椰扁甲啮小蜂寄生后椰心叶甲蛹血淋巴蛋白质双向电泳图谱分析

分析昆虫被寄生蜂寄生后血淋巴蛋白质组分及组分含量变化,从蛋白质水平分析寄生蜂寄生引起寄主昆虫的生理变化,有利于探索寄生蜂调控寄主的机理。以椰心叶甲Brontispa longissima和椰扁甲啮小蜂Tetrastichus brontispae为研究材料,采用蛋白质双向电泳技术及图像分析技术分析了椰心叶甲蛹被椰扁甲啮小蜂寄生后不同时期血淋巴蛋白质图谱及其变化情况。结果表明:寄生后0.5, 1, 2, 3和4 d,被寄生蛹血淋巴共检测到的蛋白斑点数分别为982, 926, 712, 636和680个,其中特异性蛋白斑点的数量分别为650, 400, 336, 229和150个;同期未被寄生蛹血淋巴共检测到的到的蛋白斑点数分别为645, 817, 640, 873和940个,其中特异性蛋白斑点的数量分别为313, 291, 264, 466和410个;被寄生蛹和同期未被寄生蛹能匹配的蛋白斑点分别为332, 526, 376, 407和530对。被寄生蛹血淋巴蛋白斑点数量的异常变化表明寄主的生理功能受到了严重的影响。     

椰心叶甲啮小蜂的繁殖生物学研究

对椰心叶甲啮小蜂羽化、求偶、交配、产卵、繁殖现象进行了观察研究。结果表明,补充营养对雌蜂的寿命和繁殖力均有影响。没有补充营养的情况下,雌蜂的平均寿命为2.48 d,一生可产21.43粒卵;每天补充10%蜜糖水,且雌蜂与寄主(椰心叶甲初蛹)比例为1:3时,雌蜂平均寿命为13.43 d,一生可产53.6粒卵;每天补充10%蜜糖水,且雌蜂与寄主(椰心叶甲初蛹)比例为1:1时,雌蜂平均寿命为14.78 d,一生可产42.5粒卵。椰心叶甲啮小蜂对水椰八角铁甲蛹的寄生率为82.25%,平均每寄主出蜂量为12.24头。     

低温贮存对椰心叶甲啮小蜂品质的影响

研究低温贮存椰心叶甲蛹Brontispa longissima(Gestro)和椰心叶甲僵蛹对椰心叶甲啮小蜂Tetrastichus brontispae Ferriere种群品质的影响。寄生10℃低温贮存1~4日龄椰心叶甲蛹结果表明,1日龄椰心叶甲蛹的寄生率、羽化率、出蜂率、单蛹出蜂数、后代寄生能力均优于其他日龄,其次为2、3日龄的椰心叶甲蛹,最差为4日龄的椰心叶甲蛹。1日龄椰心叶甲蛹在10℃下贮存5,10,15,20d后,上述生物学指标和种群参数均随贮存天数增加而降低,其中以5d和10d的净生殖率、内禀增长率和周限增长率较未经低温贮存的差异小。1~11日龄椰心叶甲僵蛹于10℃贮存10d的结果表明,8日龄椰心叶甲僵蛹的雌蜂寿命、羽化率、单蛹出蜂数、寄生能力均优于其他日龄僵蛹。8日龄椰心叶甲僵蛹在10℃下贮存5,10,15,20d后,上述生物学指标和种群参数均随贮存天数增加而显著低于对照。     

