【作者】 杨小龙；

【导师】 刘敬泽；

【作者基本信息】 河北师范大学 ， 动物学， 2003， 硕士

【摘要】 蜱类是重要的媒介动物，传播多种人、畜疾病，造成极大危害。卵黄发生是蜱类生殖的中心内容，是决定产卵和种群数量的关键。因此，对其研究具有重要的理论和实际意义。 本文以长角血蜱Haemaphysalis longicornis Neumann为研究对象，利用电镜技术、Bradford法、SDS-PAGE、分子筛与离子交换层析、局部点滴和体外培养等现代生物学技术对其雌性生殖系统结构与蛋白组成、卵巢结构、蛋白含量与成份及其动态变化、卵黄蛋白的纯化及其部分性质、以及保幼激素和蜕皮激素对卵黄发生的影响进行了较系统的研究，为深入开展蜱类生殖生物学研究提供依据。主要研究结果如下： 1．长角血蜱雌性生殖系统由卵巢、输卵管、阴道、受精囊、附腺和吉氏器组成。与其它硬蜱基本相似，但其输卵管近末端处呈明显的壶腹状，吉氏器的角呈球囊状。雌性生殖系统的蛋白成分复杂，具有多样性，而在各部分结构之间蛋白既有同一性又有特异性。 2．卵巢随发育阶段发生明显变化。饥饿期卵巢呈管状结构，卵母细胞直径小(5.90μm)；吸血初期卵巢明显增大，卵母细胞直径增加(38.70μm)，纵沟外侧出现大量发育的卵母细胞；交配期卵巢继续延长，变粗，部分区段呈回折状，卵母细胞血径缓慢增加(73.33μm)；饱血后，卵巢快速发育，纵沟内外紧密排布大量的卵母细胞，越靠外侧其发育程度越高，卵母细胞直径迅速增加(270.00μm)，为交配期的3.7倍，产卵时卵母细胞直径达到最大值(286.33μm)，卵巢占据了蜱体腔的大部分。 3．卵巢蛋白水平在不同发育阶段呈现明显变化。饥饿期卵巢中蛋白水平低(1.73μg／tick)；吸血后迅速提高(15.60μg／tick)，约为饥饿期的9倍；进入交配期蛋白水平持续增加(26.72μg／tick)；饱血当天和饱血后2d蛋白 如夕l‘万艺论义.!，之摘要含量分别为99.33 pg八i。k和372.49“g八iek:刚产卵时达到址大值(1512.91pg/tiek):产卵Zd以后，蛋白含量降至1184.18pg八iek。 4.饥饿期卵巢中蛋白带少，只有l条主带，吸血初期和交配期明显增多，刚饱血时最多，这三个阶段的一L带基本相同，共巧条主带:饱血以后至产卵期.主带发生变化，相对分子量为97 KD、9一KD、64 KD、 54 KD、53 KD的主带缺失，饱血后Zd、3d、刚产卵和，，’，:卵后Zd四个时期的主带基本相同，有9条。 5.首次确定长角血蟀的卵黄蛋自只有一种，经苏月·黑B和希夫试hlJ染色呈阳性，是一种血.糖脂复合蛋自，山H个亚从组成，亚荃相对分子员分别为1 12 KD、103 KD、80 KD、78 KD、71 KD、68 KD、62 KD和52 KD。 6.保幼激素和蜕皮激素均影响长角血蝉的卵黄发生。局部施用保幼激素类似物法尼醇，10“g和50“g显著提.岛交配期雌虫卵巢中蛋白含员;50 pg使饱血当天雌虫卵巢蛋白含量明显增加，显著提高卵巢中vn的含‘量;剂量大于100“g，呈现毒副作用;法尼醇对卵巢中蛋自的亚基组成没有影响。体外培养饱血当天雌虫的卵巢，培养液中含有不同浓度的20一轻基蜕皮酮(20一E)。1.00 ng/pl的20一E能显著提高卵巢蛋白含量，促进卵巢对卵黄原蛋白的吸收:浓度为5.00 ng/pl时，作用相反，并且2.50 ng/pl和5.00 ng/pl的20一E使vn在电泳中的迁移率发生·定的变化。_!几述结果表明，保幼激素和蜕皮激素共同参与调控蟀类的生殖，均对卵黄发生有作用。 上述研究结果显示，雌性生殖系统由多个部分组成;卵巢的结构、蛋白含量和成分随发育的不同阶段发生一系列动态变化;卵黄蛋白是一种含血红素、搪类、脂类和各种氮基酸的复杂性复合蛋白:在一定剂量范围内，保幼激素和蜕皮激素均对卵黄发生有影响。更多还原

