Proteome Analysis Of Panonychus Citri Expose To Avermectin

The citrus red mite, Panonychus citri (McGregor), is one of the most important citrus pests. It is distributed all over the world, and serious occurs in the main citrus producing areas in China. The mites have developed severe resistance to various acaricides based on the previous reports. As a member of biogenic pesticides, avermectin has been widely used in pest mites control for its good acaricidal activity. Due to its heavy use, the resistance risk has become worse and worse. In the post genomics era, the proteomics offers a powerful method to investigate the resistance mechanisms of this pest mite on protein level comprehensively.In this study, we determined the P. citri resistance levels of two geographic populations (Beibei and Wanzhou) and susceptible strains to fenpropathrin, avermectin, chlorpyrifos and pyridaben using leaf disc dip method. As the results indicated, the acaricidal activity of four acaricides was ranked as avermectin> pyridaben fenpropathrin> chlorpyrifos. Moreover, beside avermectin the resistance of Wanzhou population has developed quicker comparing to Beibei population.Based on the bioassay, mites were exposed to avermectin for24hours at a concentration of LC30. Then, a two-dimensional gel electrophoresis method was employed to investigate the differentially expressed proteins. As a result,622protien spots were isolated based on the2-DE. Twenty-six of them were captured by PDQuest software as their expression changed significantly. They were identified as23proteins by mass spectroscopy. They were further catergorized due to their involvement in different biological process including growth and development, energy metabolism, cytoskeleton and detoxifying metabolism and other physiological functions. Those proteins at least2-fold up-regulated were vitellogenin (Vg), ATP synthase D chain (ATPsyn-D), lambda-crystallin homolog (CRY), glyceraldehyde3-phosphate dehydrogenase (GAPDH), esterase CarE type B (CarE-B), ferritin (Fer), vacuolar ATPase B subunit (V-ATPsyn-B), translation elongation factor2(EEF2) and ATP synthase subunit alpha (ATPsyn-a). Meanwhile, calreticulin (CALR), fumarylacetoacetase (FAH) and40S ribosomal protein SA (RPSA) were found to be down-regulated more than2folds. The result showed that various metabolic pathways are involved in the response of P.citri to avermectin exposure. According to the different functions of these proteins,16genes out of23mentioned above were selected for further investigation. Using Real-Time Quantitative PCR (qPCR), we determined mRNA expression patterns responded to the24-hour exposure at the dose of LC30. We detected three genes (CRY, Fer and Vg) up-regulated more than10folds, while the genes of RPSA, EEF2and transketolase (TKT) were significantly dow-regulated. Furthermore, the transcriptional response of eleven genes were further characterized under different doses (LC10, LC30, LC50) for24h. In addition, their mRNA expression was also tested at the dose of LC10with different-time exposure (12h,24h,36h). The results of dose-effect showed that RPSA, EEF2and TKT were down-regulated dramatically after treatment, while the others were up-regulated moderately. In the time-course effects experiments CarE-B, Actin, Fer and Vg increased their expression significantly when compared to the control. However, the TKT was significantly down regulated. Combine our proteomic analysis and qPCR confirmation, we concluded that Vg, CRY, Fer, Acitn, CarE-B, SRM and V-ATPsyn-B have been studied above played key roles in the avermectin metabolism.For the future studies on the molecular mechanisms of the avermectin metabolism, the full-length cDNA of RPSA, TKT, ATPsyn-a, Actin and Vg genes were cloned using rapid amplification of cDNA ends (RACE). Sequences were submitted to GeneBank. Their accession numbers are KJ641584, KJ641585, KJ641586, KJ641587and KC978893, respectively. Sequence alignment showed the conserved domains. The phylogenetic analysis indicated those proteins had the closest relationship with the proteins in Tetranychus urticae, since they are grouped into the same cluster.The results above provide a theoretical basis for later study about the P.citri reponse to avermectin and the molecular mechanism of resistance.