印楝素对中华稻蝗血细胞的毒性影响
|
摘要
【目的】近年来,农药的长期使用使害虫产生抗性,同时造成了环境污染,目前多数研究侧重于化学农药对昆虫酶活性的影响,而植物源农药对昆虫的免疫毒理研究较少。【方法】通过给中华稻蝗Oxya chinensis(Thunberg)注射不同浓度的印楝素,用Wright-Giemsa染液染色和光学显微镜观察感染后血细胞的形态,对各种类型细胞进行数量统计;酶联免疫测定SOD,POD,PO,AchE,P450氧化酶和CES的活性变化。【结果】实验组血细胞发生固缩、空泡、脱粒和细胞变形等现象;与空白对照组相比,感染后不同时间段浆血胞的数量先升高后降低,粒血胞的数量先降低后升高,感染后期囊血胞数量显著增加;与溶剂组相比,60μg/L和120μg/L的SOD活性被极显著(P<0.01)激活;60μg/L和120μg/L的POD活性在注射后6-24 h间被显著(P<0.05)激活;120μg/L的PO活性在6-24 h被极显著(P<0.01)激活;实验组中AchE活性均在10-24 h被极显著(P<0.01)激活;30μg/L的CES活性被极显著(P<0.01)激活;P450活性均被激活,但激活效果不明显。【结论】研究表明:植物源农药使稻蝗血细胞的形态和数量发生显著变化,血细胞中相关酶活性的变化情况可推测出稻蝗对印楝素的毒害产生免疫反应,进一步探讨了稻蝗的免疫机理。本研究不仅为稻蝗的预防和治理提供了理论依据,也为昆虫的免疫毒理学提供了参考。
[Objectives] Long-term use of pesticides has selected for pesticide resistance in pests and environmental pollution. Currently most studies focus on the effects of chemical pesticides on insect enzymes and there have been relatively little research on the immunotoxicity of plant-derived pesticides to insects. [Methods] Different concentrations of azadirachtin were injected into Oxya chinensis(Thunberg) and hemocyte morphology and the number of various cell types were observed at different times under an optical microscope after Wright-Giemsa dyeing. An enzyme linked immunosorbent assay(ELISA) was used to detect the activity of SOD, POD, PO, AchE, P450 oxidase and CarE. [Results] The hemocytes of treatment groups showed signs of contracting, vacuole development, threshing and cell deformation. Compared to the control group, the number of plasma cells at different times after injection first increased then decreased, and the number of granulocytes first decreased then increased. The number of hemocytes dramatically increased. SOD and POD activity of the 60 μg/L and 120 μg/L treatment groups were significantly(P<0.01 and P<0.05, respectively) higher than those of the control group between 6 h and 24 h. PO activity of the 120 μg/L treatment group was also significantly(P<0.01) higher than that of the control group over the same time period and AchE activity in all treatment groups was significantly(P<0.01) higher than that in the control group between 10 h and 24 h. CES activity in the 30 μg/L treatment group was also significantly(P<0.01) higher than that of the control group. Although P450 activity was also higher, it was not significantly different to that in the control.[Conclusion] Treatment with plant-derived pesticides changed the morphology, and quantity, of hemocytes in O. chinensis.Changes in related enzymes activity in hemocytes could be used to infer the immune response of O. chinensis to azadirachtin,and further explore the immune response mechanisms of this species. These results not only provide a theoretical basis for the prevention and control of O. chinensis, but also general a reference for insect immunotoxicology.
