美洲大蠊对金龟子绿僵菌CQMa102菌株的免疫反应
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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。
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. anisopliae
The 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 injected
Cell 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 the
cockroach.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. anisopliae
No 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. anisopliae
Dripping 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.
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。
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. anisopliae
The 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 injected
Cell 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 the
cockroach.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. anisopliae
No 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. anisopliae
Dripping 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.
引文
1．蔡保灵，刘安西．白僵菌和绿僵菌对蜚蠊的杀虫作用．中国虫生真菌研究与应用：第一卷．北京：学术期刊出版社．1988，101-103．
2．陈建新，刘泉．昆虫生长调节剂在仿制室内卫生昆虫中的应用．中国媒介生物学剂控制杂志，1999，10(2)：Ⅱ-Ⅴ．
3．陈留存，王金星．昆虫抗菌肽研究现状．生物工程进展，1999，19(5)：55-60
4．陈守同．蜚蠊的危害、防治和利用．中央民族大学学报(自然科学版)，1996，5(2)：113-116．
5．陈学新，胡义镰，何俊华，陈卫良．中蜂抗菌物质的诱导．昆虫知识，1999，36(4)：215-218．
6．樊美珍．球孢白僵菌胞外蛋白酶及其与毒力关系的研究．微生物学通报，1994，21(4)：201-206．
7．费守华．蜚蠊的鉴别诊断．中国媒介生物学剂控制杂志，1998a，9(4)：317-320．
8．费守华．蜚蠊的鉴别诊断．中国媒介生物学剂控制杂志，1998b，9(5)：389-393．
9．何复梅，罗玉钏，梁锦英．蟑螂防治问题探讨．昆虫天敌，1997，19(4)：188-189．
10．胡景江等．球孢白僵菌对马尾松毛虫的毒力与有关生化指标的关系Ⅱ：不同世代白僵菌胞外蛋白酶及其与毒力的关系．西北林学院学报(自然版)，1993，8(4)：36-40．
