四种Bt毒素蛋白对二点委夜蛾幼虫毒力测定及中肠组织病理学变化
|
摘要
为更好地了解苏云金芽胞杆菌Bacillus thuringiensis毒素蛋白对二点委夜蛾Athetis lepigone的毒力以及作用机理,通过饲喂含有Cry1Ac、Cry1Ab、Cry2Ab和Vip3Aa四种不同Bt毒素蛋白饲料,测定Bt毒素蛋白对二点委夜蛾幼虫的毒力,并观察取食4种毒素蛋白后幼虫中肠组织的病理学变化。结果显示,二点委夜蛾幼虫取食毒素蛋白后72 h,Cry1Ab和Cry1Ac毒素蛋白对二点委夜蛾幼虫的杀虫活性较高,校正死亡率为84.7%和76.4%;Vip3Aa和Cry2Ab毒素蛋白的毒力较弱。二点委夜蛾幼虫取食4种Bt毒素蛋白后,中肠柱状细胞微绒毛脱落,杂乱地分散在肠腔内,杯状细胞变形和腔内微绒毛脱落,线粒体和内质网等变形破裂,细胞核的核膜消失、核质凝聚和形状发生变化,经Cry1Ab和Cry1Ac毒素蛋白处理后中肠细胞的病变症状和速度明显高于Cry2Ab和Vip3Aa毒素蛋白处理。表明Cry1Ab和Cry1Ac毒素蛋白对二点委夜蛾幼虫杀虫活性较高,显著高于Cry2Ab和Vip3Aa毒素蛋白,且对其中肠细胞的破坏作用也较强。
In order to examine the action mechanism of Bacillus thuringiensis toxin against Athetis lepigone larvae, A. lepigone larvae were fed on Cry1Ac, Cry1Ab, Cry2Ab and Vip3A toxins, and the toxicity of Bt toxin proteins to A. lepigone larvae was detected, and the cellular morphological change in the midgut of larvae was observed by transmission electron microscope(TEM). The results showed that Cry1Ab and Cry1Ac toxins had higher toxicity against the larvae of A. lepigone than Cry2Ab and Vip3Aa toxins, and the corrected mortality were 84.7% and 76.4%, respectively. Obvious histopathological changes were observed in the midgut cells after the larvae were fed on Bt toxins. Microvillis of column cells and goblet cells dropped off. Mitochondria and endoplasmic reticulum were dissolved and mixed disorderly. Wiredraw of nucleus, condensation of nuclear, and disappearance of nuclear membrane were also observed. The pathological changes of column cells and goblet cells in the midgut of A.lepigone larvae induced by Vip3Aa and Cry2Ab were slower than that by Cry1Ab and Cry1Ac. The results indicated that Cry1Ab and Cry1Ac toxins had higher toxicity against the larvae of A. lepigone than Vip3 Aa and Cry2Ab toxins. Similar to the changes of histopathology, Cry1Ab and Cry1Ac toxins disrupted the midgut epithelium cells faster than Vip3Aa and Cry2Ab toxins. Lysis of gut epithelium cells resulting in larval death might be the mode of action of Cry toxin and Vip toxin.
In order to examine the action mechanism of Bacillus thuringiensis toxin against Athetis lepigone larvae, A. lepigone larvae were fed on Cry1Ac, Cry1Ab, Cry2Ab and Vip3A toxins, and the toxicity of Bt toxin proteins to A. lepigone larvae was detected, and the cellular morphological change in the midgut of larvae was observed by transmission electron microscope(TEM). The results showed that Cry1Ab and Cry1Ac toxins had higher toxicity against the larvae of A. lepigone than Cry2Ab and Vip3Aa toxins, and the corrected mortality were 84.7% and 76.4%, respectively. Obvious histopathological changes were observed in the midgut cells after the larvae were fed on Bt toxins. Microvillis of column cells and goblet cells dropped off. Mitochondria and endoplasmic reticulum were dissolved and mixed disorderly. Wiredraw of nucleus, condensation of nuclear, and disappearance of nuclear membrane were also observed. The pathological changes of column cells and goblet cells in the midgut of A.lepigone larvae induced by Vip3Aa and Cry2Ab were slower than that by Cry1Ab and Cry1Ac. The results indicated that Cry1Ab and Cry1Ac toxins had higher toxicity against the larvae of A. lepigone than Vip3 Aa and Cry2Ab toxins. Similar to the changes of histopathology, Cry1Ab and Cry1Ac toxins disrupted the midgut epithelium cells faster than Vip3Aa and Cry2Ab toxins. Lysis of gut epithelium cells resulting in larval death might be the mode of action of Cry toxin and Vip toxin.