[Objectives] Long-term use of pesticides has selected for pesticide resistance in pests and environmental pollution. Currently most studies focus on the effects of chemical pesticides on insect enzymes and there have been relatively little research on the immunotoxicity of plant-derived pesticides to insects. [Methods] Different concentrations of azadirachtin were injected into Oxya chinensis(Thunberg) and hemocyte morphology and the number of various cell types were observed at different times under an optical microscope after Wright-Giemsa dyeing. An enzyme linked immunosorbent assay(ELISA) was used to detect the activity of SOD, POD, PO, AchE, P450 oxidase and CarE. [Results] The hemocytes of treatment groups showed signs of contracting, vacuole development, threshing and cell deformation. Compared to the control group, the number of plasma cells at different times after injection first increased then decreased, and the number of granulocytes first decreased then increased. The number of hemocytes dramatically increased. SOD and POD activity of the 60 μg/L and 120 μg/L treatment groups were significantly(P<0.01 and P<0.05, respectively) higher than those of the control group between 6 h and 24 h. PO activity of the 120 μg/L treatment group was also significantly(P<0.01) higher than that of the control group over the same time period and AchE activity in all treatment groups was significantly(P<0.01) higher than that in the control group between 10 h and 24 h. CES activity in the 30 μg/L treatment group was also significantly(P<0.01) higher than that of the control group. Although P450 activity was also higher, it was not significantly different to that in the control.[Conclusion] Treatment with plant-derived pesticides changed the morphology, and quantity, of hemocytes in O. chinensis.Changes in related enzymes activity in hemocytes could be used to infer the immune response of O. chinensis to azadirachtin,and further explore the immune response mechanisms of this species. These results not only provide a theoretical basis for the prevention and control of O. chinensis, but also general a reference for insect immunotoxicology.
引文
Brehelin M,Hoffmann JA,Matz G,Porte A,1975.Encapsulation of implanted foreign bodies by hemocytes in Locusta migratoria and Melolontha melolontha.Cell Tiss.Res.,160(3):283-289.
Cheung PYK,Grula EA,Burton RL,1978.Hemolymph responses in Heliothiszea to inoculation with Bacillus thuringiensis or Micrococcus Iysodeikticus.J.Invertebr.Pathol.,31(2):148-156.
CJ,2018.β-1,3-Glucan recognition protein 3 activates the prophenoloxidase system in response to bacterial infection in Ostrinia furnacalis Guenee.Dev.Comp.Immunol.,79(2):31-43.
Cha LC,Hu CX,Cao CW,2011.Effect of three pesticides on superoxide dismutase activity of caterpillars,J.Dongbei Fore.Univ.,39(5):105-107.[查黎春,胡春祥,曹传旺,2011.三种农药对天幕毛虫超氧化物歧化酶活性的影响.东北林业大学学报,39(5):105-107.]
Fang GF,2011.Toxicity of three pesticides and their effects on carboxylesterase activity of Propsilocerus akamusi.Acta Ecologica Sinica,31(17):4914-4918.[方国飞,2011.三种农药对红裸须摇蚊毒力和羧酸酯酶活性的影响.生态学报,31(17):4914-4918.]
Gupta AP,1929.Insect Hemocytes:Developent,Forms,Functions and Technigues.Cambridge:Cambridge Univ.Press.6-14.
Gillespie JP,Burnett C,Charnley AK,2000.The immune response of the desert locust Schistocerca gregaria during mycosis of the entomopathogenic fungus,Metarhizium anisopliae var acridum.J.Insect Physiol.,46(4):429-437.
Gao ZX,2004.Azadirachtin bioactivity,mechanism of action,and application techniques of rice stem borer.Master’s dissertation.Yangzhou:Yangzhou University.[高振兴,2004.印楝素对水稻二化螟的生物活性、作用机制和应用技术研究.硕士学位论文.扬州:扬州大学.]
Gao SJ,Liu AP,Xu LB,Cao YX,Te ME,Cui ZL,2010.Experiment on tne efficacy of azadirachtin and abamectin+b.t.for controlling grasshoppers.Modern Agrochemicals,9(2):44-46.[高书晶,刘爱萍,徐林波,曹艺潇,特木儿,崔志玲,2010.印楝素和阿维苏云金对草原蝗虫的防治效果试验.现代农药,9(2):44-46.]
Heather MC,Killian KA,2015.Response of the insect immune system to three different immune challenges.J.Insect Physiol.,81(10):97-108.
Hou J,Ma ZQ,Feng JT,Zhang X,2007.Bioactivity of podophyllotoxin against Plutella xylostella and its effect on metabolic enzymes.Acta Entomol.Sin.,50(9):895-899.[侯军,马志卿,冯俊涛,张兴,2007.鬼臼毒素对小菜蛾的生物活性及对其几种代谢酶系的影响.昆虫学报,50(9):895-899.]
