【作者】 钟伟；

【导师】 殷幼平； 石福明；

【作者基本信息】 西南农业大学 ， 农业昆虫及害虫防治， 2003， 硕士

【摘要】 蜚蠊是一类重要的世界性卫生害虫。本研究以重要的室内蜚蠊种类——美洲大蠊(Periplaneta americana)为实验材料，采用蜚蠊的非致病菌——金龟子绿僵菌(Metarhizium anisopliae)CQMa102菌株作为供试菌，较系统地研究了绿僵菌孢子进入蜚蠊体腔后(点滴和注射处理)，蜚蠊血淋巴某些理化性质的改变、血淋巴的免疫反应以及蜚蠊的行为反应等，有助为研究昆虫的抗病机理和生物杀虫剂的杀虫机理，同时也为生物杀虫剂的开发提供了必要的基础。 1 注射绿僵菌孢子后蜚蠊血淋巴物理性质的变化 蜚蠊若虫血淋巴pH值为pH6.8，注射4×10~7个/ml浓度的绿僵菌孢子到蜚蠊体内后，蜚蠊血淋巴的pH值上升到pH 7.2。若虫和10日龄雌成虫对绿僵菌孢子比较敏感，注射4×10~7个/ml浓度的孢子后其血细胞数量均降低显著，最低时分别为空白对照的4.6％及8.5％。30日龄雌成虫对绿僵菌孢子不敏感，其血细胞数量的变化幅度较小。注射生理盐水注射后可使血细胞数量在2h内增加，但随后下降至正常值附近波动。 以不同浓度的绿僵菌孢子注射蜚蠊后4h，较高浓度(4×10~7及4×10~9个/ml)注射均导致了蜚蠊血细胞数量显著降低，而低浓度(4×10~5个/ml)注射后蜚蠊血细胞数量却大量增加。 2 蜚蠊血细胞对注射绿僵菌孢子的免疫反应 蜚蠊对绿僵菌孢子的免疫反应主要由血细胞完成。当蜚蠊体腔内注入绿僵菌孢子后，5-10min即可见血细胞向真菌孢子靠近，30min后有孢子被吞噬入血细胞，同时血淋巴中出现了大量的、由血细胞包围真菌孢子而形成的结节。蜚蠊可以通过血细胞的吞噬作用及结节化等方式使绿僵菌孢子失去萌发能力并被杀死黑化。注射较高浓度的孢子后，许多参与免疫的血细胞也会黑化，最后裂解，血淋巴中也出现了较多血细胞和组织的碎片。这种反应导致蜚蠊的免疫体系受到破坏，生活力降低，易于感染细菌死亡。但注射低浓度孢子后，由于孢子的刺激，反而增强了蜚蠊的免疫机能，蜚蠊的死亡率甚至低于生理盐水对照。 3 绿僵菌孢子对蜚蠊血淋巴中抗菌物质及蛋白质的诱导 注射绿僵菌后的不同时期提取蜚蠊血淋巴，以多种不同类型的植物病原菌、昆虫病原菌及腐生菌作为检测菌，均未检测出蜚蠊血淋巴中有抗菌物质存在，即绿僵菌不能诱导蜚蠊产生抗菌物质。用Broadfold法测定绿僵菌孢子诱导后的蜚蠊血淋巴蛋白质的浓度，发现注射后4h内蜚蠊血淋巴蛋白质浓度显著下降，但4h后开始回升，24h时就超出空白对照约60％。 美洲大娩对金龟子绿僵菌CQMal02菌株的免疫反应 经SDS－PAGE电泳发现，注射诱导后的蜚蛾血淋巴在43kDa处出现了一条新的蛋白带，但含量很低。注射诱导后血淋巴蛋白质最明显的变化是一些原有蛋白质浓度发生改变，如88k蛋 白质显著增加，而42k蛋白质含量减少等。 4点滴接种绿僵菌抱子后蜚镰的兔疫反应 从体壁点滴接种较较高浓度绿僵菌抱子（10’个浊），发现绿僵菌抱子能够通过蜚煽的体壁侵入到蜚煽的体内，少数抱子还能萌发，却不能大量繁殖，对蜚煽血细胞数量及蜚煽生活力的影响均很小。蜚贱的血细胞通过吞噬及形成结节有效地抑制了抱子的萌发生长，使大多数绿僵菌殉子黑化、裂解。更多还原

【Abstract】 American cockroach, Periplaneta americanna is a significant house pest in the world. The hemolymph immune reaction of American cockroach to the entomopathogenic fungus, Metarhizium anisopliae isolate CQMa102, which is broadly used in locust control, was studied in the research. The results will enrich our knowledge about insect immune mechanism and the insecticidal mechanism of entomofungus. They will also be the fundamental work for insecticide development.1. Physical changes of hemolymph of P. americanna after injection with M. anisopliaeThe pH value of hemolymph of P. americanna increased rapidly from pH6.8 to pH 7.0 and the number of hemocytes(NH) of P. americanna at all the developing stages became lower than the blank control(BC) after injection with 4+107/ml fungal spores. The nymphae and ten-day old female adult were more sensitive to the fungal spores than the thirty-day old female adult, and both then- NHs decreased significantly after injection with 4+107 spores/ml. Injection of saline stimulated the increase of NH 2h postinjection(PI) and then declined to the BC.Injected with different doses of spores, the NH of nymphae changed differently. Injection of 4 +107 spores/ml and 4+109 spores/ml caused the significant decrease of the NH 4h postinjection. On the contrary, 4+105 spores/ml injection induced significant increase of the NH.2. Hemolymph immune reaction of P. americanna to the M. anisopliae injectedCell immune is the main reaction of P. americanna to the injected M. anisopliae. The hemocytes approached to the fungal spores 5-10min postinjection and some phagocytosed spores were observed 30min postinjection. At the same time, there appeared lots of nodules composed by numerous hemocytes in the hemolymph of the cockroach. P. americanna elemilated large amount of the fungal spores efficiently by phagocytosing and nodulation and the germinating of the spores was inhibited. These spores disintegrated or menalized finally. The experiment showed that injection of high dose spores destroyed the immune system of the cockroach and the cockroach was easy to be invaded by bacterial pathogens. The higher dose of spores injected,the higher mortality of thecockroach.To be interesting, low dose spores injection strengthened the immune ability of the insect in certain degree and the motaliry of the insect was even lower than that of saline injection control.3 Antimicrobiol substance and protein in the hemolymph of nymphae induced by M. anisopliaeNo antimicrobiol substance was detected in the hemolymph of nymphae by different kinds of micropathogens after injection with M. anisopliae, which meant that the fungus could not induce P. americanna to produce antimicrobiol substance. Injection of fungal spores caused the decline of protein concentration(PC) at the begaining, but after that the PC increased rapidly. The SDS-PAGE electrophoretic patterns of the hemolymph protein showed that the proteins types changed slightly, and only one new protein with 43kDa molecular weitht appeared. However the concentrations of some proteins increased or declined obviously.4 Hemolymph immune reaction of P. americanna after nature infection with M. anisopliaeDripping the cockroach body with M. anisopliae showed that the M. anisopliae spores can invade into the hemolymph coelom of P. americanna through cockroach cuticle and produce blastospores. Some blastspores even can expanded, germinated as time went on. The cockroach restrained the blastospores by phagocytosing and nodulation,and many of them melanized 5d postinfection. The number of hemocytes in the hemolymph of the P. americanna changed slightly and the vital force had no significantly change within one month postinfection with M. anisopliae.更多还原