Fenpropathrin And Avermectin Resistance, And Esterase Gene Cloning And Expression In Panonychus Citri (McGregor)

The citrus red mite, Panonychus citri (McGregor) (Acari:Tetranychidae), is an important pest mite in the world that devastates citrus trees and thus badly affects yield and quality of orange. To date, P. citri has developed severe resistance to most acaricides, which has resulted in extensive attentions and concerns of scientific researchers and administrative authority. In the current thesis, the effects of two acaricides (fenpropathrin and avermectin) on development and reproduction of P. citri and the biochemical mechanism of the mite resistance to the acaricides were systematically studied, and the molecular cloning and expression of esterase gene (PCE1) from the mite was also carried out. The study was supported by the Grants-in-Aid from the Ministry of Agriculture (nyhyzx07-057), Chongqing Natural Science Fund for Distinguished Young Scholars (CSTC, 2009BA1042) and the earmarked fund for Modern Agro-Industry (Citrus) Technology Research System of China. The main results are as follows:1 Sublethal effects of two acaricides on P. citri1.1 Effects of fenpropathrin with sublethal concentration on development and reproduction of P. citriThe effects of fenpropathrin with a sublethal concentration (the LC20 was chose as a representative) on the development and reproduction of P. citri were evaluated. The results showed that after the treatment of fenpropathrin at the sublethal dose, the emergence rate of the nymphs and sex ratio of offspring both decreased, while the number of eggs laid per female increased in F0 generation. The sublethal dose of fenpropathrin expressed different effects on the offspring (F1 and F2 generations) of the mite. However, there was no significant difference between two treatments (that is, exposed to sublethal concentration of fenpropathrin during the nymphal stages and the adult stages). After treatment with the sublethal dose of fenpropathrin, the pre-oviposition duration was significantly shortened (P< 0.05), and the sex ratio of offspring increased both in F1 and F2 generations. The hatching rate of eggs and the emergence rate of the nymphs decreased while the number of eggs laid per female and the adult longevity both increased in F1 generation, which were totally in verse in F2 generation. Furthermore, the net reproductive rate(Ro), intrinsic rate of increase (rm) and finite rate of increase (λ) values all increased, and the population doubling time (Dt) shortened in F1 generation; while Ro, Dt and the generation time (T) decreased, rm andλsignificantly increased in F2 generation. Generally, fenpropathrin with a sublethal concentration of LC20 facilitated reproduction of the mite population and there was no relationship between its effects and treatment period (adult and nymphal stages). 1.2 Effects of avermectin with sublethal concentration on development and reproduction of P. citriThe effects of avermectin with sublethal concentration on the development and reproduction of P. citri were evaluated and the results showed the sublethal dose of avermectin had no significant effect on the hatching rate of eggs and the emergence rate of the nymphs in F0 generation. However, after exposure to avermectin, progeny (F1 and F2 generations) produced fewer eggs, the pre-oviposition duration and the adult longevity shortened, resulting in a decrease in the mean generation time. The sublethal dose of avermectin expressed different effects on the offspring (F1 and F2 generations) of the mite and the effect of nymphal stage and adult stage treatments on the population life parameters of the offspring was more significant in F2 generation. After treatment during nymphal stages, higher rm, R0, andλvalues while lower Dt value were observed. However, the treatment during adult stages exhibited negative effects on population increase (i.e. lower rm, Ro, andλvalues while longer Dt).1.3 Effects of fenpropathrin and avermectin with sublethal concentrations on activities of detoxifying enzymes in P. citriAfter adult mites were treated applying fenpropathrin and avermectin with LC20 concentration, there were different induction effects on the activities of main detoxifying enzymes such as carboxylesterase (CarE), gutathione S-transferases (GSTs) and mixed-function oxidase (MFO) from the mite. After the treatment with fenpropathrin, the specific activity of CarE fluctuated a lot, increased firstly, then decreased and finally increased again, with the values at 12 h,36 h and 48 h significantly higher than the control (P< 0.05); the induction effects on GST activities were quite obvious and the specific activity at 36 h was 3.31 fold of control; there was no significant effects on MFO activity. For avermectin sublethal treatment, CarE specific activity ascended in the first 12 h, then decreased to a state value; the induction effect on GSTs was less significant than fenpropathrin treatment; the activities of O-demethylation of MFOs were distinctly inhibited by sublethal concentration of avermectin, especially after 24 h of the