菜蛾盘绒茧蜂多分DNA病毒的特性及其对小菜蛾幼虫的生理效应

对菜蛾盘绒茧蜂Cotesia plutellae多分DNA病毒的特性及其对寄主小菜蛾Plutella xylostella幼虫的生理效应进行了研究。结果表明：菜蛾盘绒茧蜂雌蜂输卵管萼中含有大量的多分DNA病毒（polydnavirus, PDV）;一个PDV内含多个核衣壳,最多可达16个;核衣壳长40～168 nm,直径39～40 nm;PDV仅在输卵管萼细胞内复制;雌蜂产卵时,随蜂卵将PDV注入寄主血腔,并扩散到寄主的许多组织中;PDV可能先通过脱膜再侵染寄主组织。雌蜂经Co⁶⁰辐射处理后再寄生（即假寄生）小菜蛾2龄、3龄和4龄初期的幼虫,被寄生后的寄主幼虫几乎全部不能化蛹,但末龄（即4龄）幼虫期显著延长,并在寄生后期,幼虫胸部有褐色的短翅芽出现;即将化蛹的4龄末小菜蛾幼虫被假寄生后,即使每头寄主被过寄生9次,依然能正常化蛹,但不能羽化。假寄生与正常寄生后寄主的脂肪体数量和形态结构有明显的不同,推测在正常寄生的情况下蜂卵孵化时释放的畸形细胞及随后的幼蜂可能对脂肪体的结构产生了作用。     

Baculoviral p94 homologs encoded in Cotesia plutellae bracovirus suppress both immunity and development of the diamondback moth,Plutellae xylostella

Polydnaviruses (PDVs) are a group of insect DNA viruses, which exhibit a mutual symbiotic relationship with their specific host wasps. Moreover, most encapsidated genes identified so far in PDVs share homologies with insect‐originated genes, but not with virus‐originated genes. In the meantime, PDVs associated with 2 wasp genera Cotesia and Glytapanteles encode some genes presumably originated from other viruses. Cotesia plutellae bracovirus (CpBV) encodes 4 genes homologous to baculoviral p94: CpBV‐E94k1, CpBV‐E94k2, CpBV‐E94k3, and CpBV‐E94k4. This study was conducted to predict the origin of CpBV‐E94ks by comparing their sequences with those of baculoviral orthologs and to determine the physiological functions by their transient expressions in nonparasitized larvae and subsequent specific RNA interference. Our phylogenetic analysis indicated that CpBV‐E94ks were clustered with other E94ks originated from different PDVs and shared high similarity with betabaculoviral p94s. These 4 CpBV genes were expressed during most developmental stages of the larvae of Plutella xylostella parasitized by C. plutellae. Expression of these 4 E94ks was mainly detected in hemocytes and fat body. Subsequent functional analysis by in vivo transient expression showed that all 4 viral genes significantly inhibited both host immune and developmental processes. These results suggest that CpBV‐E94ks share an origin with betabaculoviral p94s and play parasitic roles in suppressing host immune and developmental processes.     

Parasitism by Cotesia plutellae alters the hemocyte population and immunological function of the diamondback moth, Plutella xylostella

Cotesia plutellae, a solitary endoparasitoid wasp, parasitizes the diamondback moth, Plutella xylostella, and induces host immunosuppression and lethality in the late larval stage. This study focused on changes of cellular immunity in the parasitized P. xylostella in terms of hemocyte composition and cellular functions. In third and fourth instar larvae of nonparasitized P. xylostella, granular cells represented the main hemocyte type (60-70%) and plasmatocytes were also present at around 15% among the total hemocytes. Following parasitization by C. plutellae, the relative proportions of these two major hemocytes changed very little, but the total hemocyte counts exhibited a significant reduction. Functionally, the granular cells played a significant role in phagocytosis based on a fluorescence assay using fluorecein isothiocyanate-labeled bacteria. The phagocytic activity of the granular cells occurred as early as 5 min after incubation with the bacteria, and increased during the first 40 min of incubation. The parasitism by C. plutellae significantly inhibited phagocytosis of the granular cells. Plasmatocytes also exhibited minor phagocytic activity. Moreover, plasmatocyte phagocytosis was not inhibited by parasitism. On the other hand, hemocyte-spreading behavior in response to pathogen infection was significant only for plasmatocytes, which exhibited a characteristic spindle shape upon infection. A significant spreading of the plasmatocytes was found as early as 5 min after pathogen incubation and their ratio increased during the first 40 min.An insect cytokine, plasmatocyte-spreading peptide 1 (PSP1) from Pseudoplusia includens, was highly active in inducing plasmatocyte-spreading behavior of P. xylostella in a dose-dependent manner. P. xylostella parasitized by C. plutella was significantly inhibited in plasmatocyte-spreading in response to an active dose of PSP1. An in vivo encapsulation assay showed that the parasitized P. xylostella could not effectively form the hemocyte capsules around injected agarose beads. This research demonstrates that the parasitism of C. plutellae adversely affects the total hemocyte populations in number and function, which would contribute to host immunosuppression.     

