颗粒体病毒对小菜蛾幼虫血淋巴蛋白、免疫系统、细胞微丝骨架及保护酶的影响
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摘要
小菜蛾是十字花科植物的世界性主要害虫之一,严重影响了十字花科蔬菜的产量与品质,因其对多种化学杀虫剂产生了抗药性,防治很困难。小菜蛾颗粒体病毒(Plutella xylostella granulvirus,PlxyGV)对小菜蛾有较强致病力,是一种有效的生物杀虫剂,但小菜蛾感染病毒至死亡历时较长,且幼虫感病后仍可取食为害。有关PlxyGV对小菜蛾的免疫系统和生理生化的机制的资料较少,本实验以PlxvGV-小菜蛾幼虫这一体系为研究对象,主要研究了PlxyGV对小菜蛾幼虫血淋巴蛋白、免疫系统、细胞微丝骨架及保护酶活力的影响,主要实验结果如下:
1.血淋巴是昆虫代谢的主要场所,也是代谢过程中各种物质储存和交换的主要场所。其中含有的多种蛋白质不仅与组织形成的物质代谢有关,而且与虫体生长发育的激素调节、抗药性的产生和免疫机制等都有密切关系。蛋白质含量测定结果表明:对照组中随着幼虫的发育大体上是不断增加的;感染组在第1天较对照组稍高,第2-7天一直低于对照组;PAGE表明:第1天感染组蛋白条带较对照组略多,第2-4天血淋巴蛋白条带和条带浓度差异不大,但从第5天开始血淋巴蛋白条带及浓度都明显减少。
2.昆虫免疫系统较独特,主要是由昆虫血淋巴承担的,分为细胞免疫和体液免疫;血细胞主要承担细胞免疫,血浆中酚氧化酶(phenoloxidase,PO)、凝集素、抗菌肽等承担体液免疫,酚氧化酶原激活系统和血细胞在防御外源异物的过程中起重要的作用。在本实验中随PlxyGV感染,浆血胞延展被抑制,血细胞和胞核均变大,随后,有的血细胞裂解释放出大颗粒、细胞空泡化,有些细胞内出现大量的致密细小颗粒(可能是病毒包涵体)。感染组血细胞总数前3天均高于对照组,且浆血胞所占比例在前4天显著增加,而粒血胞所占比例几乎没变化。对照组虫体内PO活力随日龄增大而下降;除第5天外,感染组虫体内PO活力与对照组相比相对升高,但感染进程中PO活力起伏变化。
3.资料表明,许多病毒的感染会破坏细胞骨架系统。本实验中对照组随着幼虫的发育浆细胞微丝骨架几乎没有变化;而感染组随着感染的延长微丝骨架发生异常:第1天浆血胞微丝骨架仅有细短丝状伪足;第2天有些出现黑色小空泡,第3天出现树枝状或丝状伪足;第4天有的一端有毛刷状伪足,有的出现较大空泡且周围有细长伪足;第5天微丝骨架凝集,第6天出现黄色颗粒;第7天有较大空泡,变得不完整。
4.昆虫体内存在保护酶:即过氧化物酶(POD)、过氧化氢酶(CAT)和超氧化物歧化酶(SOD),它们可清除细胞内的自由基,防止毒害以维持昆虫的正常生理活动。PlxyGV对小菜蛾幼虫的三种保护酶影响程度是各不相同的。其中,CAT活力在对照组和感染组虫体中均随日龄增大而升高;感染组虫体CAT活力显著高于对照组。相比之下,PlxyGV对小菜蛾幼虫体内SOD和POD活力变化的影响较小。
The diamondback moth,Plutella xylostella(L.) is one of the most harmful pests of Brassicaceae in the world,greatly affecting the quality of cruciferous vegetable products and production.The pest is difficult to control since it is resistant to many insecticides. PlxyGV is pathogenic to the insect with high specificity,and is considered a useful biocontrol agent.However,the larvaes of diamondback moths infected by PlxyGV take long time to die.During the period,the larvaes still feed on vegetables and make dameges.Currently,datas about interaction between PlxyGV and diamondback moth are limited.In this study,experiments were performed to analyze changes in haemolymph proteins,haemocytes,activities of phenoloxidase and protective enzymes,the microfilament cytoskeleton,in the bodies of the larvae of diamondback moth,resulting from infection by PlxyGV.The results are below:
1.Changes in hemolymph proteins.