treatment, significant different from the control (P<0.05)2 Resistance selection and risk assessment of resistance to fenpropathrin and avermectin in P. citriAfter the further resistance selection in laboratory, the realized heritability of P. citri resistance to acaricides were conducted via threshold trait analysis, the resistance development ratio of the mite was forecast and the cross-resistance between the acaricides was also studied. After discontinuous selection with fenpropathrin and avermectin 16 and 11 times during 19 generations, resistance ratio of the mite to the acaricides amounted to 29.92 and 3.80 fold, respectively. The realized heritability of resistance to fenpropathrin and avermectin accounted for 0.1544 and 0.0475, respectively. Theoretically, in laboratory, to obtain a 10 fold resistance ratio requires 8 to 16 generations for fenpropathrin and 12 to 26 generations for avermectin under selective pressure of 50% -90% mortality for each selective generation. For field populations, more generations were required to obtain the same resistance levels. Bioassay revealed that the fenpropathrin resistant strain developed obvious cross-resistance to pyridaben, dicofol, azocyclotin and the avermectin resistant strain obtained cross-resistance to emamectin benzoate. The results of resistance risk assessment suggested fenpropathrin has higher resistance risk than avermectin in P. citri. The current results provide some information for the resistance management in P. citri.3 Biochemical mechanism of P. citri resistance to two acaricides3.1 Synergism effects of synergists on fenpropathrin and avermectinThe synergism of PBO, TPP and DEM all on fenpropathrin and avermectin respectively was test in susceptible and resistant strains of P. citri with the relative synergism ratio (the ratio between the synergism ratio in resistant and susceptible strains, SRFeR(AvR)/SRss) as the criterion. The results showed that PBO, TPP and DEM all possessed obvious synergism effects on fenpropathrin with the relative synergism ratio (SRFeR/SRss) of 3.18,2.90 and 1.68, respectively, the relative synergism ratios of PBO, TPP and DEM on avermectin was 1.81,1.44, and 0.84, respectively.3.2 Comparison of carboxylesterase (CarE) in different strains of P. citriCompared to their susceptible counterparts, the activities of CarE in FeR and AvR were significantly higher (P< 0.05). Kinetic parameters comparison showed that CarE from FeR expressed highest Km value to the substrate a-NA, while both two resistant strains exhibited lower Km values toβ-NA. For Vmax, the values in resistant strains were significantly higher.In vitro inhibition study showed that paraoxon-methyl, carbosulfan and malathion expressed significant inhibitory effects on CarE in three strains of P. citri. Beta-cypermethrin and fenpropathrin also exhibited obvious inhibition effects, while avermectin expressed limited inhibition effect. For pyridaben, the inhibition effects were not obvious and some facilitated effects appeared at lower concentrations. The comparison analysis revealed that except malathion, CarE from SS strain were most sensitive (lowest I50 values) and the sensitivity of CarE from FeR was lowest (highest I50 values). 3.3 Comparison of acid phosphatase (ACP) and alkaline phosphatase (ALP) in different strains of P. citriCompared to their susceptible counterparts, the specific activities of ACP and Vmax value of ALP from FeR expressed significantly higher than SS (P< 0.05), while the specific activities, the Vmax and Km values of of ACP and ALP had no significant difference between SS and AvR (P> 0.05). The results indicated that phosphatase might play some roles in fenpropathrin resistance of P. citri, but played little role in P. citri resistance to avermectin.3.4 Comparison of acetylcholinesterase (AChE) in different strains of P. citriAmong three strains, the activity per mite and specific activity of AChE, as well as the Vmax value were highest in FeR. Compared to SS strain, these values in FeR and AvR were significantly higher(P< 0.05), and significant difference was observed between two resistant strains (P< 0.05). However, there was no significant difference of Km value among three strains (P> 0.05). The result indicated that increased activity of AChE plays important roles in P. citri resistant to fenpropathrin and avermectin.3.5 Comparison of glutathione S-transferase (GSTs) in different strains of P. citriThe result revealed that the activity per mite and specific activity of GSTs in FeR were significantly higher than other two strains (SS and AvR) (P< 0.05). GSTs from FeR expressed highest Km and Vmax values to the substrate CDNB while exhibited lowest Km and Vmax values to GSH. There were significant difference between FeR and other two strains (P< 0.05) while no significant difference existed between AvR and SS strains (P> 0.05). The results indicated that GSTs was important in fenpropathrin resistance development, but played little role in P. citri resistance to avermectin.3.6 Comparison of mixed function oxidases (MFO) in different strains of P. citriCompared to SS strain, the