引文
Bravo A,Gill SS,Soberón M.2007.Mode of action of Bacillus thuringiensis Cry and Cyt toxins and their potential for insect control.Toxicon,49(4):423-435
Bravo A,Likitvivatanavong S,Gill SS,Soberón M.2011.Bacillus thuringiensis:a story of a successful bioinsecticide.Insect Biochemistry and Molecular Biology,41(7):423-431
Burkness EC,Cira TM,Moser SE,Hutchison WD.2015.Bt maize seed mixtures for Helicoverpa zea(Lepidoptera:Noctuidae):larval movement,development,and survival on non-transgenic maize.Journal of Economic Entomology,108(6):2761-2769
Chai TH,Mei CB,Zhai H,Huo LQ,Guo LW,Liu KS,Liang JH,Ma JF.2012.Chemical control methods of Athetis lepigone.Plant Protection,38(2):167-170(in Chinese)[柴同海,梅成彬,翟晖,霍立强,郭丽伟,刘奎胜,梁建辉,马继芳.2012.二点委夜蛾化学防治方法研究.植物保护,38(2):167-170]
Crespo AL,Spencer TA,Tan SY,Siegfried BD.2010.Fitness costs of Cry1Ab resistance in a field-derived strain of Ostrinia nubilalis(Lepidoptera:Crambidae).Journal of Economic Entomology,103(4):1386-1393
Estruch JJ,Warren GW,Mullins MA,Nye GJ,Craig JA,Koziel MG.1996.Vip3A,a novel Bacillus thuringiensis vegetative insecticidal protein with a wide spectrum of activities against lepidopteran insects.Proceedings of the National Academy of Sciences of the United States of America,93(11):5389-5394
Gao Z,Liang GM,Wei JZ,Zhang LL.2013.Pathological changes in larval midgut tissues of the cotton bollworm,Helicoverpa armigera(Lepidoptera:Noctuidae),after feeding Cry2Ab.Acta Entomologica Sinica,56(8):934-944(in chinese)[高珍,梁革梅,魏纪珍,张丽丽.2013.取食Cry2Ab蛋白后棉铃虫幼虫中肠组织的病理变化.昆虫学报,56(8):934-944]
Gill SS,Cowles EA,Pietrantonio PV.1992.The mode of action of Bacillus thuringiensis endotoxins.Annual Review of Entomology,37:615-636
H?fte H,Whiteley HR.1989.Insecticidal crystal proteins of Bacillus thuringiensis.Microbiology Reviews,53(2):242-255
Jackson RE,Marcus MA,Gould F,Bradley JR,van Duyn JW.2007.Cross-resistance responses of Cry1Ac-selected Heliothis virescens(Lepidoptera:Noctuidae)to the Bacillus thuringiensis protein Vip3A.Journal of Economic Entomology,100:180-186
Jakka SR,Gong L,Hasler J,Banerjee R,Sheets JJ,Narva K,Blanco CA,Jurat-Fuentes JL.2015.Field-evolved mode 1 resistance of the fall armyworm to transgenic Cry1Fa-expressing corn associated with reduced Cry1Fa toxin binding and midgut alkaline phosphatase expression.Applied and Environmental Microbiology,82(4):1023-1034
Jiang JY,Li XQ,Xu YH,Li ZH,Zhang ZY,Xu H.2008.Preliminary studies on Athetis(Proxenus)lepigone.Plant Protection,34(3):123-126(in Chinese)[姜京宇,李秀芹,许佑辉,李智慧,张志英,许昊.2008.二点委夜蛾研究初报.植物保护,34(3):123-126]
Lei LP,Nangong ZY,Li LT,Dong ZP,Wang QY.2013.Efficacy of entomopathogenic nematodes against Athetis lepigone.Journal of Environmental Entomology,35(2):171-175(in Chinese)[雷利平,南宫自艳,李立涛,董志平,王勤英.2013.昆虫病原线虫对二点委夜蛾致病力的研究.环境昆虫学报,35(2):171-175]
