菜蛾盘绒茧蜂寄生对寄主小菜蛾幼虫丝腺的影响

菜蛾盘绒茧蜂Cotesia vestalis寄生对小菜蛾Plutella xylostella幼虫丝腺形态结构、丝腺内蛋白组分及含量有显著的影响。寄生后的小菜蛾幼虫的丝腺细胞不经历膨大期即开始凋亡,无法大量分泌蛋白,因此蛋白含量显著低于未寄生的小菜蛾丝腺内的蛋白,且蛋白组分也有一定程度的差异,主要是50 kDa以上的蛋白含量和种类的差别较大。     

寄主龄期、过寄生和寄主饥饿处理对菜蛾盘绒茧蜂幼蜂及畸形细胞发育的影响

过寄生、寄生时寄主龄期和寄生后寄主饥饿处理影响菜蛾盘绒茧蜂Cotesia plutellae（Kurdj.）幼蜂及畸形细胞的发育。显微解剖和观察表明,4龄小菜蛾Plutella xylostella L.幼虫被寄生后,其体内菜蛾盘绒茧蜂幼蜂发育不整齐、假寄生比例增高。过寄生后,每头被寄生的寄主血腔中畸形细胞数量明显增多,但直径变小;随着过寄生程度的加剧,幼蜂发育严重受阻。寄主营养显著影响体内幼蜂及畸形细胞的发育,被寄生的小菜蛾经饥饿处理62 h后,体内畸形细胞的数量、活性明显降低,与此同时,幼蜂的发育也受到明显抑制,寄主发育与寄生蜂和畸形细胞的发育呈正相关性。由此可见,寄主不同龄期、过寄生及寄主营养状况均对寄主体内幼蜂和畸形细胞发育产生影响。     

菜蛾盘绒茧蜂卵携带的免疫抑制因子

抑制寄主昆虫的免疫反应是内寄生蜂存活的关键。菜蛾盘绒茧蜂Cotesia vestalis（Haliday）寄生小菜蛾Plutella xylostella （L.）幼虫后,蜂卵如何逃避和抑制寄主的免疫攻击,尚未得到全面揭示。本文采用电镜技术系统观察了菜蛾盘绒茧蜂卵表面的超微结构。结果显示：蜂卵表面覆盖有纤维层和絮状的类病毒样纤丝（VLFs）,同时携带了含多分DNA病毒粒子（PDV）的萼液。在寄生初期,包裹在蜂卵表面的纤维层和VLFs首先起到保护蜂卵不被小菜蛾血细胞包囊的被动防御作用。随后,PDV发挥主动的免疫抑制作用。通过假寄生手段,证明了菜蛾盘绒茧蜂PDV (CvBV) 具有较持久的克服寄主免疫攻击的能力,是主要的免疫抑制因子。在假寄生后连续8 d的观察时间内,菜蛾盘绒茧蜂的蜂卵均未被包囊。结果提示,在菜蛾盘绒茧蜂-小菜蛾寄生体系中,菜蛾盘绒茧蜂采取被动防御和主动攻击两种方式应对寄主小菜蛾的免疫攻击。     

菜蛾盘绒茧蜂主要寄生因子导致的寄主小菜蛾幼虫脂肪体结构的变化

在不同的寄生状态下,菜蛾盘绒茧蜂Cotesia plutellae不同的寄生因子可引起寄主小菜蛾Plutella xylostella幼虫脂肪体结构发生相应的改变。显微和亚显微形态结构显示： 假寄生后多分DNA病毒和毒液对脂肪体结构的完整性没有显著影响,但细胞内脂质体变得小而密集,线粒体和内质网丰富,并有糖原积累; 正常寄生后,脂肪体结构被破坏,多数线粒体内嵴紊乱,脂质体也变得不规则,特别是当幼蜂完成在寄主体内发育时,寄主体内几乎无完整脂肪体存在。与此同时,同批未被寄生的小菜蛾幼虫发育到4龄末期时,体内脂肪体细胞发育正常,已开始向蛹期细胞形态转化,细胞内脂质体很大,细胞器数量较多、糖原积累丰富, 而且部分细胞已成为游离态细胞。由此证明,寄生蜂携带的寄生因子,如多分DNA病毒、毒液、畸形细胞和幼蜂等,均对寄主脂肪体结构的改变产生影响,但程度明显不同。     