入侵害虫椰心叶甲的研究进展

近30年来,椰心叶甲Brontispa longissima(Gestro)在亚太椰子产区扩散蔓延,成为棕榈植物上主要害虫。2002年,该害虫入侵我国海南并暴发成灾。化学防除是控制椰心叶甲危害的首要措施,其中包括挂药包等农药缓释技术,而生物防治是控制该虫扩散蔓延的经济有效措施。从感病椰心叶甲虫体上分离的微生物杀虫剂绿僵菌Metarhizium anisopliae被我国和其他多个国家应用。椰心叶甲天敌寄生蜂的引进利用是可持续控制该害虫发生的重要措施。幼虫寄生蜂椰甲截脉姬小蜂Asecodes hispinarum和蛹寄生蜂椰心叶甲啮小蜂Tetrastichus brontispae是椰心叶甲的两种重要天敌。2004年,椰甲截脉姬小蜂和椰心叶甲啮小蜂被引入到我国,经扩繁释放后,取得了良好的控害效果,其中有些地区的椰甲截脉姬小蜂寄生率可达90%,椰心叶甲啮小蜂寄生率可达100%。连续放蜂,且椰心叶甲种群有合适虫龄被寄生蜂寄生是控害效果的关键。气候等生态因子也可能是影响寄生蜂控制靶标害虫效果的因素,应进一步加强研究。论文最后提出了未来椰心叶甲防控的研究的方向。     

椰心叶甲蛹寄生蜂——椰心叶甲啮小蜂的生物学特性

椰心叶甲啮小蜂Tetrastichus brontispae Ferrière是椰心叶甲Brontispa longissima（Gestro）蛹的重要内寄生蜂,2004年从台湾引入到海南。在实验室条件下,对椰心叶甲啮小蜂的形态、行为、发育、存活、繁殖等生物学特性进行了观察研究。结果表明,雌蜂个体比雄蜂大,腹部明显可见产卵器。每头寄主蛹出蜂量平均为21.5头,其中雌蜂约占77.4%。椰心叶甲啮小蜂羽化不久即能交配,交配时间约为1～2 min。椰心叶甲啮小蜂的产卵高峰期在交配后的24 h内,且产卵量受温度的影响较大; 蜂蜜、蔗糖、葡萄糖均能有效地延长其成虫的寿命,并能提高其怀卵量和寄生率,其中以取食蜂蜜的效果最好。该蜂发育、存活和繁殖的适宜温度为20℃～28℃,低于16℃或高于30℃对其存活不利。利用16℃、20℃、24℃、28℃、30℃下的发育历期和有效积温公式计算出椰心叶甲啮小蜂的发育起点温度（C）为9.6℃,有效积温（K）为324.9日·度。     

椰心叶甲啮小蜂(Tetrastichus brontispae)实验种群生命表的编制

通过解剖椰心叶甲被寄生僵虫统计椰心叶甲啮小蜂未成熟期的发育历期、存活率与生殖力数据,编制了椰心叶甲啮小蜂的实验种群生命表.常温下椰心叶甲啮小蜂卵、幼虫和蛹的历期分别为3.1、5.0 d和9.3 d,啮出前期为2.1 d.卵、幼虫和蛹的存活率分别为0.9184、0.9521和0.9818,椰心叶甲啮小蜂的成虫羽化率为0.9,单雌产卵量为23.78粒,种群趋势指数为12 45.通过此生命表的组建,讨论了生命表中种群趋势指数(I)与生殖力表中净增殖力(R0)的关系,认为这两个概念本为同一含义,应该合并.此外,在成虫寿命大于等于2d的情况下,成虫逐日存活率与逐日产卵概率乘积的和小于1,且成虫寿命越长其值越小,结果导致在不考察成虫逐日存活率与逐日产卵概率的情况下种群趋势指数被严重高估,因此组建任何形式的生命表都应考察并计入成虫逐日存活率与逐日产卵概率.     

