Cotesia plutellae, an endoparasitoid wasp, parasitizes larvae of Plutella xylostella, and disrupt immune response of the host through parasitic factors. These immune disruption factors are maternal (venom proteins, polydnavirus, and ovary proteins) and embryonic (teratocytes) factors. In this study, we performed transcriptome analysis of venom glands of C. plutellae and identified neprilysin-1 (NEP1) known to be potential immunosuppression gene. Cp-NEP1 encoded 451 amino acid and belongs to hymeopteran NEP1 via phylogenetic analysis. Based on the structural comparison Cp-NEP1 lacks in conserved motifs such as substrate binding (NAYY/F), zinc-binding site (HExxH), zinc-binding site, protein folding and maturation (CxxW). To investigate function of Cp-NEP1, we constructed a recombinant Cp-NEP1 harboring N-terminally fused 6X His tag. Peptide sequencing revealed successful expression of the recombinant Cp-NEP1 in Escherichia coli. Pre-heated E. coli as antigen induced spike of nodule formation whereas co-injection of the recombinant Cp-NEP1 and pre-heated E. coli exhibited suppression of nodule formation in the host. Quantitative real-time PCR revealed that expression of phenoloxidase related to nodule formation was suppressed under co-injection of the recombinant Cp-NEP1 and E. coli. These results suggest that Cp-NEP1 contributes to immunosuppression of P. xylostella via phenoloxidase suppression and conserved motifs of neprilysin family are not required for host immune suppression.

One of the endoparasitoid wasp, Cotesia plutellae (Braconidae), parasitizes young larvae of the diamondback moth, Plutella xylostella. For the successful parasitization, C. plutellae required suppression of immune response in P. xylostella. Maternal (polydnavirus, venom proteins and ovary proteins) and embryonic (teratocytes) factors have been involved in immune-suppression. In this study, we performed transcriptome analysis of venom of C. plutellae and identify neprilysin-1 (Cp-NEP1) as a potential immunosuppressive protein. Cp-NEP1 encoded 451 amino acids and largely belongs to the hymeopteran neprilysin family via phylogenetic analysis. It is of interest that Cp-NEP1 has no conserved motifs such as zinc-binding domain (HExxH), substate binding domain (NAYY/F) and protein folding and maturation domain (CxxW) generally identified in other neprilysin family. In order to examine the biochemical function of Cp-NEP1, the recombinant Cp-NEP1 tagged with N-terminally 6X His was constructed and expressed in Escherichia coli. Expression of Cp-NEP1 was confirmed with SDS-PAGE and peptide sequencing. Recombinant Cp-NEP1 significantly suppressed nodule formation when the co-injection with E. coli. These results suggest that Cp-NEP1 contributes to suppression of immune response in P. xylostella and that the conserved motifs reported from other neprilysin do not involve immunosupperssion.

Translational control is a strategy for various viruses to manipulate their hosts to suppress any acute antiviral activity. Some cys-motif genes encoded in polydnaviruses or teratocytes act as host translation inhibitory factor (HTIF) to defend the host antiviral activity. A novel cys-motif gene, TSP13, was encoded in the genome of an endoparasitoid wasp, Cotesia plutellae. TSP13 consists of 129 amino acid residues with a predicted molecular weight of 13.987 kDa and pI value at 7.928. Genomic DNA region encoding open reading frame is interrupted with three introns. TSP13 was expressed in Plutella xylostella larvae parasitized by C. plutellae. C. plutellae bracovirus (CpBV) was purified and injected to nonparasitized P. xylostella. In the virus-injected P. xylostella, TSP13 was shown to be expressed by RT-PCR analysis. Thus, TSP13 was turned out to be encoded in the proviral CpBV genome. TSP13 was cloned into a eukaryotic expression vector, which was then used to infect Sf9 cells to transiently express TSP13. The synthesized TSP13 was detected in the culture broth. Purified TSP13 significantly inhibited cellular immune responses. Furthermore, TSP13 entered the target cells and was localized in the cytosol. This study reports a novel cys-motif gene, which is encoded in CpBV genome localized on chromosome(s) of C. plutellae and replicated to be encapsidated in the episomal viral particles during parasitization.

