Ultrastructure of Cotesia plutellae Bracovirus in Its Replication at Wasp Ovarian Calyx

Cotesia plutellae bracovirus (CpBV) is a polydnavirus symbiotic to an endoparasitoid, C. plutellae. Despite rich information on CpBV genome, there has been little known on its viral replication mode from proviral to episomal form. This study illustrates fine structures of the epithelial cells producing CpBV with a reference to non-producing ovarian epithelial cells. The ovarian epithelial cells of teneral females (within 12 h after emergence) were characterized by large nucleus and rich rough endoplasmic reticulum. CpBV particles were present only at the calyx region, in which follicle epithelial cells exhibited virogenic structures. Though a matured CpBV particle found in the calyx lumen was encapsidated in a single envelop containing multiple nucleocapsids, numerous free nucleocapsids were observed in the calyx epithelial cells and appeared to undergo assembly step to a final multiple capsid form. The multiple capsid forms appeared to be released into the oviduct lumen. The epithelial cells bordering the oviduct lumen showed phagocytosis presumably due to clearing cellular debris. At the calyx area close to the common oviduct, the epithelial cells appeared to maintain protein synthetic activity due to highly developed rough endoplasmic reticulum, but showed a marked decrease in the viral production.     

Genome size estimation of an endoparasitoid wasp,Cotesia plutellae,using quantitative real-time polymerase chain reaction

An endoparasitoid wasp, Cotesia plutellae, is a natural enemy against the diamondback moth, Plutella xylostella, which is the most destructive insect pest of cruciferous crop plants. The wasp genome contains genetic information of several parasitic factors, such as its symbiotic virus (C. plutellae bracovirus), venom, teratocyte as well as the parasitoid itself. These parasitic factors interfere with physiological processes of the immature stages of P. xylostella and need to be analyzed concerning their genetic components. A full genome sequence of C. plutellae would be highly informative to determine functional genes associated with these parasitic factors. Before a full genome sequence analysis of C. plutellae can be undertaken, an estimate of genome size is needed. In this study, we used a strategy using a quantitative real-time polymerase chain reaction (qPCR) to measure the wasp genome size. qPCR determined the number of a single copy gene hexokinase in a total mass of genomic DNA. The resulting molecular weight of the DNA sample was used to calculate the genome size in base pair (bp). This estimation approach indicated a genome size of C. plutellae of 186.06 ± 1.21 Mb.     

Electroantennogram and behavioral responses of Cotesia plutellae to plant volatiles

Plant volatiles have been demonstrated to play an important role in regulating the behavior of Cotesia plutellae, a major larval parasitoid of the diamondback moth (DBM), Plutella xylostella, but little is currently known about the function of each volatile and their mixtures. We selected 13 volatiles of the DBM host plant, a cruciferous vegetable, to study the electroantennogram (EAG) and behavioral responses of C. plutellae. EAG responses to each of the compounds generally increased with concentration. Strong EAG responses were to 100 μL/mL of trans‐2‐hexenal, benzaldehyde, nonanal and cis‐3‐hexenol, and 10 μL/mL of trans‐2‐hexenal and benzaldehyde with the strongest response provoked by trans‐2‐hexenal at 100 μL/mL. In the Y‐tube olfactometer, C. plutellae, was significantly attracted by 1 μL/mL of trans‐2‐hexenal and benzaldehyde. β‐caryophyllene, cis‐3‐hexenol or trans‐2‐hexenal significantly attracted C. plutellae at 10 μL/mL, while nonanal, benzyl alcohol, cis‐3‐hexenol or benzyl cyanide at 100 μL/mL significantly attracted C. plutellae. Trans‐2‐hexenal significantly repelled C. plutellae at 100 μL/mL. EAG of C. plutellae showed strong responses to all mixtures made of five various compounds with mixtures 3 (trans‐2‐hexenal, benzaldehyde, nonanal, cis‐3‐hexenol, benzyl cyanide, farnesene, eucalyptol) and 4 (trans‐2‐hexenal, benzaldehyde, benzyl alcohol, (R)‐(+)‐limonene, β‐ionone, farnesene, eucalyptol) significantly attracting C. plutellae. These findings demonstrate that the behavior of C. plutellae can be affected either by individual compounds or mixtures of plant volatiles, suggesting a potential of using plant volatiles to improve the efficiency of this parasitoid for biocontrol of P. xylostella.     

