Parasitism of Cotesia spp. Enhances Susceptibility of Plutella xylostella to Other Pathogens

Two endoparasitoids, Cotesia plutellae and C. glomerata, parasitize the diamondback moth, Plutella xylostella, and induce significant host immunosuppression. This study analyzed the susceptibility changes of the parasitized P. xylostella against other pathogens using an entomopathogenic bacterium, Xenorhabdus nematophila (Xn), and a viral pathogen, Autographa californica nucleopolyhedrosis virus (AcNPV). The P. xylostella parasitized by either C. plutellae or C. glomerata exhibited higher susceptibilities to both microbial pathogens than the nonpara-sitized. To determine the parasitism factors inducing the enhanced susceptibility, three polydnaviral genes so far successfully cloned were selected from C. plutellae bracovirus (CpBV). CpBV-lectin and CpBV15 α/β were inserted into AcNPV under a CpBV promote and analyzed in their pathogenicities against P. xylostella larvae. Two AcNPVs recombined with CpBV15α/β were more potent than the control AcNPV recombined with an enhanced green fluorescent protein gene or the AcNPV recombined with CpBV-lectin. These results suggest that the wasp parasitization enhances other pathogen susceptibilities by inducing host immunosuppression, in which the symbiotic polydnavirus can play significant role in the enhanced susceptibility.     

Transient expression of specific Cotesia plutellae bracoviral segments induces prolonged larval development of the diamondback moth, Plutella xylostella

A polydnavirus, Cotesia plutellae bracovirus (CpBV), possesses a segmented and dispersed genome that is located on chromosome(s) of its symbiotic endoparasitic wasp, C. plutellae. When the host wasp parasitizes larvae of the diamondback moth, Plutella xylostella, at least 27 viral genome segments are delivered to the parasitized host along with the wasp egg. The parasitized P. xylostella exhibits significant immunosuppression and a prolonged larval development. Parasitized larvae take about 2 days longer than nonparasitized larvae to develop until the wandering stage of the final larval instar, and die after egress of the full grown wasp larvae. Developmental analysis using juvenile hormone and ecdysteroid analogs suggests that altering endocrine signals could induce the retardation of larval developmental rate in P. xylostella. In this study we used a transient expression technique to micro-inject individual CpBV genome segments, and tested their ability to induce delayed larval development of P. xylostella. We demonstrated that a CpBV segment was able to express its own encoded genes when it was injected into nonparasitized larvae, in which the expression patterns of the segment genes were similar to those in the larvae parasitized by C. plutellae. Twenty three CpBV genome segments were individually cloned and injected into the second instar larvae of P. xylostella and their effects assessed by measuring the time taken for host development to the cocooning stage. Three CpBV genome segments markedly interfered with the host larval development. When the putative genes of these segments were analyzed, it was found that they did not share any common genes. Among these segments able to delay host development, segment S27 was predicted to encode seven protein tyrosine phosphatases (CpBV-PTPs), some of which were mutated by insertional inactivation with transposons, while other encoded gene expressions were unaffected. The mutant segments were unable to induce prolonged larval development of P. xylostella. These results suggest that CpBV can induce prolonged larval development of P. xylostella, and that at least some CpBV-PTPs may contribute to the parasitic role probably by altering titers of developmental hormones.     

IkB genes encoded in Cotesia plutellae bracovirus suppress an antiviral response and enhance baculovirus pathogenicity against the diamondback moth, Plutella xylostella

An endoparasitoid wasp, Cotesia plutellae, parasitizes larvae of the diamondback moth, Plutella xylostella, with its symbiotic polydnavirus, C. plutellae bracovirus (CpBV). This study analyzed the role of Inhibitor-kB (IkB)-like genes encoded in CpBV in suppressing host antiviral response. Identified eight CpBV-IkBs are scattered on different viral genome segments and showed high homologies with other bracoviral IkBs in their amino acid sequences. Compared to an insect ortholog (e.g., Cactus of Drosophila melanogaster), they possessed a shorter ankyrin repeat domain without any regulatory domains. The eight CpBV-IkBs are, however, different in their promoter components and expression patterns in the parasitized host. To test their inhibitory activity on host antiviral response, a midgut response of P. xylostella against baculovirus infection was used as a model reaction. When the larvae were orally fed the virus, they exhibited melanotic responses of midgut epithelium, which increased with baculovirus dose and incubation time. Parasitized larvae exhibited a significant reduction in the midgut melanotic response, compared to nonparasitized larvae. Micro-injection of each of the four CpBV genome segments containing CpBV-IkBs into the hemocoel of nonparasitized larvae showed the gene expressions of the encoded IkBs and suppressed the midgut melanotic response in response to the baculovirus treatment. When nonparasitized larvae were orally administered with a recombinant baculovirus containing CpBV-IkB, they showed a significant reduction in midgut melanotic response and an enhanced susceptibility to the baculovirus infectivity.     

