蝶蛹金小蜂寄生对菜粉蝶血细胞骨架相关蛋白基因的转录调控

为揭示蝶蛹金小蜂Pteromalus puparum抑制寄主菜粉蝶Pieris rapae血细胞免疫反应的分子机制, 克隆了菜粉蝶肌动蛋白、肌动蛋白解聚因子及微管蛋白cDNA, 并研究了蝶蛹金小蜂寄生对其转录水平的影响。结果显示： 菜粉蝶肌动蛋白cDNA ORF为1 131 bp, 编码377 aa, 预测分子量为41.78 kDa, pI为5.29, 与其他昆虫肌动蛋白的相似性非常高, 在90%以上。氨基酸组成特点和系统进化分析表明, 克隆到的菜粉蝶肌动蛋白基因属细胞质型肌动蛋白。菜粉蝶肌动蛋白解聚因子cDNA全长为1 243 bp, ORF为 447 bp, 编码149 aa, 预测分子量为16.97 kDa, pI为7.11,与家蚕Bombyx mori、赤拟谷盗Tribolium castaneum、意大利蜜蜂Apis mellifera和桑粉介壳虫Maconellicoccus hirsutus肌动蛋白解聚因子的相似性分别为97%, 87%, 89%和72%。菜粉蝶微管蛋白cDNA全长为1 757 bp, ORF为1 344 bp, 编码448 aa, 预测分子量为50.38 kDa, pI为4.86, 与家蚕β型微管蛋白1, 2, 3和4的相似性分别为97%, 97%, 87%和93%。系统进化分析表明, 克隆到的菜粉蝶微管蛋白基因属β型微管蛋白。RT-PCR分析表明, 蝶蛹金小蜂寄生能抑制肌动蛋白、肌动蛋白解聚因子及微管蛋白基因在菜粉蝶蛹血细胞中的转录水平。由此推断蝶蛹金小蜂调控寄主血细胞骨架相关蛋白基因的转录是该蜂抑制寄主血细胞免疫反应的分子机制之一。     

菜粉蝶蛹体型大小对蝶蛹金小蜂后代数量、性比及体型大小的影响

为检验“寄主体型大小-质量假说”,采取饥饿方法处理菜粉蝶(Pieris rapae)高龄幼虫,以获得体型大小(用体质量表示)差异大于同一自然地理种群内个体间差异的寄主蛹,然后供聚寄生蜂-蝶蛹金小蜂(Pteromalus puparum)寄生,观察寄生蜂在不同大小寄主蛹内的后代数量、性比以及体型大小。结果表明,蝶蛹金小蜂1次攻击产出的后代蜂数随寄主蛹体质量增加而显著增多,蛹中平均出蜂314.97头·g^-1。蝶蛹金小蜂后代的雌性比例随寄主蛹质量的增大而显著提高,后代雌蜂和雄蜂的体型大小(后足胫节长度)均随寄主蛹质量的增大而显著增大。可见,蝶蛹金小蜂母蜂能够根据寄主蛹质量来调整后代数量和性比,以使后代适应度最大化。最后,就蝶蛹金小蜂产卵策略及其影响因素进行了讨论。     

蝶蛹金小蜂在杭州的年生活史

蝶蛹金小蜂是菜粉蝶蛹期的主要寄生蜂。在杭州,绝大多数以老熟幼虫在寄主蛹内越冬。田间养虫室内饲养结果:1979年发生11—12代;1981年早、中、迟三组分别为12—13代、10—11代以及9—10代,三组合计,非越冬代成虫自5月上旬至12月初始终不断,一年之内,前后(连续或不连续)两代成虫重叠发生的天数至少175天,前后(连续或不连续)三代成虫重叠发生的天数至少49天。 2、3月间小蜂即可羽化,远远早于田阔第一代菜粉蝶蛹的出现,惟不能寄生越冬后的越冬代菜粉蝶蛹,但越冬代蜂的羽化盛期仍处于田间第一代菜粉蝶蛹初见至化蛹始盛期之间,且喂食后寿命很长,故越冬代蜂与第一代菜粉蝶蛹之间的发生期基本上是吻合的。其后,两者代数既多(菜粉蝶一年发生9代),发生又都不整齐,小蜂成虫与菜粉蝶蛹均连绵不断,时间上彼此也是吻合的。这是此蜂自然寄生率高并成为其重要天敌的一个主要原因。 对发生代数多、世代重叠的重要天敌建议分期分批饲养,以深入掌握其生活史及与寄主发生的关系。     