小菜蛾主要寄生性天敌——菜蛾绒茧蜂与菜蛾啮小蜂间的相互作用

在28℃下,以小菜蛾3龄幼虫作寄主,研究了菜蛾绒茧蜂与菜蛾啮小蜂间的相互关系．当寄主供2种蜂同时产卵寄生时,与只供1种蜂时相比。绒茧蜂的寄生率无显著变化,而啮小蜂的寄生率则显著下降;2种蜂的合计寄生率与任一种蜂单独存在时相比无显著差异．当寄主先供绒茧蜂寄生,再供啮小蜂寄生时,绒茧蜂的成功寄生率不受影响,而啮小蜂的寄生率仅为8％～13％;啮小蜂能寄生在寄主体内的绒茧蜂高龄幼虫．绒茧蜂能寄生已被啮小蜂寄生的寄主幼虫,其子代部分个体能正常发育至成虫羽化．当已被绒茧蜂寄生和未被寄生的寄主同时存在时,啮小蜂主要寄生未被寄生的寄主．表明绒茧蜂具有竞争优势。但这种优势可因啮小蜂的寄生而被削弱．     

Host physiological changes due to parasitism of a braconid wasp, Cotesia plutellae, on diamondback moth, Plutella xylostella

Braconid wasps, Cotesia plutellae (Kurdjumov), were collected from parasitized host larvae of diamondback moth, Plutella xylostella (L.) in Korea. Virus particles were found in the oviduct lumen of C. plutellae females. Multiple nucleocapsids with approximately 30-nm diameter and variable length (30-80 nm) were surrounded with a single unit membrane envelope. The parasitization of C. plutellae completely inhibited pupal metamorphosis. The parasitized larvae showed significant decrease in feeding activity and total hemolymph proteins, especially as larval storage proteins. They also showed a significant decrease in immune capacity as evidenced by reduced ability to form hemocyte nodules and reduced phenoloxidase and lysozyme activity. Here, we show that C. plutellae has an endosymbiotic virus like other reported species in Microgastrinae, and suggest that it causes host developmental arrest and immune-depression at parasitization.     

小菜蛾的耐热性

小菜蛾是世界范围内十字花科蔬菜上的重要害虫.临界高温(critical thermal maxi-mum,CTMax)是昆虫耐热性的常用指标.采用动态加热方法,利用自行组装的装置测定了小菜蛾的临界高温,以此作为其耐热性指标,研究发育阶段、饲养温度、世代、性别和热激对小菜蛾耐热性的影响.结果表明:25℃下饲养的小菜蛾4龄幼虫的CTMax均值为50.31℃,显著高于1龄幼虫(43.03℃)、2龄幼虫(46.39℃)、3龄幼虫(49.67℃)以及雌性成虫(45.76℃)和雄性成虫(47.73℃);不同饲养温度(20、25和30℃)下成虫耐热性无显著差异;30℃下饲养1代、3代及6代的不同世代成虫CTMax也无显著变化;所有处理雌雄成虫的CTMax无显著差异;40℃下45 min热激可使5日龄雄成虫的CTMax值从45.51℃增加到46.49℃.     

