【作者】 封云涛；

【导师】 张友军；

【作者基本信息】 中国农业科学院 ， 农业昆虫与害虫防治， 2009， 博士

【摘要】 B型烟粉虱Bemisia tabaci（Gennadius）biotype B是近年来传入我国的重要农业害虫。该害虫在世界范围内对多种杀虫剂（尤其新烟碱类杀虫剂）产生了抗性,难以治理。噻虫嗪是目前防治这一害虫效果最好的农药品种之一。为了延缓B型烟粉虱对噻虫嗪的抗性发展,延长噻虫嗪的田间使用寿命,本研究通过B型烟粉虱室内抗性筛选及适合度、形态学分析,系统的评估了B型烟粉虱对噻虫嗪产生抗性的风险;同时,从生化与分子生物学的水平,分析了B型烟粉虱对噻虫嗪的抗性机制。主要研究内容及结论如下:一、B型烟粉虱对噻虫嗪抗性的室内连代筛选与抗性风险评估本研究利用噻虫嗪对B型烟粉虱进行了连续36代的抗性筛选,成功获得了B型烟粉虱抗噻虫嗪室内品系（TH-R）。对种群抗性监测、抗性现实遗传力（h²）评估及抗性风险预测研究发现:1)在最初筛选的17代(T₁-T₁₇),抗性发展非常缓慢,抗性只缓慢增长至约10.0倍。第18代到第23代抗性增长加快,由第18代的12.2倍增长至第23代的20.4倍;随后抗性稳定了6代(T₂₄-T₂₉),维持在20倍左右。从第30代(T₃₀)开始,抗性迅速增长,增长速度高于之前所有阶段,抗性倍数由T₃₀的25.8倍上升至第33代(T₃₃)的66.3倍;之后,抗性基本稳定在60倍。2)抗性现实遗传力分别为0.0909（F₀-F₄）,0.0877(F₅-F₁₇),0.0591(F₁₈-F₂₃),0.0269(F₂₄-F₂₉),0.1703(F₃₀-F₃₆),整个汰选阶段的抗性现实遗传力为0.1309。3)假设遗传力为室内筛选估算值的一半,当死亡率分别为50%-90%时,预计B型烟粉虱对噻虫嗪的抗性增长10倍,需要约3-21代,而噻虫嗪抗性为隐性单基因控制。二、噻虫嗪抗性对B型烟粉虱种群适合度的影响本研究通过构建种群生命表,比较了敏感与抗性(T₃₃)品系B型烟粉虱一系列生长发育与繁殖特征。结果表明,与敏感品系相比,抗性T₃₃代的卵、各龄若虫平均发育历期都显著长于敏感品系,成虫寿命显著比敏感品系短,存活率与单雌产卵量也显著下降。用种群趋势指数（I）来确定抗性品系的相对适合度发现,T₃₃代抗性品系的适合度（0.53）下降到敏感品系的1/2。因此推断,抗性适合度的下降是生产上B型烟粉虱对噻虫嗪抗性发展缓慢的原因之一。此外,对T₃₃代进行了形态学研究,结果表明抗性品系烟粉虱形态上发生了一定改变。与敏感品系相比,抗性品系的第一、二、三龄若虫体长显著短于敏感品系,第一、四龄的体宽也显著小于敏感品系。而两品系雌、雄成虫体长之间没有差异。三、B型烟粉虱抗噻虫嗪的生化机理分析以敏感品系和噻虫嗪抗性筛选过程中代表性世代烟粉虱（25.6倍与66.3倍）为试虫,通过增效实验,交互抗性测定、代谢解毒酶活力分析,就B型烟粉虱对噻虫嗪的抗性生化机理进行了分析。测定了敏感和抗性烟粉虱的羧酸酯酶（CarE）、谷光甘肽-S-转移酶（GST）和多功能氧化酶对硝基苯甲醚-O-脱甲基（PNOD）及7-乙氧香豆素-O-脱乙基（ECOD）活力。结果表明,在两个抗性水平下,与敏感品系相比,抗性品系PNOD活性分别升高了1.21和3.23倍,ECOD活性分别升高了1.68和2.30倍,表明多功能氧化酶活性增强是产生抗性的重要原因;羧酸酯酶只在低水平抗性时升高了2.96倍,表明其只在较低抗性水平时发挥作用;而谷光甘肽-S-转移酶与抗性形成关系不大,该结果与增效实验结果一致。