Julián FH,2016.Insect immunology and hematopoiesis.Dev.Comp.Immunol.,58(5):102-118.
Jamileh A,Javad K,Mohammad G,Mahbubeh S,Ahmad A,2017.Physiological defence of the white grub,Polyhylla adspersa Motschulsky(Col.,Scarabaeidae)against entomopathogenic nematodes.Journal of Asia-Pacific Entomology,20(3):878-885.
Lavine MD,Strand MR,2002.Insect haemocytes and their role in immunity.Insect Biochem.Mol.Biol.,32(10):1295-1309.
Li Q,Zheng WY,Wang ZG,Yu Q,Chen JX,2003.Study on effect of perioxidase in Porphyra haitanensis exposed to organophosphorus pesticides.J.Xiamen Univ.,42(2):201-204.[李钦,郑微云,王重刚,余群,陈纪新,2003.有机磷农药对坛紫菜过氧化物酶活性影响的研究.厦门大学学报,42(2):201-204.]
Li ZZ,Shen HJ,Jiang QG,Ji BZ,1994.Study on the activity of protective enzyme system in several insects.Acta Entomol.Sin.,37(4):399-403.[李周直,沈惠娟,蒋巧根,嵇保中,1994.几种昆虫体内保护酶系统活力的研究.昆虫学报,37(4):399-403.]
Marmaras VJ,Lampropoulou M,2009.Regulators and signalling in insect haemocyte immunity.Cell Signal.,21(2):186-195.
Mohammed SA,Siddiqui MI,2012.Circulating haemocytes in insects:Phylogenic review of their types.Pakistan J.Zool.,44(6):1743-1750.
Mi Z,Ruan CL,Li JR,Fu QJ,Du WH,Li GN,Long YH,Zhu Y,2013.Influence of fluoride on activity of carboxylesterase and esterase in hemolymph of Bombyx mori.Chin.J.Ecology,33(4):1134-1141.[米智,阮成龙,李姣蓉,付巧娟,杜文华,李冠楠,隆耀航,朱勇,2013.氟化物对家蚕血液羧酸酯酶及全酯酶活性的影响.生态学报,33(4):1134-1141.]
Mo F,Bao HE,2001.Observation on hemocytes of culex pipiens quinquefasciatus larvae infected by Lagenidium giganteum.Chin.J.Parasitol Parasit Dis.,19(6):330-332.[莫非,包怀恩,2001.感染大链壶菌后致倦库蚊幼虫血淋巴细胞的观察,中国寄生虫学与寄生虫病杂志,19(6):330-332.]
Ma ZQ,Feng JT,Guo ZB,Zhang X,2008.Effects of terpinen-4-ol on four kinds of metabolizing enzymes and polyphenol oxidase in Muscadomestica.J.Zhengjiang Univ.,34(5):509-515.[马志卿,冯俊涛,郭志波,张兴,2008.松油烯-4-醇对家蝇几种代谢酶及酚氧化酶的影响.浙江大学学报,34(5):509-515.]
Miranpuri GS,Khachatourians GG,1993.Hemocyte surface changes in the migratory grasshopper Melanoplus sanguinipes in response to wounding and infection with Beauveria bassiana.Entomol.Exp.Appl.,68(2):157-164.
Ribeiro C,Brehelin M,2006.Insect haemocytes:what type of cell is that?J.Insect Physiol.,52(5):417-429.
Ren CH,2014.The morphology study on hemocytes of Oxya chinensis.Master’s dissertation.Taiyuan:Shanxi University.[任彩红,2014.中华稻蝗血细胞形态学的研究.硕士学位论文.太原:山西大学.]
Shu YA,2008.Effects of several pesticides on hepatic cytochrome P450 in Curassius auratus.Master’s dissertation.Wulumuqi:Xinjiang Agricultural University.[舒耀皋,2008.几种农药对鲫鱼肝脏微粒体P450酶系的影响.硕士学位论文.乌鲁木齐:新疆农业大学.]
Strand MR,2008.The insect cellular immune response.Insect Sci.,15(1):1-14.