菜粉蝶蛹血细胞体外细胞免疫反应及蝶蛹金小蜂毒液的影响

为探明蝶蛹金小蜂Pteromalus puparum毒液对其寄主菜粉蝶Pieris rapae蛹颗粒血细胞和浆血细胞的包囊与吞噬能力的影响, 本研究分别采用Na₂-EDTA处理和尼龙毛法对菜粉蝶蛹颗粒血细胞与浆血细胞进行分离纯化; 再采用离体细胞培养方法, 研究了菜粉蝶蛹颗粒血细胞、 浆血细胞各自在包囊和吞噬反应中的作用, 以及蝶蛹金小蜂毒液对其所产生的影响。结果表明： 颗粒血细胞和浆血细胞均参与了包囊反应, 其中前者包囊作用明显, 后者作用微弱, 但两者同时存在时包囊作用最为明显; 血淋巴浆质对颗粒血细胞和浆血细胞包囊反应均无显著影响。毒液对颗粒血细胞和浆血细胞的包囊能力均存在显著的抑制作用, 且抑制作用具有明显的剂量效应特征。此外, 菜粉蝶蛹颗粒血细胞和浆血细胞均具吞噬能力, 其中前者吞噬能力显著强于后者; 毒液对颗粒血细胞与浆血细胞的吞噬能力亦均存在显著的抑制作用, 且该抑制作用亦具有显著的剂量效应特征。结果说明, 菜粉蝶蛹颗粒血细胞及浆血细胞均参与寄主的细胞免疫反应, 蝶蛹金小蜂毒液对其寄主颗粒血细胞和浆血细胞的包囊与吞噬能力均存在显著的抑制作用。     

蝶蛹金小蜂的卵巢发育在不同寄主之间差异性的比较

为明确不同寄主对蝶蛹金小蜂Pteromalus puparum卵巢发育的影响,以柑橘凤蝶Papilio xuthus和菜粉蝶Pieris rapae这两种常见的寄主为研究对象,调查了两种寄主对蝶蛹金小蜂卵巢管长度、不同发育级别卵的卵室数,以及各自占整个卵巢卵室总数的比例进行了调查.结果表明,从两种寄主中羽化出的寄生蜂在卵巢管长度间差异显著,而在Ⅲ级卵的卵室比例上以柑橘凤蝶为寄主的寄生蜂明显高于以菜粉蝶为寄主的寄生蜂,而Ⅰ级和Ⅱ级的卵比例则低于后者.     

两种金小蜂毒液对菜粉蝶蛹血细胞延展、存活及包囊作用的影响

活体微注射测定结果表明,将0.5毒囊当量（venom reservoir equivalent, VRE）的蝶蛹金小蜂毒液注射于其寄主菜粉蝶蛹体内,注射后4～24 h寄主浆血细胞和颗粒血细胞的延展、存活和对Sephadex A50微珠的包囊能力显著下降;以0.002～0.02 VRE/μL的该蜂毒液处理其离体寄主血细胞均能产生同样的生理效应。该毒液抑制90%菜粉蝶蛹浆血细胞和颗粒血细胞延展的浓度各为0.00076 VRE/μL和0.00804 VRE/μL。可见,蝶蛹金小蜂毒液能显著抑制其寄主血细胞的延展和包囊作用,并导致血细胞的死亡。然而,同样条件下丽蝇蛹集金小蜂毒液对其非自然寄主菜粉蝶蛹的血细胞延展、存活和包囊作用则无任何效应。可见,寄生蜂毒液的生理作用具有明显的寄主特异性。     

蝶蛹金小蜂毒液蛋白不同提取分离方法的比较

为了建立蝶蛹金小蜂Pteromalus puparum毒液抑制寄主血细胞免疫活性组分合适的分离纯化方法,就等电点沉淀法、乙醇沉淀法、75%硫酸铵沉淀法、75%硫酸铵沉淀法+40℃加热处理法,以及75%硫酸铵沉淀法分别与3种不同滤膜的分子大小截留法的组合等7种方法对毒液蛋白分离效果及活性的影响进行了比较。结果表明：等电点沉淀法获得的组分抑制寄主菜粉蝶Pieris rapae离体血细胞延展和包囊的活性最强,乙醇沉淀法次之,75%硫酸铵沉淀法最弱。从蛋白组分的SDS-PAGE图谱来看,等电点沉淀法获得毒液组分相对最纯,仅有3条主要谱带,分子量大小在45～116.2 kDa范围内;乙醇沉淀法次之,有5条主要谱带,分子量大小在24～116.2 kDa范围内;硫酸铵沉淀法的谱带组成与毒液蛋白粗提液相似。3种分子大小截留法获得的毒液组分的活性分析表明,强活性组分分子量大小可能都大于100 kDa。综合认为,7种方法中以等电点沉淀法提取分离蝶蛹金小蜂毒液蛋白相对为最适。     

