丽蝇蛹集金小蜂卵巢发育与卵黄发生的时间动态
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摘要
昆虫卵黄发生及其内分泌调控一直是昆虫生殖生理的研究核心之一，至今已就10个目60余种昆虫不同程度开展地了这方面的研究。但是，就膜翅目昆虫而言研究较少，关于膜翅目的寄生蜂则更少，且对寄生蜂是否拥有卵黄蛋白存有争议。迄今为止，就外寄生蜂是否拥有卵黄蛋白尚乏任何研究。为此，本论文就蝇类蛹期外寄生蜂——丽蝇蛹集金小蜂Nasoniavitripennis(膜翅目：金小蜂科)，卵巢发育、卵子发生，卵黄蛋白的分子特性，及卵黄原蛋白的合成、卵巢对卵黄原蛋白的摄取及卵子内卵黄磷蛋白的降解动态作了研究，结果如下：
丽蝇蛹集金小蜂卵巢83％由4对卵巢管组成，17％由3对卵巢管组成。同一根卵巢管中的卵子可分为三级，即1级卵(无卵黄)、2级卵(含有部分卵黄)和3级卵(充满卵黄)。卵巢管从寄生后第10天开始出现(此时，卵巢管是透明的，卵室尚未形成)，1级卵从寄生后第11天开始出现，2级卵从寄生后第12天开始出现，3级卵从羽化后第一天开始出现；喂20％蜂蜜水对卵巢发育有一定促进作用，能显著延长丽蝇蛹集金小蜂的寿命，不喂食或仅喂蒸馏水，其寿命仅4天，而喂食20％蜂蜜水，从第11天开始出现死亡，大部分可存活15天，另有少部分生存达一个月之久。同一种喂食处理中，卵巢长度的时间动态几乎都无变化；1级卵子数、2级卵子数和成熟卵子数的变化，是抛物线变化趋势，即先增加，然后减少；基部卵子短径和长径也是抛物线变化趋势。
PAGE电泳结果表明：丽蝇蛹集金小蜂明显存在2条雌特异性带-卵黄蛋白，分子量分别为181 KD和136 KD；SDS-PAGE电泳分析：存在3条雌特异性带，其分子量为123 KD、120 KD和91 KD，由此，可推定卵黄原蛋白(vitellogenin，Vg)和卵黄磷蛋白(vitellin，Vn)由2个蛋白组成，其中分子量较大的蛋白由2个亚基组成；双向电泳结果显示，在120KD附近有两个特异性点，其等电点为5.5和5.7；双扩散表明，丽蝇蛹集金小蜂卵黄磷蛋白的抗血清与雌隐成虫虫体、脂肪体、血淋巴和卵巢匀浆液均有免疫沉淀反应，而与雄蜂血淋巴无免疫反应，说明了Vg与Vn具有免疫同源性，是雌特异性蛋白，且由脂肪体合成。
采用ELISA测定法检测了丽蝇蛹集金小蜂卵黄原蛋白合成与摄取，及卵黄磷蛋白降解的时间动态。结果表明，该蜂卵黄原蛋白的合成与摄取分别起始于寄生后的第10 d和第13 d，即正处于该蜂的蛹初期和隐成虫期。该蜂卵内的卵黄磷蛋白随其胚胎发育过程而降解，寄生后第3 d，即胚胎发育结束时则全部降解完毕。此外，尚探讨了卵黄蛋白合成、摄取和降解过程中，血淋巴、卵巢或卵内可溶性蛋白含量的时间动态变化。
Vitellogenesis and its endocrine regulation in insects are always the key problems of insect reproductive physiology, which had been researched in about 60 species of 10 orders. However, they have been studied in few species of Hymenoptera parasitoids, especially, were undefined in ectoparasitic wasps. In this paper, the ovarian development, oogenesis, vitellogenin molecular characteristic, vitellogenin synthesis and uptake, and vitellin degradation in Nasonia vitripennis (Hymenoptera: Pteromalidae), are studied. The detailed results are shown as follows:
83% of ovaries were found to be composed of eight ovarioles with four on each side and 17% of ones to be composed of six ovarioles with three on each side. Ovarioles appeared on the 10th day after parasitization, which were transpearant and had no egg chambers. On the same ovariole, the development of egg chambers could be divided into three grades, namely, the 1st grade was yolkless, the 2nd was imcomplete with yolk protein, and the 3rd was full with yolk protein. Egg chambers with the 1st grade appeared on the 11th day after parasitization (the pupa was white, and its eyes were scarlet), egg chambers with the 2nd grade appeared on the 12th day after parasitization ad egg chambers with the 3rd grade occurred on the 1st day after emergence. Feeding adults of this parasitoid with 20% honey solution showed no marked effects on the ovarian development except significantly prolonging their life span (most adults can survive for half a month and even to about a month). In contrast, feeding with nothing or only wa
ter, the wasps only survived for four days. No difference in ovariole lengh of female adults was found among different feedig treatments. For each fedding treatment, the longitude and latitude of the root egg, the number of egg chambers with each developmental grade all changed in a parabola model.
