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 encoding 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. ORF301 and ORF302 were transiently expressed in hemocyte, fat body, gut, and epidermis of P. xylostella. To analyze effects of these genes on the parasitism, the segment of CpBV-S3 was injected to non parasitized larvae of P. xylostella, in which the two genes were expressed at least for four days post-injection. The P. xylostella larvae injected with CpBV-S3 exhibited significant immunosuppression, such as reduction in total hemocyte population, suppression of immune associated genes including cecropin, pro-phenoloxidase (PO) and serpin1, 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 by the viral segmenttreated larvae, but not by ORF302 specific dsRNA. The larval injected with CpBV-S3 exhibited an enhanced susceptibility to baculovirus infection. These results indicate that ORF301 of CpBV-S3, which containing BEN domain, suppresses both cellular and humoral immune responses in P. xylostella.

A genome project has been launched and aims to sequence total genome of Cotesia plutellae bracovirus. On this process, several open reading frames (ORFs) have been identified. This study was intended to clone and express protein tyrosine phosphatase genes, PTP1 and PTP6. The ORFs of these two genes consist of 900 and 891 bp, respectively. PTP1 and PTP6 are genes of a group of genes that has been implicated as important regulatory components in cell growth, differentiation and malignant transformation by certain viruses. In this work,we studied the cloning and expression patterns of these genes in Plutella xylostella, a lepidopteran host of C. plutellae. A polymerase chain reaction (PCR) produced the corresponding products of PTP1/6. These PCR products were cloned and expressed using an expression vector pBAD-TOPO, and then over-expressed using an inducer, L-arabinose. Then the purified proteins were confirmed using Western blotting (immunoblotting using V5 antibody) and the apparent molecular weights of both proteins were about 40 kDa. Expression of PTP genes were analyzed in the parasitized P. xylostella by realtime RT-PCR, which indicated late expression pattern of both PTPs during parasitization. We are pursuing to elucidate their physiological function in the parasitized host insect.

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 and antimicrobial responses. 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. The transiently expressed CpBV-IkB3 inhibited expression of hemolin, but did not those of lysozyme and cecropin in P. xylostella, while both lysozyme and cecropin were inhibited in the treated Spodoptera exigua. When the recombinant AcNPV was mixed with Bacillus thuringiensis subsp. kurstaki (Bt), the bacterial pathogenicity was significantly enhanced in a dose-dependent manner, compared to a Bt mixture with an AcMNPV recombined with an enhanced green fluorescence protein gene.

Viruses employ host translational machinery to synthesize their own proteins while negatively controling host protein translation. Endoparasitoid wasp (Cotesia plutellae) parasitizes young larvae of the diamondback moth (Plutella xylostella) larvae and possesses at least 27 genome segments. Two viral genes (CpBV15α and CpBV15 β) were obtained from cDNA library of the parasitized larvae showing no homology with any known polydnaviral genes. The parasitized larvae did not produce storage protein 1 (SP1) among at least three polypeptides (SP1, SP2, and SP3) at the stage of polydnaviral CpBV15β synthesis. When CpBV15β protein, which was expressed in Sf9 cells, was incubated with fat body isolated from nonparasitized larvae, SP1 synthesis was markedly inhibited. In vitro translation of mRNAs from nonparasitized larvae using rabbit reticulocyte lysate with CpBV15β significantly resulted in inhibition of SP1 synthesis, suggesting a negative role of CpBV15β in host protein synthesis.

A cysteine- rich protein encoded by Cotesia plutellae bacovirus (CpBV) was identified in the parasitized Plutella xylostella. The gene, called CpBV-CRP, encodes 189 amino acids with a signal peptide of 20 residues at N-terminus determined by bioinformatic analysis, suggesting a secretory protein. High CpBV-CRP expression in the parasitized P. xylostella was observed at early days after parasitization and decreased with the course of parasitization. Expression of CpBV-CRP was tissue-specific in the fat body/epidermis, but not in hemocyte and gut. Its physiological function was analyzed by transient expression of a CpBV segment containing CpBV-CRP. The treated larvae underwent an immunosuppression in terms of hemocyte-spreading behavior. When the treated larvae were also co-injected with dsRNA against CpBV-CRP, the suppressed hemocyte behavior was significantly recovered. This study reports a cysteine-rich protein encoded in CpBV genome and its physiological function to be an immunosuppressant.

DNA in the eukaryotic nucleus is packaged into highly organized chromatin. The basic structural unit of chromatin is the nucleosome, which consists of approximately 146 base pairs of DNA wrapped around a histone octamer core containing two molecules each of core histones H2A, H2B, H3, and H4. Histone covalent modification at the protruding N-terminal region from the nucleosomal core can change the chromatin conformation in order to regulate gene expression. A viral H4 was found in the genome of Cotesia plutellae bracovirus (CpBV). The obligate host of the virus is an endoparasitoid wasp, C. plutellae, which parasitizes the diamondback moth, Plutella xylostella, and interrupts host development and immune reactions. CpBV-H4 has been regarded as an immunosuppressive gene. Its extended N-terminal region contains nine lysine residues which are the target for modification. Previous report showed that CpBV-H4 inhibited hemocyte-spreading after transient expression. Here, transient expression of truncated CpBV-H4 (without N-terminal region) did not show high inhibitory effects on hemocyte-spreading. Moreover, the truncated CpBV-H4 induced acetylation of nucleus histone H4. Host H4 was found to be decreased in transcription after parasitization compared to nonparasitized larvae. Atransient expression of CpBV-H4 significantly inhibited host H4 transcription, suggesting a role of CpBV-H4 in controlling gene expression. Point mutagenesis study showed that two lysines (K6 and K16) of CpBV-H4 were found to have high inhibitory effects on hemocyte spreading. These results indicate the importance of CpBV-H4 and its N-terminal region to control gene expression and suppress host immunity.

