【作者】 刘广生；

【导师】 牟志美；

【作者基本信息】 山东农业大学 ， 特种经济动物饲养， 2005， 硕士

【摘要】 蚕业生产中,病原微生物对家蚕的混合感染广泛存在,严重影响蚕茧优质高产,阻碍蚕业的健康发展。家蚕(Bombyx mori)是鳞翅目昆虫的典型代表和重要的模式生物,以家蚕为生物材料的生防研究在农林害虫生物防治中具有重要的参考价值。细菌性中毒病和核型多角体病是蚕业生产上比较常见的病害,苏云金杆菌是生防上应用最广泛的微生物农药。目前对苏云金杆菌(Bacillus thuringiensis)和家蚕核型多角体病毒(Bombyx mori nuclear polyhedrosis virus, BmNPV)对家蚕和其他昆虫联合感染的研究甚少。为了系统地研究联合感染对家蚕的致病性,探讨联合感染中病原致病作用变化的规律,为蚕病综合防治和农业害虫生物防治提供理论依据,本文采用生物统计分析方法、生化技术、光镜技术以及电镜技术对B.t 和BmNPV对家蚕的联合致病作用、联合感染后家蚕生理生化的变化以及中肠、血淋巴组织细胞的病变进行了研究,主要取得以下研究成果: 1 联合感染后相继出现两个发病高峰期和两种典型病征;与单独感染相比,联合感染后家蚕病程缩短,发病高峰期提前,死亡率增加,然而不同联合感染方式(同时感染、先感染、后感染)之间对于家蚕致死率和存活家蚕体重的影响均没有显著差异;联合感染明显减少存活家蚕体重,B.t对此的影响比BmNPV更明显;联合感染比单独感染缩短LT50 0.1d~1.8 d ;不同浓度的BmNPV 均可增强B.t 毒力,2.4×104、1.2×105 个/ml B.t 对BmNPV毒力有增效作用。2 与单独感染相比,联合感染后家蚕肠液淀粉酶、蛋白酶、中肠碱性磷酸酶活性、肠液pH 值和细胞浓度明显降低,血淋巴中CAT、SOD 活性和pH 值明显升高,原白血球比例减少,浆细胞和颗粒细胞比例增加。3 BmNPV与亚致死浓度B.t 联合感染后B.t 增殖速度比单独感染快,NPB 增殖速度比单独感染慢,3 种B.t 形态中营养菌体比例比单独感染大,芽孢比例比单独感染小。4 感染后20h,联合感染导致中肠微绒毛、内质网以及线粒体发生严重病变,尤其是细胞核病变突出;B.t 单独感染后中肠组织细胞核病变不明显;BmNPV单独感染后中肠组织细胞内质网、线粒体和核膜均发生病变。感染后84h,联合感染致使中肠组织细胞内质网和所有线粒体发生病更多还原

【Abstract】 During silkworm breeding, it existed wide that mixed infection of pathogenic microbes resulted in worse quality and less quantity of silkworm cocoon and inhibiting sericultural development well. Silkworms was representative of Lepidopterous insect and important model creature, research of biological control on wich could provide much reference value for biological control of insects in agriculture and forestry. Bacterial poisoning and nuclear polyhedrosis often happened during silkworm breeding, and Bacillus thuringiensis (B.t)was applied in biological control most wide, however, research on joint infection with B.t and Bombyx mori Nuclear Polyhedrosis Virus (BmNPV) to silkworms was reported little yet. In order to research joint pathogenicity to silkworms, and discuss change laws of pathogenicity, providing theoretical terms for integrated control of silkworm diseases and biological control of agricultural insects, joint pathogenicity of B.t and BmNPV to silkworms, change of physiology and biochemistry and pathologic midgut and hemolymph histiocyte in silkworm were researched by using biological statistical analysis, technology of high physiology and biochemistry , and technology of optical microscope and TEM, and the main results were showed as the followings: 1. After joint infection with B.t and BmNPV, both two fastigiums of disease occurrence and two typical symptoms of disease appeared one after another; joint infection advanced time of death and fastigium of disease occurrence, increased mortality; there was no significant difference of silkworm mortality and weight of surviving silkworms between simultaneous infection, earlier infection and later infection; Joint infection decreased weight of surviving silkworms significantly, in which B.t played a more important part than BmNPV, and LT50 by 0.1 days~1.8 days; Different concentrations of BmNPV could increased virulence of B.t while 2.4×10~4、1.2×10~5 per ml B.t has synergistic action on virulence of BmNPV respectively. 2, After joint infection, activities of amylase and protease, pH in intestinal juice, alkaline phosphatase of larva midgut, and concentration of blood cells decreased more remarkably while pH and activity of CAT and SOD in larva hemolymph increased more obviously, and proportion of original blood cells decreased while that of plasma cells and granular cells increased more obviously than single infection. 3, After joint infection with BmNPV and sublethal concentration of B.t , proliferation speed of B.t was much quicker, but that of NPB was slower; Joint infection caused higher proportion of vegetative cells, but lower proportion of spores than single infection. 4, At 20 hours after infection, joint infection caused serious pathology of microvilli, endoplasmic reticulum, nucleus and mitochondria, especially of nucleus, but single infection with B.t didn’t cause remarkable pathology of nucleus of midgut histiocyte, and with BmNPV it happened to endoplasmic reticulum, mitochondria and nuclear membrane. At 84 hours after infection, serious pathology of endoplasmic reticulum and all mitochondria of midgut histiocyte happened in joint infection, especially nuclear membrane cracked, and virions appeared both within nucleus and outside nucleus, and cells decomposed, however, most of pathologic cellular organs was found and virions appeared only within nucleus in single infection with BmNPV. 5, At 60 hours after infection, joint infection led to serious pathology of all kinds of blood cells, pathology outside and within nucleus was serious, many virions appeared, and cytoplasm decomposed before nuclear polyhedral body(NPB) was released, even blood cells decomposed before NPB was formed, however, after single infection with B.t, pathology of blood cells happened from outside nucleus to within nucleus, and pathology of different kinds of blood cells was different, and with BmNPV, all kinds of blood cells became abnormal, but could be distinguished, and nucleus and cytoplasm didn’t decompose before NPB was released. At 96 hours after infection, joint infection led to intensified pathology of blood cells, which showed that all kinds of blood cells couldn’t be distinguished, much more virions and NPBs更多还原