Tojo S,Naganuma F,Arakawa K,Yokoo S,2000.Involvement of both granular cells and plasmaocytes in phagocytic reactions in the greater wax moth Galleria mellonella.J.Insect Physiol.,46(7):1129-1135.
Wu TY,Zhao Y,Wang ZY,Song QS,Wang ZX,Xu QW,Wang YG,Wang LB,Zhang YQ,Feng CJ,2018.β-1,3-Glucan recognition protein 3 activates the prophenoloxidase system in response to bacterial infection in Ostrinia furnacalis Guenee.Dev.Comp.Immunol.,79(2):31-43.
Wang Y,2008.The effect of four pesticides on Paratrioza sinsca Yang et Li and the activity of acetylcholinesterase.Master’s dissertation.Huhehaote:Neimenggu Agriculture University.[王莹,2008.四种农药对枸杞木虱及其乙酰胆碱酯酶活性的影响.硕士学位论文.呼和浩特:内蒙古农业大学.]
Wang SD,2010.Study on the effects of four plant-derived compounds on phenoloxidase of Spodotera exigua(Hubner)and immunology of the enzyme.Doctor’s dissertation.Tai’an:Shandong Agriculture University.[王树栋,2010.四种植物源化合物对甜菜夜蛾酚氧化酶的影响及酶免疫学研究.博士学位论文.泰安:山东农业大学.]
Yan JG,Ru SG,Wang W,2006.On effects of monocrotophos on AchE,CaE,ALP and ACP activities of Monopterus albus.Joumal of Safety and Environment,6(3):61-63.[闫建国,汝少国,王蔚,2006.久效磷对黄鳝乙酰胆碱酯酶、羧酸酯酶和磷酸酶活性的影响.安全与环境学报,6(3):61-63.]
Yan R,Liu L,Liu H,He LF,2012.Study on hemocytes classification and immunologic function of Musca domestica larva.Sichuan J.Zool.,31(1):104-107.[晏容,刘流,刘晖,贺莉芳,2012.家蝇幼虫血细胞类型及免疫功能的初步研究.四川动物,31(1):104-107.]
Ying Y,Gao YQ,Xia,YX Liu FH,2016.Wright-Giemsa staining to observe phagocytes in Locusta migratoria infected with Metarhizium acridum.J.Invertebr.Pathol.,139(9):19-24.
Zhang KH,Qian XJ,Ji GH,Liu CZ,2012.Changes of the activites of protective enzymes in Pierisrapae infected by Heterorhabditis megidis.J.Gansu Agri.Univ.,47(2):58-61.[张奎花,钱秀娟,姬国红,刘长仲,2012.异小杆线虫对菜青虫体内保护酶活力的影响.甘肃农业大学学报,47(2):58-61.]
Cheung PYK,Grula EA,Burton RL,1978.Hemolymph responses in Heliothiszea to inoculation with Bacillus thuringiensis or Micrococcus Iysodeikticus.J.Invertebr.Pathol.,31(2):148-156.
CJ,2018.β-1,3-Glucan recognition protein 3 activates the prophenoloxidase system in response to bacterial infection in Ostrinia furnacalis Guenee.Dev.Comp.Immunol.,79(2):31-43.
Cha LC,Hu CX,Cao CW,2011.Effect of three pesticides on superoxide dismutase activity of caterpillars,J.Dongbei Fore.Univ.,39(5):105-107.[查黎春,胡春祥,曹传旺,2011.三种农药对天幕毛虫超氧化物歧化酶活性的影响.东北林业大学学报,39(5):105-107.]
Fang GF,2011.Toxicity of three pesticides and their effects on carboxylesterase activity of Propsilocerus akamusi.Acta Ecologica Sinica,31(17):4914-4918.[方国飞,2011.三种农药对红裸须摇蚊毒力和羧酸酯酶活性的影响.生态学报,31(17):4914-4918.]
Gupta AP,1929.Insect Hemocytes:Developent,Forms,Functions and Technigues.Cambridge:Cambridge Univ.Press.6-14.