烟粉虱两种寄生蜂生物学特性及寄主竞争关系研究

室内比较了寄生烟粉虱Bemisia tabaci的两种寄生蜂——丽蚜小蜂Encarsia formosa和浅黄恩蚜小蜂En. sophia的生物学特性,并研究了以烟粉虱为寄主时两种寄生蜂间的竞争关系。结果表明：两者的后足胫节长度间无显著差异。浅黄恩蚜小蜂的产卵器及卵均长于丽蚜小蜂的,但浅黄恩蚜小蜂的待产卵量显著少于丽蚜小蜂。浅黄恩蚜小蜂检测寄主所花时间短于丽蚜小蜂,但检查圈数显著多于丽蚜小蜂,产卵时间以丽蚜小蜂所花时间为多。以4龄初期的烟粉虱为寄主时,丽蚜小蜂的卵期为48 h,而浅黄恩蚜小蜂24 h即完成卵期的发育。后者的蛹历期也显著短于丽蚜小蜂的蛹历期。丽蚜小蜂由卵到成虫的总发育历期比浅黄恩蚜小蜂的要长72 h左右。一头丽蚜小蜂与一头浅黄恩蚜小蜂组合后的总产卵量为14.0粒,略高于两头丽蚜小蜂组合的总产卵量（10.2粒）,显著高于两头浅黄恩蚜小蜂组合的产卵量（9.5粒）;两种蜂组合的处理中被寄生寄主体内的着卵量为1.73粒,显著高于单独一种蜂组合中被寄生寄主的着卵量,后两者分别为1.29和1.39粒。在寄主竞争情况下,在被两种蜂均寄生的寄主体内丽蚜小蜂和浅黄恩蚜小蜂产入的卵量分别为1.21和1.43粒,显著高于仅被一种蜂寄生的寄主体内的对应蜂种的卵量,后者分别为1.06和1.19粒。结果提示两种蜂均能识别对方的存在,且浅黄恩蚜小蜂表现出更强的竞争能力。     

中国隐毒甲属分类研究及四新种记述

记述中国隐毒甲属Cryphaeus Klug, 1833的4新种: 短角隐毒甲C. brevicornus sp. nov.,长角隐毒甲C. longicornus sp. nov.,短毛隐毒甲C. barbellatus sp. nov.和歪角隐毒甲C. obliquicornus sp .nov.。模式标本保存在河北大学博物馆。给出中国及部分周边地区已知种雄性和雌性检索表。     

椰心叶甲寄生蜂——椰甲截脉姬小蜂的生物学特性

椰甲截脉姬小蜂Asecodes hispinarum Boucek是椰心叶甲Brontispa longissima（Gestro）幼虫的重要内寄生蜂,2004年3月从越南引进到海南省。在实验室条件下,对椰甲截脉姬小蜂的形态、发育、繁殖、寄生潜能等生物学特性进行了初步研究。结果表明,椰甲截脉姬小蜂在24℃±2℃,RH 75%±10%条件下,卵期平均为2.8天,幼虫期6.7天,蛹期(含预蛹期)7.5天;成蜂在没有营养补充的情况下,平均存活2.5天。椰甲截脉姬小蜂的寿命受温度和补充营养的影响,雌蜂寿命长于雄蜂寿命。雌蜂平均怀卵量为43粒,产卵高峰期在交配后的12 h内。椰甲截脉姬小蜂对椰心叶甲4龄幼虫的功能反应属 HollingⅡ型,随自身密度的增加其寄生效果呈下降的趋势。     

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。     

蝶蛹金小蜂寄生对菜粉蝶体液免疫反应的影响(英文)

以蝶蛹金小蜂及其寄主菜粉蝶为研究对象 ,研究了内寄生蜂对寄主体液免疫反应的影响。当寄主蛹被寄生后 1 2h或第 4和 5d时 ,血淋巴中酚氧化酶活性明显增高。寄生蛹血淋巴中血细胞凝集素活性始终高于针刺和未寄生蛹 ;同样 ,寄生蛹血淋巴的抗菌活性也明显增强 ,而后两者处理蛹的活性则很微弱。由此可知 ,该蜂寄生能引起寄主体液免疫因子活性的不同程度的变化     

颈双缘姬蜂毒液对寄主小菜蛾的免疫抑制作用

对颈双缘姬蜂Diadromus collaris (Gravenhorst)及其毒液引起寄主小菜蛾Plutella xylostella的一些生理效应进行了研究。结果表明,颈双缘姬蜂寄生寄主后可引起寄主小菜蛾蛹总血细胞及浆血细胞和颗粒血细胞数量的上升。寄生后1天观察,血细胞延展行为受到影响,表现在颗粒血细胞放射状丝的产生及浆血细胞伪足的形成受到抑制。通过毒液对寄主离体幼虫血细胞延展行为、形态及活性影响的研究,发现毒液抑制了寄主离体浆血细胞的延展,但对颗粒血细胞的影响不明显;毒液引起寄主浆血细胞和颗粒血细胞的破裂和死亡,毒液对寄主幼虫血淋巴酚氧化酶活性有一定的抑制作用,当反应至40、60及80 min时,毒液处理和未经毒液处理的寄主血淋巴在490 nm处的吸光值差异比较明显。对毒液蛋白成分的聚丙烯酰胺凝胶电泳分析发现,毒液中有9种多肽,分子量介于9～50.2 kD,其中50.2、30.5、28.2、25.1 和12.6 kD的多肽含量较高, 与其他蜂毒液的一些作用已知的蛋白条带相似,因而推测它们同样具有免疫及发育抑制作用。结果证明颈双缘姬蜂毒液能破坏寄主细胞及体液因子调节的免疫反应。     