11．胡修元．蜚蠊可能引起/传播的疾病．中国媒介生物学及控制杂志，1999，10(3)：168-130．
12．胡修元．蟑螂世家．中国医药科技出版社．北京．1988．
13．黄钢，高慧敏，李玉兰等．河北省城市蜚蠊携带寄生虫及病毒的调查．中国媒介生物学及控制杂志，1999，10(3)：211-212．
14．江雪峰，蟑螂的危害和杀灭蟑螂的方法．人民军医出版社．北京．1985．
15．蒋书楠．城市昆虫学．重庆出版社．重庆．1992：246-259．
16．李增智，樊美珍．真菌生物技术与真菌杀虫剂的发展．微生物农药及其产业化(喻子牛等主编)．科学出版社．北京．2000：115-120．
17．林立辉，傅庭容．十四珠绿僵菌对文幼\家蝇\蟑螂的毒力实验．中国虫生真菌研究与应用：第一卷．学术期刊出版社．北京．1988，179．
18．陆龙喜，时连根．家蚕血细胞对病原白僵菌的防御反应研究．浙江农业学报，2001，13(4)：201-204．
19．农向群，高松，邓春生，李俊国，袁京京．球孢白僵菌对德国小蠊侵染力的初步研究．中国生物防治，1996，12(3)：97-99．
20．蒲蛰龙，李增智．昆虫真菌学．安徽科学技术出版社．合肥．1996．
21．蒲蛰龙．昆虫病理学．广东科技出版社．广东．1994：341-368．
22．齐静娇，曲传智．不同诱导源有家蝇抗菌物质的动力学研究．洛阳医专学报，1999，17(1)：35-
36．
23．时超美，昆虫酚氧化酶原活化及其在免疫中的作用．昆虫知识．2000，37(5)：310-314．
24．宋漳，冯丽贞，景云．马尾松毛虫感染绿僵菌后某些生化指标的变化．昆虫知识，2002，39(4)：297-300．
25．汪家政，范明主编．蛋白质技术手册．科学出版社．北京．2000．
26．王海川，尤民生．绿僵菌对昆虫的入侵机理．微生物学通报，1999，26(1)：71-73．
27．王远程，刘伟，杨峰．家蝇血淋巴的提取及抗菌物质的诱导．微生物学报，1992，32(6)：439-444。
28．姚剑，李曾智，樊美珍等．虫生真菌入侵过程中酶的研究(综述)．安徽农业大学，1996，23(3)：303-308．
29．耶夫拉霍娃著，黄传贤译．昆虫病原真菌．科学出版社．北京．1982，39-72．
30．翟锦彬，黄秀梨．球孢白僵菌对棉铃虫细胞防御反应的影响．北京师范大学学报(自然科学版)，1996，32(4)：541-545．
31．翟锦彬．杀虫真菌--球孢白僵菌的昆虫致病机理研究近况．微生物学通报，1995，22(1)：45-47．
32．张爱文，邓春生，农向群，任淑仙，徐睢仁．亚洲玉米螟感染白僵菌后组织病理学研究．生物防治通报，1994，(10)4：151-156．
33．张大羽，程家安．德国小蠊的抗性水平和主要机制．中国媒介生物学剂控制杂志，1997，8(3)：236-238．
34．张刚应，杨怀文，黄钢．绿僵菌防治德国小蠊的初步研究．中国生物防治，1996，12(3)：100-102．
35．张克斌，谭六谦．昆虫生理．陕西科学技术出版社．西安．1989．
36．张然，陈濂生等．蜚蠊灭菌肽的诱导及初步分离分析．昆虫学报，1990，33(1)：9-12．
37. Amirante G A. Production of heteroagglutinins in haemocytes of Leucophaea maderae. L. Experientia., 1976,32:526-529.
38. Baines D,Downer R G H. 5-Hydroxytryptamine-sensitive adenylate cyclase affects phagocytosis in cockroach hemocytes. Arch. Insect Biochem. Physiol., 1992, 21(4): 303-316.
39. Baines D,Downer R G H. Octopamine enhances phagocytosis in cockroach hemocytes: involvement of inositol trisphosphate. Arch. insect biochem.physiol., 1994,26(4):249-261.
40. Broome J R, Sikorowski P P,Norment B R. A mechanism of pathogenicity of Beauveria bassiana on larvae of the imported fire ant Solenopsis richteri. J. Invertebr. Pathol. 1976, 28:87-91.
41. Chain B M, Leyshon-Soland K, Siva-Jothy M T. Haemocyte heterogeneity in the cockroach Periplaneta americanna analyzed using monoclonal antibodies. J. Cell Sci., 1992, 103: 1261-1267.
42. Cleonor da Silvaa, Gary B, Dunphyb M E. Interaction of hemocytes and prophenoloxidase system of fifth instar nymphs of Acheta domesticus with bacteria. Dev. Comp. Immunol., 2000, 24(4):367-379.
43. Cochran D G. Monitor for insecticide risistance in fieldcollected strains of the German
cockroach. J.Econ.Entomol,1989,82(2):336.
44. Fearon D T. Seeking wisdom in innate immunity. Nat.,1997,388:323-324.
45. Fehlbaum P,Bulet P, Chernysh S. Structureactivity analysis of thanatin, a 21-residue inducible ingsect defense piptide with sequence homology to froh skin antimicrobial piptides. Proc. Natl. Acad. Sci. USA. 1996, 93:1221-25.
46. Fisher C W, Brady U E. Activation, properties and collection of haemolymph phenoloxidase of the Americann cockroach, Periplaneta americanna. Comp. Biochem. Physiol., 1983, 75(1): 111-114.
47. Garry B T. Studies on the cellular immune responses of insects toward the insect pathogen Trypanosoma rangeli. J. Invertebr. Pathol.,1988,51:64-72.
48. Gunnarsson S G S.Hemocytis aggregation in schistocerca gregaria and Periplaneta americanna as a response to injected substances of microbial origin. J. Invertebr. Pathol. 1985,46:312-319.
49. Han Y S, Chnn J, Schwartz A, Nelson S, Paskewitz S M. Induction of mosquito hemolymph proteins in response to immune challenge and wounding. Dev. Comp. Immunol., 1999, 23: 553-562
50. Hemingway J, Small G J, Mono A G. Possible mechanisms of organophosphorus and carbamate insecticide resistance in German Cockroaches from different geographical areas. J. Econ. Entomol., 1993,86(6):1623.
51. Hilary J D,Norma A R, Charles R H, Anna A, Kenneth S.Purification of the pro-phenol oxidase enzyme from haemocytes of the cockroach Blaberus discoidalis. Biochem. J., 1993, 289: 87-91.
52. Horohov D W, Dunn P E. Changes in the circulating hemotyte population of Manducasexta lavae following injection of bacteria. J. Invertebr. Pathol., 1982, 40: 327-339.
53. Hung H S, Hung J C, Cheng H H, Jen C C, Yen L S. Phenoloxidase Activity of hemocytes derived from penaeus monodonand Macrobrachium rosenbergii, J. Invertebr. Pathol., 1998, 71(1):26-33.