Gillespie JP,Burnett C,Charnley AK,2000.The immune response of the desert locust Schistocerca gregaria during mycosis of the entomopathogenic fungus,Metarhizium anisopliae var acridum.J.Insect Physiol.,46(4):429-437.
Gao ZX,2004.Azadirachtin bioactivity,mechanism of action,and application techniques of rice stem borer.Master’s dissertation.Yangzhou:Yangzhou University.[高振兴,2004.印楝素对水稻二化螟的生物活性、作用机制和应用技术研究.硕士学位论文.扬州:扬州大学.]
Gao SJ,Liu AP,Xu LB,Cao YX,Te ME,Cui ZL,2010.Experiment on tne efficacy of azadirachtin and abamectin+b.t.for controlling grasshoppers.Modern Agrochemicals,9(2):44-46.[高书晶,刘爱萍,徐林波,曹艺潇,特木儿,崔志玲,2010.印楝素和阿维苏云金对草原蝗虫的防治效果试验.现代农药,9(2):44-46.]
Heather MC,Killian KA,2015.Response of the insect immune system to three different immune challenges.J.Insect Physiol.,81(10):97-108.
Hou J,Ma ZQ,Feng JT,Zhang X,2007.Bioactivity of podophyllotoxin against Plutella xylostella and its effect on metabolic enzymes.Acta Entomol.Sin.,50(9):895-899.[侯军,马志卿,冯俊涛,张兴,2007.鬼臼毒素对小菜蛾的生物活性及对其几种代谢酶系的影响.昆虫学报,50(9):895-899.]
Julián FH,2016.Insect immunology and hematopoiesis.Dev.Comp.Immunol.,58(5):102-118.
Jamileh A,Javad K,Mohammad G,Mahbubeh S,Ahmad A,2017.Physiological defence of the white grub,Polyhylla adspersa Motschulsky(Col.,Scarabaeidae)against entomopathogenic nematodes.Journal of Asia-Pacific Entomology,20(3):878-885.
Lavine MD,Strand MR,2002.Insect haemocytes and their role in immunity.Insect Biochem.Mol.Biol.,32(10):1295-1309.
Li Q,Zheng WY,Wang ZG,Yu Q,Chen JX,2003.Study on effect of perioxidase in Porphyra haitanensis exposed to organophosphorus pesticides.J.Xiamen Univ.,42(2):201-204.[李钦,郑微云,王重刚,余群,陈纪新,2003.有机磷农药对坛紫菜过氧化物酶活性影响的研究.厦门大学学报,42(2):201-204.]
Li ZZ,Shen HJ,Jiang QG,Ji BZ,1994.Study on the activity of protective enzyme system in several insects.Acta Entomol.Sin.,37(4):399-403.[李周直,沈惠娟,蒋巧根,嵇保中,1994.几种昆虫体内保护酶系统活力的研究.昆虫学报,37(4):399-403.]
Marmaras VJ,Lampropoulou M,2009.Regulators and signalling in insect haemocyte immunity.Cell Signal.,21(2):186-195.
Mohammed SA,Siddiqui MI,2012.Circulating haemocytes in insects:Phylogenic review of their types.Pakistan J.Zool.,44(6):1743-1750.
Mi Z,Ruan CL,Li JR,Fu QJ,Du WH,Li GN,Long YH,Zhu Y,2013.Influence of fluoride on activity of carboxylesterase and esterase in hemolymph of Bombyx mori.Chin.J.Ecology,33(4):1134-1141.[米智,阮成龙,李姣蓉,付巧娟,杜文华,李冠楠,隆耀航,朱勇,2013.氟化物对家蚕血液羧酸酯酶及全酯酶活性的影响.生态学报,33(4):1134-1141.]
Mo F,Bao HE,2001.Observation on hemocytes of culex pipiens quinquefasciatus larvae infected by Lagenidium giganteum.Chin.J.Parasitol Parasit Dis.,19(6):330-332.[莫非,包怀恩,2001.感染大链壶菌后致倦库蚊幼虫血淋巴细胞的观察,中国寄生虫学与寄生虫病杂志,19(6):330-332.]
