Insecticide Resistance Monitoring And Resistance Allele Frequency Detection In Q-biotype Bemisia Tabaci From Eastern China

The whitefly, Bemisia tabaci Gennadius (Homoptera:Aleyrodidae) is one of the most devastating insect pests worldwide in tropical and subtropical regions. This pest causes losses both directly by feeding and indirectly by vectoring several hundreds of plant viruses. It is considered a highly cryptic species complex of diverse biotypes with differences in host range, geographic distribution, insecticide resistance and virus-transmission capabilities. The two most widespread biotypes are referred as "B" and "Q". Use of chemical insecticides has been the primary strategy for controlling B. tabaci. As a consequence of extensive exposure to insecticides, B. tabaci has developed resistance to a wide range of insecticides including organophosphates (OPs), carbamates. pyrethroids (Pyr). cyclodienes, neonicotinoids, and insect growth regulators (IGRs). In this paper, the resistance levels, resistance allele frequencies, single-nucleotide polymorphisms (SNPs) associated with resistance to neonicotinoid insecticide in first intron of cytochrome P450CYP6CM1gene in Q-biotype B. tabaci field populations from eastern China were determined. Fragments of insecticide target site gene and5’ flanking sequence of CYP6CM1gene in Q biotype B. tabaci were also cloned and analyzed.The resistance levels of Q-biotype Bemisia tabaci (Gennadius) in five field populations including Wuxi, Yangzhou, Dongtai in Jiangsu province, Hangzhou in Zhejiang province and Shanghai to six representative insecticides were determined by leaf dipping method. The results showed that, compared with the reference strain, these field populations had developed low to moderate levels of resistance to cypermethrin, imidacloprid and nitenpyram with the LC50value ranging between570.41-2413.93mg/L、44.48-653.51mg/L and8.65-103.17mg/L, respectively. Low susceptibility to dichlorvos and carbosulfan was also observed in these field populations with the LC50value ranging between460.86-1066.99mg/L and50.18-349.12mg/L, respectively. However, these field populations remained high susceptible to abamectin with the LC50value ranging between0.0006-0.02mg/L.Two fragments of acetylcholinesterase enzyme acel gene and para-type voltage gated sodium channel gene in Q-biotype B. tabaci from eastern China were amplified by PCR, with the length being287bp and184bp, respectively. Sequence analysis suggested the presence of F331W mutation in acetylcholinesterase acel and L925I and T929V mutations in sodium channel in these Q biotype field populations from eastern China, which are associated with organophosphate and pyrethroid insecticides, respectively.The PCR-RFLP protocol was used to detect the allele frequency of the OP resistance-associated F331W mutation of acetylcholinesterase enzyme acel gene in Q-biotype B. tabaci. All tested individuals were homozygous for the F331W mutation, suggesting that this mutation was fixed in Q-biotype B. tabaci from eastern China. Frequencies of two mutations in the IIS4-5linker of para-type voltage gated sodium channel gene, L925I and T929V, were determined by PCR-RFLP and PASA, respectively. The result showed that the frequency of the L925I mutation and T929V mutation were in the range of0.394-0.633and0.559-0.904, respectively. The average T929V mutation frequency (0.721) was significantly higher than the average L925I mutation frequency (0.589). However, no correlation was found between the frequency of L925I or T929V mutations and the resistance level of Q-biotype B. tabaci to cypermethrin, indicating the presence of other metabolic resistance mechanisms.The genomic fragment of cytochrome P450gene CYP6CM1in Q-biotype B. tabaci from Yangzhou population was amplified by PCR, which contains the last63bp of the first exon of the CYP6CM1gene together with the first826-829bp of the adjacent intron. Three single-nucleotide polymorphisms(SNPs) associated with resistance to neonicotinoid insecticide were identified by multiple sequences alignment, which were located in positions195、230and242of the intron, respectively. In addition, the5’flanking sequence of P450CYP6CM1gene of Q-biotype B. tabaci was cloned by genome walking protocol, with the length being1226bp. The NNPP online analysis software predicted the transcription initiation site as the nucleotide A, which is located57bp upstream of the initiation codon. The TFSEARCH1.3software predicted several basic transcriptional promoter elements together with multiple transcription factor binding sites in this region.