The results from native PAGE indicated that there were two specific protein i.e. vitellogenin (Vg) or vitellin (Vn) in the female wasp of N. vitripennis. The Vg or Vn was composed of two proteins with molecular weight of 181 KD and 136 KD. The results from SDS-PAGE presented that there were three female specific protein subunits with molecular weights of 123 KD, 120 KD and 91 KD, respectively. We can conclude the higher molecular compose of two subunits; The results from two dimension electrophoresis showed the isoelectric points of two female-specific
spots with molecular weight of about 120KD were 5.5 and 5.7. Immunodiffusion reactions demonstrated that Vg existed both in female fat body and hemolymph, which as Vn was deposited in the ovary, while not in the male.
Temporal patterns of vitellogenin (Vg) synthesis and uptake as well as vitellin (Vn) degradation in N. vitripennis, were studied using the method of enzyme-linked immunosorbent assay (ELISA). The results were showed as follows: the synthesis and uptake of Vg of this parasitic wasp respectively initiated since the 10th and 13th day after parasitism, which were equivalent to the first pupal stage and the pharate adult stage of this parasitic wasp. The degradation of Vn in eggs of this parasitic wasp was simultaneous with their embryo development in their hosts. The Vn thoroughly degraded on the 3rd day after parasitism, just being at the end of embryo development of this parasitic wasp. In addition, the temporal pattern in the levels of soluble proteins in hemolymph, ovaries or eggs of this parasitic wasp was also defined during the processes of Vg synthesis and uptake as well as Vn degradation.
丽蝇蛹集金小蜂卵巢83％由4对卵巢管组成，17％由3对卵巢管组成。同一根卵巢管中的卵子可分为三级，即1级卵(无卵黄)、2级卵(含有部分卵黄)和3级卵(充满卵黄)。卵巢管从寄生后第10天开始出现(此时，卵巢管是透明的，卵室尚未形成)，1级卵从寄生后第11天开始出现，2级卵从寄生后第12天开始出现，3级卵从羽化后第一天开始出现；喂20％蜂蜜水对卵巢发育有一定促进作用，能显著延长丽蝇蛹集金小蜂的寿命，不喂食或仅喂蒸馏水，其寿命仅4天，而喂食20％蜂蜜水，从第11天开始出现死亡，大部分可存活15天，另有少部分生存达一个月之久。同一种喂食处理中，卵巢长度的时间动态几乎都无变化；1级卵子数、2级卵子数和成熟卵子数的变化，是抛物线变化趋势，即先增加，然后减少；基部卵子短径和长径也是抛物线变化趋势。
PAGE电泳结果表明：丽蝇蛹集金小蜂明显存在2条雌特异性带-卵黄蛋白，分子量分别为181 KD和136 KD；SDS-PAGE电泳分析：存在3条雌特异性带，其分子量为123 KD、120 KD和91 KD，由此，可推定卵黄原蛋白(vitellogenin，Vg)和卵黄磷蛋白(vitellin，Vn)由2个蛋白组成，其中分子量较大的蛋白由2个亚基组成；双向电泳结果显示，在120KD附近有两个特异性点，其等电点为5.5和5.7；双扩散表明，丽蝇蛹集金小蜂卵黄磷蛋白的抗血清与雌隐成虫虫体、脂肪体、血淋巴和卵巢匀浆液均有免疫沉淀反应，而与雄蜂血淋巴无免疫反应，说明了Vg与Vn具有免疫同源性，是雌特异性蛋白，且由脂肪体合成。
采用ELISA测定法检测了丽蝇蛹集金小蜂卵黄原蛋白合成与摄取，及卵黄磷蛋白降解的时间动态。结果表明，该蜂卵黄原蛋白的合成与摄取分别起始于寄生后的第10 d和第13 d，即正处于该蜂的蛹初期和隐成虫期。该蜂卵内的卵黄磷蛋白随其胚胎发育过程而降解，寄生后第3 d，即胚胎发育结束时则全部降解完毕。此外，尚探讨了卵黄蛋白合成、摄取和降解过程中，血淋巴、卵巢或卵内可溶性蛋白含量的时间动态变化。
Vitellogenesis and its endocrine regulation in insects are always the key problems of insect reproductive physiology, which had been researched in about 60 species of 10 orders. However, they have been studied in few species of Hymenoptera parasitoids, especially, were undefined in ectoparasitic wasps. In this paper, the ovarian development, oogenesis, vitellogenin molecular characteristic, vitellogenin synthesis and uptake, and vitellin degradation in Nasonia vitripennis (Hymenoptera: Pteromalidae), are studied. The detailed results are shown as follows:
83% of ovaries were found to be composed of eight ovarioles with four on each side and 17% of ones to be composed of six ovarioles with three on each side. Ovarioles appeared on the 10th day after parasitization, which were transpearant and had no egg chambers. On the same ovariole, the development of egg chambers could be divided into three grades, namely, the 1st grade was yolkless, the 2nd was imcomplete with yolk protein, and the 3rd was full with yolk protein. Egg chambers with the 1st grade appeared on the 11th day after parasitization (the pupa was white, and its eyes were scarlet), egg chambers with the 2nd grade appeared on the 12th day after parasitization ad egg chambers with the 3rd grade occurred on the 1st day after emergence. Feeding adults of this parasitoid with 20% honey solution showed no marked effects on the ovarian development except significantly prolonging their life span (most adults can survive for half a month and even to about a month). In contrast, feeding with nothing or only wa
ter, the wasps only survived for four days. No difference in ovariole lengh of female adults was found among different feedig treatments. For each fedding treatment, the longitude and latitude of the root egg, the number of egg chambers with each developmental grade all changed in a parabola model.
The results from native PAGE indicated that there were two specific protein i.e. vitellogenin (Vg) or vitellin (Vn) in the female wasp of N. vitripennis. The Vg or Vn was composed of two proteins with molecular weight of 181 KD and 136 KD. The results from SDS-PAGE presented that there were three female specific protein subunits with molecular weights of 123 KD, 120 KD and 91 KD, respectively. We can conclude the higher molecular compose of two subunits; The results from two dimension electrophoresis showed the isoelectric points of two female-specific
spots with molecular weight of about 120KD were 5.5 and 5.7. Immunodiffusion reactions demonstrated that Vg existed both in female fat body and hemolymph, which as Vn was deposited in the ovary, while not in the male.
Temporal patterns of vitellogenin (Vg) synthesis and uptake as well as vitellin (Vn) degradation in N. vitripennis, were studied using the method of enzyme-linked immunosorbent assay (ELISA). The results were showed as follows: the synthesis and uptake of Vg of this parasitic wasp respectively initiated since the 10th and 13th day after parasitism, which were equivalent to the first pupal stage and the pharate adult stage of this parasitic wasp. The degradation of Vn in eggs of this parasitic wasp was simultaneous with their embryo development in their hosts. The Vn thoroughly degraded on the 3rd day after parasitism, just being at the end of embryo development of this parasitic wasp. In addition, the temporal pattern in the levels of soluble proteins in hemolymph, ovaries or eggs of this parasitic wasp was also defined during the processes of Vg synthesis and uptake as well as Vn degradation.
引文
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