54. Hung S H, Boucias D G. Influence of Beauveria bassiana on the cellular defense response of the beet armyworm, Spodoptera exigua. J. invertebr. Pathol., 1992,60: 152-158.
55. Huxham I M, Lackie A M, McCorkindale N J. Inhibitory effects of cyclodepsi peptides, destruxins, from the fungus Metarhizium anisopliae, on cellular immunity in insects. J. Insect Physiol., 1989, 35(2):97-105.
56. Jeremy P, Gillespie, Claire Burnett, Charnley A K. The immune response of the desert locust Schistocerca gregaria during mycosis of the entomopathogenic fungus, Metarhizium anisopliae var acridum. J. Insect Physiol., 2000,46:429-437.
57. Jomori T, Kubo T, Natori S. Purification and characterization of lipopolysaccharide-binding protein from hemolymph of Americarm cockroach Periplaneta arnericanna. European J. Biochem./FEBS.,1990,190(1):201-206.
58. Jones J C. Current conception concerning insect hemocytes. Amer. Zoologist., 1962, 2(1): 209-246.
59. Kaakeh W, Reid B L, Bennett G W. Horizontal transmission of the entomopatho-genic fungus Metarhizium anisopliae (imperfect fungi: Hyphomycetes) and hydra-methylnon among German cockroaches (Dictyoptera: Blattellidae). J. Entomological Sci., 1996, 31(4):378-390.
60. Kaakeh W, Reid B L, Bohnert T J, Bennett G W. Toxicity of imidacloprid in the German cockroach (Dictyoptera: Blattellidae), and the synergism between imidacloprid and Metarhizium anisopliae (imperfect fungi: Hyphomycetes). J. Econ. Entornol., 1997, 90(2): 473-482.
61. Lackie A M, Takle, G; Tetley, L. Haemocytic encapsulation in the locust Schistocerca gregaria (Orthoptera) and in the cockroach Periplaneta arnericanna (Dictyoptera). Cell Tissue Res., 1985, 240(2): 343-351.
62. Lackie A M. Haemocyte behaviour, Adv. Insect Physiol., 1988, 21:85-178.
63. Lavine M D, Strand M R. Insect hemocytes and their role in immunity. Insect Biochem. Mol. Biol., 2002,32:1295-1309.
64. Lavine M D, Strand M R. Surface characteristics of foreign targets that elicit an encapsulation response by the moth Pseudoplusia includens. J. Insect Physiol., 2001,47:965-974.
65. Lebestky T, Chang T, Hartenstein V, Banerjee U. Specification of Drosophila hematopoietic lineage by conserved transcription factors. Sci., 2000,288:146-149.
66. Li D, Scherfer C, Korayem A M, Zhao Z, Schmidt O, Theopold U. Insect hemolymph clotting: evidence for interaction between the coagulation system and the prophenoloxidase activating cascade. Insect Biochem. Mol. Biol., 2002,32: 919-928.
67. Ludek Z, Watson D W, Schall C. Synergism between Metarhizium anisopliae (Deuteromycota: Hyphomycetes) and Boric Acid against the German cockroach (Dictyoptera: Blattellidae). Biol. Control., 2002,23:296-302.
68. Martin F R, Nolan R A. Americann cockroach (Periplaneta americanna) hemocyte response to Entornophaga aulicae protoplasts. Can. J. Zool., 1986,64(6): 1369-1372.
69. Mazet I, Boucias D G. Effects of the fungal pathogen, Beauveria bassiana on protein biosynthesis of infected Spodoptera exigua larvae. J. Insect Physiol., 1996,42(2): 91-99.
70. McDonald D. Cockroaches and mosquitoes-the most serious urban insect pests. Intl. Pest Control., 1996, 38(6): 185-188.
71. Michael J, Bidochkaa L, Raymond J, St. Legera, Donald W. Induction of novel proteins in Manduca Sexta and Blaberus giganteusas a response to fungal challenge, J. Invertebr. Pathol. 1997, 70(3): 184-189.
72. Micheal J B, George G K. purification and properties of an extracellular protease produced by the entomopathogenic fungus Beauveria bassiana., Appl. environ.l microbiol., 1987, 1679-1684.
73. Pachamuthu P, Kamble S T. In vivo study on combined toxicity of Metarhizium anisopliae (Deuteromycotina: Hyphomycetes) strain ESC-1 with sublethal doses of chlorpyfifos, propetamphos, and cyfluthrin against German cockroach (Diptyoptera:Blattellidae). J. Econ. entomol., 2000,93(1):60-70.