Cotesia plutellae known as an endoparasitoid parasitizes larvae of the diamondback moth, Plutella xylostella which is a major pest in cruciferous crops. For the successful parasitization, maternal and embryonic factors of C. plutellae such as polydnavirus, ovarian proteins, teratocytes and venom are required. In this study, we identified calreticulin (Cp-CRT) gene from transcriptome data of the venom gland in C. plutellae. cDNA of CRT was cloned from total RNA of the venom gland via PCR and encodes 403 amino acids harboring several structural motifs such as a signal peptide sequence, a repetitive sequence, a putative coiled-coil sequence encompassing, and endoplasmic reticulum-recognizing domain (-KDEL). Phylogenetic analysis showed that the Cp-CRT gene formed a unique cluster with other hymenopteran CRT genes, indicating that the Cp-CRT belongs to the CRT family. To examine the physiological function of Cp-CRT, recombinant Cp-CRT, fused with 6X-His at N-terminal was constructed and expressed in E. coli. Recombinant Cp-CRT was successfully expressed via Western blot analysis and suppressed significant nodule formation when co-injected with E. coli as immune response inducer. These results suggest that the Cp-CRT involves in suppression of cellular immune response in the host

In insects, the sense of smell is a complex and highly sensitive modality, governing essential decisions such as choice of food and oviposition sites. Plants emit substantial amounts of volatile organic compounds (VOCs), and the characteristic scent represents a dynamic communication channel. Understanding this odor-mediated system is critically important in the habitat management and in the largest view of the conservation biological control. Here we suggest that the fitness of the egg parasitoid ,an important biological control agent of the green vegetable bug may be improved through the understanding of chemical communication in the biological control system. Initially, the attractiveness of four flowering plant species, chosen from apanelofplants based on the longevity of the eggparasitoid on these plants, to T. basalis was assessed, which indicated significant behavioral attraction of T. basalis to the buckwheat flowers. Subsequently, and GC-MS analysis were carried out to identify the olfactory-active VOC semanated from buck wheat, demonstrating that the antennalol factory receptor neurons of T. basalis were responsive to some aliphatic acids as well as a few common plant volatiles. In behavioral bioassays using synthetic formulation based on the chemical and electrophysiological analysis, T. basalis exhibited significant behavioral attraction to the synthetic blend at optimum dose. The findings, in a wider perspective, form the basis for further improvement of the use of the companion plants that may increase the insects’ communities’ ability to persist in an environment.

Cotesia plutellae as an endoparasitoid wasp is the natural enemy against the diamondback moth, Plutella xylostella. For successful parasitism of the offspring, the female parasitoid always injects venom with other parasitic factors such as polydnavirus, teratocytes and ovary proteins. These venom products prevent their eggs from host immune system as well as modulate their host. To identify of constituents of venom in relation of host-parasitoid interaction, total RNA of the venom gland were extracted and analyzed with RNA-seq. RNA-seq reveals that 5.5 Gbp were read and approximately 3322 transcripts were identified. The transcripts related to the biological process, cellular components and molecular functions occupied about 20%, 15% and 18%, respectively. Among these transcripts, 301 genes were related to the immune activities such as calreticulin, defensing, phosphatases and serpin etc. These results suggest that some proteins exhibiting biological function related to the immune response can be contribute to the development of wasp in the host.

