Resistance Of Lygus Pratensis To Lambda-cyhalothrin And Its Physiological,Biochemical And Molecular Mechanisms

The plant bug,Lygus pratensis Linnaeus（Hemiptera:Miridae）is one of the most important omnivorous insects in northwestern China.In recent years,with the increasing intensive planting and crop rotation of alfalfa in the ecotone of agriculture and animal husbandry in northern China,the occurrence of plant bug and damage degree on grass were aggravated,and the yield and quality of agricultural products decreased.In this study,the resistance levels of L.pratensis to five commonly used insecticides were monitored,and the resistant strain breeding,risk assessment and resistance mechanism of L.pratensis to lambda-cyhalothrin were studied in the laboratory.The results provide a theoretical basis for sustainable and efficient scientific control of L.pratensis.1.Monitoring of resistance of L.pratensis to five insecticides.The study documented resistance levels in seven field populations of L.pratensis to phoxim,methamphenyl,lambda-cyhalothrin,imidacloprid and abamectin in northern China from 2015 to 2019 using glass-vial bioassay approach.Low to medium level of resistance to lambda-cyhalothrin were detected in samples collected from all collection sites except the He Lin county population.And medium resistance level to imidacloprid in the Tuoketuo county population in five years.However,the5-year resistance investigation showed that resistance levels of L.pratensis at most collection sites were very low for phoxim,methomyl and avermectin.2.Resistant strain breeding,cross-resistance and risk assessment of resistance to lambda-cyhalothrin in L.pratensis.A field population of L.pratensis was selected in the laboratory for 14consecutive generations with lambda-cyhalothrin to generate 42.56 fold resistance.The cross-resistance tests and resistance risk assessment experiments were carried out.The results showed the lambda-cyhalothrin resistant strain showed no cross-resistance to anti-acetylcholinesterase agents methomyl（RR=1.474）,phoxim（RR=2.662）and biological pesticide avermectin（RR=1.346）,low level of cross-resistance to nicotinic acetylcholine receptor agonist imidacloprid（RR=5.203）and beta-cypermethrin（RR=4.854）,and a medium level of cross-resistance to deltamethrin（RR=10.040）.Realized heritability（h²）of lambda-cyhalothrin resistance was 0.339.3.Synergistic effect of different synergist on lambda-cyhalothrin and determination of P450s contents.Toxicity of insecticide in the presence and absence of synergist PBO,DEF,DEM for both of susceptible and resistant strains was tested.The detoxification enzyme activity of susceptible and resistant strains was compared by measuring heme peroxidase activity,which indirectly reflected the activity of P450.Compared with the susceptible strain,the synergism ratio of PBO to the resistant strain was 21.38,and the resistance ratio was reduced to 7.94 fold.The enzyme activity of the resistant strain was significantly increased and was 2.4 fold higher than that of the susceptible strain.The results showed that the increase of P450 enzyme activity was related to the resistance on lambda-cyhalothrin of Lygus pratensis.At the same time,the cytochrome P450 expression profiling of cyhalothrin resistant and susceptible strains was determined by q PCR technique.There were nine genes（CYP4C1,CYP4C3,CYP6A2,CYP6A13,CYP6A14,CYP6A20,CYP6B7,CYP6J1,CYP6K1,CYP303A1）with significantly high expression in the resistant strains.Seven genes（CYP4C21,CYP4V2,CYP6B3,CYP6D5,CYP9E2,CYP12,CYP305A1）were significantly lower expressed in resistant strains.4.Cloning and prokaryotic expression of P450 CYP6A13 gene in L.pratensis.The full length sequence of its c DNA was obtained by RT-PCR and RACE.The expression profiling of P450 CYP6A13 gene was performed by real-time fluorescence quantitative PCR（q PCR）in different adult genders,tissues（head,thorax,and abdomen）,strains（resistant and susceptible）,and different instar nymphs（1-5 instar）.The length of CYP6A13（Gen Bank No.:MN782520）is 2003 bp with an ORF（Open reading frame）of 1503 bp,encoding 500 amino acids,and there is no transmembrane region and no signal peptide.The q PCR results showed that the gene expression of CYP6A13 in different instars ranked as:5^th instar>4^th instar>3^rd instar>2^nd instar>1^st instar;the expression level of CYP6A13 in the head was higher than that in the thorax and abdomen;the expression of CYP6A13 in the lambda-cyhalothrin resistant strain was 2.11 fold higher than that in the susceptible strain;there was no significant difference between males and females on the expression level of CYP6A13.The full-length sequence of CYP6A13 was inserted into p ET28a（+）vector by restriction enzyme digestion and transformed into competent cell to construct the p ET28-CYP6A13 expression vector.5.Cloning of sodium ion channel gene and analysis nucleotide mutations in L.pratensisThe 4601 bp c DNA sequence of sodium ion channel gene was cloned（Gen Bank No.:MW821485）.The open reading frame of sodium ion channel sequence is 6072bp,encoding 2024 amino acids,and the predicted molecular weight of the protein is228.94 k Da with the isoelectric point（p I）of 4.99.There are multiple transmembrane regions without signal peptide and conservative structure and characteristics.Homology analysis showed that the sodium channel protein of L.pratensis was the most closely related to Apolygus lucorum,followed by Cimex lectularius.Sequencing analysis showed that no nucleotide mutations in theⅡS4-ⅡS6 region of the sodium channel sequences were found between the resistant and susceptible strains,which might lead to the resistance.