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.     

IMPACT OF IMMUNE RESPONSE OF A PARASITIC BEETLE Dastarcus helophoroides ON ITS HOST BEETLE Monochamus alternatus

Dastarcus helophoroides is an ectoparasitoid beetle of Monochamus alternatus, and the parasitism by D. helophoroides larvae remarkably influenced on the immune responses of M. alternatus larvae in many aspects. The hemolymph melanization reactions in the hosts were inhibited 1 h and 24 h postparasitization. The phenoloxidase activities of hemolymph were significantly stimulated 4 h postparasitization and inhibited 12 h postparasitization, and back to control level. The antibacterial activities of hemolymph in the parasitized hosts were significantly lower than that in the unparasitized ones 1 h postparasitization. By 72 h postparasitism, the total hemocyte numbers of the parasitized larvae declined to not more than one‐seconds of the number collected from the unparasitized larvae. All sampled hemolymph held the capability of nodulation, and there were fluctuations in the number of nodules the hemocytes made. However, there were no significant differences between unparasitized and parasitized larvae at each time point in the hemagglutination activity and the ratios of spreading hemocytes. In conclusion, D. helophoroides larvae could regulate M. alternatus immune system and resulted in the changes in host immune responses.     

Parasitism of Pieris rapae (Lepidoptera: Pieridae) by a pupal endoparasitoid, Pteromalus puparum (Hymenoptera: Pteromalidae): effects of parasitization and venom on host hemocytes

In contrast to the situation with egg-larval and larval endoparasitic wasps, little is known about the effects of pupal endoparasitoids and their secretions on the hemocytes of their insect hosts. This study focuses on the pupal endoparasitoid, Pteromalus puparum, and its host, the small white butterfly, Pieris rapae. Parasitism by P. puparum, resulted in a significant increase in the total number of host hemocytes up to day five after parasitization. From day one to day four after parasitization, the percentage of plasmatocytes significantly decreased, and the proportion of granular cells increased. Moreover, from 12 h to day three after parasitization, hemocyte mortality in parasitized pupae was noticeably higher. When P. rapae pupae were parasitized by adult females of P. puparum irradiated by gamma-ray (pseudoparasitization), it was clear that the treated wasps could induce similar hemocyte changes. However, such phenomena did not occur in punctured host pupae (mimic-parasitization). After treatment with P. puparum venom, both the percentages of spreading plasmatocytes and encapsulated Sephadex G-25 beads were lessened significantly in vitro. Electron microscopy analysis and visualization of hemocyte F-actin with phalloidin-FITC showed that hemocytes treated with venom had a rounded configuration and neither spread nor extended pseudopods, while there was no marked alteration of hemocyte cytoskeletons after venom treatment. The results suggested that venom of P. puparum could actively suppress the hemocyte immune response of its host, but not by destroying the host hemocyte cytoskeleton.     