74. Pal R, Brown A W A. Problems of insecticide resistance. Zeitschrifi fur Parasitenkunde., (Abstract). 1974, 45(2):211-221.
75. Paul S,Khan A M, Baqui MA. Evaluation of the common cockroach Periplaneta americanna (L.) as carrier of medically important bacteria. Muhibullah M J. Communicable Diseases., (Abstract) 1992, 24(4):206-210.
76. Pech L L, Strand M R. Granular cells are required for encapsulation of foreign targets by insect haemocytes. J. Cell Sci., 1996,109:2053-2060.
77. Pech L L, Strand M R. Plasmatocytes from the moth Pseudoplusia includens induce apoptosis of granular cells. J. Insect Physiol., 2000, 46: 1565-1573.
78. Pendland J C, hung S Y. Boucias D G. Evasion of host defence by in vivo-produced protoplast-like cells of the insect mycopathogen Beauveria bassiana. J. bacteriol., 1993, 175: 5962-5969.
79. Phillipe B, Charles H, Jean Luc Dimarcq, Danie le Homann. Antimicrobial peptides in insects; structure and function. Dev. Comp. Immunol., 1999,23: 329-344.
80. Rahmet Alla M,Rowley A F, Alia M Rahmet. Studies on the cellular defense reaction of the Madeira cockroach, Leucophaea maderae: nodule formation in response to injected bacteria. J. Invertebr. Pathol., 1989, 54(2):200-207.
81. Rahmet Alla M,Rowley A F. Studies on the cellular defense reactions of the Madeira cockroach, Leucophaea maderae: in vitro phagocytosis of different strains of Bacillus cereus and their effect on hemocyte viability. J. Invertebr. Pathol., 1990, 55(3):350-356.
82. Rahmet Alla M,Rowley A F. Studies on the cellular defense reaction of the Madeira cockroach, Leucophaea maderae: nodule formation in response to injected bacteria. J. Invertebr. Pathol. 1989, 54(2): 200-207.
83. Ryan M, Nicholas W L. The reaction of the cockroach Periplaneta americanna to the injection of foreign particulate material. J. Invertebr. Pathol., 1972, 19(3):299-307;
84. Ryan N A, Karp R D. Stimulation of hemocyte proliferation in the Americann cockroach (Periplaneta americanna) by the injection of Enterobacter cloacae, J. Insect Physiol. 1993, 39(7):601-608.
85. Seamen G, Robert N L. Immunological response of male cockroaches to infection of Tetrahymena pyriformis. Sci. 1968,161:1359-1361.
86. Shelby K S. Webb B A. Polydnaverus infection inhibits synthesis of an insect plasma protein, arylphorin, J. Gen. Virol. 1994,75:2285-2292.
87. Sophia E, Dan H. Drosophila cecropin as an antifungal agent. Insect Biochem. Mol. Biol., 1999, 29:965-972.
88. Steenberg T, Vagn Jensen K M, Jensen K M V, Smits P H. Entomopathogenic fungi for control of German cockroach (Blattella germanica) and other synanthropic cockroaches. Bulletin OILB SROP., 1998, 21(4):145-150.
89. Strand M R, Pech L L. Immunological basis for compatibility in parasitoid-host relationships. Annu. Rev. Entomol., 1995,40:31-56.
90. Strand M R., Clark K D. Plasmatocyte spreading peptide induces spreading of plasmatocytes but represses spreading of granulocytes. Arch. Insect Biochem. Physiol., 1999,42:213-223.
91. Wheeler M B, Stuart G S, Hapner K D. Agglutin mediated opsonization of fungal blastospores in belanoplus differentialis. J. Insect. Physiol., 1993,39(6):477-483.
92. Wilson R, Chen C W, Ratcliffe N A, Chen C W. Innate immunity in insects: the role of multiple, endogenous serum lectins in the recognition of foreign invaders in the cockroach, Blaberus discoidalis. J. Immunol. Baltim., 1999, 162(3): 1590-1596.
93. Wilson R, Ratcliffe N A. Effect of lysozyme on the lectin-mediated phagocytosis of Bacillus cereus by haemocytes of the cockroach, Blaberus discoidalis. J. Insect Physiol., 2000,46(5): 663-670.
94. Witting. Phagocytosis by blood cells in healthy and disease dcaterpillards. I. phatocytosis of Bacilus thuringiensis Berliner in Pseudale tiarnipuncta (Haworth). J. Invertebr. Pathol., 1965,7: 474-488.
