergism effect to the resistant strain. We draw the conclusion that multi-function oxidase has a more significant impact on resistance formation while esterase and glutathions-transferase might also be the promoting factor.4. Transcriptome sequencing for Epacromius coerulipesTranscriptome sequencing was conducted using the second generation of Illumina/Solexa HiseqTM2500 sequencing platform.13.4 Gb data was collected and later assembled into 63,033 unigenes with average length of 773bp and 1589bp N50.39.8% of the unigenes (which is 25132 unigenes) were successfully annotated. KEGG analysis showed that 218 metabolic/information channels were formed by these 7507 unigenes, among which 189 unigenes were included in metabolic channels for exogenous agents. In addition,5696 simple sequences repeats were detected. This sequencing provides further molecular foundation for researches on the resistance mechanism and genetic function of Epacromius coerulipes.5. Analysis on differential expression geneGO enrichment analysis and metabolic pathway enrichment analysis on differential expression gene was carried out on gene expression level of both PS and PR strains using Epacromius coerulipes transcriptome. The results show 2568 differential expression genes (1646 up-regulated genes and 922 down-regulated genes). GO enrichment analysis on differential expression gene showed 61 significantly enriched genes in cell component,150 in biological process. Among the 127 significantly enriched genes in molecular function, there are 37 gene sequences showing REDOX enzyme activity (23up-regulated genes and 14 down-regulated genes),5 of them were annotated as P450 gene in Nr data set. Analysis on differential expression gene annotated in GO dataset was carried out using topGO software. The results showed that the nodal point GO0052689 which was significantly enriched in molecular function was active to Carboxylic ester hydrolases while GO:0004553 was active to hydrolase.2.98% of the COG catalogue of differential expression gene (28 genes) are Secondary metabolites biosynthesis, transport and catabolismdifferential expression gene.Differential expression genes were distributed around 130 metabolic pathways. The top 7 richest pathways are ribosome, starch and sucrose metabolism, ascorbic acid metabolism, P450 drug metabolism with the aid of cytochrome, P450 allogenic material metabolism, glycolysis/gluconeogenesis and glutathione metabolism (MAPEG). P450 drug metabolism with the aid of cytochrome, P450 allogenic material metabolism and MAPEG are among the most important metabolism pathways of pesticide.23 differential expression genes were found annotated to these pathways separately (11up-regulated genes and 12 down-regulated genes). Lots of proteins were related to the up-regulated genes in metabolic pathways figures. Glutathion S-transferase [EC:2.5.1.18] appeared most frequently and were found up-related in all three metabolic pathways. There were also many other dehydrogenase and transferase that might be related to locusts’metabolism of pesticide which set foundation for later research on genes function.6. Annotation and classification of genes related to pesticideGene sequences screening of Epacromius coerulipes transcriptome data showed that 316 encoding genes for P450, CarE and GSTs were annotated related to pesticide while 76 encoding genes for pesticide-targeted protein were also annotated. Annotation of GO dataset showed that 213 unigenes were related in detoxification of allogenic material, while 34 unigenes were related in encoding pesticide-targeted proteins. Statistical analysis of SNP Locus showed that there are different types of locus, namely AT、AC、AG、GT、CG、CT、416 SNP Locus were found which can be used to research on mutation of resistance-related genes.We also established phylogenetic tree of P450, CarE and GSTs using NJ method and compared the Epacromius coerulipes transcriptome data with existingtranscriptome data of other species. The results showed that the 58 P450 genes belong to 18 CYP families,52 CarE genes belong to 5 families, while 24 GSTs genes belong to 6 GSTs families and Micromal. Most of the detoxification enzyme are annotated in CO and COG dataset as related to pesticide metabolism with 48 differential expression genes expressed in them (23up-regulated genes and 25 down-regulated genes). These genes lay the foundation of further research in resistance mechanism.7. Quantitative determination of overexpressing genes for detoxification enzymeRelative transcript level of significantly up-related genes sequences in Epacromius coerulipes transcriptome was measured in both PS and PR strains using RT-PCR method with GAPDH as the reference genes. The results showed higher expression level of 5 P450 sequences,3 CarE sequences and 3 GSTs sequences in PR strains, most of them are extraordinary significant. Similar results of RT-PCR measurement and transcriptome sequencing data proved the accuracy of Epacromius coerulipes transcriptome, which can be used for further molecular biology research. The overexpression of detoxification enzyme in Epacromius coerulipes was also further testified by the above results. In conclusion, resistance of Epacromius coerulipes to butane-fipronil is closely related to the overexpression of detoxification enzyme (namely P450, CarE and GSTs).