天蚕卵黄原蛋白的合成、运转与沉积

系统测定了天蚕Antheraea yamamai吐丝结茧至成虫期脂肪体、血淋巴和卵巢中卵黄蛋白和可溶性蛋白总含量的动态变化。结果表明,脂肪体是卵黄原蛋白(Vg)合成场所,Vg合成始于吐丝结茧后第4天;脂肪体、血淋巴中Vg滴度在吐丝结茧后第4天开始上升,化蛹后第6天或第8天达高峰,成虫羽化第1天则明显下降。卵巢对Vg摄取始于化蛹第1天,此后随蛹日龄逐渐上升,并渐趋平稳。同一卵巢管中卵黄蛋白(Vt)含量自顶端至基端随卵室增大而逐渐升高,不同日龄蛹中相应序号卵室的Vt含量以日龄大者为高;卵室中Vt含量与卵室体积大小呈正线性关系。电镜观察表明,Vg被卵母细胞摄入后以卵黄体形式存在,不同发育阶段卵巢中卵母细胞内卵黄体大小不同,以早期者为小;同一卵巢管中不同卵母细胞内卵黄体以顶端为小,基端明显增大,且卵黄体呈网状。     

蝶蛹金小蜂卵黄蛋白单克隆抗体的制备及其应用方法的建立

为深入研究寄生蜂卵黄发生及其内分泌调控,特采用杂交瘤细胞技术,制备4株能稳定分泌抗蝶蛹金小蜂Pteromalus puparum卵黄蛋白（vitellin, Vt）的单克隆抗体（mAb）,即PpVt mAb1,PpVt mAb2,PpVt mAb3和PpVt mAb4。这4株单克隆抗体的重链和轻链的亚类均分别为IgG1和κ类型,不仅特异性识别Vt,而且识别雌蜂血淋巴中卵黄原蛋白（vitellogenin,Vg）,但与雄蜂体液无反应。通过比较4种不同的ELISA方法,确定了微量检测蝶蛹金小蜂体内Vg/Vt的最适ELISA法,即双夹心ELISA法。该方法可用于单头雌蜂体内Vg/Vt的检测,其检测灵敏度为20 ng/mL。用Western 免疫印迹的方法证实了该蜂Vg的合成始于刚羽化的成虫,并在羽化后12～36 h内含量达到高峰。     

天蚕卵黄发生的激素调控

报告了蜕皮激素和保幼激素对天蚕Antheraea yamamai卵黄发生的调控作用。当单独以20－羟基蜕皮酮或保幼激素类似物methoprene处理,以及同时用这两种激素处理天蚕蛹时,蛹期脂肪体和血淋巴中卵黄原蛋白（Vg)含量明显高于对照,即二对Vg的合成起促进作用。然而,卵巢中卵黄蛋白（Vt)含量则因激素种类而异,以保幼激素处理时明显低于对照,以20－羟基蜕皮酮处理则反之,即前抑制卵巢对Vg的摄取,而后则起促进作用。离体培养脂肪体并以激素处理的结果表明,20－羟基蜕皮酮和methoprene均能促进Vg合成,但前作用更。综合考虑上述结果可以认为蜕皮激素对该蚕的卵黄发生起主要调控作用。     

龟纹瓢虫卵黄蛋白的分子特性及发生动态

研究了龟纹瓢虫Propylea japonica (Thunberg) 卵黄蛋白的基本特性以及卵黄发生过程中卵黄蛋白的动态变化。PAGE和SDS-PAGE实验表明,龟纹瓢虫卵黄蛋白分子量为294.81±40.70 kD,并由分子量分别为144.68±0.03 kD和51.23±0.27 kD的两种亚基组成。对卵黄蛋白的氨基酸组成和含量分析发现,其必需氨基酸总量占57.48％,略高于非必需氨基酸,其中谷氨酸（Glu）含量最高,为15.26％;色氨酸（Trp）和蛋氨酸（Met）含量较低,分别为0.50％和0.11％。采用间接竞争ELISA法,系统测定了龟纹瓢虫成虫期脂肪体、血淋巴和卵巢中卵黄蛋白的动态变化,结果表明：脂肪体是卵黄原蛋白合成的场所,卵黄原蛋白的合成始于羽化后第2天;脂肪体、血淋巴和卵巢中卵黄原蛋白的滴度在羽化后第4天开始迅速上升,至成虫期的第8天左右达到高峰期。     