Teratocytes (TCs) are the cells derived from the embryonic serosal membrane of some parasitic hymenopteran insects. As a parasitic factor, TCs are multifunctional in host regulation by inducing nutritional, immune, and developmental alterations. However, little is understood about their genetic constituents. This study reveals a comprehensive view of the genes expressed by TCs through a transcriptome analysis based on RNAseq technology. More than 6.29 Gb sequences were used to assemble 34,686 contigs (>200 bp) and annotated into different functional categories. The TC transcriptome profile was clearly distinct from those of hemocytes and the fat body. The TC transcriptome contained components of insulin signaling and biosynthesis of juvenile hormone and 20-hydroxyecdysone. TCs also expressed various groups of digestive enzymes, supporting its nutritional role for the growing parasitoid larvae in parasitism. Furthermore, this transcriptome analysis annotated two kinds of immunosuppressive serine protease inhibitors (serpins) and Rho GTPase-activating proteins (RhoGAPs). To determine the biological functions of these factors, we devised ex vivo RNA interference (RNAi) by conducting knockdown of gene expression in in vitro cultured TCs followed by injection of the treated TCs to test insects. Ex vivo RNAi revealed that some serpins and RhoGAPs expressed in TCs inhibited host cellular immunity. This study reports a transcriptome of the unique TC animal cell, and its immunosuppressive genetic factors using ex vivo RNAi technology.

A viral histone H4 is encoded in a polydnavirus called Cotesia plutellae bracovirus (CpBV), which is symbiotic to an endoparasitoid wasp, C. plutellae. Compared to general histone H4, the viral H4 possesses an extra N-terminal tail containing 38 amino acid residues, which has been presumed to control host gene expression in an epigenetic mode. This study addressed the mutational analysis of extra N-terminal amino acid residues of a viral histone H4 and their epigenetic control efficacy. Mutational analysis was performed by serially deleting each of the nine amino acid residues from N-terminal tail of a viral histone H4. Transient expression of each truncated mutants (K1M-K19) in diamondback moth, Plutella xylostella, was performed by microinjection of a recombinant expression vector and confirmed by RT-PCR. Under transient expression, we analysed the effect of these mutations on target gene, transferin. Interestingly, we found that truncated mutants (K1M-K15) did not inhibit the expression of target gene but mutations thereafter (K6M-K9M) significantly alter its expression. As expected these truncated mutants (K1M-K5M) also inhibit hemocyte nodule formation and development of Plutella xylostella. This suggest that lysine residue (K6) in the N-terminal tail is very crucial for the epigenetic control efficacy of viral histone H4.

Cotesia plutellae has been known as a natural enemy against the Diamondback moth, Plutella xylostella via laying eggs into a larva. When the larva hatches from the egg, teratocytes also are released and expected to work as immune suppressor via secreting immune suppressive factors such as venom proteins, teratocytes and polydnavirus. In order to identify immune suppressive factors from teratocytes, we collected the supernatant from serum-free culture media of teratocytes. Concentration of secreted proteins from teratocytes was successfully performed with using centricons among tested methods such proteins precipitation and electrophoresed in sodium dodecyl sulfate polyacrylamide gel. The gel slices were directly digested with trypsin using in-gel digestion method and analyzed via LC-Ms-Ms. Molecular weight of peptide fragments were compared with protein database predicted by full-genome sequences of C. plutellae. We identified two immune responsive proteins, which are calreticulin, host cellular response-related gene and neprilysin 2, immune regulator. This result suggests that host immune response is suppressed or regulated by the immune suppressive factors of teratocytes.

A polydnavirus, Cotesia plutellae bracovirus (CpBV), is symbiotic to an endoparasitoid wasp, C. plutellae, which specifically parasitizes young larvae of the diamondback moth, Plutella xylostella. Parasitization by an endoparasitoid wasp, Cotesia plutellae, delays the larval development and metamorphosis in Plutella xylostella. Nutritional deprivation by the wasp may induce these developmental alterations in growing host. This study focussed on the change of insulin signaling of the parasitized host. The parasitized larvae exhibit a significant suppression in insulin-like peptide (ILP) expression, which was induced only by the injection of the CpBV. Reduced ILP expression significantly increased the blood sugar level (trehalose) level in the parasitized host, which was mimicked by starvation. Foxo was expressed in the parasitized larvae, but localized mostly in the nucleus. Overexpression of ILP gene in the parasitized larvae induced translation of Foxo to cytoplasm and significantly decreased trehalose level in the plasma. Interestingly, the overexpression of ILP gene significantly prevented the successful parasitization and allowed the host metamorphosis.