Lemes ARN,Davolos CC,Legori PCBC,Fernandes OA,FerréJ,Lemos MVF,Desiderio JA.2014.Synergism and antagonism between Bacillus thuringiensis Vip3A and Cry1 proteins in Heliothis virescens,Diatraea saccharalis and Spodoptera frugiperda.PLoSONE,9(10):e107196
Li FF,Ye GY,Wu Q,Peng YF,Chen XX.2007.Histopathological changes in the midgut of the rice leaf folder,Cnaphalocrocis medinalis(Lepidoptera:Pyralidae)larvae feeding on transgenic Bt rice.Acta Entomologica Sinica,50(10):1070-1076(in Chinese)[李芳芳,叶恭银,吴琼,彭予发,陈学新.2007.取食转Bt基因水稻对稻纵卷叶螟幼虫中肠的组织病理学效应.昆虫学报,50(10):1070-1076]
Li N,He KL,Cui L,Wang ZY.2011.Environmental safety of genetically modified insect resistant maize and future perspectives for implementation in China.Plant Protection,37(6):18-26(in Chinese)[李宁,何康来,崔蕾,王振营.2011.转基因抗虫玉米环境安全性及我国应用前景.植物保护,37(6):18-26]
McNeil BC,Dean DH.2011.Bacillus thuringiensis Cry2Ab is active on Anopheles mosquitoes:single D block exchanges reveal critical residues involved in activity.FEMS Microbiology Letters,325(1):16-21
Pan Z,Xu L,Zhu Y,Shi H,Chen Z,Chen M,Chen Q,Liu B.2014.Characterization of a new cry2Ab gene of Bacillus thuringiensis with high insecticidal activity against Plutella xylostella L.World Journal of Microbiology and Biotechnology,30(10):2655-2662
Siebert MW,Nolting SP,Hendrix W,Dhavala S,Craig C,Leonard BR,Stewart SD,All J,Musser FR,Buntin GD,et al.2012.Evaluation of corn hybrids expressing Cry1F,CRy1A105,Cry2Ab2,Cry34Ab1/Cry35Ab1,and Cry3Bb1 against southern United States insect pests.Journal of Economic Entomology,105(5):1825-1834
Song F,Lin Y,Chen C,Shao E,Guan X,Huang Z.2016.Insecticidal activity and histopathological effects of Vip3Aa protein from Bacillus thuringiensis on Spodoptera litura.Journal of Microbiology and Biotechnology,26(10):1774-1780
Sousa MEC,Santos FAB,Wanderley-Teixeira V,Teixeira AAC,de Siqueira Há,Alves LC,Torres JB.2010.Histopathology and ultrastructure of midgut of Alabama argillacea(Hübner)(Lepidoptera:Noctuidae)fed Bt-cotton.Journal of Insect Physiology,56(12):1913-1919
Székács A,Lauber E,Juracsek J,Darvas B.2010.Cry1Ab toxin production of MON 810 transgenic maize.Environmental Toxicology and Chemistry,29(1):182-190
Wang ZY,Shi J,Dong JG.2012.Reason analysis on Proxenus lepigone outbreak of summer corn region in the Yellow River,Huai and Hai Rivers plain and the countermeasures suggested.Journal of Maize Science,20(1):132-134(in Chinese)[王振营,石洁,董金皋.2012.2011年黄淮海夏玉米区二点委夜蛾爆发危害的原因与防治对策.玉米科学,20(1):132-134]
Xu YL,Wang ZY,He KL,Bai SX.2009.Histopathological changes in the midgut of larvae of the Asian corn borer,Ostrinia furnacalis(Lepidoptera:Crambidae),fed on Bt-transgenic corn expressing Cry1Ab protein.Acta Entomologica Sinica,52(9):1034-1038(in Chinese)[徐艳聆,王振营,何康来,白树雄.2009.取食转Bt基因抗虫玉米后亚洲玉米螟幼虫中肠的组织病理变化.昆虫学报,52(9):1034-1038]