闭弯尾姬蜂与菜蛾盘绒茧蜂寄生菜蛾幼虫时的种间竞争

在室内25℃下,以菜蛾3龄初幼虫作寄主,研究了菜蛾盘绒茧蜂Cotesia plutellae和半闭弯尾姬蜂Diadegma semiclausum的种间竞争。当寄主供2种蜂同时产卵寄生时,2种蜂各自的寄生率与其单独寄生时无显著差异,合计寄生率比一种蜂单独存在时有所提高,但差异不显著。2种蜂均能产卵寄生已被另一种蜂寄生了的寄主幼虫。当寄主被2种蜂寄生的间隔时间很短（少于10 h）时,所育出的蜂绝大部分（80%以上）为绒茧蜂;当寄主先被绒茧蜂寄生,并饲养2天以上再供弯尾姬蜂寄生时,所育出的全为绒茧蜂;当寄主先被弯尾姬蜂寄生,并饲养2天以上再供绒茧蜂寄生时,寄主幼虫绝大部分不能存活,只有少部分能育出寄生蜂,且多为弯尾姬蜂。当2种蜂的幼虫存在于同一寄主体内时,2种蜂的发育均受到另一种蜂的抑制;绒茧蜂1龄幼虫具有物理攻击能力,能将弯尾姬蜂卵或幼虫致死。这些结果表明,菜蛾盘绒茧蜂与半闭弯尾姬蜂在同一寄主中发育时,前者具有明显的竞争优势。     

小菜蛾及菜蛾绒茧蜂乙酰胆碱酯酶敏感性的相关变化

用生物测定和生化检测的方法,对福州地区小菜蛾Plutella xylostella和菜蛾绒茧蜂Apanteles plutellae的抗药性及两种昆虫乙酰胆碱酯酶对杀虫剂的敏感性进行了田间监测。结果显示,从1998年9月至1999年4月,小菜蛾乙酰胆碱酯酶对6种有机磷和氨基甲酸酯杀虫剂敏感性逐渐恢复,寄生于同一虫源的菜蛾绒茧蜂乙酰胆碱酯酶敏感性的变化也呈明显的相关性,但菜蛾绒茧蜂乙酰胆碱酯酶的敏感性高于其寄主小菜蛾。脱离选择压力后,两种昆虫对杀虫剂的敏感性迅速恢复,乙酰胆碱酯酶的K_i值显著增高。对乙酰胆碱酯酶的K_m、V_max和K_i值测定结果表明,两种昆虫对有机磷和氨基甲酸酯杀虫剂的抗性与乙酰胆碱酯酶对杀虫剂的不敏感性有关。此外还研究了不同发育期小菜蛾乙酰胆碱酯酶活性及其K_i值的变化。探讨了在杀虫剂选择压力下,两种昆虫乙酰胆碱酯酶敏感性的环境适应性变化机制。     

菜蛾盘绒茧蜂对寄主小菜蛾幼虫营养的调节和利用

寄主小菜蛾Plutella xylostella被内寄生蜂菜蛾盘绒茧蜂Cotesia plutellae寄生后,其取食、发育及营养代谢在各种寄生因子的作用下伴随幼蜂的发育而发生很大的变化,畸形细胞作为调节因子之一也发挥了重要的作用。本实验通过比较被寄生和未被寄生小菜蛾血淋巴蛋白浓度以及两种血淋巴对菜蛾盘绒茧蜂幼蜂进行体外培养的培养液的蛋白浓度,发现被寄生小菜蛾血淋巴比未被寄生小菜蛾血淋巴的蛋白浓度略低但差异不显著,而未被寄生小菜蛾血淋巴幼蜂培养液的蛋白浓度显著低于被寄生小菜蛾血淋巴幼蜂培养液的蛋白浓度,证明畸形细胞的蛋白质分泌功能。被寄生后期, 小菜蛾体重明显大于未被寄生的小菜蛾体重,而脂肪体重量相比正好相反;通过显微染色观察,在小菜蛾念珠状脂肪体表面粘附有畸形细胞,对脂肪体进行分解破坏而使其成颗粒状; 蛋白含量和脂滴浓度测定也表明,脂肪体的可溶性蛋白含量和脂滴浓度也迅速降低,同比低于未被寄生小菜蛾。而与此同时,幼蜂正处在快速生长阶段,中肠酯酶的活性逐步上升,幼蜂得以快速消化吸收小菜蛾体内的营养直到完成幼虫发育,整个幼蜂的脂滴浓度也达到了最大值。因此寄生后期,推测在畸形细胞的协助下,幼蜂吸收了寄主小菜蛾体内的营养为自身生长发育所用。     