95. Zukowski K, Bajan C, Popowska Nowak E. The effect of infestation by mixed culture of Beauveria bassiana and Poecilomycess farinosus [sic] on reduction in numbers of experimental cultures of Blatella germanica L. Roczniki Panstwowego Zakladu Higieny. (Abstract).1999, 50(3):321-327.
2．陈建新，刘泉．昆虫生长调节剂在仿制室内卫生昆虫中的应用．中国媒介生物学剂控制杂志，1999，10(2)：Ⅱ-Ⅴ．
3．陈留存，王金星．昆虫抗菌肽研究现状．生物工程进展，1999，19(5)：55-60
4．陈守同．蜚蠊的危害、防治和利用．中央民族大学学报(自然科学版)，1996，5(2)：113-116．
5．陈学新，胡义镰，何俊华，陈卫良．中蜂抗菌物质的诱导．昆虫知识，1999，36(4)：215-218．
6．樊美珍．球孢白僵菌胞外蛋白酶及其与毒力关系的研究．微生物学通报，1994，21(4)：201-206．
7．费守华．蜚蠊的鉴别诊断．中国媒介生物学剂控制杂志，1998a，9(4)：317-320．
8．费守华．蜚蠊的鉴别诊断．中国媒介生物学剂控制杂志，1998b，9(5)：389-393．
9．何复梅，罗玉钏，梁锦英．蟑螂防治问题探讨．昆虫天敌，1997，19(4)：188-189．
10．胡景江等．球孢白僵菌对马尾松毛虫的毒力与有关生化指标的关系Ⅱ：不同世代白僵菌胞外蛋白酶及其与毒力的关系．西北林学院学报(自然版)，1993，8(4)：36-40．