Transient expression of a viral histone H4, CpBV-H4, suppresses immune-associated genes of Plutella xylostella and Spodoptera exigua

A viral histone H4, CpBV-H4, is encoded in the Cotesia plutellae bracovirus (CpBV) genome. This polydnavirus is symbiotic with C. plutellae, an endoparasitoid wasp. When the wasp parasitizes its host, Plutella xylostella, the symbiotic CpBV is delivered to host hemocoel and infects different internal tissues. CpBV-H4 encoded in the virus exhibits high sequence similarity to host histone H4, except for an extended N-terminal tail (38 amino acids long). When the CpBV-H4 cloned in a eukaryotic expression vector was transiently expressed in P. xylostella and a nonhost, Spodoptera exigua, it clearly inhibited several immune-associated genes, including cecropin, gloverin, serpin, apolipophorin III, and transferrin. However, its truncated construct, prepared by deleting 38 amino acids at the N-terminal tail, lost its inhibitory activity against immune-associated genes of the both species. This study has verified an inhibitory activity of CpBV-H4 against host immune-associated genes and has provided a possibility to expand its activity spectrum to the genes of other insect species.     

Cotesia plutellae Bracovirus Genome and Its Function in Altering Insect Physiology

Polydnavirus is a group of animal DNA virus mutually associated with some ichneumonoid wasp. Its relatively large size of genome has been considered as a major source of the parasitoid function to manipulate developmental and immunological processes of target parasitized insects. Cotesia plutellae bracovirus (CpBV) is a polydnavirus derived from C. plutellae, which parasitizes the diamondback moth, Plutella xylostella. Parasitized P. xylostella exhibits altered physiological symptoms in development and immune reactions. Though several other parasitic factors such as ovarian proteins, venom, and teratocytes are identified, CpBV has been more focused on elucidating various host physiological alterations occurring due to the parasitism, which has driven the CpBV genome project. CpBV attains a typical bracovirus structure by its single unit membrane envelope, in which multiple nucleocapsids are enclosed. Its genome DNAs are segmented and located on the genome of C. plutellae. Its replication begins at adult tissue development during pupal stage. An apparent genome size is 471 kb estimated from 27 segments separated on 5% agarose gel. A current work on the genome has been completely sequenced 24 genomic segments and analyzed their genomic structure. The aggregated genome size is 351, 299 bp long and exhibits an average GC content of approximately 34.6%. Average coding density is about 32.3% and 125 putative open reading frames are predicted. Though more than half (52.5%) of predicted genes are annotated as hypothetical, the annotated CpBV genes share amino acid sequence homologies with those of other bracoviral genomes. The annotated genes are classified into the known bracoviral families, in which a family of protein tyrosine phosphatase is the largest including 36 ORFs, suggesting a significant role during parasitization. In addition, 8 and 7 ORFs encode Iκβ-like and EP1-like, respectively. Some predicted genes are known only in Cotesia-associated bracoviral genomes. Finally, two homologous genes, CpBV15α/β, are unique in CpBV genome, which are not matched to any other known polydnaviral genes. Their homology with malarian circumsporozoite toxin and eukaryotic translation inhibition factors suggests their function in host translation inhibitory factor. This review discusses CpBV genes on their putative physiological functions based on the molecular interactions between the host-parasite.     

Parasitism by Cotesia glomerata Induces Immunosuppression of Pieris rapae: Effects of Ovarian Protein and Polydnavirus

A gregarious endoparasitoid wasp, Cotesia glomerata, parasitizes the cabbage butterfly, Pieris rapae. During wandering larval stage for pupal metamorphosis, the parasitoid larvae egress from the parasitized host to form cocoons thus eventually leading to death of the host. This study focused on the effect of C. glomerata parasitization on cellular immune response of P. rapae. For this purpose, an ideal anticoagulant buffer was formulated to procure the hemocytes in native form with morphological, behavioral, and functional characteristics. The hemocytes selectively encapsulated only DEAE beads under in vitro conditions and a quantitative study revealed about 70% of the beads being encapsulated. On the other hand, calyx fluid from C. glomerata injected to P. rapae markedly inhibited the spreading ability of the hemocytes in a dose-dependent manner and also attenuated the in vitro encapsulation response of the hemocytes against the cationic bead. The calyx fluid contained polydnavirus as well as ovarian proteins. The isolated polydnavirus genome consisted of variously sized-segments with their unequal amounts. The P. rapae injected with the calyx fluid expressed several polydnaviral genes within 2 h. These results suggest that the immunosuppression of the parasitized P. rapae may be induced by the polydnaviral gene products as well as ovarian proteins.     

