| Carmine spider mite Tetranychus cinnabarinus (Boisduval) belongs to Arachnida, Tetranychidae, Tetranychus. It is a phytophagous mite distributed worldwide, and feeds on a large number of host plants including cotton, flowers, fruits and vegetables, as well as the mulberry. In recent years, T. cinnabarinus has caused severe damage in the field. The management of this mite still mainly depends on acaricides, while the resistance of T. cinnabarinus could develop quickly for its high reproductive potentials, short life cycle, and the unreasonable use of acaricides. Nowadays, the resistance problem of T. cinnabarinus has been the severest in the mites.As a traditional acaricide, propargite has been wide used in control of pest mites in the fields and forests since1964for its high efficiency, low toxicity, and broad-spectrum. Although propargite has been used for a long history, the resistance monitoring showed that the citrus red mite and the carmine spider mite were still sensitive to this acaricide. In future, propargite will still be frequently used in the management of mites. Currently, researches about propargite were mainly focused on its residues and environmental toxicology, whereas basic systematic research on pest resistance was still insufficient. As a result, the mechanism study of propargite, and its resistance risk evaluation are basic works to understand its resistance development and conduct effective resistance management..This study focused on the important pest mite, T. cinnabarinus, paid attention to its resistance problem, worked on the resistance monitoring of different populations of T. cinnabarinus in mulberry fields in Yunnan province, and reported the resistance level for the first time. A propargite-resistant strain was selected in the laboratory, and its realized heritability and resistance risk were evaluated to reveal the rules of resistance development. Based on the biochemistry study of resistance strain, GSTs were identified as an important factor for the resistance development of propargite. The molecular characterization of GSTs were further analyzed. Genes encoding GSTs of T.cinnabarinus were cloned and their specific expressions were compared between susceptible and resistance strains. Heterogenous expression was also conducted. The resistance mechanism of propargite were synthetically analyzed by population biology and molecular biology methods, and the contribution of GSTs in the resistance mechanism were clarified. The results are important reference to solve production problems in agriculture, and can enrich the resistance theory of pest mites. Through the research work of three years, the findings are presented as follows:1Acaricide susceptibility of seven T. cinnabarinus strains from mulberry fields in YunnanTo gain primary data that reflects the resistance levels of CMS in mulberry plantations, we evaluated and compared the susceptibility of one susceptible strain and7field-collected populations to7acaricides of5chemistry classes using FAO (1980) recommended slide dipping method. The activities and kinetics of major detoxification enzymes (cytochrome P450, esterases and glutathione s-transferases) of each population were also determined to understand their potential role in acaricide resistance. The results showed that most of T. cinnabarinus populations in mulberry trees exhibited low and moderate levels of resistance. Mengzi and Xiangyun population had moderate resistance to phoxim (RR=31.45and26.22, respectively). The Qiaojia, Luliang, Mengzi and Heqing population showed moderate resistance to chlorfenapyr (RR=14.98-22.52). T. cinnabarinus was still sensitive to propargite (the LC50was87.34-243.65mg/L and the resistance ratio (RR) was3.62-10.08). The esistant of field populations to insecticides correlation analysis showed that abamectin and methomyl was significantly and negatively correlated (rs=-0.857,p<0.05). Comparing the enzymes activity between the field isolates and the susceptible strain of CMS. All the tesed enzymes were more active in the field populations than that in the susceptible strain, GSTs was6.44-117.96folds, CarE was1.53-29.87folds, and the P450was1.12-2.67folds, which indicating that GSTs played an important role in the detoxification of mulberry T. cinnabarinus. The results provide useful reference data for acaricides application and spider mite management in mulberry plantations.2Resistance selectivity and characteristics of T. cinnabarinus against Propargite2.1Resistant strain selectivity of Propargite against carmine spider miteThe susceptible strain of T. cinnabarinus was original collected from Beibei District, Chongqing, China in2000and maintanct in laboratory without exposure to aracricides for10years. The susceptible strain was screened continuously with propargite to obtain the resistant strain and the basic characteristics were investigated in this study. After discontinuous (F1-F23) and continuous (F24-F34) selection over34generations, the resistance ratio increased30.669folds. T. cinnabarinus had a slow rate in resistance development to propargite. The breeding procedure of the T. cinnabarinus resistant strain