11．胡修元．蜚蠊可能引起/传播的疾病．中国媒介生物学及控制杂志，1999，10(3)：168-130．
12．胡修元．蟑螂世家．中国医药科技出版社．北京．1988．
13．黄钢，高慧敏，李玉兰等．河北省城市蜚蠊携带寄生虫及病毒的调查．中国媒介生物学及控制杂志，1999，10(3)：211-212．
14．江雪峰，蟑螂的危害和杀灭蟑螂的方法．人民军医出版社．北京．1985．
15．蒋书楠．城市昆虫学．重庆出版社．重庆．1992：246-259．
16．李增智，樊美珍．真菌生物技术与真菌杀虫剂的发展．微生物农药及其产业化(喻子牛等主编)．科学出版社．北京．2000：115-120．
17．林立辉，傅庭容．十四珠绿僵菌对文幼\家蝇\蟑螂的毒力实验．中国虫生真菌研究与应用：第一卷．学术期刊出版社．北京．1988，179．
18．陆龙喜，时连根．家蚕血细胞对病原白僵菌的防御反应研究．浙江农业学报，2001，13(4)：201-204．
19．农向群，高松，邓春生，李俊国，袁京京．球孢白僵菌对德国小蠊侵染力的初步研究．中国生物防治，1996，12(3)：97-99．
20．蒲蛰龙，李增智．昆虫真菌学．安徽科学技术出版社．合肥．1996．
21．蒲蛰龙．昆虫病理学．广东科技出版社．广东．1994：341-368．
22．齐静娇，曲传智．不同诱导源有家蝇抗菌物质的动力学研究．洛阳医专学报，1999，17(1)：35-
36．
23．时超美，昆虫酚氧化酶原活化及其在免疫中的作用．昆虫知识．2000，37(5)：310-314．
24．宋漳，冯丽贞，景云．马尾松毛虫感染绿僵菌后某些生化指标的变化．昆虫知识，2002，39(4)：297-300．
25．汪家政，范明主编．蛋白质技术手册．科学出版社．北京．2000．
26．王海川，尤民生．绿僵菌对昆虫的入侵机理．微生物学通报，1999，26(1)：71-73．
27．王远程，刘伟，杨峰．家蝇血淋巴的提取及抗菌物质的诱导．微生物学报，1992，32(6)：439-444。
28．姚剑，李曾智，樊美珍等．虫生真菌入侵过程中酶的研究(综述)．安徽农业大学，1996，23(3)：303-308．
29．耶夫拉霍娃著，黄传贤译．昆虫病原真菌．科学出版社．北京．1982，39-72．
30．翟锦彬，黄秀梨．球孢白僵菌对棉铃虫细胞防御反应的影响．北京师范大学学报(自然科学版)，1996，32(4)：541-545．
31．翟锦彬．杀虫真菌--球孢白僵菌的昆虫致病机理研究近况．微生物学通报，1995，22(1)：45-47．
32．张爱文，邓春生，农向群，任淑仙，徐睢仁．亚洲玉米螟感染白僵菌后组织病理学研究．生物防治通报，1994，(10)4：151-156．
33．张大羽，程家安．德国小蠊的抗性水平和主要机制．中国媒介生物学剂控制杂志，1997，8(3)：236-238．
34．张刚应，杨怀文，黄钢．绿僵菌防治德国小蠊的初步研究．中国生物防治，1996，12(3)：100-102．
35．张克斌，谭六谦．昆虫生理．陕西科学技术出版社．西安．1989．
36．张然，陈濂生等．蜚蠊灭菌肽的诱导及初步分离分析．昆虫学报，1990，33(1)：9-12．
37. Amirante G A. Production of heteroagglutinins in haemocytes of Leucophaea maderae. L. Experientia., 1976,32:526-529.
38. Baines D,Downer R G H. 5-Hydroxytryptamine-sensitive adenylate cyclase affects phagocytosis in cockroach hemocytes. Arch. Insect Biochem. Physiol., 1992, 21(4): 303-316.
39. Baines D,Downer R G H. Octopamine enhances phagocytosis in cockroach hemocytes: involvement of inositol trisphosphate. Arch. insect biochem.physiol., 1994,26(4):249-261.
40. Broome J R, Sikorowski P P,Norment B R. A mechanism of pathogenicity of Beauveria bassiana on larvae of the imported fire ant Solenopsis richteri. J. Invertebr. Pathol. 1976, 28:87-91.
41. Chain B M, Leyshon-Soland K, Siva-Jothy M T. Haemocyte heterogeneity in the cockroach Periplaneta americanna analyzed using monoclonal antibodies. J. Cell Sci., 1992, 103: 1261-1267.
42. Cleonor da Silvaa, Gary B, Dunphyb M E. Interaction of hemocytes and prophenoloxidase system of fifth instar nymphs of Acheta domesticus with bacteria. Dev. Comp. Immunol., 2000, 24(4):367-379.
43. Cochran D G. Monitor for insecticide risistance in fieldcollected strains of the German
cockroach. J.Econ.Entomol,1989,82(2):336.
44. Fearon D T. Seeking wisdom in innate immunity. Nat.,1997,388:323-324.
45. Fehlbaum P,Bulet P, Chernysh S. Structureactivity analysis of thanatin, a 21-residue inducible ingsect defense piptide with sequence homology to froh skin antimicrobial piptides. Proc. Natl. Acad. Sci. USA. 1996, 93:1221-25.
46. Fisher C W, Brady U E. Activation, properties and collection of haemolymph phenoloxidase of the Americann cockroach, Periplaneta americanna. Comp. Biochem. Physiol., 1983, 75(1): 111-114.
47. Garry B T. Studies on the cellular immune responses of insects toward the insect pathogen Trypanosoma rangeli. J. Invertebr. Pathol.,1988,51:64-72.
48. Gunnarsson S G S.Hemocytis aggregation in schistocerca gregaria and Periplaneta americanna as a response to injected substances of microbial origin. J. Invertebr. Pathol. 1985,46:312-319.
49. Han Y S, Chnn J, Schwartz A, Nelson S, Paskewitz S M. Induction of mosquito hemolymph proteins in response to immune challenge and wounding. Dev. Comp. Immunol., 1999, 23: 553-562
50. Hemingway J, Small G J, Mono A G. Possible mechanisms of organophosphorus and carbamate insecticide resistance in German Cockroaches from different geographical areas. J. Econ. Entomol., 1993,86(6):1623.
51. Hilary J D,Norma A R, Charles R H, Anna A, Kenneth S.Purification of the pro-phenol oxidase enzyme from haemocytes of the cockroach Blaberus discoidalis. Biochem. J., 1993, 289: 87-91.
52. Horohov D W, Dunn P E. Changes in the circulating hemotyte population of Manducasexta lavae following injection of bacteria. J. Invertebr. Pathol., 1982, 40: 327-339.
53. Hung H S, Hung J C, Cheng H H, Jen C C, Yen L S. Phenoloxidase Activity of hemocytes derived from penaeus monodonand Macrobrachium rosenbergii, J. Invertebr. Pathol., 1998, 71(1):26-33.
54. Hung S H, Boucias D G. Influence of Beauveria bassiana on the cellular defense response of the beet armyworm, Spodoptera exigua. J. invertebr. Pathol., 1992,60: 152-158.