A viral lectin encoded in Cotesia plutellae bracovirus and its immunosuppressive effect on host hemocytes

An endoparasitoid wasp, Cotesia plutellae, induces immunosuppression of the host diamondback moth, Plutella xylostella. To identify an immunosuppressive factor, the parasitized hemolymph of P. xylostella was separated into plasma and hemocyte fractions. When nonparasitized hemocytes were overlaid with parasitized plasma, they showed significant reduction in bacterial binding efficacy. Here, we considered a viral lectin previously known in other Cotesia species as a humoral immunosuppressive candidate in C. plutellae parasitization. Based on consensus regions of the viral lectins, the corresponding lectin gene was cloned from P. xylostella parasitized by C. plutellae. Its cDNA is 674 bp long and encodes 157 amino acid residues containing a signal peptide (15 residues) and one carbohydrate recognition domain. Open reading frame is divided by one intron (156 bp) in its genomic DNA. Amino acid sequence shares 80% homology with that of C. ruficrus bracovirus lectin and is classified into C-type lectin. Southern hybridization analysis indicated that the cloned lectin gene was located at C. plutellae bracovirus (CpBV) genome. Both real-time quantitative RT-PCR and immunoblotting assays indicated that CpBV-lectin showed early expression during the parasitization. A recombinant CpBV-lectin was expressed in a bacterial system and the purified protein significantly inhibited the association between bacteria and hemocytes of nonparasitized P. xylostella. In the parasitized P. xylostella, CpBV-lectin was detected on the surface of parasitoid eggs after 24 h parasitization by its specific immunostaining. The 24 h old eggs were not encapsulated in vitro by hemocytes of P. xylostella, compared to newly laid parasitoid eggs showing no CpBV-lectin detectable and easily encapsulated. These results support an existence of a polydnaviral lectin family among Cotesia-associated bracovirus and propose its immunosuppressive function.     

A putative protein translation inhibitory factor encoded by Cotesia plutellae bracovirus suppresses host hemocyte-spreading behavior

An endoparasitoid, Cotesia plutellae (Hymenoptera: Braconidae), possesses a mutualistic bracovirus (CpBV), which plays significant roles in the parasitized host, Plutella xylostella (Lepidoptera: Plutellidae). CpBV15beta, a viral gene encoded by CpBV, is expressed at early and late parasitization periods, suggesting that it functions to manipulate the physiology of the parasitized host. This paper reports a physiological function of CpBV15beta as an immunosuppressive agent. The effect of CpBV15beta on cellular immunity was analyzed by assessing hemocyte-spreading behavior. Parasitization by C. plutellae caused altered behavior of hemocytes of P. xylostella, in which the hemocytes were not able to attach and spread on glass slides. CpBV15beta was expressed in Sf9 cells using a baculovirus expression system and purified from the culture media. When hemocytes of nonparasitized P. xylostella were incubated with purified CpBV15beta protein, spreading behavior was impaired in a dose-dependent manner at low micro-molar range. This inhibitory effect of CpBV15beta could also be demonstrated on hemocytes of a non-natural host, Spodoptera exigua. CpBV15beta protein significantly inhibited F-actin growth of hemocytes in response to an insect cytokine. Similarly, cycloheximide, a eukaryotic translation inhibitor, strongly inhibited the spreading behavior and F-actin growth of P. xylostella hemocytes. Under in vitro condition, hemocytes of nonparasitized P. xylostella released proteins into the surrounding medium. Upon incubation of hemocytes with either CpBV15beta or cycloheximide, their ability to release protein molecules was markedly inhibited. This study suggests that CpBV15beta suppresses hemocyte behavior by inhibiting protein translation.     

Effects of selected insecticides on Cotesia plutellae, endoparasitoid of Plutella xylostella

Effects of field dosages ofselected insecticides to Cotesiaplutellae (Kurdjumov) (Hymenoptera: Braconidae), a larval endoparasitoidof Plutella xylostella L. (Lepidoptera: Plutellidae), wereinvestigated under laboratory conditions.Emergence of adult C. plutellae frominsecticide-treated pupae was not significantlydifferent from the control treatment. Contacttoxicity to C. plutellae adults variedgreatly among the insecticides in a paperresidue contact bioassay. Threeazadirachtin-based insecticides, Agroneem(4.8 mg a.i.liter^–1), Neemix (20 mga.i.liter^–1) and Ecozin (20 mgai.liter^–1) caused 11.1, 16.7 and 5.6%adult mortality, respectively. Of fourcommercial Bacillus thuringiensis (Bt)insecticides examined (all at 1.2 mga.i.liter^–1), Crymax and Xentari had noeffect on adult parasitoids, whereas Mattchcaused 5.6% mortality, and Dipel caused 11.1%mortality. Indoxacarb (53 mg a.i.liter^–1),-cyhalothrin (28 mg a.i.liter^–1) andspinosad (53 mg a.i.liter^–1) caused 100,88.5 and 50% adult mortalities, respectively.Low adult mortality (0–5.6%) was recorded fromingestion of azadirachtin-based, Btinsecticides and indoxacarb, compared with100% adult mortality in treatments of spinosador -cyhalothrin. Compared with the watercontrol, ingestion of azadirachtin-basedinsecticides significantly reduced parasitismby 50–57%, and Bt insecticides by 8–25%.However, ingestion of these insecticides didnot affect longevity of male and femaleparasitoid adults with one exception; femalelongevity was significantly reduced in theindoxacarb treatment. Insecticide residuescaused considerable mortality of C.plutellae adults, 39 and 44% mortality causedby 10 d old indoxacarb and -cyhalothrin,respectively, and 24 and 0% mortality causedby 7 and 10 d old residues of spinosad,respectively.     