温度对菜蛾绒茧蜂功能反应的影响

在室内１５℃、２０℃、２５℃、３０℃、３２．５℃下测定了菜蛾绒茧蜂寄生数量随寄主密度的变化情况．结果表明,在１５～３２．５℃范围内的各个温度下的功能反应均能用Ｈｏｌｉｎｇ圆盘方程很好地拟合,但各温度间功能反应的参数存在着显著差异．２５℃、３０℃、３２．５℃下的瞬时攻击率比１５℃、２０℃下显著要高,而处理时间则随温度升高而缩短．各温度下在供寄主的５ｈ内,均有部分雌蜂不产卵寄生小菜蛾幼虫．产卵寄生的雌蜂百分率随温度升高和寄主密度增加而增加     

二化螟盘绒茧蜂触角感器的超微结构

采用扫描电子显微镜对二化螟盘绒茧蜂Cotesia chilonisMunakata触角感器进行了观察和研究。结果表明,二化螟盘绒茧蜂触角上共存在6种感器,分别为板形感器、毛形感器、刚毛型感器、柱形感器、钟形感器、锥形感器。对各种触角感器的形态、分布特点进行了描述,并对两性间的差异及其功能进行了探讨。     

寄主抗药性对菜蛾绒茧蜂抗药性发展的影响

室内筛选小菜蛾中抗品系（SRP）、高抗品系（RP）、以敏感小菜蛾幼虫（SP）为寄主的菜蛾绒茧蜂SRC品系和以SRP幼虫为寄主的菜蛾绒茧蜂RSC品系对氰戊菊酯的抗性。分别筛选了13、14、14和13代。小菜蛾SRP和RP品系分别获得了68.9和605.8倍的抗性,菜蛾绒茧蜂SRC和RSC品系分别获得了4.3和11.0倍的抗性。上述结果表明通过施药于体内有寄生蜂的小菜蛾幼虫筛选寄生蜂抗性,可以获得具有抗性的寄生蜂。以SRP为寄主的RSC品系的抗性水平高于以SP为寄主的SRC品系的抗性水平,表明和抗性较高的寄主同步筛选,寄生蜂的抗性发展更快。小菜蛾SP、SRP和RP三个品系幼虫的多功能氧化酶（MFO）活性比为1∶1.15∶1.50;菜蛾绒茧蜂SC、SRC和RSC三个品系幼虫的MFO活性比为1∶1.10∶1.49,成蜂的MFO活性比为1∶1.18∶1.54;而每种昆虫不同品系的羧酸酯酶（CarE）、总酯酶（Es）活性水平与其抗性水平变化不一致,表明抗性与MFO活性升高有关,而与CarE和Es的活性无关。     

Plant Resistance Affects the Olfactory Response and Parasitism Success of Cotesia vestalis

Understanding the factors influencing host-selection behavior of parasitoids is essential in studies on host-parasitoid ecology and evolution, and in combining sustainable strategies of pest management, such as host-plant resistance and biological control. The effects of host-plant resistance on the olfactory response and parasitism success by Cotesia vestalis, a parasitoid of diamondback moth (Plutella xylostella) larvae were examined. Here, it was demonstrated that host-plant resistance can strongly influence foraging behavior and parasitism success of the parasitoid. In olfactometer experiments, C. vestalis did not differentiate between crucifer plant types with similar levels of susceptibility or resistance to P. xylostella but showed a strong preference for susceptible compared with partially-resistant host plants. The influence of previous oviposition activity varied with the host-plant type experienced by the parasitoid. In cage experiments, C. vestalis preferred to parasitize P. xylostella larvae on a susceptible plant compared with larvae on a partially resistant host plant when exposed to hosts for 24 h. However, this preference appeared to be transitory, and was not found after 96 h exposure. The present study suggests that combining partial host-plant resistance with biological control by C. vestalis for the control of P. xylostella may in some circumstances be antagonistic and negatively affect parasitism success.     

Additive effect of teratocyte and calyx fluid from Cotesia plutellae on immunosuppression of Plutella xylostella