showed the slope of F34strain was smaller than the forgoing strain (F1) according to log concentration-probit curve. The realized heritability (h2) in the continuous (F24-F34) propargite-selection was greater than the discontinuous one (F1-F23), and the values were0.055and0.047, respectively. According to the realized heritability (h2), theoretically, to obtain a10-fold increase in resistance requires53-21generations under discontinuous selective pressure of propargite, and46-18generations continuous selection under selective pressure of propargite of50%-90%for each selective generation.2.2Relative fitness of propargite-resistant strains of T. cinnabarinusUsing the leaf disc feeding method, The life table of T. cinnabarinus susceptible strain (F0) and resistant strain (F34) at25℃were constructed, the ecological aspects of two strains were investigated. The age-specific fecundity and survival curves showed no significant difference between two strains, so were the developmental duration, eggs per female and hatchability, etc. The fitness of propargite-resistance strain was also not significant changed compared to SS.2.3Biochemical mechanism of T. cinnabarinus against propargiteThe F26and F34generations of propargite-resistant strain showed increased resistance against propargite11folds and31folds respectively using slide-dip method. Toxicity of other pesticides/acaricides to the propargite-resistance strain (F26and F34) were assayed using same method. The LC50values and resistance ratios to these pesticides/acaricides indicated that the F26and F34still sensentive to avermectin, but exhibit cross-resistance to phoxim, pyridaben and dichlorvos, chlorfenapyr and diafenthiuron. PBO, TPP and DEM were the specific inhibitors of cytochrome P450monooxygenases (P450), carboxylesterase (CarE) and glutathione S-transferase (GSTs), respectively. The synergistic ratios of PBO, TPP and DEM to propargite were1.06,1.47and2.58fold in resistant strain (F34) of female adult T. cinnabarinus, respectively. None of those three synergists showed significant synergistic effect in susceptible strain of T. cinnabarinus. Based on this result, the mite of four developmental stages (eggs, larvae, nymph, adults) were exposed to the synergist DEM in combination with propargite using potter spray method. There were no significant synergistic effect on susceptible among different life-stages in carmin spider mite. The synergistic resistance ratios of the propargite-resistant strain (F34) with DEM were calculated to be1.27,1.16,1.09, and1.99folds respectively in eggs, larvae, nymph and adults. Compared to susceptible strain, the activities of GST in F34increased by1.98and2.72folds to different substrate GSH and CDNB, respectively. In conclusion, the results indicated GSTs might play an important role in carmine spider mite resistance to propargite.3cDNA cloning and mRNA expression profiles of GSTs genes in T. cinnabarinus3.1cDNA cloning and characterizationBased on the genome data of Tetranychus urticae,12novel GST genes were cloned from T. cinnabarinus using the reverse transcriptase-PCR (RT-PCR) techniques. Putative proteins have been predicted by Protparam and Scanprosite softwares. Based on sequence similarity and phylogenetic analysis, two genes were classified to the Delta gene family, five genes were classified to the Mu family, two genes were classified to the Omega family, two genes were classified as Kappa and Zeta family genes, and the remaining one gene did not belong to any of known family, temporarily be named as an unclassified GST. The nomenclature of these T. cinnabarinus GSTs cDNA and their deduced amino acid sequences have been deposited in Genbank with the following accession numbers:TcGSTd1(KF421142), TcGSTd2(KF421143), TcGSTm1(KF421144), TcGSTm2(KF421149), TcGSTm3(KF421146), TcGSTm4(KF421147), TcGSTm5(KF421148), TcGSTo1(KF421150), TcGSTo2(KF421151), TcGSTz1(KF421153), TcGSTk1(KF421152), TcGSTu2(KF421145). The length of cDNA of these12GSTs gene contained were ranged from651to735bp, encoding proteins with216-244amino acids. Predicted molecular weight was24.19-28.95kDa and the isoelectric point (pI) was between5.97and8.99. It was predicted that there was no transmembrane domain present in these twelve proteins using TMHMM2.0bio-prediction software suggesting that they could be easily purified.ScanProsite showed that two distinct functional domains were found in10proteins except TcGSTkl and TcGSTul. N-terminal domains were highly conserved, which was binding sites of GSH, were found both in TcGSTd1,TcGSTd2(residues1-82) and TcGSTml, TcGSTm2, TcGSTm3, TcGSTm4and TcGSTm5(1-88,1-90,1-88,1-88, 1-82). Other TcGSTo1, TcGSTo2and TcGSTz1were:22-101,23-101and3-90. Also, C-terminal region were located at position86-219,88-207,90-211,92-220,90-214,90-209,90-211,105-240and105-234in TcGSTdl, TcGSTd2, TcGSTm1, TcGSTm2, TcGSTm3, TcGSTm4, TcGSTm5, TcGSTol, TcGSTo2and TcGSTzl. Delta family GSTs in T. cinnabarinus had the same catalytic domain as other species containing Serine, and Asparagine in the center and to determine the same amino acids regarding protein folding including Proline,Leucine,Glycine,Aspartic acid. Mu