交互抗性研究结果表明抗性品系对与噻虫嗪同为新烟碱的吡虫啉、啶虫眯以及烯啶虫胺均表现出交互抗性,交互抗性倍数分别为47.28、35.82及9.99倍;对阿维菌素和丁硫克百威分别表现出5.33和4.43倍的交互抗性;而对氟虫腈、溴氰菊酯、毒列蜱未表现出交互抗性,揭示了噻虫嗪抗性可能存在靶标不敏感性。四、B型与ZHJ-1型烟粉虱生物学、形态学及解毒酶活力比较通过构建种群生命表,比较了B型敏感（棉花）品系与ZHJ-1型品系烟粉虱一系列生长发育与繁殖特征。结果表明,ZHJ-1型品系的卵、各龄若虫平均发育历期都显著比B型敏感品系长,成虫寿命显著比B型敏感品系短,单雌产卵量显著比敏感品系少,存活率也显著下降。各种生物学参数分析表明B型的内禀增长率、净增殖率等都大于ZHJ-1型,表现了强劲的增殖潜力。形态学研究表明,ZHJ-1型烟粉虱一龄到三龄若虫的体长、体宽都显著长于B型,而两品系四龄若虫的体长、体宽差异不显著。比较了B型敏感（棉花）品系与ZHJ-1品系烟粉虱羧酸酯酶、谷光甘肽-S-转移酶和多功能氧化酶对硝基苯甲醚-O-脱甲基（PNOD）的活性。结果表明两生物型之间3大解毒酶活性没有差异。五、B型烟粉虱烟碱型乙酰胆碱受体亚基的克隆采用RT-PCR技术克隆了B型烟粉虱乙酰胆碱受体（nAChR）α亚基全长基因。克隆的烟粉虱敏感品系的nAChRα亚基包含了1935个核苷酸,其开放阅读框的（ORF）的起点是第257位核苷酸,终点是第1861位核苷酸,长度为1605bp。开放阅读框编码534个氨基酸nAChR亚基前体,它包含了nAChR家族的一些典型结构:α亚基具有的保守的两个相邻的半胱氨酸,以及4个跨膜结构域等。该基因与其它昆虫nAChRs亚基具有很高同源性,最高达79%,说明克隆的亚基是烟粉虱的乙酰胆碱受体亚基基因。该基因序列Genebank登录号为:DQ480159。B型敏感与抗性品系的α亚基全长基因蛋白经比对表明,未发现突变位点,推测由于筛选品系中抗性杂合子仍占很大比例。六、噻虫嗪对B型烟粉虱乙酰胆碱受体α亚基mRNA表达的影响利用荧光定量PCR技术（Q-PCR）对B型烟粉虱乙酰胆碱受体（nAChRs）α亚基转录水平的表达进行了定量分析。以烟粉虱β-actin为内参基因,结果显示,相对于敏感品系TH-S,抗性噻虫嗪品系TH-R的nAChRsα亚基的表达量上调,其相对表达量是敏感品系的3.6倍,但方差分析筹异不显著。原因及其意义,尚有待进一步研究验证。综上所述,本研究首次评价了B型烟粉虱对噻虫嗪的抗性风险。对抗性品系进行了适合度研究,首次发现了B型烟粉虱对噻虫嗪产生抗性后会表现一系列生长、生殖劣势。代谢抗性解毒酶活性测定与增效实验相结合,揭示了抗性主要是多功能氧化酶在起作用。交互抗性的研究揭示了噻虫嗪抗性可能存在靶标不敏感性,同时为田间抗性治理提供了一定的理论基础。成功获得了受体亚基全长基因序列,但敏感和抗性品系的全长没有差异,进一步荧光定量PCR研究结果表明噻虫嗪抗性会引起nAChRsα亚基的表达量上调,为研究B型烟粉虱乙酰胆碱受体基因的功能奠定了基础。更多还原

【Abstract】 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.更多还原