An Evidence for Host Translation Inhibitory Factor Encoded in a Polydnavirus,Cotesia glomerata Bracovirus,Genome and Its Expression in Parasitized Cabbage White Butterfly,Pieris rapae

Polydnavirus is a DNA virus symbiotic to some endoparasitic wasps and plays a critical role in accomplishing successful parasitic life cycle of host wasps. Host translation inhibitory factor (HTIF) has been found in some polydnaviral genomes and performs parasitic functions leading to host immunosuppression and redirecting host nutrient usage to wasp development. The cabbage white butterfly, Pieris rapae, parasitized by a gregarious endoparasitoid, Cotesia glomerata, undergoes several physiological alterations including immune malfunctioning and failure of pupal metamorphosis. C. glomerata possesses its own symbiotic polydnavirus, C. glomerata bracovirus (CgBV). Its genome consisted of at least 12 segments in unequal amounts. Parasitized P. rapae hemolymph contained HTIF-like protein, which was determined through an immunoblotting assay using HTIF antibody of C. plutellae bracovirus (CpBV). RT-PCR using HTIF primers of CpBV produced an HTIF-like gene in P. rapae larvae parasitized by C. glomerata. Also, this HTIF-like gene was encoded in CgBV genome and its partial sequence of CgBV showed highly homology (98.5%) to amino acid sequence of an HTIF of CpBV, called CpBV15a. These results suggest that a common HTIF-like moiety may be shared among Cotesia-associated bracovirus.     

The Bracovirus Genome of the Parasitoid Wasp Cotesia congregata Is Amplified within 13 Replication Units,Including Sequences Not Packaged in the Particles

The relationship between parasitoid wasps and polydnaviruses constitutes one of the few known mutualisms between viruses and eukaryotes. Viral particles are injected with the wasp eggs into parasitized larvae, and the viral genes thus introduced are used to manipulate lepidopteran host physiology. The genome packaged in the particles is composed of 35 double-stranded DNA (dsDNA) circles produced in wasp ovaries by amplification of viral sequences from proviral segments integrated in tandem arrays in the wasp genome. These segments and their flanking regions within the genome of the wasp Cotesia congregata were recently isolated, allowing extensive mapping of amplified sequences. The bracovirus DNAs packaged in the particles were found to be amplified within more than 12 replication units. Strikingly, the nudiviral cluster, the genes of which encode particle structural components, was also amplified, although not encapsidated. Amplification of bracoviral sequences was shown to involve successive head-to-head and tail-to-tail concatemers, which was not expected given the nudiviral origin of bracoviruses.     

Genomic segments cloning and analysis of Cotesia plutellae polydnavirus using plasmid capture system

Cotesia plutellae polydnaviruses (CpBV) has a segmented genome consisting of multiple circular double stranded DNAs. Recently, we have developed an easy, simple, and convenient system based on Tn7 transposition in order to clone genomic segments of CpBV in Escherichia coli cell and designated plasmid capture system (PCS). The PCS donor-S transferred a pUC19 origin of replication and an ampicillin resistance marker into CpBV genomic DNA by in vitro transposition. Through PCS system, we were able to clone 53 genomic clones ranging from 0.1 to 25.5 kb and further they were classified into 29 segments by their sizes and restriction endonuclease patterns. Among them, a complete nucleotide sequence of CpBV-S28 segment was determined and 10 putative genes were predicted from this segment. Interestingly, 9 of 10 putative ORFs had high level of similarities with catalytic domain of protein tyrosine phosphatase. Also, ORF2807 showed similarity with EP1-like proteins of C. congregata polydnavirus.     

