Study On Resistance Mechanism Of Tetranychus Urticae (Koch) To Avermectin And Spirotetramat

As a worldwide pest mite, the two-spotted spider mite, Tetranychus urticae, is one of theorganisms currently found in arthropods that can best resist the pesticide. Its control greatlyrelies on the use of chemical pesticides. Currently avermectin is an important antibioticspesticide variety for the control of the spider mites, and spirotetramat is by far the first newinsecticide and acaricide with bidirectional and systemic conduction. The single, long-termand high-frequent use of any pesticide will inevitably result in the risk of pesticide resistance.In this dissertation, and avermectin-resistant (Av-R) and spirotetramat-resistant (Sp-R) strainsof T. urticae were obtained by using the sensitive strain reared indoors for years. Changes inmajor detoxification enzymes were determined from the physiological and biochemical levelwhile target gene mutations were detected from the molecular level. At last, the expressions ofmRNA transcription level of28detoxifying enzyme genes were analyzed by using thescreened stable reference gene from6housekeeping genes of T. urticae. The main results areas follows:1. Resistance selection of T. urticae to avermectin and spirotetramat.The avermectin-resistant (Av-R) and spirotetramat-resistant (Sp-R) strains were obtainedafter18generations of selection as a result of some individuals separated from SS strain ofT.urticae being treated with the spray of avermectin and spirotetramat respectively. By indoorBioassay, the resistance index of Av-R and Sp-R strains were180.5and90.58folds,respectively, indicating that a high level of resistance to avermectin and spirotetramat wereproduced in T. urticae. From the perspective of the selecting process, the resistance formationand development of T. urticae to spirotetramat were relatively slow, but there is littledifference in the resistance development trend of T. urticae to avermectin and spirotetramat.2. Analysis of detoxification enzymes activity in Av-R and Sp-R strains of T. urticae.The activities of MFOs, GSTs and CarEs in Av-R and Sp-R strains of T. urticae weremeasured via the microplate reader method. The results showed that among3detoxificationenzymes the biggest change in activity was MFOs. The specific activity of MFOs in Av-R andSp-R strains of T. urtica were significantly increased3.06and2.20folds than that in SS. Thechanges of GSTs and CarEs were small, the specific activity of GSTs in Av-R and Sp-R strains increased1.26and1.40folds while the specific activity of CarEs were1.34and1.27folds than that in SS. The increase of activity with varying degrees in GSTs, MFOs and CarEsof resistant strains indicated that the enhanced activity was related to the resistance toavermectin and spirotetramat in T. urtica and the enhancement of MFOs is one of the mainreasons for the generation of resistance in Av-R and Sp-R strains.3. Detection of target mutations in Av-R and Sp-R strains of T. urticaeUsing direct sequencing of PCR products, GABA receptor gene and ACCase gene wereamplified from SS and Av-R, SS and Sp-R strains, respectively. The results showed that noresistance-associated mutations existed in GABA receptor gene of Av-R and ACCase gene ofSp-R when compared with SS strain. However, only one synonymous mutation was found at1914thbase of GABA01gene and3597thbase of ACCase gene, indicating that the formationof resistance to avermectin and spirotetramat in T. urticae was not associated with target-siteresistance in the current resistance level conditions.4. Screening of the control gene in different strains of T. urticaeThe mRNA expression stability of6housekeeping genes, i.e., GAPDH, SDHA, ELF,5.8S rRNA, α-tubulin and β-actin of T. urticae were detected via geNorm and Normfindersoftware by qRT-PCR. The evaluation and analysis by geNorm showed that the ranking ofgene expression stability value (M) were GAPDH=SDHA﹤ELF﹤5.8S rRNA﹤α-tubulin﹤β-actin. The stability of the internal genes was evaluated according to the M value; the lowerM value is, the more stable the reference gene is, and vice versa. Therefore, the referencegenes with better stability were GAPDH, SDHA and ELF. After analysis by Normfinder, thestable value of6housekeeping genes from small to large were ELF(0.050)﹤SDHA(0.143)﹤GAPDH(0.207)﹤5.8S rRNA(0.337)﹤α-tubulin(0.351)﹤β-actin(0.370). ELF was the moststable gene that can be used as an internal gene while β-actin, α-tubulin and5.8S rRNAcannot do so because of their poor stability. Comprehensive comparison of the evaluation ofgeNorm and Normfinder, the most stable gene in SS, Av-R and Sp-R strains of T. urticae wasELF.5. Analysis of the mRNA transcription level of detoxifying enzyme genes in resistantstrains of T. urticae.When ELF was used as a reference gene, the mRNA transcription level of CYP392E subfamily genes of P450s, Delta family genes of GSTs and CarEs genes in Av-R and Sp-Rstrain were analyzed. The results showed that the relative expression levels of CYP392E7,TuGSTd16and TuCCE35genes in Av-R strain were significantly higher (2.18,1.12and1.59folds, respectively) than that in SS strain while CYP392E4, CYP392E9、CYP392E10,TuGSTd04, GSTd15and TuCCE36genes decreased significantly (0.48-0.74folds). Together,these results indicated that significant changes in mRNA transcription levels of these genesmay play an important role in the formation of the resistance to avermectin in T. urticae. InSp-R strain, only the relative expression levels of TuCCE35gene increased1.62folds thanthat in SS strain while CYP392E1, CYP392E9, TuGSTd04and GSTd15genes decreasedsignificantly (0.40-0.73folds). These results indicated that significant changes in mRNAtranscription levels of these genes may be related to the formation of the resistance tospirotetramat in T. urticae.In conclusion, the resistance of T. urticae to avermectin and spirotetramat were primarilyrelevant to the enhanced enzyme activity of MFOs in the physiological and biochemical level.To a certain extent, GSTs and CarEs were also involved in the formation of resistance.However, at the molecular level, significant increase of the gene transcription expression levelof CYP392E7, TuGSTd16and TuCCE35gene in Av-R strain, TuCCE35gene in Sp-R strainmay be relevant to the formation of resistance to avermectin and spirotetramat, respectively.