Resistance Mechanism Of B Biotype Bemisia Tabaci To Thiamethoxam

Bemisia tabaci is an important pest of arable and horticultural crops in the temperate regions of theworld. In resent years, pesticide resistance has been developed in B. tabaci in many parts of the world.Thiamethoxam as a second-generation neonicotinoid which has been widely used B. tabaci control. Tounderstand the molecular basis of thiamethoxam resistance,“omics” and molecular analysis werecarried out to examine the risk of B biotype B. tabaci to develop high resistance to thiamethoxam. It’sshowed that cytochrome P450gene over-expressed has responsible for the thiamethoxam resistance,also provided foundation for pest control in the field. The main results and conclusions were as follows:1. Biochemical mechanisms for thiamethoxam resistance in B. tabaciResistant B. tabaci strains （TH-R and TH-2000） were derived from a field-collected susceptiblestrain （TH-S）, which had no previous exposure to any insecticides. Both of these resistant strains havebeen exposed to thiamethoxam continuously for over60generations. Samples collected from TH-2000were LC80survivors, whereas TH-R were LC50survivors. Theoretically, individuals from TH-2000have a higher level of tolerance to thiamethoxam treatment, and this difference was reflected in themetabolic enzyme activity assays. For example, GST activity of TH-R strain was slightly higher thanthat of the susceptible TH-S strain, however, the difference was not substantial （one-way ANOVA, P <0.05; LSD test）. In contrast, GST activity was significantly elevated in TH-2000strain, which exhibiteda1.79-fold higher GST activity than TH-S when using CDNB and GSH as substrates. The same trendwas observed in P450monooxygenase activities as well. The PNOD activities of the two resistant TH-Rand TH-2000strains are1.67and2.82-fold, respectively, greater than that of the susceptible TH-S strain.Furthermore, both GST and P450activities between the two resistant strains （TH-R and TH-2000） werealso significantly different （one-way ANOVA, P <0.05; LSD test）.2. Microarray analysis of thiamethoxam resistance mechanism in B. tabaciThiamethoxam susceptible （TH-S） and resistant （TH-R） B. tabaci strains were maintained in agreenhouse, insects were collected from both strains at the following life stages:（i） eggs,（ii）4th instarnymphs （iii） and one day-old unmated adult females. The array design is based on8,394B. tabaci ESTsfrom gene-families with putative association to insecticide resistance. A new B. tabaci8x15k Agilentmicroarray was designed for this experiment. In this study we have shown that the expression of434,553and360ESTs were altered in the egg, nymph and female adult stages, respectively. Gene ontologyand bioinformatic analyses revealed that in all life stages many of the differentially expressed transcriptsencoded enzymes involved in metabolic processes and/or metabolism of xenobiotics. Several of theseare candidate resistance genes and include the cytochrome P450CYP6CM1, which has been shown toconfer resistance to several neonicotinoids previously, a P450belonging to the CYP4family and a GSTbelonging to the sigma class. Finally several ATP-binding cassette transporters of the ABCG subfamilywere highly overexpressed in the adult-stage of the TH-R strain and may play a role in resistance byactive efflux.3. Transcriptome research of B. tabaci Thiamethoxam susceptible and resistant B. tabaci transcriptomes were sequenced individually,generating approximately12million raw reads for each library. Among the two RNA-seq libraries,77.69%and79.03%of reads were mapped to a gene in the reference database with a perfect match ratioof64.55%and65.45%, respectively. A total of1,338mRNAs were differentially expressed betweenresistant and susceptible B. tabaci. KEGG analysis mapped318differentially expressed genes to160pathways （p<0.05）. Using Blast2GO,186differentially expressed transcripts were able to assign to37GO classes. Majority of these genes were putatively linked to insecticide resistance, includingglutathione-S-transferase, cytochrome P450, UDP glucuronosyltransferase, and glucosyl/glucuronosyltransferase.4. Proteome research of B. tabaciThe total number of mass spectrometry detected in B. tabaci proteomes was39316, representing5765peptide spectra and2226distinct peptides. Of the1005peptides identified, more than70%（711）were assigned to a putative protein by homology search against non-redundant （NR） database. Afterchallenged with thiamethoxam,52differentially expressed proteins （p-value≤0.05） were identifiedbetween susceptible （TH-S） and resistant （TH-2000） B. tabaci. Among them,38proteins wereup-regulated （≥1.2^Fold, p-value≤0.05） and14proteins were down-regulated （≤0.8^Fold,p-value≤0.05）. Among the52differentially expressed proteins,28were sub-categorized into31hierarchically-structured GO classes, and35differentially expressed proteins to14pathways （p<0.05）,including “Metabolism of xenobiotics by cytochrome P450”,“Drug metabolism-cytochrome P450”,and “Drug metabolism-other enzymes”. Among them,“Metabolism of xenobiotics by cytochromeP450”, and “Drug metabolism-cytochrome P450” had the lowest p-value. KEGG pathway analysis alsorevealed that the most-enriched peptides, including GSTs, UDP-glucuronosyltransferases,glucosyl/glucuronosyl transferases, and cytochrome P450s, were involved in xenobiotic metabolism.5. Correlation between the transcriptome and proteomeTo correlate protein with mRNA expression profiles, accession numbers from the proteomic datasetwas extracted and compared to the annotated RNA-seq libraries.11differentially proteins and theircorresponding mRNAs in response to thiamethoxam exposure, and the correlation coefficient betweenthe proteins and gene expression profiles was0.6643. Expression profiles of representative transcriptsencoding UDP-glucuronosyltransferase2B10-like isoform1（XP₀02704642.1）, glucosyl/glucuronosyltransferases （XP₉69321.2）, GST （ACH90394.1）, and cytochrome P450CYP6CX1（ACT78507.2）showed significantly higher expressions （p-value<0.05） in the resistant TH-2000strain in comparison tothe susceptible TH-S strain, in which CYP6CX1had the highest differential expression （¹1-fold）.