The diamondback moth, Plutella xylostella, is reluctant to a baculovirus, Autographa california nucleopolyhedrosis virus (AcNPV) at its oral administration. However, parasitization by an endoparasitoid wasp, Cotesia plutellae, enhances the viral susceptibility. This study analyzed an antiviral activity of P. xylostella in response to the viral infection and determined the parasitic factor inhibiting the antiviral mechanism. For the analysis of antiviral activity of P. xylostella, a recombinant AcNPV expressing enhanced green fluorescence (AcNPV-EGFP) was orally adminstered to lavae of P. xylostella. After 24 h, EGFP expression was observed in the midgut tissue at a confocal-FITC mode. At the same time, a characteristic midgut melanotic response (MMR) was observed in some midgut regions under a phase contrast microscope. Thereafter, the EGFP signal was attenuated, while MMR spread on most midgut region. When the MMR was scored from 0 to 5 by the intensity of melanized cell density, it increased in time- and dose-dependent manners at the viral administration per os. These results suggest that the MMR is an antiviral activity of P. xylostella. This antiviral activity was significantly attenuated by C. plutellae parasitism. The parasitized P. xylostella showed significant decrease in the MMR score compared to nonparasitized larvae when they were orally administered with the same dose of AcNPV. To determine the parasitic factor(s) inhibiting the antiviral activity from the symbiotic polydnavirus of C. plutellae (C. plutellae bracovirus: CpBV), CpBV-IkB, which is a viral homolog of NFkB inhibitor and has been considered as an antiviral factor as in other polydnaviruses, was tested. A recombinant AcNPV expressing CpBV-IkB (AcNPV-IkB) was constructed and administered to P. xylostella larvae. As expected, AcNPV-IkB significantly decreased the antiviral activity measured by the MMR score compared to AcNPV-EGFP treatment. This study suggests that CpBV-IkB plays an antiviral parasitic role in the molecular interactions between P. xylostella and C. plutellae.

Reproductive incompatibility is an important factor to select a specific biological control agent for successful augmentation of the corresponding endogenous population. An endoparasitoid, Cotesia plutellae (Kurdjumov), is an effective control agent to diamondback moth, Plutella xylostella (L.) and has been known to be classified into two groups in terms of reproductive incompatibility. This study analyzed an Korean population of C. plutellae in terms of morphological characters and mitochondrial DNA marker, which did not match with either of two reproductive incompatibility groups. These results suggest that a Korean population of C. plutellae can be involved in a novel reproductive group. For any augmentation program of C. plutellae in Korea, reproductive incompatibility should be seriously considered to select a particular exotic population.

A solitary endoparasitoid, Cotesia plutellae, parasitizes diamondback moth, Plutellae xylostella. It bas been suggested that its antennae are a major sensory organ to search host and recognize host developmental and other physiological condition during parasitization. This research was performed to understand the parasitic behavior of C. plutellae by analyzing sensory types and their numbers on the antennae using scanning electron microscope. There was no significant difference in antennal length in both male and female C. plutellae, in which both sexes had 16 flagellomeres. Three different types of sensilla (trichoid, seta, and placodea sensilla) were located mostly on flagella and analyzed in their density on the different antennomeres. Trichoid sensillum was the major sensory type and showed about 87% density among all sensilla. Both trichoid and placodea types of sensilla exhibited even numbers on all flagellomeres with some decrease at terminal segments. In contrast, seta form of sensilla showed drastic increase in its density at distal part after 9th flagellomere. When distal half of flagellomeres were cut off, the C. plutellae could not parasitize host larvae. Even when only four distal flagellorneres were removed, the parasitism showed only 30%. These results indicate that C. plutellae antennae are required for parasitism and suggest that seta form of sensilla may play significant roles in recognizing host for parasitization.

Inhibitor <SUB>K</SUB>B (I<SUB>K</SUB>B)-like gene has been found ill the genome of Cotesia plutellae bracovirus (CpBV), which is the obligatory symbiont of an endoparsitoid wasp, C. plutellae. The open reading frame of CpBV-I<SUB>K</SUB>B was 417 bp and encoded 138 amino acids. Four ankyrin repeat domains were found in CpBV-I<SUB>K</SUB>B, which shared high homology with other known polydnavirus I<SUB>K</SUB>Bs. Considering a presumptive cellular I<SUB>K</SUB>B based on Drosophila Cactus, CpBV-I<SUB>K</SUB>B exhibited a truncated structure with deletion of signal-receiving domains, which suggested its irreversible inhibitory role in NF<SUB>K</SUB>B signal transduction pathway of the parasitized host in response to the wasp parasitization. CpBV-I<SUB>K</SUB>B was expressed only in the parasitized diamondback moth, Plutella xylostella. Its expression was estimated by quantitative RT-PCR during parasitization period, showing a constitutive expression pattern from the first day of parasitization. An indirect functional analysis of CpBV-I<SUB>K</SUB>B was conducted and suggested a hypothesis of host antivirus inhibition.