【作者】 张倩倩；

【导师】 叶恭银；

【作者基本信息】 浙江大学 ， 农业昆虫与害虫防治， 2009， 博士

【摘要】 蝶蛹金小蜂Pteromalus puparum是菜粉蝶Pieris rapae蛹期优势内寄生蜂，该蜂仅以毒液抑制寄主免疫。本论文就蝶蛹金小蜂寄生对寄主细胞免疫与生理代谢的影响进行了研究。结果概述如下：1．蝶蛹金小蜂寄生对菜粉蝶蛹血细胞的影响光学显微镜观察结果表明，受蝶蛹金小蜂寄生影响最大的是寄主颗粒血细胞与浆血细胞。寄生后这两种血细胞变圆，粘附与延展行为受到抑制，而其它类型血细胞形态上未受影响。然而透射电镜观察结果表明，除囊血细胞外，蝶蛹金小蜂寄生对菜粉蝶蛹各类型血细胞均造成不同程度的损伤。2．蝶蛹金小蜂毒液对菜粉蝶蛹血细胞包囊与吞噬反应的影响采用尼龙毛法分离纯化了菜粉蝶蛹颗粒血细胞与浆血细胞。研究了毒液对菜粉蝶蛹包囊反应关键步骤及颗粒血细胞与浆血细胞吞噬反应的影响。结果表明血细胞数量、毒液浓度均显著影响包囊指数。菜粉蝶蛹体外包囊反应试验中，包囊反应的第一步为颗粒血细胞粘附在异物上，毒液的存在虽会减少粘附在异物上的颗粒血细胞数量，但不会完全阻止包囊反应第一步的发生。但毒液的存在能使包囊反应第二步浆血细胞的募集阶段无法进行，导致包囊反应第三步即颗粒血细胞粘附在包囊最外层的浆血细胞上，形成包囊的外围无法实现，最终导致无法形成完整的包囊。菜粉蝶蛹血淋巴浆质对包囊指数无显著影响。菜粉蝶蛹颗粒血细胞是主要具有吞噬能力的血细胞，浆血细胞多少也能吞噬大肠杆菌，毒液能显著抑制颗粒血细胞与浆血细胞吞噬能力，并且这种抑制能力与毒液的剂量正相关。3．蝶蛹金小蜂寄生对菜粉蝶蛹血淋巴和脂肪体糖代谢的影响研究了蝶蛹金小蜂寄生24 h，36 h，48 h，60 h和72 h后菜粉蝶蛹血淋巴与脂肪体糖代谢功能。结果表明，经蝶蛹金小蜂寄生的菜粉蝶蛹血淋巴与脂肪体中丙酮酸含量显著降低，乳酸含量显著升高，乳酸脱氢酶活性显著降低，表明进入三羧酸循环的丙酮酸量减少。苹果酸脱氢酶与琥珀酸脱氢酶是线粒体中参与三羧酸循环的关键酶。在被蝶蛹金小蜂寄生的初始阶段，菜粉蝶蛹血淋巴与脂肪体苹果酸脱氢酶活性升高，随着寄生时间的延长，这两种酶活性被显著抑制，说明寄生导致菜粉蝶蛹血淋巴与脂肪体呼吸率降低，菜粉蝶蛹设法从无氧糖酵解获得能量以抵御毒液所带来的压力。4．蝶蛹金小蜂寄生对菜粉蝶蛹抗氧化酶活性与抗氧化剂水平的影响寄主昆虫在防御寄生蜂等入侵异物时产生氧胁迫，释放对细胞有毒的活性氧（ROS）。测定了蝶蛹金小蜂寄生24 h，36 h，48 h，60 h和72 h后菜粉蝶蛹血淋巴及脂肪体与氧胁迫相关的抗氧化酶活性与抗氧化剂水平。蝶蛹金小蜂寄生导致菜粉蝶蛹脂肪体超氧化物歧化酶与血淋巴过氧化氢酶活性及血淋巴与脂肪体谷胱甘肽-S-转移酶活性显著下降，说明由于寄生而注入的毒液及菜粉蝶蛹随之产生的大量ROS钝化了上述抗氧化酶活性；而菜粉蝶蛹血淋巴与脂肪体谷胱甘肽还原酶活性与还原型谷胱甘肽水平在整个试验中与对照基本无显著差异，表明寄生并未诱导谷胱甘肽还原酶与还原型谷胱甘肽产生对抗防卫。5．蝶蛹金小蜂寄生对菜粉蝶蛹质膜上磷酸酶与转氨酶活性的影响测定了蝶蛹金小蜂寄生24 h，36 h，48 h，60 h和72 h后菜粉蝶蛹血淋巴与脂肪体结合在质膜上磷酸酶与转氨酶活性。经蝶蛹金小蜂寄后生菜粉蝶蛹血淋巴与脂肪体结合在质膜上的Na⁺／K⁺／ATPase，Ca²⁺／Mg²⁺／ATPase以及总ATPase活性显著降低，丙氨酸转氨酶与天冬氨酸转氨酶活性显著高于对照。这说明经蝶蛹金小蜂寄后生菜粉蝶蛹血细胞与脂肪体细胞质膜流动性降低，并且上述组织可能已产生大范围损伤。更多还原