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。     

聚寄生性蝶蛹金小蜂雌蜂体型大小对产卵策略的影响

在寄生蜂行为生态学研究中, 通常将寄主体型大小作为寄主品质的主要性状来探究寄生蜂的搜寻行为机理, 而忽略寄生蜂体型大小的意义。为揭示聚寄生蜂雌蜂体型大小对其产卵决策的影响, 在严格控制寄主菜粉蝶Pieris rapae蛹体型大小（体重）的情况下, 于室内观察了不同体型大小的蝶蛹金小蜂Pteromalus puparum雌蜂的产卵行为, 并调查了子代蜂数量（窝卵数）、 性比和体型大小的变化。结果表明： 雌蜂在寄主上的驻留时间随其自身体型增大而缩短, 但随寄主体重增大而延长。窝卵数和余卵量受到雌蜂体型大小的显著影响, 均随雌蜂体型增大而显著增加（P<0.05）; 但子代蜂性比不受雌蜂体型大小的显著影响 (P>0.05)。子代雌、 雄性体型大小均与雌蜂体型大小无关, 但子代雌蜂体型随寄主体重增大而增大。结果证实, 雌性蝶蛹金小蜂体型大小影响其部分产卵决策。因此, 在建立聚寄生蜂产卵决策模型中应考虑雌蜂体型大小这一重要变量因素。     

三种寄生蜂虫体可溶性蛋白组成及蝶蛹金小蜂卵黄蛋白的分子特性分析(英文)

聚丙烯凝胶电泳PAGE和毛细管电泳分析结果表明 ,蝶蛹金小蜂雌蜂个体和卵巢中明显存在雌性特异蛋白 ,即卵黄蛋白。但茧蜂科的螟长距茧蜂Macrocentruslinears和菜蛾盘绒茧蜂Cotesiaplutella雌雄虫体可溶性蛋白间无明显差异。SDS PAGE梯度电泳结果表明 ,蝶蛹金小蜂卵黄原蛋白 (Vg)和卵黄磷蛋白 (Vt)均由二个分子量接近的亚基组成 ,分子量各为 74 4和 5 2 8KD。双向免疫扩散和PAGE梯度电泳都显示该蜂隐成蜂的雌性虫体及雌蜂血淋巴、脂肪体和卵巢中都有Vg或Vt,且卵黄原蛋白是由雌蜂脂肪体合成     

EFFECT OF PARASITIZATION BY THE PUPAL ENDOPARISITOID,PTEROMALUS PUPARUM (HYMENOPTERA: PTEROMALIDAE) ON HUMORAL IMMUNE REACTIONS OF PIERIS RAPAE (LEPIDOPETERA: PIERIDAE)*

Abstract Studies on the effect of parasitization by the endoparasitoid on host humoral immune reactions are carried out with the pupal endoparisitic wasp, Pteromalus puparum, and its host, Pieris rapae. Phenoloxidase (PO) activity of parasitized hosts hemolymph increased significantly at 12 h, day four and day five after parasitization. Hem‐agglutination activity of parasitized hosts hemolymph was always higher than that of wounded and unparasitized ones. Moreover, antibacterial activity of parasitized hosts hemolymph became more and more stronger, whilst wounded and unparasitized pupae only owned a weak antibacterial activity. It suggested that activities of humoral immune factors of Pieris rapae could be influenced to some degrees by P. puparum.     

Venom of Pteromalus puparum (Hymenoptera: Pteromalidae) induced endocrine changes in the hemolymph of its host,Pieris rapae (Lepidoptera: Pieridae)