Transient expression of a polydnaviral gene, CpBV15beta, induces immune and developmental alterations of the diamondback moth, Plutella xylostella

The diamondback moth, Plutella xylostella, parasitized by its endoparasitoid wasp, Cotesia plutellae, undergoes various physiological alterations which include immunosuppression and an extended larval development. Its symbiotic virus, C. plutellae bracovirus (CpBV), is essential for their successful parasitization with more than 136 putative genes encoded in the viral genome. CpBV15β, a CpBV gene, has been known to play significant role in altering host physiological processes including hemocyte-spreading behavior through inhibition of protein synthesis under in vitro conditions. In the current study, we investigated its specific involvement in physiological processes of the host by transient expression and RNA interference techniques. The open reading frame of CpBV15β was cloned into a eukaryotic expression vector and this recombinant CpBV15β was transfected into nonparasitized 3rd instar P. xylostella by microinjection. CpBV15β was expressed as early as 24 h and was consistent up to 72 h. Due to the expression of this gene, plasma protein levels were significantly reduced and the ability of the hemocytes to adhere and spread on extracellular matrix was inhibited, wherein CpBV15β was detectable in the cytoplasm of hemocytes based on an indirect immunofluorescence assay. To confirm the role of CpBV15β, its double stranded RNA could efficiently recover the hemocyte-spreading behavior and synthesis of plasma proteins suppressed by the transient expression of CpBV15β. In addition, the larvae transfected with CpBV15β significantly suffered poor adult development probably due to lack of storage proteins. Thus these results demonstrate the role of CpBV15β in altering the host physiological processes involving cellular immune response and metamorphic development, which are usually induced by wasp parasitization.     

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.     

The role of host species, age and defensive behaviour on ovipositional decisions in a solitary specialist and gregarious generalist parasitoid (Cotesia species)

The main objective of this study was to determine the extent to which host acceptance behaviour as related to host species, age, and defensive behaviour might explain the differences in host use that exist between two congeneric and sympatric species of parasitic wasps. Cotesia glomerata (L.) (Hymenoptera: Braconidae) is gregarious and generalist on several species of Pieridae, whereas C. rubecula (Marshall) is solitary and specific to Pieris rapae (L.). Cotesia species differed in their responses to host species (P. brassicae (L.), P. napi (L.) and P. rapae) and developmental stage (early and late 1st, 2nd and 3rd instars). In no-choice tests, host acceptance by C. rubecula was higher for p. rapae and females did not distinguish among the 6 host ages. In contrast, when foraging for P. brassicae and P. napi, C. rubecula females more readily attacked early first instar. Cotesia glomerata showed a higher degree of behavioural plasticity towards acceptance of Pieris host species and host age than did C. rubecula. Cotesia glomerata females parasitized the three Pieris species and showed higher acceptance of first and second instars over third instar. Oviposition success was also influenced by host defensive behaviour. The frequency and the effectiveness of defensive behaviour rose with increasing age of the host, P. brassicae being the most aggressive Pieris species. Furthermore, the mean duration of C. glomerata oviposition was significantly reduced by the defensive reactions of P. brassicae, which would likely affect parasitoid fitness as oviposition time is positively correlated to clutch size in C. glomerata. Acceptance frequencies corresponded well to field reports of Pieris-Cotesia associations and to patterns of parasitoid larval performance, suggesting that the acceptance phase might be used as a reliable indicator of Cotesia host-specificity.     

Demonstration of Cytotoxicity against Wasps by Pierisin-1: A Possible Defense Factor in the Cabbage White Butterfly

The cabbage white butterfly, Pieris rapae, produces pierisin-1, a protein inducing apoptosis of mammalian cells. In the present study, the biological activity of pierisin-1 as a protective agent against parasitic wasps for P. rapae was examined. Pierisin-1 caused detrimental effects on eggs and larvae of non-habitual parasitoids for P. rapae, Glyptapanteles pallipes, Cotesia kariyai and Cotesia plutellae at 1–100 µg/ml, levels essentially equivalent to those found in P. rapae larvae. In contrast, eggs and larvae of the natural parasitoid of P. rapae, Cotesia glomerata proved resistant to the toxicity of pierisin-1 through inhibition of pierisin-1 penetration of the surface layer. The expression level of pierisin-1 mRNA in the larvae of P. rapae was increased by parasitization by C. plutellae, whereas it was decreased by C. glomerata. In addition, C. plutellae was associated with elevation of activated pierisin-1 in the hemolymph. From these observations, it is suggested that pierisin-1 could contribute as a defense factor against parasitization by some type of wasps in P. rapae.     