Abstract.  Teratocytes are cells that originate from the extra-embryonic tissues of some hymenopteran parasitoids, typically dissociate upon hatching, and develop in the host haemolymph. They are considered to be involved in parasitoid larval nutrient uptake, host immunosuppression and/or repression of competing parasitoid development. Teratocytes of the parasitoid, Cotesia plutellae (Kurdjumov) (Hymenoptera: Braconidae) are found in its natural host, Plutella xylostella (Linnaeus) (Lepidoptera: Yponomeutidae) and can be cultured in vitro . The present study demonstrates that teratocytes of C. plutellae possess a significantly depressive effect on host cellular immunity. When the hosts are preinjected with 200 cultured teratocytes (corresponding to the normal number of teratocytes released during wasp hatching), haemocyte nodulation is inhibited by approximately 40%, with younger teratocytes being more potent than older ones. Similarly, the medium in which teratocytes are cultured has similar immunosuppressive properties. In comparison, calyx fluid extracted from the C. plutellae ovary also has an immunosuppressive effect on P. xylostella . These two maternal (calyx fluid) and embryonic (teratocytes) factors are additive and result in a reduced level of nodule formation equivalent to that induced by natural parasitization. However, the immunosuppression of the parasitized P. xylostella does not appear to be due to inhibition of phospholipase A₂, an immune mediator, because injection of arachidonic acid failed to restore haemocyte nodulation capability.     

Organic and conventional fertilizer effects on a tritrophic interaction: parasitism,performance and preference of Cotesia vestalis

Interest in sustainable farming methods that rely on alternatives to conventional synthetic fertilizers and pesticides is increasing. Sustainable farming methods often utilize natural populations of predatory and parasitic species to control populations of herbivores, which may be potential pest species. We investigated the effects of several types of fertilizer, including those typical of sustainable and conventional farming systems, on the interaction between a herbivore and parasitoid. The effects of fertilizer type on percentage parasitism, parasitoid performance, parasitoid attack behaviour and responses to plant volatiles were examined using a model Brassica system, consisting of Brassica oleracea var capitata, Plutella xylostella (Lepidoptera) larvae and Cotesia vestalis (parasitoid). Percentage parasitism was greatest for P. xylostella larvae feeding on plants that had received either a synthetic ammonium nitrate fertilizer or were unfertilized, in comparison to those receiving a composite fertilizer containing hoof and horn. Parasitism was intermediate on plants fertilized with an organically produced animal manure. Male parasitoid tibia length showed the same pattern as percentage parasitism, an indication that offspring performance was maximized on the treatments preferred by female parasitoids for oviposition. Percentage parasitism and parasitoid size were not correlated with foliar nitrogen concentration. The parasitoids did not discriminate between hosts feeding on plants in the four fertilizer treatments in parasitoid behaviour assays, but showed a preference for unfertilized plants in olfactometer experiments. The percentage parasitism and tibia length results provide support for the preference–performance hypothesis.     

Diploid males sire triploid daughters and sons in the parasitoid wasp Cotesia vestalis

In the Hymenoptera, males develop as haploids from unfertilized eggs and females develop as diploids from fertilized eggs. In species with complementary sex determination (CSD), however, diploid males develop from zygotes that are homozygous at a highly polymorphic sex locus or loci. We investigated mating behavior and reproduction of diploid males of the parasitoid wasp Cotesia vestalis (C. plutellae), for which we recently demonstrated CSD. We show that the behavior of diploid males of C. vestalis is similar to that of haploid males, when measured as the proportion of males that display wing fanning, and the proportion of males that mount a female. Approximately 29% of diploid males sired daughters, showing their ability to produce viable sperm that can fertilize eggs. Females mated to diploid males produced all-male offspring more frequently (71%) than females mated to haploid males (27%). Daughter-producing females that had mated to diploid males produced more male-biased sex ratios than females mated to haploid males. All daughters of diploid males were triploid and sterile. Three triploid sons were also found among the offspring of diploid males. It has been suggested that this scenario, that is, diploid males mating with females and constraining them to the production of haploid sons, has a large negative impact on population growth rate and secondary sex ratio. Selection for adaptations to reduce diploid male production in natural populations is therefore likely to be strong. We discuss different scenarios that may reduce the sex determination load in C. vestalis.     

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.     

菜蛾盘绒茧蜂多分DNA病毒EP-1-like基因克隆、原核表达与多克隆抗体制备方法

本实验提取被菜蛾盘绒茧蜂寄生后24h的小菜蛾幼虫体内总RNA,反转录合成cDNA,以此为模板PCR扩增出菜蛾盘绒茧蜂多分DNA病毒（Cotesia vestalis polydnavirus,CvBV）EP-1-like基因,将其分别克隆到表达载体pET30a、pET28a中,转化宿主菌BL21（DE3）,获得单克隆重组质粒pET-30a-EP1L和pET-28a-EP1L。经IPTG诱导,pET-28a-EP1L成功表达了约34.8kDa的包涵体蛋白。将诱导条件优化以后,采用割胶回收的方法纯化包涵体,将纯化后的表达蛋白免疫新西兰大耳兔制备多克隆抗体。ELISA分析表明制备的抗体效价达1：128000,Western blot检测证明抗体具有良好的免疫反应特异性。