Polydnaviruses (PDVs) are a group of insect viruses and symbiotic to some endoparasitoid wasps classified in to Braconidae and Ichneumonidae. Though a lot of PDV genes are identified and analyzed in the host-parasitoid molecular interactions, PDV replication is still far from our understanding. PDVs are replicated in the wasp ovary during late pupal stage. A PDV, Cotesia plutellae bracovirus (CpBV), is symbiotic to Cotesia plutellae. The C. Plutellae ovary was analyzed in transcriptome by 454 pyrosequencing. The ovarian transcriptome provided several major DNA polymerases including Pol α, Pol δ, and Pol ε. All contigs matched to these polymerases were expressed in C. plutellae. Especially DP1 contig homologous to Pol α was highly expressed during late pupal and female adult stages. Its RNA interference significantly suppressed CpBV viral titre in the ovary. This study suggests a hint that CpBV replication uses a host DNA polymerase, in which Pol α may play a specific role in the viral replication in the ovary.

Cotesia plutellae has been known as a natural enemy against the Diamondback moth, Plutella xylostella via laying eggs into a larva. When the larva hatches from the egg, teratocytes also are released and expected to work as immune suppressor via secreting immune suppressive factors. In order to analyze the gene expression in teratocytes, total RNAs were isolated and genes expressed in the teratocyte were sequenced by Illumina HiSeq2000 RNASeq analysis. The information on RNA sequences was assembled by Trinity and contigs were annotated by Blast analysis. The levels of gene expression were calculated by FPKM. Approximately, 6.3 Gbs were obtained and 34,686 contigs were found and annotated. Forty two percent of contigs were homologous to previously reported genes and classified by gene ontologies: the highly abundant components are metabolic process, biological regulation and cellular process in biological function; binding, catalytic activity and transporter activity in molecular function; cell part, membrane part and organelle in cellular function, respectively. In addition, some teratocyte transcripts of C. plutellae are related to host regulation such as immunosuppression and nutrition: Ankyrin repeat proteins, Serpin, protease, lipase, chitinase and scavenger receptor.

국내 배추좀나방(Plutella xylostella) 집단은 피레스로이드 농약에 대해서 저항성을 보이며, 이는 이 살충제의 작용점인 소듐이온채널 유전자의 돌연변이에 기인된다. 더욱이 배추좀나방은 대부분 상용화된 살충제에 대해서 저항성을 발달시킬 수 있다. 본 연구는 배추좀나방을 효과적으로 방제하기 위해 내부기생성 천적인 프루텔고치벌(Cotesia plutellae)과 미생물농약인 Bacillus thuringiensis의 혼합처리 기술을 개발하기 위해 수행되었다. 프루텔고치벌이 감수성과 저항성 배추좀나방에 대한 기생 선호성에 차등이 있는 지 조사하기 위해 다섯 개 서로 다른 집단에 대해서 살충제 감수성과 프루텔고치벌 기생성 차이를 비교하였다. 이들 배추좀나방 집단들은 피레스로이드, 유기인계, 네오니코틴계 및 곤충성장조절제를 포함하는 세 종류의 상용 살충제에 대한 약제 감수성에서 뚜렷한 차이를 보였다. 그러나 이들 집단들은 프루텔고치벌에 의한 기생률에서는 차이를 보이지 않았다. 더욱이 기생된 배추좀나방은 B. thuringiensis에 대해서 감수성이 증가되었다. 프루텔고치벌이 갖는 면역억제인자 가운데 바이러스 유래 ankyrin 유전자(vankyrin)를 비기생된 배추좀나방에 발현시켰다. Vankyrin의 발현은 배추좀나방 3령충의 B. thuringiensis에 대한 감수성을 현격하게 증가시켰다. 즉, 프루텔고치벌에 의해 야기된 면역저하가 B. thuringiensis의 살충력을 증가시켰다. 이러한 결과들은 프루텔고치벌과 미생물농약인 B. thuringiensis의 혼합처리가 살충제 저항성 배추좀나방을 효과적으로 방제할 수 있다고 제시하고 있다.