family GSTs in T. cinnabarinus had the center Tyrosine as the catalytic site and amino acids determing protein folding, like Threonine, Phenylalanine as well as the "AILRYLARKH" motif. Omega GSTs contained the key residue, Cysteine as the catalytic site and a very conserved N-terminal, in which characteristic motif is "ESLIIAEYL"3.2Stability evaluation of housekeeping genesThe stability of reference genes used for the quantification of mRNA can be affected by the experimental condition. Therefore, the evaluation of reference genes is critical for gene expression profiling. The stabilities of six candidate reference genes (a-TUB, RPS18, EFlα,5.8SrRNA, SDHA, GAPDH) of T.cinnabarinus were analyzed at different developmental stages and strains using geNorm and NormFinder software, respectively. RPS18and GADPH were identified as the most stable genes in defferent developmental stages, while a-TUB and RPS18were found to be the most stable in different strains. Thus, RPS18genes was used as reference gene in RT-qPCR in this study.3.3Developmental stages expression profiles of the eight GSTs genesThe expression of eight GSTs genes in T. cinnabarinus were analyzed by real-time quantitative PCR (RT-qPCR) using RPS18as an reference gene. The assayed developmental stages included eggs, larvae, nymph and female adults. The results showed that2Delta family genes,5Mu family genes and TcGSTzl were differently expressed at different stages of mites. The relative expression level of TcGSTdl and TcGSTd2were increased in the larvae and nymph stage compared with egg stage, peaked in nymph stage and declined in adult stage in susceptable strain. But in the resistant strain, the results indicated that the trend of relative expression rose along with growth and development, and reached maximum point in the adult stage. In suscepitable strains, the highest expression levels of four Mu family GST genes(TcGSTm1, TcGSTm2, TcGSTm3and TcGSTm4) were observed at larvae stage. Another Mu family GST genes TcGSTm5showed higher expression level in the nymph stage. In resistant strains, The expression level of three Mu GST genes(TcGSTml,TcGSTm2and TcGSTm4) increased at larvea, showed highest expression in nymph, but reduced in adult stage. Another TcGSTm3showed higher expression levels in the larvae stages, followed by a sudden reduction in the nymph stage, and a recovery in adult. The expression level of TcGSTzl were not significantly different during the development in susceptible strain but showed higher expression in nymph stage in resistant strain. The result indicated that two Delta family GST genes(TcGSTdl and TcGSTd2) and five Mu family GST genes(TcGSTm1,TcGSTm2,TcGSTm3and TcGSTm4) might not only involved in T. cinnabarinus growth and development, but also may played an important role in propargite resistant development in T.cinnabarinus.3.4Expression profiles of the eight GSTs genes between two different strainsIn order to verify whether GSTs are over-expressed in propargite-resistant strain, qPCR with RPS18as the reference gene was employed to determine the relative expression quantity of two Delta GST genes(TcGSTd1,TcGSTd2) and five Mu GST genes(TcGSTml,TcGSTm2,TcGSTm3and TcGSTm4in four life-stage (eggs, larvae, nymph and adults) of T. cinnabarinus in both susceptible and propargite-resistant strain. Expression for eight GST genes in propargite-resistant strain of T. cinnabarinus showed down regulation in eggs and larvae stage, but up regulation in nymph and adult stage in comparison with susceptible strain. In eggs, the relative expression of TcGSTd2was significantly lower than susceptible strain. In larvae, the expression of TcGSTd1,TcGSTd2,TcGSTm1,TcGSTm2,TcGSTm3and TcGSTm4were significantly lower than susceptible strain. In nymph and adults, all of eight GST genes were up regulated than susceptible strain, which implied that these GST genes were possibly involved in propargite resistance.3.5Prokaryotic expression vector of TcGSTm2The cDNA of TcGSTm2was cloned into expression vector pET30a (+) by the restriction sites of Nde I and Xho I, which provided the basis for GSTs expression in E. coli.In summary, this study determined the resistant levels of T. cinnabarinus in Yunnan mulberry plantations. A resistance strain ofpropargite was selected in the laboratory.. After34generations, resistance ratio has increased to30.89folds. The reality of resistant riskment, physiological and biochemical synergist characteristic were analyzed.Biochemical and toxicological characterization of GSTs of T. cinnabarinus were studied; based on genome of T. urticae,12GST genes were cloned from T. cinnabarinus using RT-PCR technique; GSTs genes expression profiles were assayed using qPCR technique from different strains T. cinnabarinus at developmental stages; the expression vector for TcGSTm2was constructed. The results provide insights into exploring the functions of GSTs system in physiology, and it showed evidence for clarifying the resistance mechanisms of carmine spider mite to propargite. Meanwhile, the results also enlighten the resistance mechanism of other insect/mite pests. |
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