| The citrus red mite, Panonychus citri(Mc Gregor)(Acari: Tetranychidae) is a kind of polyphagous spider mite distributed worldwide and can feed with 106 host plants, which containing both the herbage and xylophyta plants. Until now, chemical control has still being the primary strategy for the management of citrus red mite in the field. However, due to some nature characteristics of the citrus red mite, such as short life cycle, abundant progeny and generation overlapping, as well various objective factors including the unreasonable spraying of acaricides in the field, the resistance of P. citri to various class of acaricides has developing rapidly in the large scale. The resistance monitoring of P. citri in the field and research on the resistance mechanism would be helpful for the design of efficient management strategies. Up to now, the study on the resistance mechanisms of insects/mites mainly focus on the target-site insensitivity and enhanced detoxification of pesticides. Glutathione S-transferases(GSTs; EC 2.5.1.18), one major group of detoxification enzymes, play a key role in the detoxification of both endogenous and xenobioticcompounds, and also involved in protection against oxidative stress. It has been proved that GSTs participate in the detoxification of insecticides/acaricides, and possible further mediate the resistance of insects/mites to insecticides/acaricides. Currently, however, the study about the GSTs of P. citri has still been staying on a relative behindhand stage. It is more rarely reported about the molecular characterizations and function research of GSTs in P. citri.Therefore, for the purpose of comprehensively understanding the molecular mechanism of acaricides resistance in P. citri. We firstly collected the resistant population after resistance monitoring of P. citri sampled from the field around Chongqing. On the basis of transcriptome data, the full length of GSTs c DNA sequence were cloned through the method of RACE and their molecular characteristics were analyzed subsequently. The recombinant proteins of those GSTs were produced in E. coli and their enzymatic characteristics were comparatively analyzed. The interaction between GSTs and acaricides were further investigated using high performance liquid chromatogram(HPLC). The expression profiles of 10 GSTs under the condition that different developmental stages, acaricides stress, and extreme environmental stress were analyzed by real time q PCR(RT-q PCR) and their potential physiological function were predicted. RNAi system for P. citri has been successfully established, through which the detoxification role of GSTs against acaricides was investigated. Through the next-generation high throughput sequencing technology, various genes associated with fenpropathrin resistance were identified. On the basis of up-regulated genes identified in fenpropathrin-resistant population, the potential detoxification pathway of fenpropathrin in P. citri was discussed. Those results obtained from this study may contribute to comprehensively understand the detoxification role of GSTs in P. citri, and those conclusions may support the design of management strategies to control the resistance development in the field.The main results are as follows:1. Acaricides resistance monitoring of P. citri in the field The relative resistance level of P. citri sampled from the four populations(Wanzhou, Fengjie, Wulong and Beibei) were investigated by the leaf-soaking method. The bioassay results show that the citrus red mite from four populations are all resistant at various level to fenpropathrin. In term of the fenpropathrin resistance, the mites from Fengjie and Beibei populations were more resistant to fenpropathrin, and their median lethal concentration value(LC50) for fenpropathrin are 7.14 mg/L(310-fold) and 5.30 mg/L(230-fold), respectively. Although the LC50 value for Wanzhou population was relative low, it showed that this population are also resistant to fenpropathrin with an approximately 53-fold resistant ratio value. The LC50 value for Wulong population is comparable to that of susceptible strain and shows little resistant to fenpropathrin.The susceptibilities of four population to abamectin are similar with that of susceptible strain. It is only the mites from Wanzhou population that shows resistant(14-fold) to abamectin with a relative high LC50 value. Similarly, the mites from Fengjie population shows a relative weakly resistant(12-fold) to pyridaben. As expected, cyflumetofen, known as a novel kind of acaricide, has an excellent performance in the control of mites. The cyflumetofen-resistant mites haven’t been detected in all the four populations.2. Molecular cloning and sequence analyses of GST genes from P. citri On the basis of transcriptome data, the c DNA full-length sequence of 10 GSTs were cloned by