七星瓢虫的卵黄发生:脂肪体与卵巢中卵黄原蛋白的合成与分泌

本文利用[³H]亮氨酸参入及特异性抗体沉淀等方法,研究了七星瓢虫体外培养的脂肪体中卵黄原蛋白合成与分泌的动力学,以及不同发育期脂肪体与卵巢中卵黄原蛋白合成的定量变化。脂肪体中卵黄原蛋白的合成与分泌在培养1—4小时内直线上升,到6小时稍下降。保留在脂肪体内的卵黄原蛋白缓慢积累,但一直水平很低。卵黄原蛋白合成的最初30分钟,分泌速率较慢,60%以上的卵黄原蛋白保留在脂肪体内。1小时后分泌速率加快,70%以上的卵黄原蛋白被分泌,保留的卵黄原蛋白在4小时中逐渐被释放。在4小时,被分泌的卵黄原蛋白超过80%,最高可达92%。 在雌虫发育过程中,脂肪体中卵黄原蛋白合成的高峰在羽化后11—15天,所合成的卵黄原蛋白占整个发育期合成总量的80%。在合成高峰期分泌的卵黄原蛋白高达90%以上,但在发育的早期和晚期分泌的卵黄原蛋白仅占30%或稍多。 卵黄发生前的卵巢就开始合成卵黄原蛋白,但卵巢中卵黄原蛋白的合成高峰期与脂肪体中大致相同。与脂肪体相反,卵巢合成的卵黄原蛋白大部分保留在卵巢内。在卵黄发生盛期,卵巢合成的卵黄原蛋白为脂肪体合成的卵黄原蛋白的20%。     

家蝇的卵黄发生及其激素调节

用5—15%SDS-PAGE分析表明,家蝇Musce domestica viaina卵黄蛋白由三个亚基组成,其亚基分子量分别为58KD、50KD、48KD.火箭免疫电泳的结果表明,脂肪体、血淋巴和卵巢内卵黄原蛋白的变化具有密切的相关性,卵黄原蛋白在体内最早出现在羽化后30小时左右,然后迅速增加,在羽化后48小时,脂肪体和血淋巴中卵黄原蛋白含量达到最大值,卵巢开始沉积卵黄蛋白在羽化后30小时,到产卵前达到最大值,脂肪体在离体培养条件下,通过测定³H-亮氨酸掺入卵黄原蛋白的量,对不同发育时期家蝇脂肪体合成卵黄原蛋白的能力及激素的调节作用进行了研究,结果表明,羽化12小时后,合成能力迅速上升,48小时时形成高峰,60小时后迅速下跌直至产卵,其合成能力一直维持在低水平,产卵后合成能力又迅速回升,激素处理结果表明,保幼激素可以促进卵黄发生前期和后期家蝇脂肪体的卵黄原蛋白合成,20-羟基蜕皮酮可以大幅度促进卵黄发生期家蝇脂肪体的卵黄原蛋白合成.当二种激素共同处理时,对卵黄发生前期和卵黄发生期的家蝇脂肪体有协同促进作用,而对卵黄发生后期的脂肪体没有这种作用.本文还对家蝇卵黄发生过程中脂肪体、血淋巴和卵巢三者之间的关系及家蝇卵黄发生的激素调节进行了讨论.     

蜜蜂卵黄原蛋白的作用

卵黄蛋白不仅为胚胎发生提供营养物质,它在生物体内还具有其他的生物学功能。昆虫卵黄蛋白是昆虫卵内的营养储备,它的前体主要来源于脂肪体的雌性特异血蛋白——卵黄原蛋白(Vg),它是近年来昆虫生理学和生化学最活跃的领域之一,文章介绍蜜蜂卵黄原蛋白在蜜蜂生殖过程中的作用,以及与蜜蜂社会性生活及寿命的关系。     

不同类群寄生蜂卵黄蛋白的检测和免疫相关性分析

为了探讨寄生蜂种类与卵内蛋白质存在与否及免疫相关性, 采用蛋白质电泳和Western免疫印迹的方法对7科12种寄生蜂卵内蛋白进行了检测和分析。结果表明：小蜂科和姬蜂科寄生蜂与其他昆虫一样, 卵内含有分子量约为200 kD的卵黄原蛋白; 而茧蜂科寄生蜂不含有卵黄原蛋白, 但存在分子量为40～80 kD的多肽, 且在所有供试的寄生蜂卵内均存在一个分子量约为62 kD的多肽。     