Ma ZQ,Feng JT,Guo ZB,Zhang X,2008.Effects of terpinen-4-ol on four kinds of metabolizing enzymes and polyphenol oxidase in Muscadomestica.J.Zhengjiang Univ.,34(5):509-515.[马志卿,冯俊涛,郭志波,张兴,2008.松油烯-4-醇对家蝇几种代谢酶及酚氧化酶的影响.浙江大学学报,34(5):509-515.]
Miranpuri GS,Khachatourians GG,1993.Hemocyte surface changes in the migratory grasshopper Melanoplus sanguinipes in response to wounding and infection with Beauveria bassiana.Entomol.Exp.Appl.,68(2):157-164.
Ribeiro C,Brehelin M,2006.Insect haemocytes:what type of cell is that?J.Insect Physiol.,52(5):417-429.
Ren CH,2014.The morphology study on hemocytes of Oxya chinensis.Master’s dissertation.Taiyuan:Shanxi University.[任彩红,2014.中华稻蝗血细胞形态学的研究.硕士学位论文.太原:山西大学.]
Shu YA,2008.Effects of several pesticides on hepatic cytochrome P450 in Curassius auratus.Master’s dissertation.Wulumuqi:Xinjiang Agricultural University.[舒耀皋,2008.几种农药对鲫鱼肝脏微粒体P450酶系的影响.硕士学位论文.乌鲁木齐:新疆农业大学.]
Strand MR,2008.The insect cellular immune response.Insect Sci.,15(1):1-14.
Tojo S,Naganuma F,Arakawa K,Yokoo S,2000.Involvement of both granular cells and plasmaocytes in phagocytic reactions in the greater wax moth Galleria mellonella.J.Insect Physiol.,46(7):1129-1135.
Wu TY,Zhao Y,Wang ZY,Song QS,Wang ZX,Xu QW,Wang YG,Wang LB,Zhang YQ,Feng CJ,2018.β-1,3-Glucan recognition protein 3 activates the prophenoloxidase system in response to bacterial infection in Ostrinia furnacalis Guenee.Dev.Comp.Immunol.,79(2):31-43.
Wang Y,2008.The effect of four pesticides on Paratrioza sinsca Yang et Li and the activity of acetylcholinesterase.Master’s dissertation.Huhehaote:Neimenggu Agriculture University.[王莹,2008.四种农药对枸杞木虱及其乙酰胆碱酯酶活性的影响.硕士学位论文.呼和浩特:内蒙古农业大学.]
Wang SD,2010.Study on the effects of four plant-derived compounds on phenoloxidase of Spodotera exigua(Hubner)and immunology of the enzyme.Doctor’s dissertation.Tai’an:Shandong Agriculture University.[王树栋,2010.四种植物源化合物对甜菜夜蛾酚氧化酶的影响及酶免疫学研究.博士学位论文.泰安:山东农业大学.]
Yan JG,Ru SG,Wang W,2006.On effects of monocrotophos on AchE,CaE,ALP and ACP activities of Monopterus albus.Joumal of Safety and Environment,6(3):61-63.[闫建国,汝少国,王蔚,2006.久效磷对黄鳝乙酰胆碱酯酶、羧酸酯酶和磷酸酶活性的影响.安全与环境学报,6(3):61-63.]
Yan R,Liu L,Liu H,He LF,2012.Study on hemocytes classification and immunologic function of Musca domestica larva.Sichuan J.Zool.,31(1):104-107.[晏容,刘流,刘晖,贺莉芳,2012.家蝇幼虫血细胞类型及免疫功能的初步研究.四川动物,31(1):104-107.]
Ying Y,Gao YQ,Xia,YX Liu FH,2016.Wright-Giemsa staining to observe phagocytes in Locusta migratoria infected with Metarhizium acridum.J.Invertebr.Pathol.,139(9):19-24.
Zhang KH,Qian XJ,Ji GH,Liu CZ,2012.Changes of the activites of protective enzymes in Pierisrapae infected by Heterorhabditis megidis.J.Gansu Agri.Univ.,47(2):58-61.[张奎花,钱秀娟,姬国红,刘长仲,2012.异小杆线虫对菜青虫体内保护酶活力的影响.甘肃农业大学学报,47(2):58-61.]