the method of RACE. The alignment results classified those GST genes into four families: delta(3), mu(5), omega(1) and zeta(1). The lengths of the deduced amino acid sequences of 10 GSTs varied from 217 to 224 amino acid, andthe predicted protein molecular weight ranged from 23.9 to 27.7 k Da, with theoretical isoelectric points of5.18-7.65. The nucleotide sequences identities ranged from 33.1% to 66.4%,and their deduced amino acid sequence similarities were 10.9-60.8%.32 GSTs from Tetranychus urticae, 16 GSTs from Ixodes scapularis, 20 GSTs from Bombyx mori and 10 GSTs in this study were chosen to conduct the phylogenetic tree using the maximum likelihood method by MEGA 6.0 software. The phylogenetic analysis suggest that 10 GSTs of P. citri are highly similar with the GSTs form T. urticae, and be distributed closely with the according GSTs belong to the same family.In addition that, on the basis of associated references, Ser11(delta class), Tyr33(mu class), Cys32(omega class) and Ser12(zeta class) were supposed to be the catalytic active sites for each family GSTs after the multiple alignment with GSTs from T. urticae.The c DNA and deduced protein sequences were deposited in Gen Bank under the following accession numbers: Pc GSTm1(JQ069034), Pc GSTm2(JQ069035), Pc GSTm3(JX846609), Pc GSTm4(JX846610), Pc GSTm5(KT717379), Pc GSTd1(JQ069033), Pc GSTd2(JQ069037), Pc GSTd3(KU904396), Pc GSTz1(JQ069036) and Pc GSTo1(KU904397).3. Enzymatic characterization of GSTs from P. citri The recombinant proteins of 10 GSTs from P. citri were produced in E. coli using heterologous expression system, and their qualities were checked by SDS-PAGE and western blot. The activity assay shows that all the 10 recombinant GSTs possess various catalytic activities toward the conjugation of CDNB and GSH. Notably, both the specific activities of Pc GSTm5 and Pc GSTd1 are significantly higher than that of the other 8 GSTs. The thermo stability assay show that all GSTs can be stable under the condition of 50°C and the lower temperature. The optimal reaction p H value for all 10 GSTs is 7.5, suggest they possess their maximum activities when reaction condition is weakly alkaline or neutral. The kinetic assay shows that Pc GSTm3 are most affinity with CDNB among the 10 GSTs. Compared with the GSTs from other species, all the 10 GSTs possess peroxidase activity toward cumene hydroperoxide, which is a common oxidative compound. The in vitro inhibition assay shows that both abamectin and fenpropathrin were unable to inhibit the activity of GSTs.4. Analyses of m RNA expression profiles of GSTs from P. citri The relative expression level of GSTs at developmental stages and under the stress of chemical exposure and heat shock were investigated by real time quantitative-PCR(RT-q PCR).4.1. Analyses of expression profiles of GSTs at developmental stages Stage-dependent expression profiles show that all the 10 GSTs expression were detected at the four stages(egg, larva, nymph and adult), and the expression profiles for each GST are different. It is worthy note that both the expression levels of Pc GSTm5 and Pc GSTo1 at feeding-stages(larva, nymph and adult) are significantly higher than that of egg, suggesting their roles in the detoxification of xenobiotic compounds during the feeding-stages of citrus red mite. However, the expression of Pc GSTm2, Pc GSTm3 and Pc GSTd3 are keep consistently at all the four stages. The specific expression profiles of 10 GSTs suggest their multiple functions during the development of P. citri.4.2. Analyses of expression profiles of GSTs under the exposure of acaricides and allelochemical In order to understand the expression profile of GSTs response to synthetic and natural xenobiotics, the m RNA expression level of GSTs under the exposure of three acaricides and an allelochemical were investigated by RT-q PCR respectively.Interestingly, the expression of Pc GSTm3, Pc GSTm4, Pc GSTm5 and Pc GSTd1 were induced by the exposure of abamectin with a sub-lethal concentration, and the up-regulation of Pc GSTm5 was most obvious. Similarly, the transcripts of Pc GSTm2, Pc GSTm3 and Pc GSTd1 were also up-regulated by the exposure of fenpropathrin, which belongs to the type II pyrethroids. However, none of the tested GSTs are responsive to the exposure of pyridaben, which is a kind of mitochondrial electron transport inhibitor. In addition that, after the treatment of citral, which can be extract from lemon, most GSTs of P. citri responded rapidly. For example, at 12 h after the exposure of citral, the m RNA level of Pc GSTm1, Pc GSTm3, Pc GSTm4 and Pc GSTd1 were obviously up-regulated. According to those results described above, we can speculate that those up-regulated GSTs may contribute to the detoxification of associated acaricides and allelochemical.4.3. Analyses of expression profiles of GSTs under