Parasitism by an endoparasitoid wasp, Cotesia plutellae, results in significant immunosuppression of the diamondback moth, Plutella xylostella. Parasitized larvae significantly suffered higher susceptibility to a microbial biopesticide, Bacillus thuringiensis (Bt) than nonparasitized (NP) larvae. To find out an immunosuppressive agent causing the enhanced Bt efficacy, viral ankyrin (=vankyrin) genes encoded in C. plutellae bracovirus (CpBV) were analyzed by transient expression in NP larvae. CpBV segments containing different vankyrins were microinjected to NP larvae and expressed their encoded vankyrins. Expression of some vankyrins significantly inhibited immune response and enhanced Bt efficacy. This study suggests that expression of vankyrins suppress a cellular immune response and lose Bt tolerance of P. xylostella larvae.

An endoparasitoid wasp, Cotesia plutellae, parasitizes young larvae of the diamondback moth, Plutella xylostella, with its parasitic factors of polydnavirus, venom, ovarian proteins, and teratocytes (TC). TCs are originated from embryonic serosal membrane at hatch of C. plutellae eggs. TCs, after released in hemocoel of parasitized larvae, increased their average cell size from 20.6 μm to 77 μm during whole developmental period of the parasitoid larvae, but did not increase their cell number by maintaining about 150 cells per larvae. TCs of C. plutellae, are considered to be involved to extend the host larval development period and to arrest larval-pupal metamorphosis, were cultured in an insect cell culture medium for 21 days. Like TCs in parasitized larvae, in vitro cultured TCs showed increase in cell size, but did not show increase of cell number. Microinjection of in vitro cultured TCs significantly inhibited larva-to-pupa metamorphosis of nonparasitized P. xylostella, in which pupated host also showed extended larval period. Larvae injected with TCs exhibited alteration in expression of ecdysone receptor (EcR) and insulin receptor (InR) as well as in parasitized larvae. Teratocyte-secretory factors in culture medium showed this antimetamorphic effect on P. xylostella, while heat treated TC culture medium lost the effect. However, a successful parasitization of C. plutellae required both TCs and polydnavirus to alter host physiology.

An endoparasitoid wasp, Cotesia plutellae, contains a polydnavirus called C. plutellae bracovirus (CpBV) and induces various physiological alterations of parasitized host along with expressions of viral genes. Two host translation inhibitory factors (HTIFs) encoded in CpBV specifically inhibit host mRNAs at post-transcriptional level. They are expressed in late larval stage of Plutella xylostella parasitized by C. plutellae. To understand their late expression control, promoter region of an HTIF gene called CpBV15α was cloned by inverse PCR. The cloned HTIF upstream region (1,113 bp) possessed a putative JH response element (JHRE) and other promoter elements. The putative promoter region was rejoined with an open reading frame of enhanced green fluorescence protein (EGFP). When the recombinant vector construct was injected into early third instar larvae of nonparasitized P. xylostella, it was expressed in fourth larval instar at 72 h after injection, compared to relatively early expression in 24 h after injection of control construct containing a baculovirus immediate-early promoter. However, recombinant EGFP construct lost the late expression pattern when its promoter region was incomplete by truncating JHRE region. PYR application inhibited EGFP expression of the recombinant construct, but gave little influence on truncated constructs. Interestingly, when the complete promoter construct was injected to pupal stage, its late expression pattern was lost and showed early expression pattern. However, an addition of PYR to pupae, which had been injected with the complete promoter construct, inhibited the reporter gene expression. These results suggest that late expression of a HTIF (CpBV15α) is controlled by its promoter, which is sensitive to host JH titer.