Transient transcription of a putative RNase containing BEN domain encoded in Cotesia plutellae bracovirus induces an immunosuppression of the diamondback moth, Plutella xylostella

A polydnavirus, Cotesia plutellae bracovirus (CpBV), possesses segmented genome located on chromosome(s) of an endoparasitoid wasp, C. plutellae. An episomal viral segment (CpBV-S3) consists of 11,017 bp and encodes two putative open reading frames (ORFs). ORF301 shows amino acid sequence homologies (28-50%) with RNase T2s of various organisms. It also contains BEN domain in C-terminal region. ORF302 is a hypothetical gene, which is also found in other bracoviruses. Both genes were expressed in larvae of Plutella xylostella parasitized by C. plutellae. Their expressions were detected in all tested tissues including hemocyte, fat body, gut, and epidermis. To analyze effects of these genes on the parasitism, the segment of CpBV-S3 was injected to nonparasitized larvae of P. xylostella, in which the two genes were expressed at least for 4 days post-injection. The larvae injected with CpBV-S3 exhibited significant immunosuppression, such as reduction in total hemocyte population and impairment in nodule formation behavior of hemocytes in response to bacterial challenge. Each gene expression in the treated larvae was inhibited by co-injecting respective double strand RNA (dsRNA) specific to each ORF. Injection of dsRNA of ORF301 could rescue the immunosuppression of the viral segment-treated larvae, while dsRNA specific to ORF302 did not. These results suggest that a putative RNase fused with a BEN domain encoded in CpBV-S3 plays a parasitic role in inducing host immunosuppression in the parasitism.     

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.     

<Emphasis Type="Italic">Cotesia plutellae</Emphasis> bracovirus suppresses expression of an antimicrobial peptide,cecropin, in the diamondback Moth, <Emphasis Type="Italic">Plutella xylostella</Emphasis>, challenged by bacteria

An endoparasitoid wasp, Cotesia plutellae, induces significant immunosuppression of host insect, Plutella xylostella. This study was focused on suppression in humoral immune response of P. xylostella parasitized by C. plutellae. An EST database of P. xylostella provided a putative cecropin gene (PxCec) which is 627 bp long and encodes 66 amino acids. A signal peptide (22 amino acids) is predicted and two putative O-glycosylation sites in threonine are located at positions 58 and 64. Without bacterial infection, PxCec was expressed in pupa and adult stages but not in the egg and larval stages. Upon bacterial challenge, however, the larvae expressed PxCec as early as 3 h post infection (PI) and maintained high expression levels at 12–24 h PI. By 48 h PI, its expression noticeably diminished. All tested tissues of bacteria-infected P. xylostella showed PxCec expression. However, other microbes, such as virus and fungus, did not induce the PxCec expression. Parasitization by C. plutellae suppressed the expression of PxCec in response to bacterial challenge. Among the parasitic factors of C. plutellae, its symbiotic virus (C. plutellae bracovirus: CpBV) alone was able to inhibit the expression of PxCec of P. xylostella challenged by bacteria. These results indicate that PxCec expression is regulated by both immune and developmental processes in P. xylostella. The parasitization by C. plutellae inhibited the expression of PxCec by the wasp’s symbiotic virus.     

Immunosuppression induced by expression of a viral RNase enhances susceptibility of Plutella xylostella to microbial pesticides

Abstract Polydnaviruses are a group of insect DNA viruses and are characterized in their segmented genome that is located in the chromosome(s) of host wasps. A polydnavirus, Cotesia plutellae bracovirus (CpBV), encodes a viral ribonuclease (RNase) T2 in a specific segment #3 (CpBV‐S3). This study tested its effect on gene expression associated with host immune responses in the diamondback moth, Plutella xylostella. Micro‐injection of CpBV‐S3 into nonparasitized larvae induced expression of its two encoded genes, CpBV‐ORF301 (=CpBV‐RNase T2) and CpBV‐ORF302. In response to a bacterial challenge, four antimicrobial peptide genes (hemolin, gloverin, cecropin and lysozyme) and six phenoloxidase (PO)–associated genes (proPO‐activating proteinase, PO, serine proteinase homolog and serpins 1–3) were up‐regulated in their expressions. However, the transient expression of CpBV‐S3 suppressed the expressions of cecropin, PO and serpin 1. Double‐stranded RNA specific to the viral RNase T2 could specifically knockdown the viral gene expression and restored the three gene expressions suppressed in the larvae injected with CpBV‐S3. The inhibitory activity of the viral RNase T2 on the target genes was further proven by the suppression of PO activation in response to bacterial challenge in the larvae injected with CpBV‐S3. This immunosuppression by the expression of the viral RNase T2 resulted in significant increase of pathogen susceptibility of P. xylostella against Bacillus thuringiensis or baculovirus infection.     

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.