A food-supply device for maintaining Cotesia vestalis,a larval parasitoid of the diamondback moth Plutella xylostella,in greenhouses

The limited availability of sugar sources (e.g., flowers) in greenhouses may affect biological pest control by parasitoid wasps. However, few studies have focused on feeding devices to provide parasitoids with sugar foods. We investigated the accessibility of a yellow-colored bottle-type feeding device to adult Cotesia vestalis (Halliday), a larval endoparasitoid of the diamondback moth Plutella xylostella (L.). All parasitoids died within four days in a room with no sugar source, whereas 66.7 % of individuals survived if a bottle-type feeding device providing honey solution was installed. We also investigated female longevity in response to different sugar solutions presented in a bottle-type feeding device. Honey and sugar mixtures (glucose and fructose) improved female longevity (38.4 and 39.2 days, respectively) much more than water (3.1 days), indicating these feeding devices containing sugar foods to be potentially useful for maintaining C. vestalis in greenhouses where natural food sources are limited.     

印楝素乳油对小菜蛾种群的控制作用

通过室内外系统试验探讨了印楝素乳油对小菜蛾Plutella xylostella种群的控制作用。结果表明,其控制作用主要表现在对成虫产卵的显忌避作用。对幼虫的拒食作用以及对生长发育的影响和减少幼虫危害为等方面,印楝素乳油0.005mL/59.0%,使小菜蛾种群趋势指数（I值）降为2.4干扰作用控制指数（IIPC）为0.1079,采用状态空间分析法就印楝素乳油施用后小菜蛾种群数量动态的模拟分析亦表明,其显的控制作用表现在种群发展初期对成虫的忌避作用。     

印楝素乳油x对-小菜蛾种群的控制作用

通过室内外系统试验探讨了印楝素乳油对小菜蛾Plutella xylostella种群的控制作用.结果表明,其控制作用主要表现在对成虫产卵的显著忌避作用、对幼虫的拒食作用以及对生长发育的影响和减少幼虫危害力等方面.印楝素乳油0.005 mL/L处理对成虫的忌避率为78.3%,对幼虫的选择性和非选择性拒食率分别为71.8%和62.9%,幼虫总取食量是对照的59.0%,使小菜蛾种群趋势指数(I值)降为2.4,干扰作用控制指数(1lpc)为0.1079.采用状态空间分析法就印楝素乳油施用后小菜蛾种群数量动态的模拟分析亦表明,其显著的控制作用表现在种群发展初期对成虫的忌避作用.     

Biological control of the diamondback moth, Plutella xylostella: A review

The diamondback moth (DBM), Plutella xylostella (L.) (Lepidoptera: Plutellidae), is one of the most destructive cosmopolitan insect pests of brassicaceous crops. It was the first crop insect reported to be resistant to DDT and now, in many crucifer producing regions, it has shown significant resistance to almost every synthetic insecticide applied in the field. In certain parts of the world, economical production of crucifers has become almost impossible due to insecticidal control failures. Consequently, increased efforts worldwide have been undertaken to develop integrated pest management (IPM) programs, principally based on manipulation of its natural enemies. Although over 130 parasitoid species are known to attack various life stages of DBM, most control worldwide is achieved by relatively few hymenopteran species belonging to the ichneumonid genera Diadegma and Diadromus, the braconid genera Microplitis and Cotesia, and the eulophid genus Oomyzus. DBM populations native to different regions have genetic and biological differences, and specific parasitoid strains may be associated with the specific DBM strains. Therefore, accurate identification based on genetic studies of both host and parasitoid is of crucial importance to attaining successful control of DBM through inoculative or inundative releases. Although parasitoids of DBM larvae and pupae are currently its principal regulators, bacteria-derived products (e.g., crystal toxins from Bacillus thuringiensis) and myco-insecticides principally based on Zoophthora radicans and Beauveria bassiana are increasingly being applied or investigated for biological control. Viruses, nematodes and microsporidia also have potential as biopesticides for DBM. When an insect pest is exposed to more than one mortality factor, there is the possibility of interactions that can enhance, limit, or limit and enhance the various aspects of effectiveness of a particular control tactic. This paper reviews the effectiveness of various parasitoids and entomopathogens against DBM, interactions among them, and their possible integration into modern IPM programs.     

