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1、河北师范大学 硕士学位论文 长角血蜱卵黄发生的激素调控 姓名：杨晓红 申请学位级别：硕士 专业：动物学 指导教师：刘敬泽 20100401 III 摘 要 蜱类是广泛分布的专性吸血的外寄生物，由其传播的人畜疾病非常多，对人类及畜 牧业发展危害极大。蜱类的繁殖力极强，卵黄发生及其激素调控，是该领域研究的热点 方向，为蜱类生物防治提供了坚实的理论基础。 本研究以长角血蜱 Haemaphysalis longicornis 为研究对象，研究了其卵巢发育过程 中形态及蛋白质含量变化，并利用本实验室制备的卵黄蛋白单克隆抗体，应用 ELISA 技术，研究了 20E (20-hydroxyecdysone)
2、 和 JH III（juvenile hormone III）两种激素对长 角血蜱卵黄发生过程的调控作用。主要结果如下： 体视显微镜下观察卵巢发育特征，表明随着吸血的进行，卵巢也随之发生显著的变 化。交配期卵巢非常细，呈白色。饱血后卵巢变粗变长。饱血后 34 天，卵细胞快速 发育，卵巢增大，卵母细胞呈棕红色，卵黄粒出现。饱血后 5 天开始产卵，卵巢外观与 饱血后 4 天相似，但是在卵巢腔和输卵管中可见正在排出的卵细胞。产卵后 8 天，卵巢 开始萎缩，变细变白，只剩下很少的未排出的卵细胞。同时，卵巢重量也发生明显变化， 交配期卵巢非常轻（0.867mg），到产卵当天卵巢达到最大重量 30.540
3、mg，之后开始下 降，到产卵后 8 天卵巢重量降到最低 3.667mg。相应地，到产卵后 2 天卵巢重量占体重 比例达到最大值 0.225%，之后慢慢下降，产卵后 8 天降到 0.041%。交配期卵巢总蛋白 含量很低（33.504mg），到产卵当天达到最大值 4069.000mg，到产卵后 10 天蛋白含量 降到 72.455mg。 对吸血6天未交配雌蜱注射20E和JH III， 20E和JH III的最大注射剂量为10g/tick。 向 饱血当天雌蜱注射20E，最大注射剂量为10g/g bw，局部点滴JH III，最大施加剂量为 100g/g bw。向半饱血未交配雌蜱注射激素4天后，解剖虫体
4、，体视镜下观察卵巢形态， 测量卵细胞长度，称量卵巢重量，并应用ELISA法检测血淋巴、卵巢、脂肪体组织中卵 黄原蛋白和卵黄蛋白含量。研究结果表明，注射剂量为10g/tick 20E和10g/tick JH III 的蜱于注射后4天全部死亡。注射剂量为2g/tick 20E蜱卵巢被充满了卵黄颗粒的卵细胞 充满，卵巢体积较大，整体呈现棕黄色，而注射对照卵巢体积极小，呈白色半透明状。 20E注射剂量为2g/tick的蜱卵巢重量占体重比例，脂肪体、血淋巴和卵巢中卵黄原蛋白 白含量和卵黄原蛋白含量与对照组相比较显著增高。而JH III对半饱血未交配长角血蜱 卵巢重量、脂肪体、血淋巴和卵巢中卵黄含量和卵黄
5、原蛋白含量无影响。对饱血当天雌 蜱注射20E和局部施加JH III，4天后称量其卵巢重量，ELISA法检测脂肪体、血淋巴、 IV 卵巢组织中卵黄原蛋白和卵黄蛋白含量，结果为激素施加组与对照组相比较无显著差 异。因此得出结论，20E显著促进了半饱血未交配雌蜱的卵巢发育，刺激了卵黄发生过 程，但是JH III对半饱血未交配雌蜱卵黄发生无影响。20E和JH III对饱血期长角血蜱卵 黄发生均无影响。 关键词：长角血蜱，卵黄蛋白，卵黄原蛋白，卵黄发生，脂肪体，血淋巴，激素 V Abstract Ticks are blood sucking arthropods and they are among
6、the most important vectors of human and animal diseases. They have a super reproductive ability. Vitellogenesis and hormonal regulation are the hot topics, and they provide the theories for biological control of ticks. The morphological structure, weight and protein content of ovary in different dev
7、elopmental stages of female Haemaphysalis longicornis were investigated and the effect of 20E and JH III on vitellogenesis and Vg uptake into oocytes was tested by indirect double antibody ELISA in the current study. The main results were as follows: The morphological structure, weight and protein c
8、ontent of ovary in different developmental stages of female H. longicornis were investigated. Ovary was very thin and translucent white in hue through observing with a stereoscopic microscope at the mating stage, and became longer and thicker between days 0 and 2 post-engorgement. After considerable
9、 growth occurred to the ovary, it was completely covered with oocytes which were large, yolk-filled and reddish-brown. The ovary with its numerous bulging oocytes resembled an elongated cluster of grapes. This phase began at approximately day 4 post-engorgement and ended when oviposition started. Ov
10、iposition began at about day 5 post-engorgement; the ovary morphology was similar to that of day 4 post-engorgement, except that ovulated oocytes were visible in the lumen of the ovary and the oviducts. The number of the oocyte became much less and ovary became thiner and whiter obviously in hue aft
11、er day 8 post-oviposition. Oviposition ended at about day 10 post-oviposition. Correspondly, the ovary weight was very low (0.867mg/tick) at the mating stage and then increased greatly after day 2 post-engorgement. Ovary reached the highest weight (30.540mg/tick) on the day of oviposition, afterward
12、s, its weight decreased gradually. On day 8 post-oviposition, the ovary weight decreased to the lowest 3.667mg/tick. The ovary weight reached the highest percent (0.225%) of body weight (bw) on the day 2 post-oviposition and then decreased to 0.041% bw on day 8 post-oviposition. The protein content
13、was low (33.504mg/tick) at the mating stage and then reached the peak (4069.000mg/tick) on the day of oviposition. Then it decreased slowly after the day of oviposition. The protein content declined to 72.455mg/tick at day 10 post-oviposition. Partially fed virgin female adults of H. longicornis tic
14、ks were injected with 20E (up to 10g/tick), JH III (up to 10g/tick), while they were attached and feeding on the rabbit host for 6 days, engorged mated ticks were also injected with 20E (up to 10g/g bw) and topical VI applied with JH III (up to 100g/g bw) on the day of engorgement to test the effect
15、 of 20E and JH III on vitellogenesis. Ticks on-host were examined 4d after injection. All of the ticks (20E; 10g/tick) and (JH III; 10g/tick) were died 2d after injection. Ovary (20E; 2g/tick) was completely covered with oocytes which were large, yolk-filled and reddish-brown by observing with a ste
16、reoscopic microscope. However, Ovary of vehicle-injected tick was very thin and translucent white in hue. Ovary weight was significantly increaser in ticks treated with 20E compared to vehicle-injected controls. There was also a significant effect of 2g/tick 20E on oocyte length compared to vehicle-injected tick. The fat body Vg content , hemolymph Vg concentration and ovary Vn content of ticks injected with 2g/tick 20E is significantly higher than vehicle-injected controls. T