七星瓢虫的卵黄发生:植入雄虫的卵巢中卵黄原蛋白的合成

为了证实七星瓢虫的卵巢能合成卵黄原蛋白, 并查明雄虫体内是否具有卵黄发生所必需的激素环境, 我们将刚羽化雌虫的一侧卵巢或数个卵巢管植入雄虫体内.移植的卵巢或卵巢管在雄虫体内能够发育, 其卵母细胞能沉积卵黄, 一部分可达成熟.体外培养证明移植的卵巢可合成卵黄原蛋白, 但受体雄虫的脂肪体不合成卵黄原蛋白, 而且其血淋巴中也不存在这种蛋白.用保幼激素类似物ZR-512处理受体雄虫, 可促进移植卵巢的发育, 但不能诱导其脂肪体合成卵黄原蛋白.此结果表明, 象大多数昆虫一样, 七星瓢虫的卵黄发生的性二型现象表现在激素的靶组织——脂肪体, 而不是激素本身.     

意蜂与中蜂血淋巴蛋白质成份的研究

本实验用聚丙烯酰胺凝胶电泳分析了两个蜂种的血淋巴蛋白质成分。意蜂和中蜂都是Apis属,血淋巴蛋白质电泳谱相似。但是,两蜂种以及同一蜂种不同级别、不同发育阶段的电泳谱又各有特点。根据实验,我们把成年蜂血淋巴电泳谱分为11条带,雌蜂电泳谱上的带5是卵黄原蛋白;雄蜂卵黄原蛋白含量很少或测不出。 用Thorun方法,在聚丙烯酰胺凝胶平板电泳上测得意蜂卵黄原蛋白的分子量为185,000。     

ANALYSIS OF THE SOLUBLE PROTEINS IN THREE SPECIES OF PARASITOIDS AND MOLECULAR CHARACTERISTICS OF YOLK PROTEIN IN PTEROMALUS PUPARUM*

Abstract The results both from PAGE and capillary electrophoresis indicated that there was a female specific protein i.e. vitellogenin (Vg) or vitellin (Vt) in the female wasp of Pteromalus puparum (Hymenoptera: Pteromalidae). While there was no difference in the electrophoresis graph between soluble proteins of the female whole body and those of the male one both in two bracoids (Hymenoptera: Braconidae), i.e. Cotesia plutellae and Macrocentrus linears. According to the graph of the gradient SDS‐PAGE, it was clear that the Vg or Vt of P. puparum consisted of two subunits with approximate molecular weights, and their molecular weights were 74.4 and 52.8 KDa, respectively. Both immunological reactions between some main different tissues of the female wasps and the male whole body and the polyantibody against the Vt of this parasitoid, and the graph of the gradient SDS‐PAGE including soluble proteins sampled separately from hemolymph, fat body and ovary of the female and the whole body of the male demonstrated that Vg existed both in female fat body and hemolymph, and Vt deposited in the ovary, not in the male, as well as the Vg was synthesized in the female fat body.     

Vitellin of Pteromalus puparum (Hymenoptera: Pteromalidae), a pupal endoparasitoid of Pieris rapae (Lepidoptera: Pieridae): Biochemical characterization, temporal patterns of production and degradation

Vitellin (Vt) and vitellogenin (Vg) profiles were analyzed in Pteromalus puparum, a pupal endoparasitoid of Pieris rapae. Non-denaturing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses indicated that both native Vt and Vg were likely 370 kDa in size, consisting of two subunits of approximate 206 and 165 kDa. An indirect double antibody enzyme-linked immunosorbent assay (ELISA) for monitoring hemolymph Vg and ovarian Vt levels was developed using a monoclonal antibody and a polyclonal antibody made specially against P. puparum Vt. The synthesis and uptake of Vg in this wasp was initiated immediately after adult eclosion. The hemolymph Vg and ovarian Vt reached their highest level of 0.58 and 4.51 microg per female 24 and 48 h after adult eclosion, respectively. Both Vg synthesis and uptake were in parallel with ovarian development. The Vt levels in the developing embryos decreased progressively except 12h after parasitism. Meanwhile, nine new polypeptides with sizes ranging from 59.2 to 151 kDa, possibly resulting from the limited proteolysis of Vt originally accumulated in newly laid eggs, were detected de-novo during embryonic development using Western blotting with the monoclonal antibody against Vt. These studies provide the basis for future investigation into endocrinal regulations of vitellogenesis and understanding the reproductive strategy in this wasp.     

