Molecular Mechanisms Of Metabolic Resistance To Thiamethoxam In B-biotype Bemisia Tabaci

In recent years, Bemisia tabaci (Gennadius) B biotype has been one of the most destructive invasive pests in many cropping systems worldwide. This whitefly species has developed high resistance to several insecticides commonly used in the world. Thiamethoxam is an important insecticide which has been widely used for the control of this pest. So, the resistance mechanism of the pest to thiamethoxam is a key for sustainable control of this pest. In this study, the resistance mechanisms of B. tabaci B biotype were analyzed using molecular biology method. The main conclusions were listed as follows:1. Selection of reference gene in B. tabaciIn this study,15candidate reference genes in B. tabaci were evaluated under a diverse set of biotic and abiotic conditions using two Excel-based algorithms geNorm and Normfinder. In summary, at least two reference genes were selected to normalize gene expressions in B. tabaci under experimental conditions, respectively. Specifically, for biotic conditions including host plant, acquisition of plant virus, developmental stage, tissue, and whitefly biotype, ribosomal protein L29was the most stable reference gene. In contrast, the expression of elongation factor1alpha, peptidylprolyl isomeraseA, NADH dehydrogenase, succinate dehydrogenase complex subunit A and heat shock protein40were consistently stable across various abiotic conditions including photoperiod, temperature, and insecticide susceptibility.2. Cloning and analysis of CYP6DZ7gene in B. tabaciThe full sequence of the mRNA was obtained by RACE (rapid amplification of cDNA ends). CYP6DZ7has a1668bp open reading frame (ORF) with a putative protein size of555amino acid. Full gene sequences were named by Dr. David Nelson. The putative full-length protein sequences of CYP6DZ7were compared between the resistant and susceptible strains, and were also compared to the CYP6protein sequences in20insect species identified by BLASTX. This study found that protein sequence of CYP6DZ7in the resistant and susceptible strains were identical.3. Relative expression analysis of CYP6DZ7in TH-S, TH-R and TH-2000strain of B.tabaciRT-qPCR analyses showed that the expression of CYP6DZ7in TH-2000strain was approximately9-fold higher than that in TH-S strain and was approximately7-fold higher than that in TH-R strain. This result showed that the expression increased when TH-R strain were treated with thiamethoxam, suggesting the CYP6DZ7gene may play an important role in the thiamethoxam resistance of the TH-R strain.4. Relative expression analysis of CYP6DZ7in developmental stages and tissues in B.tabaciThe expression of CYP6DZ7gene in different developmental stages of B. tabaci was examined by RT-qPCR. The result indicated that CYP6DZ7was consistently expressed in all life stages. The expression level of CYP6DZ7was the lowest in eggs but not significantly different from that in nymph or adult. The expression of CYP6DZ7gene was high in abdomen whereas low in the chest. The tissue-dependent expression pattern was validated by RT-qPCR. The expression of CYP6DZ7in abdomen was4-fold higher than that in the chest.5. Differential expression of CYP6DZ7gene after treating with thiamethoxamTo further test our hypothesis, we quantified the expression level of CYP6DZ7of different time course after2000mg/L thiamethoxam treatment and with different treating concentrations in TH-R. At the same time, we quantified the expression level of CYP6DZ7with different treating concentrations in TH-S. The results indicated that the expression of CYP6DZ7gene correlated with thiamethoxam resistance in whiteflies.6. RNA interference (RNAi) methods were used to explore the role of P450genes in B.tabaciThe expression of CYP6DZ7gene in the TH-R strain was partially silenced by RNAi with the double-stranded RNA (dsRNA), which further confirmed the function of CYP6DZ7gene. The results showed that the gene expression of CYP6DZ7decreased>60%by72hr after CYP6DZ7dsRNA injection, which indicated that this gene was successfully knocked down by RNAi. The bioassay tests showed a significantly higher mortalities for the dsRNA-injected adults compared to the control adults injected with buffer. Taken together, our results indicated that CYP6DZ7gene plays an important role in thiamethoxam resistance of the whitefly.7. Expression of CYP6DZ7in E. coli cellsThis study constructed recombinant plasmid using PET expression system, which successfully expressed in E. coli cells. The sresult showed that a63kDa protein was examined and confirmed by SDS-PAGE. Our work laid a foundation for the next resistance mechanism study and resistance management.