Insect hemolymph is not only a main metabolism place,but also a main place to store and exchange materials in metabolic processes.Hemolymph contains a variety of proteins.These proteins closely relate to the material-metabolism in tissue formation, hormone regulation in body growth,insecticide-resistance and immune mechanism.In the experiments,hemolymph protein concentration in the control group generally increased with the development of larvae;Protein concentration in infection group was slightly more than control in the 1~(st) day post infection,and obviously reduced in the 2~(nd)-7~(th) days post infection.PAGE analysis revealed changes in protein bands in haemolymph of the larvae infected by PlxyGV.Protein bands in infection group were slightly more than control in the 1~(st) day post infection.No obvious change was observed between infected and control groups from 2 to 4 days post infection.However,both bands and contents of hemolymph protein significantly reduced from the 5 to 7 days post infection,compared to the control.
2.Changs in hemocytes and activity of Phenoloxidase.
Insects have a unique immune system.It divides into cellular immunity and humoral immunity,mainly endured by haemolymph.Hemocytes undertake for cellular immunity. Phenoloxidase,lectin,antimicrobial peptide in plasma react as humoral immunity. Prophenoloxidase activation system and haemocytes play a crucial role in the defense process of exogenous foreign bodies.In the experiments,the spreading function of plasmatocytes was inhibited with the days of infection,hemocytes and nuclei bacame larger.Some cells vacuolized,or contained dense particles(maybe occluded viruses). Some hemocytes lysed to release large particles.The number of total hemocytes in infection group was higher than control in the first three days,the percent of plasmatocytes significantly increased,but granulocytes almost unchanged.PO activity in control group kept dropping down up to the end.PO activity in infection group was always much higher than that in the control,except the 5~(th) day.PO activity in infection group was up and down.
3.Changes in cell microfilament cytoskeleton.
Datas indicate that many viruses destroy the cytoskeleton system.In this experiment, microfilament cytoskeleton of hemocytes was visualized by FITC-phalloidin staining. Microfilament cytoskeleton of hemocytes almost did not change with development of the larvae in control group,but it was abnomal in the infection group.Plasmatocyte microfilament cytoskeletons appeared only small short filopodia in the 1~(st) day.Some appeared small black vacuoles in the 2~(nd) day.There were filopodia and dendritic pseudopodium in the 3~(rd) day.Some appeared brush-like pseudopodiums on one end,and some appeared larger vacuoles and filopodia surrounding in the 4~(th) day.They aggregated in the 5~(th) day.They appeared yellow granules in the 6~(th) day.They appeared more vacuoles and became fragmentary in the 7~(th) day.
4.Changes in protective enzymes.
There are protective enzymes,namely peroxidase(POD),catalase(CAT) and superoxide dismutase(SOD) in insects.They can clear free radicals,prevent free radical toxicity,and maintain normal physiological activities of insects.In the experiments,the effects of PlxyGV to the three protective enzymes were variable.CAT activity kept rising from 1 to 7 days in both groups of infection and control.CAT in infection group was significantly higher than that in control group.In contrast,SOD and POD were less affected by PlxyGV infection.
1.血淋巴是昆虫代谢的主要场所,也是代谢过程中各种物质储存和交换的主要场所。其中含有的多种蛋白质不仅与组织形成的物质代谢有关,而且与虫体生长发育的激素调节、抗药性的产生和免疫机制等都有密切关系。蛋白质含量测定结果表明:对照组中随着幼虫的发育大体上是不断增加的;感染组在第1天较对照组稍高,第2-7天一直低于对照组;PAGE表明:第1天感染组蛋白条带较对照组略多,第2-4天血淋巴蛋白条带和条带浓度差异不大,但从第5天开始血淋巴蛋白条带及浓度都明显减少。
2.昆虫免疫系统较独特,主要是由昆虫血淋巴承担的,分为细胞免疫和体液免疫;血细胞主要承担细胞免疫,血浆中酚氧化酶(phenoloxidase,PO)、凝集素、抗菌肽等承担体液免疫,酚氧化酶原激活系统和血细胞在防御外源异物的过程中起重要的作用。在本实验中随PlxyGV感染,浆血胞延展被抑制,血细胞和胞核均变大,随后,有的血细胞裂解释放出大颗粒、细胞空泡化,有些细胞内出现大量的致密细小颗粒(可能是病毒包涵体)。感染组血细胞总数前3天均高于对照组,且浆血胞所占比例在前4天显著增加,而粒血胞所占比例几乎没变化。对照组虫体内PO活力随日龄增大而下降;除第5天外,感染组虫体内PO活力与对照组相比相对升高,但感染进程中PO活力起伏变化。
3.资料表明,许多病毒的感染会破坏细胞骨架系统。本实验中对照组随着幼虫的发育浆细胞微丝骨架几乎没有变化;而感染组随着感染的延长微丝骨架发生异常:第1天浆血胞微丝骨架仅有细短丝状伪足;第2天有些出现黑色小空泡,第3天出现树枝状或丝状伪足;第4天有的一端有毛刷状伪足,有的出现较大空泡且周围有细长伪足;第5天微丝骨架凝集,第6天出现黄色颗粒;第7天有较大空泡,变得不完整。
4.昆虫体内存在保护酶:即过氧化物酶(POD)、过氧化氢酶(CAT)和超氧化物歧化酶(SOD),它们可清除细胞内的自由基,防止毒害以维持昆虫的正常生理活动。PlxyGV对小菜蛾幼虫的三种保护酶影响程度是各不相同的。其中,CAT活力在对照组和感染组虫体中均随日龄增大而升高;感染组虫体CAT活力显著高于对照组。相比之下,PlxyGV对小菜蛾幼虫体内SOD和POD活力变化的影响较小。
