Studies On Resistance Mechanism Of Bemisia Tabaci To Chlorpyrifos

The tobacco whitefly (Bemisia tabaci) is one of the most important insect pests all over the world. It feeds many crop and economic plants including cotton and vegebables. In recent years, the occurrance of tobacco whitefly was reported in at least22provinces, autonomous regions and municipalities across China, and is stilll diffusing to other areas. B-boitype is the main type of whitefly now in China, but is being dominated by the Q-biotype, a biotype with even higher damage inducing capacity than the B-biotype. The tobacco whitefly reproduces11-21generations anually, resulting in a heavy generation overlapping. The occurance and damage of the whitefly in the open fields is most serious between July to September, and higher temprature and drought are favorable for its occurance. For decades, the control of the whitefly is heavily dependent on chemical insecticides including orgnophosphorous and carbamates, which leads to the pest resistance problem and makes more and more control failures in fields. In order to develop a rational resistance management strategy and to obtain higher control efficacy, the mechanisms of resistance to chlorpyrifos by the whitefly was explored in this paper. The main results were summarized as follows:1. Selection of resistant and susceptible strains of B. tabaci to chlorpyrifos in laboratoryBased on a B-biotype whitefly population collected from Nanjing fields in2005, a chlorpyrifos resistant strain (NJ-R) and a susceptible strain (NJ-S) were selected by using group screening method. After26generations’selection, a33.9-fold resistance was obtained compared to NJ-S strain. During the early9generations of selection, the resistance increased very slowly, while much faster in the subsequent4generations, obtaining a33.9-fold resistance after the13th generation’s selection. However, this resistance was not stable, as it quickly descreased to18-folds after4generation’s suspending of selection, and same quickly the resistance increased from18-folds to33-folds when selection was resorted for3generations. Hereafter, continued selection could not significantly increase the resistance. It is concluded that the resistance of whitefly to chlorpyrifos can be easily selected, but the resistance may decrease significantly when the selection is suspended.2. Detoxification mechanisms for resistance of B. tabaci to chlorpyrifosThe activities on3detoxification enzymes (Estersae, GST and MFO) of NJ-S and NJ-R strains were determined. Th results showed that esterase activity of NJ-R was1.53times of that in NJ-S, significantly different between two strains, while the GST and MFO enzyme activities between resistant and susceptible strains were not significantly different. To verify the enzyme activity assay, experiments with synergist TPP, DEM and PBO were carried out. TPP showed high synergy effect to chlorpyrifos in both NJ-S and NJ-R strains, but the synergy rate (SR) in NJ-R is4.46much higher than that in NJ-S (2.43). DEM and PBO displayed no obvious synergy effect to chlorpyrifos in resistant and susceptible strains. All aboved results together show that enhenced esterase activity plays key role, while the GST and MFO play minor roles, in the metabolic resistance of B. tabaci to chlorpyrifos.3. Target mechanisms for resistance of B. tabaci to chlorpyrifosAChE activities and enzyme kinetic parameters of NJ-S and NJ-R strains were compared, and the results showed no significant differenec between NJ-R and NJ-S. This indicated no contribution by target mechanism to the33.9-folds resistance in NJ-R. However, after cloning and analysing the ace1gene sequences, it was found that both resistant and susceptible individuals of B. tabaci had the F392W point mutation in ace1, which was somewhat unexpected. As F392W mutation was found to correlate to resistance to organophosphorous and carbamate insecticides in many insect pests, NJ-S actually was a susceptible strain bearing the target resistance, and NJ-R was a resistant strain of33-folds beside the target resistance. Obviously, the33-fold resistance in NJ-R was mostly (if not completely) resulted by metabolic mechanisms. These also imply that the field whitfly population collected in Nanjing fields in2005for sellecting the NJ-R and NJ-S strains, was already a resistant population with F392W mutation in acel genes.4. The biotype identification and resistant AChE-1allele frequencies of six geographical B. Tabaci populations across ChinaThe biotypes of six tabacco whiteflies populations collected from Beijing, Nanjing, Wuhan, Guangdong, Guangxi and Xinjiang were identified by analysing the mtDNA COI gene sequence. As a result, Guangdong, Guangxi and Xinjiang populations were all B biotype, while Nanjing and Wuhan populations were Q biotype, and Beijing population was mostly Q biotype. At meantime, the mutant ace1alleles carrying F392W mutation were detected in101whiteflies from six populations, by using the molecular-based RFLP-PCR method. The result showed that around90%frequencies of resistant ace1allele were found in all six whitefly populations. Among those populations, Beijing populaion had the highest frequency of resistant ace1allele (100%), while the Nanjing population had the lowest of88.23. The frequencies in Guangxi, Xinjiang, Wuhan and Guangdong populations were94.44%,93.75%,93.75%and93.75%, respectively. Among the101tested individuals,93whiteflies were resistant homozygote,4whiteflies were susceptible homozygote and4were heterozygotes, counting for92.07%,3.96%and3.96%, respectively.5. Eukaryotic expression of the resistant and the wild AChE-1in vitroSite-directed mutagenesis and baculovirus expression system were used to construct the expression plasmid with resistant and wild type of acel, and Sf9insect cells were used for expression of the recombinant plasmids. Three days after the transfecting the insect cells with recombinant bacmid virus, the culture medium and cells were detected for the expressed AChE-1by SDS-PAGE, but no obvious expected band was found in the jel, and the AChE activity measurement also showed no significant activity found in transfected medium and cells than those in control. However, obvious signal of expressed GFP protein was detected in insect cells tranfected with GFP recombinant virus, which showed that the transfection was succesful. The reason might be that the amount of expressed AChE-1was too low to be detected, which will be improved in the ongoing experiments.