EFFECT OF A HIGH TEMPERATURE ON VITELLOGE-NESIS IN THE JAPANESE OAK SILKWORM,ANTHERAEA YAMAMAI (LEPIDOPTERA: SATURNIIDAE)*

Abstract The effect of a high temperature, i. e. 32°C. on vitellogenesis of the Japanese oak silkworm, Antheraea yamamai (Lepidoptera: Saturniidae) was markedly significant. Its extent was dependent on the development stage of the silkworm exposed to 32°C. When exposed to 32 °C since the 1st day after cocooning, titres of both vitellogenin (Vg) and soluble proteins in the fat body and hemolymph of mature larvae were evidently lower than those at 26°C. When pupae were maintained at 32°C since the 1st day after pupation. the titres of Vg in the fat body showed no significant difference from those at 26°C, but those in the hemolymph and the titres of vitellin (Vt) in the ovary mostly were obviously lower in contrast to those at 26°C. While exposed to 32°C since the 6th day after pupation, at most instance the tires of Vg both in the fat body and hemolymph were not markedly different from those at 26°C, and those of Vt in the ovary were significantly higher than those at 26°C, In addition, the changes in the titres of soluble proteins in the fat body and hemolymph as well as the ovary were monitored when pupae were maintained at 32°C since the 1st or 6th day after pupation. It is recommended that both mature larvae and pupae at cocooning stage and earlier pupal stage should not be exposed to 32°C when the silkworm is reared for egg raising.     

Vitellogenesis in the hematophagous Dipetalogaster maxima (Hemiptera: Reduviidae), a vector of Chagas' disease

Oocyte extracts of anautogenous Dipetalogaster maxima were chromatographed on an ion-exchange column in order to purify vitellin (Vt), the main insect yolk protein precursor. Purified Vt (Mr ~443 kDa) was composed of four subunits with approximate molecular weights of 174, 170, 50, and 44 kDa. Polyclonal anti-Vt antibody, which cross-reacted equally with fat body extracts and hemolymph vitellogenin (Vg), was used to measure the kinetics of Vg expression in the fat body and the levels in hemolymph. In addition, morphological and immunohistochemical changes that took place in the ovary during vitellogenesis were analyzed. The study was performed between 2 and 8 days post-ecdysis and between 2 and 25 days post-blood feeding. During the post-ecdysis period, D. maxima showed decreased synthesis of Vg and concomitantly, low levels of Vg in hemolymph (4.5 x 10(-3) microg/microl at day 4). After a blood meal, Vg synthesis in the fat body and its levels in hemolymph increased significantly, reaching an average of 19.5 microg/microl at day 20. The biochemical changes observed in the fat body and hemolymph were consistent with the histological and immunohistochemical finds. These studies showed noticeable remodeling of tissue after blood feeding.     

Characterization of vitellin from the ovaries of the banana shrimp Litopenaeus merguiensis

Vitellin (Vt) was purified from ovary extracts of mature females of the banana shrimp Litopenaeus merguiensis using DEAE-Sephacel and Superdex 200 columns. Native Vt had an apparent molecular mass of 398 kDa as determined by native PAGE and by gel filtration chromatography. Under reducing and denaturing conditions (SDS-PAGE), Vt is composed of two major subunits of 87 and 78 kDa, although some faint bands were also detected. The N-terminal 10 amino acids sequence of the 78 kDa subunit is identical to that of Litopenaeus vannamei Vt and very similar to that of Litopenaeus japonicus vitellogenin (Vg) as well as Litopenaeus semisulcatus Vt, with an identity of 89%. Anti-Vt polyclonal antibody raised against purified Vt shows a high specificity with only ovarian Vt and hemolymph Vg of vitellogenic shrimps in double immunodiffusion and Western blot assays. Vg and Vt concentrations in hemolymph, hepatopancreas and ovaries were measured by ELISA. Vg concentrations increased in the hemolymph in the early stages of ovarian development and declined in the maturation stages. As there were undetectable concentrations of Vg in the hepatopancreas while an elevation of Vg levels occurred in the hemolymph, during the time that Vt was accumulating in the ovaries during oogenesis, this would suggest that the contribution of Vg synthesized by the hepatopancreas only might be not sufficient for adequate development of the oocytes in the banana shrimp L. merguiensis during vitellogenesis.     

