Identification And Functional Validation Of Carboxylesterase Genes Involved In Acaricides Resistance In Tetranychus Cinnabarinus(Boisduval)

Chemical control is one of the most important strategies to protect modern agriculture from pest,but pesticide resistance is becoming the most challenging issues for chemical control.Currently,the mites have developed resistance to as many as 93 toxicants,contributing the mite species as “resistance champion” amongst agricultural pests.Pyrethroids and METIs(mitochondrial electron transport inhibitors)are crucially important acaricides in the market.It is meaningful to reveal the resistance mechanisms to these two kinds of acaricides,which could prolong the lifecycle of acaricides and extend the effectiveness on mites control.The current investigations gained insight to the functional roles of carboxylesterase genes in the evolution of resistance formation in Tetranychus cinnabarinus.We also identified the key genes related to resistance and studied their functional roles based on reverse genetic technology and protein expression.The main results were following as below: 1.Toxicity test and synergismFenpropathrin resistant(FeR)strain has evolved 118-fold resistance to laboratory susceptible(SS)strain and cyflumetofen resistant strain has developed 34-fold resistance to SS strain.Esterase inhibitor DEF(S,S,S-tributylphosphorotrithioate)decreased the resistance fold of FeR strain,but increased the resistance fold of CyR strain,respectively,which indicated esterases could be involved in fenpropathrin resistance and cyflumetofen resistance of mites by playing different roles.2.Esterase activityThe activity of esterase in FeR and CyR strain are significantly higher than that of SS strain,which further confirmed the close relationship between esterase and resistance development.3.Profile of expression of esterase genesFour over-expressed esterase genes,TcCCE06,TcCCE13,TcCCE15 and TcCCE18,could response to fenpropathrin exposure sensitively in FeR strain,which could be regarded as resistance related genes.Moreover,three key genes,TcCCE04,TcCCE12 and TcCCE23 were identified as resistance related genes in CyR strain.Differently,over-expressed TcCCE04 increased its expression under the exposure of cyflumetofen,while down-regulated TcCCE12 and TcCCE23 decreased their expression significantly when exposed to cyflumetofen,which indicated a cooperative work of up-regulated genes and down-regulated genes in the evolution of cyflumetofen resistance in mites.4.In vivo validation of functional roles of candidate genes by RNAiThe candidate genes(related to fenpropathrin resistance)were well silenced,in which the silencing efficiency was ranging from 55% to 73% in SS strain and from 50% to 69% in FeR strain.Decreased expression of candidate genes also caused a decrease of esterase activity,ranging from 0.33 to 0.44 fold in SS strain and from 0.25 to 0.3 fold in FeR strain.Consequently,silencing the esterase genes also decreased the resistance fold of FeR strain significantly,of which TcCCE06 contributed most to the resistance and was selected as the primary target gene for functional expression.For cyflumetofen resistance,their candidate genes were also knocked down effectively by RNAi,in which the silencing efficiency was ranging from 64% to 80% in SS strain and from 55% to 64% in CyR strain.However,knocking down of over-expressed TcCCE04 did not alter the susceptibility of mites,pointing out a distant relationship between TcCCE04 and cyflumetofen resistance.After silencing another two down-regulated esterase genes(TcCCE12 and TcCCE23),the resistance fold dramatically decreased from 18.96 fold to 1.14 fold,strongly indicating a close relationship between down-regulated esterase genes and cyflumetofen resistance.5.In vitro validation of functional roles of key genes by protein expressionThe recombinant TcCCE06 protein and TcCCE12 protein were actively expressed in E.coli cells.The activity of TcCCE06 protein to its model substrates,1-naphthyl acetate(1-NA,273.5 μM)was 22.6 nM/min/mg,while the activity of TcCCE12 was 128 nM/min/mg.Fenpropathrin could inhibit the activity of TcCCE06 protein competitively and its IC50 was 146.8 μM.Cyflumetofen could inhibit the activity of TcCCE12 protein competitively,too,and its IC50 was 97.9 μM.Moreover,the fenpropathrin was decomposed effectively(41.79%)by recombinant TcCCE06 protein within 3 hours.The cyflumetofen was hydrolyzed effectively(36.9%)by recombinant TcCCE12 protein,in which the increase of metabolite confirmed a hydrolyzing effect of TcCCE12 protein.6.Changed expression of gene through alternative splicingHere is the first report of alternative splicing in resistance related esterase gene in mites.The gene structure of TcCCE23 comprised three exons and two introns,of which alternative splicing(5’ type)was discovered in the last 18 nucleotides of first exon,starting at “GT” and terminating at “AG”.These two variants of TcCCE23,named as TcCCE23-V1 and TcCCE23-V2,were identified in SS,CyR and FeR strains,but no point mutation was figured out by sequence alignment.However,the relative expression of two variants was strain specific.The expression of TcCCE23-V2 in FeR mites(93%)was significantly higher than that of SS(71%)and CyR(73%)strain,indicating a close relationship between TcCCE23-V2 and fenpropathrin resistance.Knocking down the expression of TcCCE23-V2 also altered the susceptibility of mites to fenpropathrin,suggesting that alternative splicing could be an effective way for esterase to change gene expression besides up-regulation and down-regulation in the evolution of mites resistance.