Yang YH,Song P,Wang ZY,Dong ZP,Wang QY.2014.Selection and genotype analysis of Bt isolates with high virulence against Athetis lepigone(M?schler).Chinese Journal of Applied Entomology,51(3):630-635(in Chinese)[杨云鹤,宋萍,王振营,董志平,王勤英.2014.对二点委夜蛾高毒力苏云金芽胞杆菌的筛选及其基因型分析.应用昆虫学报,51(3):630-635]
Zhang B,Yang Y,Zhou X,Shen P,Peng Y,Li Y.2017.A laboratory assessment of the potential effect of Cry1Ab/Cry2Aj-containing Bt maize pollen on Folsomia candida by toxicological and biochemical analyses.Environmental Pollution,222:94-100
Zhang HJ,Shi J,Guo N,Wang ZY.2012.Primary study on screening of high virulent strains and biological characteristics of Beauveria bassiana against Athetis lepigone(M?schler)(Lepidoptera:Noctuidae).Chinese Journal of Biological Control,28(3):439-443(in Chinese)[张海剑,石洁,郭宁,王振营.2012.二点委夜蛾幼虫高毒力球孢白僵菌菌株筛选与生物学特性初步研究.中国生物防治学报,28(3):439-443]
Zhang HJ,Song J,Du LX,Yang YH,Shi J.2016.Control efficacy of one microsporidian against Athetis lepigone larvae.Chinese Journal of Biological Control,32(4):462-467(in Chinese)[张海剑,宋健,杜立新,杨云鹤,石洁.2016.微孢子虫对二点委夜蛾致病力研究.中国生物防治学报,32(4):462-467]
Zhang Y,Zhang W,Liu Y,Wang JH,Wang GY,Liu YJ.2016.Development of monoclonal antibody-based sensitive ELISA for the determination of Cry1Ie protein in transgenic plant.Analytical and Bioanalytical Chemistry,408(28):8231-8239
Bravo A,Likitvivatanavong S,Gill SS,Soberón M.2011.Bacillus thuringiensis:a story of a successful bioinsecticide.Insect Biochemistry and Molecular Biology,41(7):423-431
Burkness EC,Cira TM,Moser SE,Hutchison WD.2015.Bt maize seed mixtures for Helicoverpa zea(Lepidoptera:Noctuidae):larval movement,development,and survival on non-transgenic maize.Journal of Economic Entomology,108(6):2761-2769
Chai TH,Mei CB,Zhai H,Huo LQ,Guo LW,Liu KS,Liang JH,Ma JF.2012.Chemical control methods of Athetis lepigone.Plant Protection,38(2):167-170(in Chinese)[柴同海,梅成彬,翟晖,霍立强,郭丽伟,刘奎胜,梁建辉,马继芳.2012.二点委夜蛾化学防治方法研究.植物保护,38(2):167-170]
Crespo AL,Spencer TA,Tan SY,Siegfried BD.2010.Fitness costs of Cry1Ab resistance in a field-derived strain of Ostrinia nubilalis(Lepidoptera:Crambidae).Journal of Economic Entomology,103(4):1386-1393
Estruch JJ,Warren GW,Mullins MA,Nye GJ,Craig JA,Koziel MG.1996.Vip3A,a novel Bacillus thuringiensis vegetative insecticidal protein with a wide spectrum of activities against lepidopteran insects.Proceedings of the National Academy of Sciences of the United States of America,93(11):5389-5394
Gao Z,Liang GM,Wei JZ,Zhang LL.2013.Pathological changes in larval midgut tissues of the cotton bollworm,Helicoverpa armigera(Lepidoptera:Noctuidae),after feeding Cry2Ab.Acta Entomologica Sinica,56(8):934-944(in chinese)[高珍,梁革梅,魏纪珍,张丽丽.2013.取食Cry2Ab蛋白后棉铃虫幼虫中肠组织的病理变化.昆虫学报,56(8):934-944]