specific activities of O-demethylation in FeR and AvR were significantly higher (1.36 and 1.31 fold of SS strain) (P< 0.05). For kinetic parameters, O-demethylation from AvR showed highest Km value while lowest Vmax value, and its Km value was significantly higher than that from SS (P< 0.05), indicating MFO might have changed in quality in AvR strain. However, there was no significant difference in the Km and Vmax values between FeR and SS strain (P> 0.05). These results indicated that MFO was essential in avermectin resistance development, and it played some roles in fenpropathrin resistance to P. citri.3.7 Comparison of adenosine triphosphatase (ATPase) in different strains of P. citri The biochemical characterization of ATPase comparison analysis showed that the specific activity of Na+-K+-ATPase from high to low was AvR> SS> FeR. The specific activity of Na+-K+-ATPase in FeR is 0.68-fold of SS, significantly lower than other two strains (P< 0.05). There was no significant difference between AvR and SS strain (P> 0.05). Further kinetic parameters comparison showed that Na+-K+-ATPase from FeR expressed significantly higher Km and Vmax values than other two strains(P< 0.05), and the Vmax value from AvR was also higher than SS strain (P< 0.05). For the activities per mite, the specific activities, the Km value and Vmax value of Mg2+-ATPase, no significant difference was observed among these strains of P. citri(P> 0.05).In vitro inhibition of insecticides on Na+-K+-ATPase of P. citri showed that fenpropathrin expressed significant inhibitory effects under tested six concentrations. The sensitivity of Na+-K+-ATPase from SS was highest while from FeR was lowest, and significance existed between FeR and other two strains (P< 0.05). In addition, avermectin also exhibited some in vitro inhibition effect, and the inhibition rate on Na+-K+-ATPase of the three strains of P. citri ranged from 3.60%to 12.76% (under 0.0229-2290.7164μM) and significant difference existed between susceptible and resistant strains under higher concentration (i.e.2290.7146μM)(P< 0.05)These results indicated that Na+-K+-ATPase played major role in fenpropathrin resistance of P. citri by its significantly lower activity and insensitivity to fenpropathrin and thus speculated that Na+-K+-ATPase is one of target enzymes of fenpropathrin. In addition, it may play some roles in avermectin resistance to P. citri.4 Molecular cloning and expression of esterase gene in P. citri4.1 Molecular cloning and sequence analysis of esterase gene in P. citriThe full length cDNA encoding esterase PCE1 (GenBank No. GQ144324) was cloned from P. citri by the method of reverse transcriptase PCR (RT-PCR) and rapid amplification of cDNA ends (RACE). The complete cDNA of this gene consists of 1892 bp with an open reading frame (ORF) of 1653 bp, encoding a protein of 550 amino acids residues with the putative signal peptide of 19 residues. The mature protein has a molecular weight of 61.1 kDa with an isoelectric point of 5.21. Based on the online software ScanProsite, two conserved regions of CarE were found:serine active site (FGGDPDQVTIFGESAG) and the conserved cysteine that compose disulfide bridge (EDCLTLNVITP). In addition, an endoplasmic reticulum targeting sequence (HEEL) as well as 14 phosphorylation sites,12 N-myristoylation sites and 4 N-glycosylation sites were also found. The sequence homology analysis showed that the esterase cloned from P. citri share low homology to other insect species, but the sequences is highly conserved in active site. Phylogenetic trees generated from the amino acid sequences revealed a closer relationship to other acaris than to insects. In addition, the PCE1 gene 3-D structure models were constructed by SWISS-MODEL.4.2 mRNA expression level of the esterase gene PCE1 from P. citriThe adult female mites of P. citri were treated with LC50 concentration of fenpropathrin using leaf dip method and then the expression levels of PCE1 gene was determined. After treated for 6 h, 12 h,24 h and 48 h, the relative quantity of PCE1 was investigated applying real-time PCR, respectively. The results showed that the relative quantity of PCE1 from P. citri fluctuated significantly. At the beginning of fenpropatrin treatment, the relative quantity of PCE1 was inhibited (compared to the control group, PCE1 transcript was only 0.74 fold after 6 h treated). It increased gradually with the increase of time treated, then decreased, and then again increased. After 48 h treated, the relative quantity of PCE 1 is significantly higher than the control (just 1.36 fold compared with the control). In general, the transcript of PCE1 from P. citri treated was higher with longer treatment time.4.3 Heterologous expression of the esterase gene PCE1 from P. citriApplying the double digestion of BamHI and Xhol restriction enzymes with the DNA recombination technology, the expression vectors for PCE1 was constructed based on pET-43.1a (+) vector. Specific band appeared on the gel of SDS-PAGE. The current result provides useful base for furthermore exploration of the property and function of the esterase gene PCE1.