【Abstract】 Pteromalus puparum is a key important pupal-specific endoparasitoid of Pieris rapae,which has evolved a unique means to suppress the host’s immune system, as no otherparasitoid-associated virulence factors other than venom is found in the femalereproductive organ. The effects of parasitization by P. puparum on host cellular immuneresponses and physiological metabolism were studied. The results were summarized asfollows:1. Hemocytes characterization of P. rapae parasitized by P.puparumThe hemocytes morphology of P. rupae responed to parasitization by P. puparumwas investigated using light microscope and transmission electron microscope. Underlight microscope, the main influence of parasitization was observed on granulocytes andplasmatocytes. Light microscope showed that most of the granulocytes andplasmatocytes turned rounded, and their adhesion and spreading behavior was inhibited,while other types of hemocytes remained unaffected. Under transmission electronmicroscope, parasitization had a differential effect on host hemocytes. Prohemocytes,granulocytes, plasmatocytes and oenocytoids were all damaged to various extents.However, no changing was observed in coagulocytes.2. The effeets of venom on the encapsulation and phagoeytie reactionsin P. rapaeUsing monolayers of plasmatocytes and granulocytes prepared from P. rapae pupaehaemolymph by lylon wool cell fractionation method, we investigated the venom on thekey process of capsule formation and phagocytic reactions of plasmatocytes andgranulocytes in vitro in P. rapae. The results showed that both hemocyte number andvenom concentration affected the encapsulation index. In this study, Although venom reduced the number of granulocytes attached to sephadex A-50 beads, it could notinhibited the first step process that granulocytes attached to beads. However, venominhibited the attachment of multiple layers of plasmatocytes, which led to thetermination of capsule formation could not occured. In the present study wedemonstrated that in vitro, both granular cells and plasmatocytes are involved inphagocytic reaction. However, granulocytes were the main phagocytic hemocytes.Venom inhibited both plasmatocytes and granulocytes phagocytic ability, and thisinhibition showed a positive correlation with venom concentration.3 The effects of parasitization by P. puparum on the carbohydratemetabolism in hemolymph and fat body of P. rapaeEffects of parasitization on the carbohydrate metabolism of hemolymph and fat bodyof P. rapae pupae on hours 24, 36, 48, 60 and 72 were investigated. The results showedthat pyruvate level and lactate dehydrogenase activity decreased with elevated lactatelevels, indicating reduced mobilization of pyruvate into the Krebs cycle. Enzymes ofKrebs cycle, namely succinate dehydrogenase and malate dehydrogenase, weresignificantly lowered, suggesting a decrease in respiration rate at the tissue level. Theseresults suggest that parasitization affect the carbohydrate metabolism in P. rapae andconsequently alter their metabolic functions to meet the required energy demands underthe parasitized stress conditions.4 The effects of parasitization by P. puparum on the activity ofantioxidant enzymes and levels of antioxidant in P. rapaeParasitization causes oxidative stress by the release of reactive oxygen species （ROS）that are toxic to the cells. The antioxidant enzymes associated with oxidative stressduring the process of parasitization in hemolymph and fat body of P. rapae werequantitatively determined at different time intervals post parasitization （24, 36, 48, 60and 72 h after parasitization）. The activities of superoxide dismutase in fat body,catalase in hemolymph and glutathione-S-transferase both in hemolymph and fat body,showed significant reductions compared to the control ones. These results showed thatthe venom injected into P. rapae followed parasitization together with ROS released by P. rapae inactivated the antioxidant enzymes. While glutathione reductase and reducedglutathione showed no differences to the controls indicated that parasitization did notinduce glutathione reductase and reduced glutathione to defend against parasitization.5 The effects of parasitization by P. puparum on activities of membranebound phosphatases and transaminases in P. rapaeThe activities of membrane bound phosphatases and transaminases were analyzed inhemolymph and fat body of P. rapae parasitized by P. puparum at different timeintervals post parasitization （24, 36, 48, 60 and 72 h after parasitization）. Our resultsshowed that the activities of membrane bound phosphatases (Na⁺ K⁺ ATPase,C^a2+ATPase, Mg²⁺ ATPase and Total ATPase) in P. rapae parasitized by P. puparumsignificantly reduced compared to these control ones, which indicated that parasitizationcauses the decrease of membrane fluidity of host hemocytes and fat body cells. Theactivities of transaminases （alanine transaminase （ALT） and aspartate transaminase（AST）） were significantly elevated. This may be due to the tissues were large rangedamaged.更多还原