| The citrus red mite,Panonychus citri（Mc Gregor）,is one of the cosmopolitan citrus mite pest,which can rapidly develop resistance to acaricides.However,chemical control is still the main method in agriculture.to maintain the effectiveness of the current acaricides,it is necessary to conduct resistance monitoring and elucidate resistance mechanisms.In this study,resistance of seven acaricides,including spirodiclofen,abamectin,and etoxazole in field populations of P.citri was investigated.The target mutation frequency and metabolic resistance of the resistant population were further studied.1.Resistance monitoring of P.citriIn this study,the resistance of P.citri to seven acaricides in four field-collected populations of China was determined by the leaf spray method.The monitoring results showed that adult mites of four populations have developed moderate to extremely high resistance to abamectin（resistance ratio,RR:15.73?162.98）,which was sensitive to bifenazate（RR:2.35?3.91）,relatively sensitive to fluazinam and propargite（RR:1.55?32.08 and 2.36?16.06,respectively）.Eggs of four populations have developed extremely high resistance levels to etoxazole（RR:502.06?5001.51）,moderate to extremely high resistance levels to fenpyroximate（RR:30.35?185.99）,low to extremely high resistance levels to spirodiclofen（7.46?63009.98）,Which was relatively sensitive to bifenazate（RR:4.79?21.25）,sensitive to fluazinam and propargite with the RR of 1.38-3.80 and 1.70-2.86,respectively.2.Analysis of target?site mutationThe above results of resistance monitoring showed that Nanning population had high resistance to abamectin,spirodiclofen and etoxazole.Based on reported literature,Targets related to resistance to abamectin,and spirodiclofen was analyzed,respectively.The mutations of P1486S and A2042V in the acetyl-Co A carboxylase gene（ACCase）were found.Three new mutations A302E（Glu Cl3）,G314E（Glu Cl1）,and G326R（Glu Cl3）in glutamate-gated chloride channel（Glu Cl）gene were detected.Mutation frequency and its relationship with resistance were further analyzed.The results indictaed that P1486S and A2042V mutations in the ACCase gene is related to the resistance of spirodiclofen and the mutations of A302E（Glu Cl3）,G314E（Glu Cl1）,and G326R（Glu Cl3）is related to abamectin resistance in P.citri.3.Transcriptome sequencing analysis between laboratory and resistance populationUsing transcriptome sequencing,the differentially expressed genes of nymphs between laboratory and Nanning population were identified.We found 398 up-regulated genes and 482 down-regulated genes.Gene Ontology（GO）database indicated that DEG is most involved in catalytic activity and metabolic processes.The pathway database of Kyoto Encyclopedia of Genes and Genomes（KEGG）indicated that DEG is most involved in the pathways of lysosome and fatty acid metabolism.9 up-regulated DEG（ID:EVM0010342,EVM0003744,EVM0002476,EVM0006560,EVM0005463,EVM0010586,EVM0005858,EVM0003202,and new Gene＿814）to were selected for further analysis of resistance molecular mechanism.4.Expression pattern analysis of candidate gene and enzyme activity determination of different populationsThe m RNA expression levels of 9 candidate genes at different developmental stages were analyzed by q PCR.Real-time PCR indicated the expression level of EVM0010342（glutathione S-transferase,PcGSTmu01）gene was significantly up-regulated at different developmental stages.Compared to control,Nanning population treated with 1000 mg/L diethyl maleate（DEM）,the LC50value was 2880.56 mg/L,Synergistic ratio（SR）was3.09.These results suggested that the up-regulation of PcGSTmu01 gene may be related to etoxazole resistance in Nanning population.5.Functional verification of candidate metabolic genesProtein character prediction indicated that PcGSTmu01 had no transmembrane domain and signal peptide,and PcGSTmu01 was high hydrophilicity.The expression level of PcGSTmu01 gene in nymphs decreased by 53.79%after feeding 1000 ng/μL of ds PcGSTmu01 for 24h,compared with nymphs feeding ds EGFP.The mortality of nymphs feeding ds PcGSTmu01 was increased by 21.51%after exposed to etoxazole（5500 mg/L）for 24h compared with nymphs feeding ds EGFP.The active soluble PcGSTmu01 protein was obtained by prokaryotic expression system and the catalytic activity of recombinant PcGSTmu01 against CDNB was 1.29±0.02 U/mg pro/min.The results of high performance liquid chromatography（HPLC）analysis showed that the PcGSTmu01 protein could metabolize 12.84%etoxazole in the 3h reaction.These results indicated that the PcGSTmu01 was related to the resistance of P.citri to etoxazole. |