Pteromalus puparum is a predominant endoparasitoid wasp of Pieris rapae. Its venom is the only active factor injected into host associated with oviposition. In this report, we explored whether the venom alone from this wasp affects the endocrine system of its host or not. We monitored the changes of hemolymph juvenile hormone (JH; only JH III detected), ecdysteroid, and juvenile hormone esterase activity (JHE) over 72 h in parasitized and venom‐microinjected P. rapae pupae. Non‐parasitized and PBS‐microinjected P. rapae served as controls. Results showed that JH titers were significantly higher in parasitized and venom‐microinjected pupae than that in control pupae during 24 to 72 h. After 12 h, JH titers were significantly promoted by parasitization and venom microinjection. JHE activities of non‐parasitized and PBS‐microinjected pupae were significantly higher than that of parasitized and venom‐microinjected pupae, which was with a peak at 12 h (parasitized pupae) or 24 h (venom‐microinjected pupae) during 6 to 48 and 12 to 36 h, respectively. The hemolymph titers of ecdysteroid in non‐parasitized and PBS‐microinjected pupae increased rapidly during 12 to 36 h with a peak at 36 h, and were higher than treatments before 48 h, while presenting a significant difference at 24 to 48 h between the treatments and controls. The results demonstrate that venom alone of this parasitoid wasp can disrupt its host's endocrine system. © 2009 Wiley Periodicals, Inc.     

中国菜粉蝶群(Pieris napi-broniae)的分类(鳞翅目:粉蝶科)

本文根据雄性粉蝶香鳞的形态特征,对粉蝶类群的种和亚种进行了修订,并用图标明其分布地区,也提出待研究的生物学及生态学的问题。     

Parasitism of Pieris rapae (Lepidoptera: Pieridae) by the endoparasitic wasp Pteromalus puparum (Hymenoptera: Pteromalidae): Effects of parasitism on differential hemocyte counts,micro‐ and ultra‐structures of host hemocytes

Abstract Parasitism by the endoparasitic wasp Pteromalus puparum (Hymenoptera: Pteromalidae) by using only its associated venom, can suppress the immunal responses of Pieris rapae (Lepidoptera: Pieridae). However, up to now, current knowledge of the mechanisms has been limited. The response of host hemocytes to parasitism was investigated using a combination of light and transmission electron microscopy (TEM). Five hemocyte types, prohemocytes (PRs), granulocytes (GRs), plasmatocytes (PLs), oenocytoids (OEs) and coagulocytes (COs), were observed and characterized from both unparasitized and parasitized Pieris rapae pupae. Light microscopy showed that both GRs and PLs became more round and spread abnormally after parasitism, whereas the shape of other types of hemocytes remained unaffected. In addition, the size of PRs and PLs became larger while OEs became smaller. The proportion of PRs significantly increased after parasitism and that of PLs decreased by 43.9%, but there was no significant increase of GRs and OEs. TEM showed that all types of hemocytes except COs were damaged to various degrees after parasitism, especially resulting in electron opaque cytoplasm and nucleus, fewer cell organelles of rough endoplasmic reticulum, mitochondria and vesicles. Our results indicate that parasitism by P. puparum affects differential hemocyte counts and structures of host hemocytes, particularly for GRs and PLs, which may be the main cause of the parasitoid suppressing host cellular immune responses.     

20-羟基蜕皮酮对蓖麻蚕蛹体液免疫的调节作用(英)

本研究指出20—羟基蜕皮酮(20-OHE)参加蓖麻蚕蛹对大肠杆菌的体液免疫反应。20-OHE的作用方式是多方面的,包括提高血淋巴蛋白质含量,产生抗菌蛋白,增加溶菌酶活性,和激活原酚氧化酶系统。这表明多个免疫控制系统参与蓖麻蚕蛹体液免疫反应的可能性。     

REGULATORY ROLE OF 20-HYDROXYECDYSONE ON HUMORAL IMMUNITY IN THE PUPAE OF THE INDIAN SILKWORM,PHZLOSAMIA CYNTHIA RICINI*

Abstract The present study shows that 20-hydroxyecdysone(20-OHE) participates in the humoral immune responses of Philosamia Cynthia ricini, either normal or debrained pupae, to the E. coli. The mode of action of 20—-OHE is multiple. It raises hemolymph protein content, makes antibacterial proteins be produced, increases lysozyme activity, and activates prophenoloxidase system. It is possible that several immune control systems are involved.     

赛阿金小蜂属分类及一新种描述(膜翅目,金小蜂科)

研究了金小蜂科、金小蜂亚科的赛阿金小蜂属Cyr-toptyx Delucchi,1956,描述1新种黄翅赛阿金小蜂Cyrtoptyx flavida sp．nov．及2新纪录种：丽躯赛阿金小蜂C．1ichten—steini(Masi)和实蝇赛阿金小蜂C．1atipes(Masi)。编制了中国赛阿金小蜂属分种检索表。新种黄翅赛阿金小蜂Cyrtop-tyx flavida sp．nov与其它两种的主要区别是：触角各索节与第1棒节均具2轮感觉毛,梗节与鞭节长之和大于头宽。模式标本保存于中国科学院动物研究所动物标本馆。