Resistance Mechanisms Of Invasive B-biotype Bemisia Tabaci To Thiamethoxam

Bemisia tabaci(Gennadius) B biotype is one of the most destructive invasive pests in many cropping systems worldwide.In recently years,whiteflies have developed high resistance to several commonly used insecticides in the world.Thiamethoxam is the key insecticide which has been widely used for control of this pest.So,study on thiamethoxam resistance is very important for sustainable control of B-biotype B.tabaci.In this paper,the resistance selection,fitness analysis and morphological properties measuring were carried out to evaluate the risk for B-biotype B.tabaci to develop high resistance to thiamethoxam.With selected susceptible strain and resistant strains,the biochemical and molecular mechanisms for the resistance were also analyzed.The main results and conclusion were as follows:1.Thiamethoxam resistance selection with B-biotype B.tabaciBased on the leaf-dip bioassay,thiamethoxam resistance in B-biotype B.tabaci was continuously selected for 36 generations.The development of thiamethoxam resistance in B-biotype B.tabaci was uneven over time.In the first 17 generations,resistance developed very slowly and resulted in about a 10-fold increase in the resistance ratio.From 18th to 23th generation,resistance reached 20.4-fold,then, after stagnating for six generations(T₂₄-T₂₉).Resistance increased quickly from 30th generation,and the resistance ratio of 33rd generation was 66.3 and remained around 60 with little variation beyond this point.Realized heritability(h²) and resistance risk were both estimated.The realized heritability was 0.0909(F₀-F₄),0.0877(F₅-F₁₇),0.0591(F₁₈-F₂₃),0.0269(F₂₄-F₂₉) and 0.1703(F₃₀-F₃₆),respectively, and the value was 0.1309 after selection for 36 generations.It required 3 to about 21 generations to obtain 10-fold increase in LC₅₀ under selective pressure of 50%to 99%mortality for each selective generation when realized heritability was 0.1309.Estimation on the numbers of gene involved in thiamethoxam suggested that the resistance was controlled by a single gene.The degree of dominance of the resistant trait in thiamethoxam selected strain was -0.95,indicating that thiamethoxam resistance was nearly completely recessive.2.The influence of thiamethoxam resistance on the fitness of B-biotype B.tabaciFitness analysis by constructing life tables,demonstrated that resistant B-biotype whiteflies had obvious fitness disadvantages in their development and reproduction.The mean developmental times of egg,nymphae of TH-R strain were all significantly longer than those of TH-S strain.The adult longevity of TH-R strain was significantly shorter than that of TH-S strain.The fecundity and the survivorship of TH-R strain were all reduced than TH-S strain.The fitness of resistant B-biotype whiteflies decreased dramatically(0.53),to only one-half that of the TH-S strain.The results suggested that lower fitness of resistant B.tabaci was one of the reasons of slower resistance development.Some changes in the morphological characteristics of the resistant strain were observed.The lengths of first, second and third instars of the resistant strain were significantly smaller than those of the susceptible strain,and the width of the first and the fourth instars were also significantly smaller than in the susceptible strain.3.Biochemical mechanisms for thiamethoxam resistance in B-biotype B.tabaciThe biochemical mechanisms of thiamethoxam resistance in B-biotype B.tabaci were studied by synergism test,cross-resistance analysis,detoxifying enzyme activity test.The results showed that compared with the susceptible strain(TH-S),the selected TH-R strain showed clear cross-resistance to imidacloprid(47.28-fold),acetamiprid(35.82-fold),nitenpyram(9.99-fold),abamectin(5.33-fold),and carbosulfan(4.43-fold).No cross-resistance was seen to fipronil,chlorpyrifos,or decamethrin. Glutathione-S-transferase was not related to thiamethoxam resistance in this pest.Piperonyl butoxide (PBO) and triphenyl phosphate(TPP) exhibited significant synergism on thiamethoxam effects in the TH-R strain(3.14- and 2.37-fold,respectively).Biochemical assays at two resistance levels(T₃₀ and T₃₆) showed that cytochrome P450 monooxygenase activities(PNOD) increased 1.21-fold and 3.23-fold respectively,and ECOD activity increased 1.68-fold and 2.30-fold respectively in the TH-R strain. Carboxylesterase activity increased 2.96-fold only at the T₃₀ level.Cytochrome P450 monooxygenase appeared responsible for the resistance,while carboxylesterase was only responsible at the low resistance level.4.Comparison of life tables,morphological properties and detoxification enzymes in B-biotype and ZHJ-1-biotype B.tabaciBy constructing life tables,we demonstrated that the mean developmental times of egg,nymphae of the ZHJ-1-biotype strain were all significantly longer than those of the B-biotype strain.There were also significant differences among the adult longevity and fecundity of the two strains.The population parameters of the two strains showed that the finite rate of increase,gross reproductive,the net reproductive rate of ZHJ-1-biotype strain were all lower than those of the B-biotype strain,and the mean generation time of the ZHJ-1-biotype strain was longer than that of the B-biotype strain.The length and the width of first,second and third nymph of the B-biotype strain were all significantly smaller than those of the ZHJ-1-biotype strain.The length and the width of the fourth nympha were not different between the two strains.Glutathione -S-transferase,carboxylesterase and cytochrome P450 monooxygenase activities were not different between the two strains.5.Cloning of nAChRs gene from B-biotype B.tabaciUsing RT-PCR technique,α-subunit of nicotinic acetylcholine receptor gene from B-biotype B. tabaci was cloned and sequenced.This gene is of 1935bp and the open reading frame is 1605bp from 257bp to 1861bp,which encoding 534 amino acid.It has the conserved amino acids and typical features shared by the nAChR family and alpha-subunit,such as a large N-terminal extracellular domain involved in agonist binding,followed by four transmembrane regions(TM1-TM4).The presence of two vicinal cysteine residues,defines nACh receptorαsubunit in insects.And the homology to the nAChRαsubunit gene of other insects is 54%-79%.The Genebank access number was DQ480159.There was no difference of the sequence between the the resistant and the susceptible strains,speculated that the heterozygote was the main part in the resistant strain.6.The influence of thiamethoxam resistance on expression in mRNA ofαsubunit of nAChRsThe expression of nicotinic acetylcholine receptorαsubunit gene of two strains was detected by the real-time PCR(Q-PCR) analysis.We designed three pair primers,selected one pair to analysis the expression ofαsubunit gene.The results showed that gene expression increased in the TH-R strain,it was 3.6 times to that of the TH-S strain,but the mean data was not different from the TH-S with statistic analysis.The reason and the role of expression of the gene need further demonstration.Collectively,the present study firstly researched the thiamethoxam resistance mechanism of Bbiotype B.tabaci.Studies showed that the risk for B-biotype B.tabaci to develop high resistance to thiamethoxam,the resistance resulted fitness cost and development disadvantage.The synergist tests and the biochemical studies showed that the Cytochrome P450 monooxygenase appeared responsible for the resistance.Cross-resistance offered the basis for control this pest in the fields.Theα-subunit of nicotinic acetylcholine receptor gene from B-biotype B.tabaci was cloned and the expressions of two strains were detected by the real-time PCR analysis.