丽蝇蛹集金小蜂寄生对寄主蛹可溶性蛋白与芳基蛋白组成与含量的影响

为了探讨蛹期寄生蜂对寄主蛋白代谢的寄生生理效应,利用Bradford蛋白含量测定法、Western免疫印迹法及酶联免疫吸附检测法研究了棕尾别麻蝇Boettcherisca peregrina蛹被丽蝇蛹集金小蜂Nasonia vitripennis寄生后其脂肪体和血淋巴中可溶性蛋白及芳基蛋白组成与含量的变化。结果表明：寄生蛹脂肪体和血淋巴中可溶性蛋白的组成与未寄生相比基本无明显差异; 不论寄生与否寄主蛹脂肪体和血淋巴中芳基蛋白亚基分子量均为80 kDa,该亚基在脂肪体中未出现降解现象,而在血淋巴中仅于寄生后12 h的寄主蛹中呈现2条分子量相近的Western免疫印迹带,说明其降解可能先于未寄生对照。就含量而言,寄生蛹脂肪体中可溶性蛋白含量除寄生后24 h外均显著低于未寄生对照,芳基蛋白含量除寄生后48 h外也均显著低于未寄生对照,其中寄生后12 h的含量仅为未寄生的32.0%。寄生蛹血淋巴中可溶性蛋白含量多低于未寄生蛹,且寄生后2,12,24 h的差异达显著水平;芳基蛋白的含量均有低于未寄生的趋势,其中寄生后12 h的含量为未寄生的17.0%。综合认为,丽蝇蛹集金小蜂的寄生可导致寄主脂肪体和血淋巴中可溶性蛋白及芳基蛋白含量下降。     

CLP gene family,a new gene family of Cotesia vestalis bracovirus inhibits melanization of Plutella xylostella hemolymph

Polydnaviruses (PDVs) are obligatory symbionts of parasitoid wasps and play an important role in suppressing host immune defenses. Although PDV genes that inhibit host melanization are known in Microplitis bracovirus, the functional homologs in Cotesia bracoviruses remain unknown. Here, we find that Cotesia vestalis bracovirus (CvBV) can inhibit hemolymph melanization of its host, Plutella xylostella larvae, during the early stages of parasitization, and that overexpression of highly expressed CvBV genes reduced host phenoloxidase activity. Furthermore, CvBV-7-1 in particular reduced host phenoloxidase activity within 12 h, and the injection of anti-CvBV-7-1 antibody increased the melanization of parasitized host larvae. Further analyses showed that CvBV-7-1 and three homologs from other Cotesia bracoviruses possessed a C-terminal leucine/isoleucine-rich region and had a similar function in inhibiting melanization. Therefore, a new family of bracovirus genes was proposed and named as C -terminal L eucine/isoleucine-rich P rotein (CLP). Ectopic expression of CvBV-7-1 in Drosophila hemocytes increased susceptibility to bacterial repression of melanization and reduced the melanotic encapsulation of parasitized D. melanogaster by the parasitoid Leptopilina boulardi. The formation rate of wasp pupae and the eclosion rate of C. vestalis were affected when the function of CvBV-7-1 was blocked. Our findings suggest that CLP genes from Cotesia bracoviruses encoded proteins that contain a C-terminal leucine/isoleucine-rich region and function as melanization inhibitors during the early stage of parasitization, which is important for successful parasitization.     

A comparison of artificial diets for mass rearing Brontispa longissima (Coleoptera: Chrysomelidae) as hosts for the larval and the pupal parasitoids

The coconut hispine beetle, Brontispa longissima (Gestro) (Coleoptera: Chrysomelidae) is a serious pest of coconut palm. In this study, we developed an artificial diet for B. longissima so that the beetle could be used as a host for rearing two of its parasitoids, Asecodes hispinarum Boucek (Hymenoptera: Eulophidae) and Tetrastichus brontispae Ferrière (Hymenoptera: Eulophidae). The new artificial diet represents an improvement of our previous diet, which we used as a control. When beetle larvae were reared on the new diet, which contains cysteine but not cellulose powder and has twice as much coconut leaf powder as in the control, the adult emergence was 71% (approximately 2 times that in the control). We also examined the suitability of beetles fed on the new diet as hosts for the larval parasitoid A. hispinarum and the pupal parasitoid T. brontispae. The percentage of wasps that emerged from hosts that were fed the new diet was higher than that from the control-fed hosts. The new diet allowed both A. hispinarum and T. brontispae to produce adult wasps of the next generation, whereas the control only allowed T. brontispae to produce the next generation. These results suggest that the new diet is suitable for B. longissima and will facilitate mass-rearing of A. hispinarum and T. brontispae.