Gill SS,Cowles EA,Pietrantonio PV.1992.The mode of action of Bacillus thuringiensis endotoxins.Annual Review of Entomology,37:615-636
H?fte H,Whiteley HR.1989.Insecticidal crystal proteins of Bacillus thuringiensis.Microbiology Reviews,53(2):242-255
Jackson RE,Marcus MA,Gould F,Bradley JR,van Duyn JW.2007.Cross-resistance responses of Cry1Ac-selected Heliothis virescens(Lepidoptera:Noctuidae)to the Bacillus thuringiensis protein Vip3A.Journal of Economic Entomology,100:180-186
Jakka SR,Gong L,Hasler J,Banerjee R,Sheets JJ,Narva K,Blanco CA,Jurat-Fuentes JL.2015.Field-evolved mode 1 resistance of the fall armyworm to transgenic Cry1Fa-expressing corn associated with reduced Cry1Fa toxin binding and midgut alkaline phosphatase expression.Applied and Environmental Microbiology,82(4):1023-1034
Jiang JY,Li XQ,Xu YH,Li ZH,Zhang ZY,Xu H.2008.Preliminary studies on Athetis(Proxenus)lepigone.Plant Protection,34(3):123-126(in Chinese)[姜京宇,李秀芹,许佑辉,李智慧,张志英,许昊.2008.二点委夜蛾研究初报.植物保护,34(3):123-126]
Lei LP,Nangong ZY,Li LT,Dong ZP,Wang QY.2013.Efficacy of entomopathogenic nematodes against Athetis lepigone.Journal of Environmental Entomology,35(2):171-175(in Chinese)[雷利平,南宫自艳,李立涛,董志平,王勤英.2013.昆虫病原线虫对二点委夜蛾致病力的研究.环境昆虫学报,35(2):171-175]
Lemes ARN,Davolos CC,Legori PCBC,Fernandes OA,FerréJ,Lemos MVF,Desiderio JA.2014.Synergism and antagonism between Bacillus thuringiensis Vip3A and Cry1 proteins in Heliothis virescens,Diatraea saccharalis and Spodoptera frugiperda.PLoSONE,9(10):e107196
Li FF,Ye GY,Wu Q,Peng YF,Chen XX.2007.Histopathological changes in the midgut of the rice leaf folder,Cnaphalocrocis medinalis(Lepidoptera:Pyralidae)larvae feeding on transgenic Bt rice.Acta Entomologica Sinica,50(10):1070-1076(in Chinese)[李芳芳,叶恭银,吴琼,彭予发,陈学新.2007.取食转Bt基因水稻对稻纵卷叶螟幼虫中肠的组织病理学效应.昆虫学报,50(10):1070-1076]
Li N,He KL,Cui L,Wang ZY.2011.Environmental safety of genetically modified insect resistant maize and future perspectives for implementation in China.Plant Protection,37(6):18-26(in Chinese)[李宁,何康来,崔蕾,王振营.2011.转基因抗虫玉米环境安全性及我国应用前景.植物保护,37(6):18-26]
McNeil BC,Dean DH.2011.Bacillus thuringiensis Cry2Ab is active on Anopheles mosquitoes:single D block exchanges reveal critical residues involved in activity.FEMS Microbiology Letters,325(1):16-21
Pan Z,Xu L,Zhu Y,Shi H,Chen Z,Chen M,Chen Q,Liu B.2014.Characterization of a new cry2Ab gene of Bacillus thuringiensis with high insecticidal activity against Plutella xylostella L.World Journal of Microbiology and Biotechnology,30(10):2655-2662