Teratocytes are originated from embryonic serosal membrane of some endoparasitoid wasps. Cotesia plutellae eggs release teratocytes in parasitoid host hemocoel at hatch in about 150 cells per egg. Teratocytes of C. plutellae were cultured in an insect culture medium for at least 14 days. Teratocytes cultured in vitro showed no increase in cell numbers but increased in cell size. Similarly,teratocytes in parasitized larvae did not increase cell numbers, but increased their cell size. Microinjection of invitro cultured teratocytes in to third instar larvae of nonparasitized Plutella xylostella showed a dose-dependently inhibitory effect on development and larval-pupal metamorphosis. In addition, teratocytes prolonged the immature developmental period and reduced the pupation rate, in which young aged host larvae were more sensitive to teratocytes treatment than old larvae. These results suggest that teratocytes play a crucial role in successful parasitization of C.plutellae by altering host developmental program.

A polydnavirus, Cotesia plutellae bracovirus (CpBV), is symbiotic to an endoparasitoid wasp, C.plutellae, which specifically parasitizes young larvae of the diamondback moth, Plutella xylostella. A recent study on CpBV replication by analysis of ovary transcriptome of C.Plutellae suggests several candidate coat protein genes. This study was conducted to confirm the coat protein genes by analyzing coat proteins of CpBV viral particles by a tandem mass MALDI-TOF. Immunoprecipitation of ovary protein extract with a polyclonal CpBV antibody captured three proteins named as p35, p60, and p70. More number of coat proteins were resolved in a protein extract directly from viral particles. All candidate coat proteins are analyzed in amino acid sequences by MALDI-TOF. A comprehensive analysis of viral proteomics and ovary transcriptome determined novel viral coat proteins from CpBV

Parasitization by an endoparasitoid wasp, Cotesia plutellae, extends a larval period of Plutella xylostella and inhibits a larva-to-pupa metamorphosis. To determine antimetamorphic parasitic factor(s) in this host-parasitoid interaction, an effect of its symbiotic polydnavirus, Cotesia plutellae bracovirus (CpBV), was investigated by injecting purified virus particles to nonparasitized larvae of P. xylostella. Larvae injected with CpBV exhibited antimetamophosis in a viral dose-dependent manner. Also, the susceptibility to the viral injection was increased at young larval stages. Parasitized or virus-injected larvae shwed significant decrease in cell size of prothoracic gland and reduction in expression of ecdysone receptor (EcR) gene. However, they increased and maintained expression of insulin receptor (InR) gene. Twenty four CpBVsegments were individually injected to nonparasitized larvae. Only two segments (S22 and S27) had significant antimetamorphic effect. Subsequent RNA interference using double stranded RNA (dsRNA) was performed in each of encoded genes in each segment. Protein tyrosine phosphatase, ELP, and three hypothetical genes were determined to be antimetamorphic factors.

Polydnaviruses (PDVs) are a group of insect double stranded DNA viruses and symbiotically associated with host endoparasitoid wasps. Their segmented genome is located in host chromosome(s) in a proviral form. Viral replication is initiated at the ovary during late pupal stages. Little is known about the factors involved in the viral replication. This study analyzed the ovarian transcripts of an endoparasitoid wasp, Cotesia plutellae, by 454 pyrosequencing and subsequent gene annotation. Out of 2,226 contigs and 12,457 singletons, 50 transcripts categorized in DNA replication, coat proteins, and viral origins were selected as putative viral replication factors. The selected genes were analyzed in their expressions according to host wasp development. Quantitative real-time RT-PCRs showed that some of the selected genes were expressed during the viral replication at late pupal stage. Using RNA interference, five putative genes were tested in their implication in the viral replication by analyzing viral DNA amplification, structure of ovarian calyx, and parasitism. RNA interference of contig#1004 (broad complex) or contig#174 (a viral DNA polymerase gene) significantly inhibited DNA amplification without any impairment of viral formation, and subsequently resulted in significant reduction in the wasp parasitism. This study reports that two wasp genes (or not encapsidated viral genes) are implicated in the viral DNA amplification and viral coat protein production during the polydnaviral replication.