| The citrus red mite,Panonychus citri(McGregor),is a major citrus pest with a worldwide distribution responsible for serious damage in China’s major citrus producing areas and has developed strong resistances to many insecticides and acaricides.It has proved that the carboxylesterases(CarEs)are involved in the resistance of insects/mites to insecticides/acaricides.In this study,synergist triphenyl phosphate(TPP)dramatically increased the toxicity of fenpropathrin,suggesting involvement of Car Es in the metabolic processof this insecticide.Subsequently,the spatiotemporal expression profiles of 11 carboxylesterase genes in different field populationsand after insecticide exposure of P.citri were analyzed using the real time qPCR(RT-qPCR),and PcE1,PcE7 and PcE9 were finally predicted to be involved in the detoxification of fenpropathrin.To further explore the link between the CarE genes and fenpropathrin in P.citri,cytotoxicity assays using Sf9 cells overexpressing PcE1,PcE7 and PcE9,along with the reverse genetic study RNA interference(RNAi)were applied.The results provided some theoretic basis to control the resistance development of P.citri in the field.The main results are as follows: 1 The synergism of TPP to fenpropathrinThe synergism of TPP to fenpropathrin were investigated by the leaf-soaking method targeting P.citri from BeiBei(BB).The results showed that the addition of TPP dramatically increased the toxicity of fenpropathrin,with SR of 3.09,suggesting that CarEs wereinvolved in the metabolic process of fenpropathrin.2 Spatiotemporal expression profiles of CarEs from P.citriThe expression profiles of CarEs genes in P.citri from different field populations were determined by RT-qPCR.The relative expression levels of PcE3 were 9.07-fold higher(P < 0.05)in BB and 3.54-fold higher in FJ than that in SS.PcE7 expressed a dramatic fluctuation among different population,ranging from 23.03-fold in WZ to 1.12-fold in FJ.As for,PcE1,WZ and FJ populations indicated 9.43-and 3.72-fold increase,respectively.The expression ratio of Pc E9 fluctuated least,while significantly higher compared to that of SS.The mRNA expression profiles of CarEs were investigated after mite exposure to a sub-lethal concentration of fenpropathrin(LC30,0.818 mg/L)in a time-dependent manner.The qPCR results showed that PcE1,PcE2,PcE4,PcE5,PcE7 and PcE9 were all induced and the up-regulation of PcE9 was most obvious,which indicated that those genes may play an important role in the detoxification of fenpropathrin.3 Eukaryotic expression of PcE1,PcE7 and PcE9 and cytotoxicity assayTo further study the function of PcE1,Pce7 and PcE9,the eukaryotic expression experiment with Bac-to-Bac baculovirus expression system was conducted.An enzymatic assay of the recombinant PcE protein was measured in vitro to determine if there was CarE-specific activity.The results showed that compared with the GFP-expression protein,the CarE-specific enzyme activities of PcE1,PcE7 and PcE9 toward the substrate of α-naphthyl acetate(α-NA)were 27.31-,9.21-and 12.17-fold higher,respectively.Cytoxicity assay was performed to examine the cytotoxicity of fenpropathrin in cells expressing PcE1,Pce7 and PcE9.The results revealed that there were higher cell viability against cytotoxic effects of fenpropathrin in PcE1-,Pc E7-or PcE9-expressing cells than that in the enhanced green fluorescent protein(GFP)-expressing cells.Effects of Pc Es silencing by RNAi on susceptibility of P.citri to fenpropathrinRNAi of PcE1,PcE7 and PcE9 combined with bioassay was performed to investigate the link between the expression of PcEs and fenpropathrin resistance.The results showed that the transcript levels of PcE1,PcE7 and PcE9 after RNAi were significantly decreased 81%,77% and 54%,respectively,compared with control dsGFP.Subsequently,the sensitivity of mites after RNAi to fenpropathrin were detected.When treated with LC50 of fenpropathrin,the mortality of mites after dsPcE1,dsPcE7 and dsPcE9 treatments increased significantly by 31%,27% and 22%,respectively.Thus,it is concluded that PcE1,PcE7 and PcE9 may contribute to the detoxification of fenpropathrin and may further mediate the resistance of P.citri to fenpropathrin.In the current study,the synergist TPP dramatically increased the toxicity of fenpropathrin,suggesting involvement of CarEs in the metabolic process of this insecticide.The subsequent spatiotemporal expression profiles of 11 carboxylesterase genes in P.citri revealed potential involvement of PcE1,PcE7 and PcE9 in fenpropathrin resistance.The further functional analysis applying RNAi and heterologous expression methods demonstrated that PcE1,PcE7 and PcE9 probably participated in the detoxification process of fenpropathrin.In conclusion,this study 3provides evidence that PcE1,PcE7 and PcE9 are functional genes mediated in fenpropathrin resistance in P.citri and enrich molecular understanding of CarEs during the resistance development of the mite. |