55. Huxham I M, Lackie A M, McCorkindale N J. Inhibitory effects of cyclodepsi peptides, destruxins, from the fungus Metarhizium anisopliae, on cellular immunity in insects. J. Insect Physiol., 1989, 35(2):97-105.
56. Jeremy P, Gillespie, Claire Burnett, Charnley A K. The immune response of the desert locust Schistocerca gregaria during mycosis of the entomopathogenic fungus, Metarhizium anisopliae var acridum. J. Insect Physiol., 2000,46:429-437.
57. Jomori T, Kubo T, Natori S. Purification and characterization of lipopolysaccharide-binding protein from hemolymph of Americarm cockroach Periplaneta arnericanna. European J. Biochem./FEBS.,1990,190(1):201-206.
58. Jones J C. Current conception concerning insect hemocytes. Amer. Zoologist., 1962, 2(1): 209-246.
59. Kaakeh W, Reid B L, Bennett G W. Horizontal transmission of the entomopatho-genic fungus Metarhizium anisopliae (imperfect fungi: Hyphomycetes) and hydra-methylnon among German cockroaches (Dictyoptera: Blattellidae). J. Entomological Sci., 1996, 31(4):378-390.
60. Kaakeh W, Reid B L, Bohnert T J, Bennett G W. Toxicity of imidacloprid in the German cockroach (Dictyoptera: Blattellidae), and the synergism between imidacloprid and Metarhizium anisopliae (imperfect fungi: Hyphomycetes). J. Econ. Entornol., 1997, 90(2): 473-482.
61. Lackie A M, Takle, G; Tetley, L. Haemocytic encapsulation in the locust Schistocerca gregaria (Orthoptera) and in the cockroach Periplaneta arnericanna (Dictyoptera). Cell Tissue Res., 1985, 240(2): 343-351.
62. Lackie A M. Haemocyte behaviour, Adv. Insect Physiol., 1988, 21:85-178.
63. Lavine M D, Strand M R. Insect hemocytes and their role in immunity. Insect Biochem. Mol. Biol., 2002,32:1295-1309.
64. Lavine M D, Strand M R. Surface characteristics of foreign targets that elicit an encapsulation response by the moth Pseudoplusia includens. J. Insect Physiol., 2001,47:965-974.
65. Lebestky T, Chang T, Hartenstein V, Banerjee U. Specification of Drosophila hematopoietic lineage by conserved transcription factors. Sci., 2000,288:146-149.
66. Li D, Scherfer C, Korayem A M, Zhao Z, Schmidt O, Theopold U. Insect hemolymph clotting: evidence for interaction between the coagulation system and the prophenoloxidase activating cascade. Insect Biochem. Mol. Biol., 2002,32: 919-928.
67. Ludek Z, Watson D W, Schall C. Synergism between Metarhizium anisopliae (Deuteromycota: Hyphomycetes) and Boric Acid against the German cockroach (Dictyoptera: Blattellidae). Biol. Control., 2002,23:296-302.
68. Martin F R, Nolan R A. Americann cockroach (Periplaneta americanna) hemocyte response to Entornophaga aulicae protoplasts. Can. J. Zool., 1986,64(6): 1369-1372.
69. Mazet I, Boucias D G. Effects of the fungal pathogen, Beauveria bassiana on protein biosynthesis of infected Spodoptera exigua larvae. J. Insect Physiol., 1996,42(2): 91-99.
70. McDonald D. Cockroaches and mosquitoes-the most serious urban insect pests. Intl. Pest Control., 1996, 38(6): 185-188.
71. Michael J, Bidochkaa L, Raymond J, St. Legera, Donald W. Induction of novel proteins in Manduca Sexta and Blaberus giganteusas a response to fungal challenge, J. Invertebr. Pathol. 1997, 70(3): 184-189.
72. Micheal J B, George G K. purification and properties of an extracellular protease produced by the entomopathogenic fungus Beauveria bassiana., Appl. environ.l microbiol., 1987, 1679-1684.
73. Pachamuthu P, Kamble S T. In vivo study on combined toxicity of Metarhizium anisopliae (Deuteromycotina: Hyphomycetes) strain ESC-1 with sublethal doses of chlorpyfifos, propetamphos, and cyfluthrin against German cockroach (Diptyoptera:Blattellidae). J. Econ. entomol., 2000,93(1):60-70.
74. Pal R, Brown A W A. Problems of insecticide resistance. Zeitschrifi fur Parasitenkunde., (Abstract). 1974, 45(2):211-221.