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.     

Complementary sex determination in the parasitoid wasp Cotesia vestalis (C. plutellae)

In the Hymenoptera, single locus complementary sex determination (sl-CSD) describes a system where males develop either from unfertilized haploid eggs or from fertilized diploid eggs that are homozygous at a single polymorphic sex locus. Diploid males are often inviable or sterile, and are produced more frequently under inbreeding. Within families where sl-CSD has been demonstrated, we predict that sl-CSD should be more likely in species with solitary development than in species where siblings develop gregariously (and likely inbreed). We examine this prediction in the parasitoid wasp genus Cotesia, which contains both solitary and gregarious species. Previous studies have shown that sl-CSD is absent in two gregarious species of Cotesia, but present in one gregarious species. Here, we demonstrate CSD in the solitary Cotesia vestalis, using microsatellite markers. Diploid sons are produced by inbred, but not outbred, females. However, frequencies of diploid males were lower than expected under sl-CSD, suggesting that CSD in C. vestalis involves more than one locus.     

高效液相色谱法检测菜蛾绒茧蜂幼虫体内多杀菌素残留

应用高效液相色谱法检测施用于寄主幼虫的多杀菌素可否传递到在其体内发育的寄生蜂幼虫。以对多杀菌素高抗的小菜蛾Plutella xylostella幼虫作为菜蛾绒茧蜂Cotesia plutellae的寄主,待绒茧蜂发育到1龄幼虫时,将浓度为50 mg/L的多杀菌素点滴到寄主幼虫背板上,随后让寄主幼虫取食经50 mg/L多杀菌素处理过的甘蓝叶片,寄主幼虫和其体内的蜂幼虫再发育3天后,将寄主幼虫解剖取出蜂幼虫,用高效液相色谱法对经多杀菌素处理的小菜蛾幼虫体液以及绒茧蜂幼虫匀浆液进行检测,结果多杀菌素的2个活性成分spinosyn A和spinosyn D均被检测到,两者的多杀菌素残留浓度分别是2.79 mg/L和0.94 mg/L。这表明,通过寄主幼虫体壁接触和取食进入其体内的多杀菌素,可通过寄生蜂幼虫体壁浸透、蜂幼虫对寄主血淋巴的取食,或这两种途经一起进入蜂幼虫的体内,对蜂幼虫产生作用。     

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.     

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。     

Seasonal changes of resistance to fenvalerate and cypermethrin in the larval parasitoid Cotesia plutellae （Hymenoptera： Braconidae）

The seasonal changes of insecticide resistance and stability in hymenopteran Cotesia plutellae, collected from Jianxin, Fuzhou-City, and Shangjie, Minhou-County, Fujian, China, were assessed by using a dry residual film method. The resistance to two insecticides in the field populations of C. plutellae was not stable under insecticide-free conditions in the insectarium. Compared with susceptible F11 progeny of C. plutellae in the insectarium, the resistance ratios （RR） in F0 parents were 18.4 for fenvalerate and 11.4 for cypermethrin based on LC50 at 9 hours, and 32.8 for fenvalerate and 28.5 for cypermethrin based on LC50 at 24 hours when the parasitoids were left in contact with the insecticides for 1 hour and mortalities were recorded at 9 and 24 hours, respectively. However, the RR in a field population of C. plutellae were 9.2 for fenvalerate and 12.7 for cypermethrin, if the parasitoids were left in contact with the insecticides for 24 hours. The resistances to the two pyrethroids in other field populations collected from Jianxin and Shangjie from November 2000 and July 2004 were also determined. Significant seasonal variations of resistance to the two insecticides in the field populations of C. plutellae were found. The RR were 3.0-18.4 for fenvalerate and 4.8-20.6 for cypermethrin in Jianxin populations from November 2000 to April 2002 based on LC50 at 9 h, and 2.3-13.6 for fenvalerate and 3.6-16.0 for cypermethrin in Shangjie populations from May 2002 to July 2004 based on LC50 at 24 hours. The resistance levels were high in spring and autumn and decreased sharply in summer. In addition, significant recovery from the knocked-down caused by the insecticides was found in the F0 and field populations of C. plutellae which were resistant to fenvalerate and cypermethrin if the parasitoids were left in contact with the pyrethroids for 1 hour. However, no recovery was found in susceptible F11 progeny.     