Effects of starvation on the vitellogenesis,ovarian development and fecundity in the ectoparasitoid,Nasonia vitripennis (Hymenoptera: Pteromalidae)

A female‐specific protein, vitellogenin (Vg), and its corresponding egg vitellin (Vt) are identified in the ectoparasitic wasp Nasonia vitripennis. Both native Vt and Vg have a molecular mass of about 350 kDa, which is composed of two subunits of approximately 190 kDa and 165 kDa under reducing and denaturing conditions (sodium dodecyl sulfate—polyacrylamide gel electrophoresis). An indirect sandwich enzyme‐linked immunosorbent assay developed with both monoclonal and polyclonal antibodies against N. vitripennis Vt. Vg was first detected in the hemolymph on the 10th day after parasitism, and was first observed in oocytes on the 12th day. In adults deprived of food, the highest hemolymph Vg level occurred at the time of adult eclosion and the highest level of Vt in ovaries was found at 30 h after eclosion. In contrast, feeding adults with 20% sucrose resulted in the reduction of Vt uptake by ovaries and the extension of life span, but had little effect on Vg production. Deprived of hosts, starvation of female wasps had no significant effects on ovariole growth and oocyte maturation before the wasps died. However, starvation of female wasps supplied with hosts accelerated the wasps laying progeny into hosts, but resulted in a decrease of total progeny production by comparison with wasps fed with 20% sucrose.     

Development of an ELISA for evaluating the reproductive status of female brown planthopper,Nilaparvata lugens,by measuring vitellogenin and vitellin levels

To evaluate the reproductive status of the female brown planthopper (BPH), Nilaparvata lugens (Stål) (Hemiptera: Delphacidae), an indirect sandwich enzyme‐linked immunosorbent assay (ELISA) for monitoring vitellogenin (Vg) and vitellin (Vt) was developed by using monoclonal antibodies and polyclonal antiserum made specifically against BPH Vt. The ovarian development of BPH was divided into five stages according to ovariole development and morphological characteristics. Stages I–III, IV, and V represented the pre‐oviposition, peak oviposition, and post‐oviposition stages, respectively. Levels of Vt in the ovary and Vg/Vt in the whole female body during the five ovary stages appeared to relate well with the corresponding ovarian stages, suggesting that ovarian development can be evaluated by measuring ovarian Vt or whole body Vg/Vt in BPH. With this ELISA protocol, the reproductive status of macropterous BPH captured in rice fields during immigration, dwelling, and emigration was determined based on the levels of Vg/Vt in individual females. The females were mainly in stages I and II, as was confirmed by ovarian dissection. Therefore, this study presented an alternative method for evaluating the reproductive status of BPH in rice fields, which is more precise, convenient, and efficient than conventional techniques, such as dissection and classification of ovaries.     

七星瓢虫卵黄原蛋白基因的表达:保幼激素类似物对mRNA的诱导

在七星瓢虫(Coccinella septempunctata)中,保幼激素调控脂肪体中卵黄原蛋白基因的表达。蛋白质合成实验证明,保幼激素类似物大幅度地促进取食人工饲料的雌虫脂肪体中卵黄原蛋白的合成。保幼激素类似物的作用有高度选择性,使卵黄原蛋白占总蛋白的百分比提高12倍。取食人工饲料的雌虫中,脂肪体RNA含量及其转译活性均极低,转译产物中不存在卵黄原蛋白多肽。保幼激素类似物能显著提高脂肪体RNA的含量及其中可转译mRNA的水平。处理后的雌虫,象蚜虫饲养的成熟雌虫一样,其脂肪体RNA能在体外转译系统中指导卵黄原蛋白多肽的合成,并在变性琼脂糖凝胶电泳上显示一条高分子量的带(约5100核苷酸),初步鉴定为卵黄原蛋白mRNA。由此证明,保幼激素类似物能诱导卵黄原蛋白mRNA的出现和积累。