75. Paul S,Khan A M, Baqui MA. Evaluation of the common cockroach Periplaneta americanna (L.) as carrier of medically important bacteria. Muhibullah M J. Communicable Diseases., (Abstract) 1992, 24(4):206-210.
76. Pech L L, Strand M R. Granular cells are required for encapsulation of foreign targets by insect haemocytes. J. Cell Sci., 1996,109:2053-2060.
77. Pech L L, Strand M R. Plasmatocytes from the moth Pseudoplusia includens induce apoptosis of granular cells. J. Insect Physiol., 2000, 46: 1565-1573.
78. Pendland J C, hung S Y. Boucias D G. Evasion of host defence by in vivo-produced protoplast-like cells of the insect mycopathogen Beauveria bassiana. J. bacteriol., 1993, 175: 5962-5969.
79. Phillipe B, Charles H, Jean Luc Dimarcq, Danie le Homann. Antimicrobial peptides in insects; structure and function. Dev. Comp. Immunol., 1999,23: 329-344.
80. Rahmet Alla M,Rowley A F, Alia M Rahmet. Studies on the cellular defense reaction of the Madeira cockroach, Leucophaea maderae: nodule formation in response to injected bacteria. J. Invertebr. Pathol., 1989, 54(2):200-207.
81. Rahmet Alla M,Rowley A F. Studies on the cellular defense reactions of the Madeira cockroach, Leucophaea maderae: in vitro phagocytosis of different strains of Bacillus cereus and their effect on hemocyte viability. J. Invertebr. Pathol., 1990, 55(3):350-356.
82. Rahmet Alla M,Rowley A F. Studies on the cellular defense reaction of the Madeira cockroach, Leucophaea maderae: nodule formation in response to injected bacteria. J. Invertebr. Pathol. 1989, 54(2): 200-207.
83. Ryan M, Nicholas W L. The reaction of the cockroach Periplaneta americanna to the injection of foreign particulate material. J. Invertebr. Pathol., 1972, 19(3):299-307;
84. Ryan N A, Karp R D. Stimulation of hemocyte proliferation in the Americann cockroach (Periplaneta americanna) by the injection of Enterobacter cloacae, J. Insect Physiol. 1993, 39(7):601-608.
85. Seamen G, Robert N L. Immunological response of male cockroaches to infection of Tetrahymena pyriformis. Sci. 1968,161:1359-1361.
86. Shelby K S. Webb B A. Polydnaverus infection inhibits synthesis of an insect plasma protein, arylphorin, J. Gen. Virol. 1994,75:2285-2292.
87. Sophia E, Dan H. Drosophila cecropin as an antifungal agent. Insect Biochem. Mol. Biol., 1999, 29:965-972.
88. Steenberg T, Vagn Jensen K M, Jensen K M V, Smits P H. Entomopathogenic fungi for control of German cockroach (Blattella germanica) and other synanthropic cockroaches. Bulletin OILB SROP., 1998, 21(4):145-150.
89. Strand M R, Pech L L. Immunological basis for compatibility in parasitoid-host relationships. Annu. Rev. Entomol., 1995,40:31-56.
90. Strand M R., Clark K D. Plasmatocyte spreading peptide induces spreading of plasmatocytes but represses spreading of granulocytes. Arch. Insect Biochem. Physiol., 1999,42:213-223.
91. Wheeler M B, Stuart G S, Hapner K D. Agglutin mediated opsonization of fungal blastospores in belanoplus differentialis. J. Insect. Physiol., 1993,39(6):477-483.
92. Wilson R, Chen C W, Ratcliffe N A, Chen C W. Innate immunity in insects: the role of multiple, endogenous serum lectins in the recognition of foreign invaders in the cockroach, Blaberus discoidalis. J. Immunol. Baltim., 1999, 162(3): 1590-1596.
93. Wilson R, Ratcliffe N A. Effect of lysozyme on the lectin-mediated phagocytosis of Bacillus cereus by haemocytes of the cockroach, Blaberus discoidalis. J. Insect Physiol., 2000,46(5): 663-670.
94. Witting. Phagocytosis by blood cells in healthy and disease dcaterpillards. I. phatocytosis of Bacilus thuringiensis Berliner in Pseudale tiarnipuncta (Haworth). J. Invertebr. Pathol., 1965,7: 474-488.
95. Zukowski K, Bajan C, Popowska Nowak E. The effect of infestation by mixed culture of Beauveria bassiana and Poecilomycess farinosus [sic] on reduction in numbers of experimental cultures of Blatella germanica L. Roczniki Panstwowego Zakladu Higieny. (Abstract).1999, 50(3):321-327.