Biochemical And Molecular Toxicological Characteristics Of Carboxylesterases From The Citrus Red Mite, Panonychus Citri (Acari:Tetranychidae)

The citrus red mite, Panonychus citri (McGregor), has a worldwide distribution, and is regarded as one of the most important citrus pests in our countries. Meanwhile, it was also reported that P. citri has already developed resistance to27acaricides. Carboxylesterases (EC3.1.1.1) have a variety of physiological functions, such as defense against xenobiotic compounds, including insecticides. Overexpression of carboxylesterase gene is associated with insecticide (acaricide) resistance. Previous studies have confirmed that the activities of carboxylesterases in mites have contributed to resistance to Bifenthrin, Fenpyroximate, Spirodiclofen, Phoxim, Azocyclotin, Malathion, and Deltamethrin.In this study, we determine the resistance levels of two geographic populations of P. citri in Chongqing. The biochemical and toxicological characteristics of CarEs from two geographic populations of P. citri are analyzed via the microplate reader method. Based on the transcriptome date,10novel CarE genes are cloned from P. citri using RACE techniques. The expression profiles of P. citri CarEs in geographic populations and treated groups (dose treatment and time treatment) are determine using qPCR method. The results offer the initial evidence to resolve the molecular resistance mechanism of P. citri, and provide strong theoretical basis for the comprehensive management of the citrus red mite. The main results are as follows:Using leaf disc dip method, we determine the resistance levels to Avermectin, Pyridaben, Fenpropathrin, and Chlorpyrifos of two geographic populations of P. citri in Chongqing. The acaricidal activity of four acaricides is ranked as Avermectin> Pyridaben> Fenpropathrin> Chlorpyrifos. The relative resistant of Wanzhou populations to Fenpropathrin and Chlorpyrifos are10-fold and35-fold.The biochemical and toxicological characteristics of CarEs from two geographic populations of P. citri are analyzed via the microplate reader method. The total vitality and michaelis constant (Km) of CarEs in Beibei and Wanzhou populations are significantly higher than those in the susceptible strain.I50of CarEs in Wanzhou population is significantly higher than that in sensitive strain. The result of biochemical and toxicological characteristics indication CarEs activity increased citrus red mite resistance to Chlorpyrifos.Based on the transcriptome date,10novel CarE genes are cloned from P. citri using RACE techniques. The Gene names and Genbank numbers are as follows: PCE2(JQ069032), PCE3(JQ951935), PCE4(JX846607), PCE5(JQ951936), PCE6(JQ951937), PCE7(JQ951938), PCE8(JX846608), PCE9(JQ951939), PCE10(JQ951940) and PCE11(JQ951941). The relative molecular weight range is51.2780.99kDa; the theoretical isoelectric point range is4.95to8.24. The nucleotide homology of P. citri CarE genes is between29.07to56.64%; the protein homology is between14.58to46.13%. The multiple alignment result of the amino acid sequence shows CarE genes conserved sequences, including:active site triad (Ser, Glu, His), two pairs of cysteine residues forming intra-chain disulfide bonds, site of forming the disulfide bond (E×CL), and serine active site (FGGDPxxxTIFG×SAG).After induced by LC50of Fenpropathrin, expression patterns of PCE4, PCE8, PCE10, and PCE11are significantly higher than that in the control group, was7.7-fold,3.1-fold,7.9-fold and7.0-fold higher than in the control, respectively; the expression profiles of PCE2, PCE3, and PCE9in LC10and LC30dose treatment are significantly lower than that in the control group, and the transcription level of PCE2in LC50treatment is still significantly lower than that in the control group; the expression level of PCE5in LC50treatment is significantly higher than that in LC30treatment. After different times (12h,24h,36h) exposed, in addition to the relative expression levels of PCE3and PCE9significantly lower, the other eight CarE genes relative expression levels owned a peak, follows a rapid decreased.8CarE genes expression levels peak don’t appear in the same time; PCE2, PCE5, PCE6, PCE7PCE8, PCE10, and PCE11expression levels peaks are appeared after12h treated; PCE4expression levels peaks are appeared after the mite was treated for24h. The10CarE genes transcription levels are changed after being treated with Fenpropathrin, these genes may play important roles in the detoxification of Fenpropathrin.Except PCE3and PCE9, expression levels of eight other CarE genes are enhanceed after the mite being treated with different dose and different time of Pyridaben. The result indicating that these eight genes are involved in detoxification of Pyridaben in the mite.Focus on the dose treatments, the expression levels of PCE5and PCE11is significantly elevated after being induced by LC10Pyridaben for24h; the expression level of PCE4is significantly higher in LC50treatment than that in the control. Focus on the time treatments, the expression levels peaks of PCE5, PCE8, PCE9, and PCE10are appeared after the mite was treated for12h; the transcription levels of PCE2and PCE4display two peaks, one in12h, and the other in36h. The expression patterns of CarE genes in different geographic populations are determined. The relative expression levels of CarE genes in Beibei population have no significant difference with susceptible strain. However, the expression patterns of CarE genes in Wanzhou population are significant higher than that in susceptible strain. Thereinto, the transcription profiles of PCE2, PCE6, PCE7, PCE8, and PCE9are5.22-,2.74-,6.27-,2.01-, and1.75-fold higher than that in susceptible strain. The result indicates that CarEs play an important role in the resistance of the mite to Chlorpyrifos.