云南弥渡小菜蛾发生及防治

通过田间调查 ,查明弥渡县小菜蛾年发生 1 0～ 1 2代 ,发生危害期有 9个月 ,一般有两个发生高峰期 ,即 4～ 6月和 9～ 1 0月 ,春季危害比秋季重 ;其发生影响因素主要有温度、降雨、十字花科蔬菜种植面积以及寄生蜂的种类和数量。针对小菜蛾发生规律及影响因素 ,提出了相应的防治措施。     

小菜蛾在温带地区越冬研究进展

小菜蛾是世界性重要害虫。在热带和亚热带地区冬季的十字花科植物上能正常发育繁殖,可见到各种虫态。但在温带冬季十字花科植物不能生长的地区,小菜蛾的越冬成为一个重要生态学问题。综述了亚洲、北美洲和欧洲小菜蛾越冬的研究进展。小菜蛾在日本的北海道、本州岛的北陆和东北大部分地区不能越冬,越冬北限相当于冬季积雪覆盖时间为60d的区域;在中国,小菜蛾不能在寒冷的东北地区越冬,在长江中下游以南地区冬季可见各虫态,但越冬北限尚不清楚;小菜蛾在冬天气候温和的韩国以及澳大利亚东南部继续发生;北美洲的加拿大西部和安大略地区大量的试验证明小菜蛾不能成功越冬,在美国南部小菜蛾冬季可正常发生,北部小菜蛾的越冬尚未见系统研究报道,但确认春季从南部运输的受小菜蛾感染的甘蓝等种苗是美国北部的重要虫源。小菜蛾在欧洲各地越冬的系统研究未见报道,没有证据表明在英国小菜蛾会发生有显著意义的越冬。目前小菜蛾越冬研究主要采用冬季直接试验观察和基于耐寒性试验的越冬预测两种方法。冬季直接试验观察法包括:(1)利用人工饲养的小菜蛾在田间各种潜在的越冬场所的越冬试验;(2)在秋播、野生或残留的十字花科植物上进行冬季种群的系统抽样调查;(3)越冬前后在前茬为十字花科植物的田块广泛搜寻普查小菜蛾的存活个体。基于耐寒性试验的越冬预测法:在获取小菜蛾越冬场所温度的基础上设计低温处理模式,试验低温处理后小菜蛾的存活率及后续发育和生殖。将试验数据和各地气温或小气候相结合,对小菜蛾在的越冬可能性进行推断。     

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.     

小菜蛾对几种常用药剂的敏感性测定

采用叶片浸溃法,在2001年10月和2002年10月分别测定了福州上街蔬菜基地的小菜蛾,对10种杀虫剂的敏感性。结果表明,上街菜区小菜蛾对灭多威、杀虫双、辛硫磷、毒死蜱、杀灭菊酯和功夫菊酯等农药极不敏感,已不能有效控制小菜蛾的危害。而小菜蛾对氟啶脲、阿维菌素、氟虫腈和虫螨腈比较敏感。通过两次毒力测定比较(2002年10月的LC50;2001年10月的LC50),结果表明,大多数杀虫剂敏感性变化较小。但小菜蛾对阿维菌素敏感性明显下降,毒力倍数达6．65倍,对氟虫腈毒力倍数相差1．48倍。此外,2002年10月小菜蛾对功夫菊酯的LC50为2001年10月的2．02倍。用浸叶法和浸溃法测定多杀菌素、氟虫腈与虫螨腈推荐浓度对小菜蛾幼虫的毒杀作用。结果表明3种药剂浸叶法处理效果均较理想。