heat shock stress Over-expression of the most GSTs were investigated when the citrus red mite were incubated at 41°C for 3 h. Especially, the expression level of Pc GSTd1 was increased up to 30-fold higher than control. These results suggest that GSTs may participate in anti-oxidative process against the oxidative stress induced by heat shock.5. Functional analysis of GSTs from P. citri5.1. The analysis of the role of Pc GSTm5 in the detoxification of abamectin in P. citri Through RT-q PCR method, the over-expression of Pc GSTm5 was discovered in abamectin-resistant strain. Furthermore, silencing of Pc GSTm5(65% knockdown) increased significantly the susceptibility of P. citri to abamectin. The mortalities of Pc GSTm5-silencing and control mites were 55.6% and 23.0%, respectively. These results suggest Pc GSTm5 may play an important role in the detoxification of abamectin. However, in vitro metabolism assay shows that there is no obvious evidence to elucidate the ability of Pc GSTm5 to metabolize abamectin directly. Thus, it is concluded that Pc GSTm5 may contribute to the detoxification of abamectin in P. citri and further mediate the resistance of P. citri to abamectin.5.2. Comparative transcriptome analyses of fenpropathrin-resistant and susceptible of mites by RNA-seq The transcriptome of resistant- and susceptible mites to fenpropathrin were sequenced by next generation high throughput sequencing technology and analyzed comparatively. The results of gene expression analyses show that the total of 861 genes, containing 499 up-regulated and 362 down-regulated genes, were expressed differently between these two strains. Function annotation of these up-regulated genes shows they include cytochrome P450s(P450s), glutathione S-transferases(GSTs), carboxylesterases(Car Es), heat shock protein(Hsp), cuticular protein(CPR), ATP-binding cassette transporters(ABC transporters), peroxidase(Px) and thioredoxin(TPx) etc. It is worthy note that Hsp, CPR and antioxidant proteins were all highly over-expressed in fenpropathrin-resistant mites. These results suggested those upregulated genes may contribute to the detoxification of fenpropathrin and the putative detoxification pathway of fenpropathrin in P. citri was deduced on the basis of obtained data.5.3. The analysis of the role of Pc GSTd1 in the detoxification of fenpropathrin The overexpression of Pc GSTm1, Pc GSTd3 and Pc GSTo1 were investigated in fenpropathrin-resistant mites using RT-q PCR. Comparative transcriptome analysis also reveal that Pc GSTd1 was over-expressed in fenpropathrin-resistant population of P. citri. However, no obvious evidence was detected by in vitro metabolism assay to suggest that Pc GSTd1 has the potential to metabolize fenpropathrin directly. Although the RNAiof Pc GSTd1 cannot increased the susceptibility of P. citri to fenpropathrin, the role of Pc GSTd1 in the regulation of lipid peroxidation level was revealed. Pc GSTd1 was speculated as a minor factor contribute to the detoxification of fenpropathrin and further mediate the resistance of P. citri to this pyrethroid.In conclusion, this study firstly obtained the molecular information of 10 GSTs genes in P. citri on the basis of numerous de novo assembled sequences. Then, their enzymatic characteristics were analyzed comparatively after those recombinant proteins were produced in E. coli. It is worthy noted that, the catalytic activities of Pc GSTm5 and Pc GSTd1 were significantly higer than the other GSTs, suggesting their primary positions in the GSTs superfamily in P. citri. Moreover, the potential functions of 10 GSTs at developmental stages and when the citrus red mites were treated with various stress were analyzed using RT-q PCR, suggesting the role of those up-regulated genes under the exposure of abamectin, fenpropathrin and citral in the detoxification of these chemical compounds accordingly. The establishment of RNAi system for P. citri contribute to the function verification of Pc GSTm5 in the detoxification of abamectin, and the potential association between Pc GSTm5 and abamectin resistance was discussed. Both the results of RT-q PCR and transcriptome analyses infer the association between the overexpression of Pc GSTd1 and the resistance of P. citri to fenpropathrin. In addition that, the possible detoxification pathway of fenpropathrin in P. citri, including the antioxidant role of Pc GSTd1, was speculated on the basis of comparative transcriptome analyses. Finally, this is the first comprehensive study on the molecular characterization and functional analysis of GSTs in P. citri. Those results and conclusions obtained in this study would be useful to provide theoretical references for the design of novel efficient strategies applied to control the resistance of P. citri in the field. |
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