Siebert MW,Nolting SP,Hendrix W,Dhavala S,Craig C,Leonard BR,Stewart SD,All J,Musser FR,Buntin GD,et al.2012.Evaluation of corn hybrids expressing Cry1F,CRy1A105,Cry2Ab2,Cry34Ab1/Cry35Ab1,and Cry3Bb1 against southern United States insect pests.Journal of Economic Entomology,105(5):1825-1834
Song F,Lin Y,Chen C,Shao E,Guan X,Huang Z.2016.Insecticidal activity and histopathological effects of Vip3Aa protein from Bacillus thuringiensis on Spodoptera litura.Journal of Microbiology and Biotechnology,26(10):1774-1780
Sousa MEC,Santos FAB,Wanderley-Teixeira V,Teixeira AAC,de Siqueira Há,Alves LC,Torres JB.2010.Histopathology and ultrastructure of midgut of Alabama argillacea(Hübner)(Lepidoptera:Noctuidae)fed Bt-cotton.Journal of Insect Physiology,56(12):1913-1919
Székács A,Lauber E,Juracsek J,Darvas B.2010.Cry1Ab toxin production of MON 810 transgenic maize.Environmental Toxicology and Chemistry,29(1):182-190
Wang ZY,Shi J,Dong JG.2012.Reason analysis on Proxenus lepigone outbreak of summer corn region in the Yellow River,Huai and Hai Rivers plain and the countermeasures suggested.Journal of Maize Science,20(1):132-134(in Chinese)[王振营,石洁,董金皋.2012.2011年黄淮海夏玉米区二点委夜蛾爆发危害的原因与防治对策.玉米科学,20(1):132-134]
Xu YL,Wang ZY,He KL,Bai SX.2009.Histopathological changes in the midgut of larvae of the Asian corn borer,Ostrinia furnacalis(Lepidoptera:Crambidae),fed on Bt-transgenic corn expressing Cry1Ab protein.Acta Entomologica Sinica,52(9):1034-1038(in Chinese)[徐艳聆,王振营,何康来,白树雄.2009.取食转Bt基因抗虫玉米后亚洲玉米螟幼虫中肠的组织病理变化.昆虫学报,52(9):1034-1038]
Yang YH,Song P,Wang ZY,Dong ZP,Wang QY.2014.Selection and genotype analysis of Bt isolates with high virulence against Athetis lepigone(M?schler).Chinese Journal of Applied Entomology,51(3):630-635(in Chinese)[杨云鹤,宋萍,王振营,董志平,王勤英.2014.对二点委夜蛾高毒力苏云金芽胞杆菌的筛选及其基因型分析.应用昆虫学报,51(3):630-635]
Zhang B,Yang Y,Zhou X,Shen P,Peng Y,Li Y.2017.A laboratory assessment of the potential effect of Cry1Ab/Cry2Aj-containing Bt maize pollen on Folsomia candida by toxicological and biochemical analyses.Environmental Pollution,222:94-100
Zhang HJ,Shi J,Guo N,Wang ZY.2012.Primary study on screening of high virulent strains and biological characteristics of Beauveria bassiana against Athetis lepigone(M?schler)(Lepidoptera:Noctuidae).Chinese Journal of Biological Control,28(3):439-443(in Chinese)[张海剑,石洁,郭宁,王振营.2012.二点委夜蛾幼虫高毒力球孢白僵菌菌株筛选与生物学特性初步研究.中国生物防治学报,28(3):439-443]
Zhang HJ,Song J,Du LX,Yang YH,Shi J.2016.Control efficacy of one microsporidian against Athetis lepigone larvae.Chinese Journal of Biological Control,32(4):462-467(in Chinese)[张海剑,宋健,杜立新,杨云鹤,石洁.2016.微孢子虫对二点委夜蛾致病力研究.中国生物防治学报,32(4):462-467]
Zhang Y,Zhang W,Liu Y,Wang JH,Wang GY,Liu YJ.2016.Development of monoclonal antibody-based sensitive ELISA for the determination of Cry1Ie protein in transgenic plant.Analytical and Bioanalytical Chemistry,408(28):8231-8239