【Abstract】 Ticks are well known vectors transmitting a great variety of infectious pathogens and causing serious damage to human and livestock. In ticks, vitellogenesis is the central events of reproduction and the key of egg production and population size. Thus, researches on vitellogenesis have important significance on the theoretical and practical level.A series of researches were conducted on the reproduction of female Haemaphysalis longicornis Neumann, including the structure and protein components of the genital system, ovary structure, protein concentration and components, as well as their variation during different developmental stages, purification and partial characterization of vitellin, as well as the effects of juvenile hormone and molting hormone on vitellogenesis, using scanning electron microscope (SEM), Bradford method, SDS-PAGE, gel filtration and ion exchange chromatography, topical application and an in vitro culture methods etc. The present research will provide foundation for further investigations on reproductive biology of ticks in the future. The main result were as follows:1. The reproductive system of female H. longicornis consisted of ovary, paired oviducts, vagina, receotaculun seminis, accessory glands and Gene’s organ, which was similar basically to other tick species reported. However, the oviducts were manifestly widened distally as ampul late and the Gene’s organ horns were saccules. The two characterizations were different from other hard ticks. The protein components of female genital system were complexity and biodiversity. However, the proteins of different parts were not only identity but also particularity.2. The ovary changed obviously with the developmental stages. In unfedadults, the ovary was a simple tube-like structure and the oocytes were small (5.90 μm). At the early stage of attachment, the ovary increased obviously and the diameter of oocytes was 38.70μ m, many developing oocytes appeared at the outside of the longitudinal groove. At mating stage, the ovary continue increased and elongated, some folds were apparent in a part of it; the diameter of oocytes increased slowly (73.33 μ m). After engorgement, the ovary developed rapidly, there were lots of oocytes collocated at the inside and outside of the longitudinal groove, oocytes which lied at the outside developed quickly. The diameter of oocytes increased rapidly (270.00μ m), which was about 3.7 times than that of mated female, and reached its maximum value (286.33 P m) at ovipositing stage. During this period, the ovary expanded into the major hemocoele.3 The protein level of the ovary varied obviously during female developmental stages. The concentration of protein was low (1.73 μ g/tick), and increased rapidly at the early stage after attachment (15.60μg/tick), which was about 9 times than that of unfed female. At the mating stage, the protein level continue increased (26.72 μg/tick). After engorgement on dO and d2, the proteins were 99.33 u g/tick and 372.49 μg/tick respectively, and reached its maximum level (1512.91 μg/tick) at ovipositing stage. On d 2 after oviposition, the protein level declined (1184.18μ g/tick).4. In unfed females, only one main ovary protein band was detected. At early feeding and mating stages, protein band multiplied obviously, and maximized on the day of engorgement. Fifteen main protein bands of the three stages were the same. From engorgement to oviposition, the main protein bands changed. Five protein bands were disappeared, the molecular weights of them were 97 KD, 91 KD,64 KD, 54 KD, 53 KD respectively. On d2, d3 after engorgement and d0, d2 after oviposition, the 9 main protein bands were the same.5. The vitellin (Vn) was firstly purified from eggs of H. longicornis, whichwas only one kind of protein. The purified protein could be stained by Black B and periodic acid-ShchifT (PAS), so it was an hcmoglycolipoprotcin. The vitellin was composed of eight polypeptides with the molecular weight of 112, 103, 80, 78, 71, 68,62 and 52 KDa.6. Juvenile更多还原