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。     

A copy of cystatin from the diamondback moth Plutella xylostella is encoded in the polydnavirus Cotesia plutellae bracovirus

Cystatins (CSTs) are reversible and competitive inhibitors of cysteine proteases. Some polydnaviruses encode viral CSTs that have been speculated to play a crucial role in viral pathology. Four CSTs have been reported in the episomal genome of a polydnavirus, Cotesia plutellae (synonymous with C. vestalis) bracovirus (CpBV). These 4 CSTs share high sequence homologies with other bracoviral CSTs. Further sequence analysis showed that 2 of the CpBV-CSTs are identical. The remaining 3 CSTs have been designated CpBV-CST1, CpBV-CST2, and CpBV-CST3. Expression analysis indicated that CpBV-CST2 was not expressed in any stage of Plutella xylostella, either parasitized or non-parasitized by C. plutellae. However, both CpBV-CST1 and CpBV-CST3 were expressed in all stages of P. xylostella. Interestingly, these 2 genes were also expressed in non-parasitized P. xylostella in all developmental stages. A CST sequence from the non-parasitized larva was 100% identical with that of CpBV-CST1 for the entire open reading frame (ORF). To understand the role of CpBV-CST1 in viral pathology, the ORF was cloned into a eukaryotic expression vector and transiently expressed in non-parasitized larvae. The in vivo transient expression lasted for at least 4 days. Under this condition, the treated larvae suffered significant suppression in immune responses and in development. These results suggest that CpBV-CSTs play a crucial role in parasitism, altering host immune and developmental processes by interrupting normal interactions between CSTs and cysteine proteases in P. xylostella.     

Cotesia plutellae parasitizing Plutella xylostella: Host-age dependent parasitism and its effect on host development and food consumption

The braconid Cotesia plutellae(Kurdjumov) (Hymenoptera: Braconidae) is amajor solitary, larval endoparasitoid of thediamondback moth, Plutella xylostella(L.) (Lepidoptera: Plutellidae). Parasitism oflarvae of different host instars and fourdevelopmental ages of the 4th instar ofthe pest was examined. The effects of hostinstar at initial parasitization on thedevelopment, survival, size and fecundity ofthe parasitoid were determined in thelaboratory at 25 °C. The effects ofparasitism on host development and foodconsumption were investigated at 28 °C.Cotesia plutellae could parasitize larvaeof all four instars of P. xylostella, butpreferred 2nd and 3rd instars. In achoice test, the relative parasitism indicesfor 2nd, 3rd and 4th instarswere 0.37, 0.39 and 0.24, respectively.Parasitism decreased sharply with increasinghost age in the 4th instar and approachedzero in host larvae that had gone beyond 37%of 4th stadium. The development time andthe final adult size of the parasitoid variedwith the host instar at initial parasitization.Parasitoids with initial parasitism in the4th instar hosts had the shortestdevelopment time, followed by those in the3rd instar, and then by those in the2nd instar. Parasitoids startingparasitism in 2nd instar hosts weresmaller in body size than those starting in the3rd or 4th instar. However, resultantfemales starting parasitism in 3rd instarhosts had the highest fecundity. Parasitizedlarvae exhibited longer development time andincreased food consumption compared withunparasitized ones. This study presents thefirst record that a solitary parasitoidregulates host behavior leading to an increasein food consumption by the host.     

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．