The diamondback moth,Plutella xylostella(L.) is one of the most harmful pests of Brassicaceae in the world,greatly affecting the quality of cruciferous vegetable products and production.The pest is difficult to control since it is resistant to many insecticides. PlxyGV is pathogenic to the insect with high specificity,and is considered a useful biocontrol agent.However,the larvaes of diamondback moths infected by PlxyGV take long time to die.During the period,the larvaes still feed on vegetables and make dameges.Currently,datas about interaction between PlxyGV and diamondback moth are limited.In this study,experiments were performed to analyze changes in haemolymph proteins,haemocytes,activities of phenoloxidase and protective enzymes,the microfilament cytoskeleton,in the bodies of the larvae of diamondback moth,resulting from infection by PlxyGV.The results are below:
1.Changes in hemolymph proteins.
Insect hemolymph is not only a main metabolism place,but also a main place to store and exchange materials in metabolic processes.Hemolymph contains a variety of proteins.These proteins closely relate to the material-metabolism in tissue formation, hormone regulation in body growth,insecticide-resistance and immune mechanism.In the experiments,hemolymph protein concentration in the control group generally increased with the development of larvae;Protein concentration in infection group was slightly more than control in the 1~(st) day post infection,and obviously reduced in the 2~(nd)-7~(th) days post infection.PAGE analysis revealed changes in protein bands in haemolymph of the larvae infected by PlxyGV.Protein bands in infection group were slightly more than control in the 1~(st) day post infection.No obvious change was observed between infected and control groups from 2 to 4 days post infection.However,both bands and contents of hemolymph protein significantly reduced from the 5 to 7 days post infection,compared to the control.
2.Changs in hemocytes and activity of Phenoloxidase.
Insects have a unique immune system.It divides into cellular immunity and humoral immunity,mainly endured by haemolymph.Hemocytes undertake for cellular immunity. Phenoloxidase,lectin,antimicrobial peptide in plasma react as humoral immunity. Prophenoloxidase activation system and haemocytes play a crucial role in the defense process of exogenous foreign bodies.In the experiments,the spreading function of plasmatocytes was inhibited with the days of infection,hemocytes and nuclei bacame larger.Some cells vacuolized,or contained dense particles(maybe occluded viruses). Some hemocytes lysed to release large particles.The number of total hemocytes in infection group was higher than control in the first three days,the percent of plasmatocytes significantly increased,but granulocytes almost unchanged.PO activity in control group kept dropping down up to the end.PO activity in infection group was always much higher than that in the control,except the 5~(th) day.PO activity in infection group was up and down.
3.Changes in cell microfilament cytoskeleton.
Datas indicate that many viruses destroy the cytoskeleton system.In this experiment, microfilament cytoskeleton of hemocytes was visualized by FITC-phalloidin staining. Microfilament cytoskeleton of hemocytes almost did not change with development of the larvae in control group,but it was abnomal in the infection group.Plasmatocyte microfilament cytoskeletons appeared only small short filopodia in the 1~(st) day.Some appeared small black vacuoles in the 2~(nd) day.There were filopodia and dendritic pseudopodium in the 3~(rd) day.Some appeared brush-like pseudopodiums on one end,and some appeared larger vacuoles and filopodia surrounding in the 4~(th) day.They aggregated in the 5~(th) day.They appeared yellow granules in the 6~(th) day.They appeared more vacuoles and became fragmentary in the 7~(th) day.
4.Changes in protective enzymes.
There are protective enzymes,namely peroxidase(POD),catalase(CAT) and superoxide dismutase(SOD) in insects.They can clear free radicals,prevent free radical toxicity,and maintain normal physiological activities of insects.In the experiments,the effects of PlxyGV to the three protective enzymes were variable.CAT activity kept rising from 1 to 7 days in both groups of infection and control.CAT in infection group was significantly higher than that in control group.In contrast,SOD and POD were less affected by PlxyGV infection.
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