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.

To clarify the molecular mechanism of metamorphosis, we analyzed the Broad-Complex (BR-C) gene in the beet armyworm, Spodoptera exigua. We obtained a partial BR-C sequence from a pyrosequencing cDNA library. The BR-C of S. exigua was expressed only in the final larval instar, at which its expression was clearly detected in the epidermis. A treatment of a juvenile hormone analog, pyriproxyfen, inhibited larval-pupal metamorphosis and suppressed the BR-C expression. The hormonal treatment also inhibited expression of two storage protein genes that were usually expressed during a final larval instar. RNA interference of the BR-C using its double strand RNA suppressed BR-C expression and inhibited the larval-pupal metamorphosis. These results suggest that the BR-C is critical to induce larval-pupal metamorphosis of S. exigua.

A polydnavirus, Cotesia plutella bracovirus (CpBV), encodes host translation inhibitory factors (HTIFs). Two HTIFs have been identified and named as CpBV15α and CpBV15β. HTIFs specifically inhibit host gene expression at post transcriptional level. This suggests that HTIFs can discriminate host mRNAs for their inhibition. To test this hypothesis, we chose a storage protein 1 (SP1) for a target and CpBV15β for non-target genes. Both target and non-target genes were constructed in a cloning vector by cloning their 5'UTR+ORF and in vitro transcribed. The capped partial mRNAs were in vitro translated using rabbit reticulocyte lysate in the presence or absence of HTIFs. Translation of SP1 mRNA was significantly inhibited by the HTIF, but those of CpBV15α and CpBV15β were not. We prepared chimeric mRNAs by exchanging 5'UTR and ORF of both target and non-target genes. Inhibitory activity of HTIFs was dependent on the 5'UTR structure. Target 5'UTR had more complicated secondary structure than non-target 5'UTR in terms of free energy required for unwinding. To identify the target molecule of HTIF to discriminate 5'UTR, an immunoprecipitation using HTIF antiserum was conducted. HTIF was co-precipitated with eIF4A that would be required for unwinding the secondary structure of mRNA. These results suggest that HTIF of CpBV can sequester host eIF4A, any mRNAs possessing complicated secondary structures in their 5'UTRs would be difficult to recruit initiation complex for efficient translation.

Sodium-potassium-chloride co-transporter (NKCC) is a membrane bound channel protein that plays a prominent role in a variety of epithelial absorptive, secretory processes and a direct role in cell volume regulation, in which NKCC transports sodium, potassium, and chloride ions across the cell membrane. It has been known that prostaglandin E2 (PGE2) induces an acute cell lysis of specific hemocyte type, oenocytoid, to release prophenoloxidase into the plasma and ouabain (a specific sodium pump inhibitor) inhibits the oenocytoid cell lysis resulting in preventing phenoloxidase activation. However, it is not clear how the intracellular signaling pathway leads to oenocytoid cell lysis in response to PGE2. This study was designed to analyze functional role of NKCC in the cell lysis to release prophenoloxidase. A gene structure of NKCC was derived from cDNA library of Spodoptera exigua hemocyte, NKCC was expressed in all developmental stages and tissues. A real time quantitative RT-PCR showed that bacterial challenge significantly induced its expression. Specific inhibitors of NKCC, bumetanide and chlorothiazide, clearly prevented the cell lysis in a dose dependent manner. When RNA interference using double stranded RNA (dsRNA) specific to NKCC suppressed its expression, the oenocytoid lysis and PO activation was significantly inhibited in response to PGE2. It also reduced nodule formation to bacterial challenge. These results indicate that NKCC is associated with oenocytoid cell lysis probably by increasing cell volume through inward transport of ions in response to PGE2.

A cDNA of PBAN receptor (Plx-PBANR) isolated from female pheromone gland of the diamondback moth encodes 338 amino acids and has 7 transmembranes, belonging to G-protein coupled receptor family. The fact that Plx-PBANR expression was only found in female pheromone gland revealed that pheromone gland is the only molecular target of Plx-PBAN. Plx-PBANR expressing cells increased level of Ca2+ influx when challenged with PBANs. When RNAi fragment for PBANR was injected into pupae, suppression of PBANR expression was maintained for at least 2 days at post-emergence. Injection of RNA fragment for inhibition of Plx-PBANR expression also inhibited mating behavior and suppressed sex pheromone production, suggesting that some molecular target was affected by reduced Plx-PBANR expression. We cloned partial Δ9 and Δ11 desaturase gene and investigated expression level in Plx-PBANR-RNAi moth. It is of interest that desaturases expression was reduced by RNA fragment injection. These results suggest of PBANR expression affects the molecular biological events of PBAN and eventually suppresses mating behavior.

Sex pheromone production in lepidopteran is stimulated and regulated by a pheromone biosynthesis activating neuropeptide (PBAN). A cDNA of PBAN receptor (Plx-PBANR) isolated from female pheromone gland of the diamondback moth (DBM, Plutella xylostella (L.) encodes 338 amino acids. Plx-PBANR has conserved biochemical motifs and 7 transmembranes, indicating it belongs to G-protein coupled receptor family. Plx-PBANR expression was only found in female pheromone gland, demonstrating that pheromone gland is the only molecular target of Plx-PBAN. Human uterus carcinoma (HeLa) was stably transfected with Plx-PBANR gene and its expression was confirmed by RT-PCR analysis. Plx-PBANR expressing cells increased level of Ca2+ influx when challenged with Plx-PBAN and Hez-PBAN from Heliothis zea. When RNAi fragment for PBANR was injected into pupae, suppression of PBANR expression was confirmed by RT-PCR and maintained for at least 2 days at post-emergence. Injection of RNA fragment into pupae for inhibition of Plx-PBANR expression also inhibited mating behavior, revealing that reproductive organ of the female has no spermatocyte and that there are no successful reproductive behaviors. These results suggest of PBANR expression affects the molecular biological events of PBAN and eventually suppresses mating behavior.

Two entomopathogenic bacteria, Xenorhabdus nematophila (Xn) and Photorhabdus temperata temperata (Ptt), are symbionts of nematodes, Steinernema carpocapsae and Heterorhabditis megidis, respectively. When the nematodes enter host insect hemocoel, the bacteria are released from the nematode intestine to insect hemocoel and cause immunosuppression, which results in septicemia. Culture broth of both bacteria had insecticidal effects when injected into hemocoel of Plutella xylostella larvae, but did not when orally administered. However, either mixture of Xn or Ptt with Bacillus thuringiensis (Bt) significantly enhanced the Bt pathogenicity against P. xylostella. The culture broth was fractionated with hexane and diethylacetate extracts. Diethylacetate extract had potent factor (s) to increase Bt pathogenicity. A compound, benzylideneacetone, identified from the diethylacetate fraction had oral toxicity against P. xylostella. This compound also showed high acaricidal effect on the two spotted spider mite, Tetranychus urticae

Two entomopathogenic bacteria, Xenorhabdus nematophila (Xn) and Photorhabdus temperata temperata (Ptt), maintain monoxenic condition within host insect cadaver by synthesizing and releasing various antibiotics. These two bacteria were cultured in tryptic soy broth during different times, which were screened in their antibacterial activities. Both bacterial culture broth had high antibacterial activities against Escherichia coli at their stationary growth phase. The potent culture broth was used to screen target plant bacterial pathogens using both inhibition zone assay and liquid culture assay. Ralstonia sp. was most susceptible, while Xanthomonas sp. was highly resistant. Pseudomonas sp. and Bacillus sp. showed hemi-susceptible. The culture broth was further fractionated into hexane and diethylether extracts. Significant antibacterial effect was found in the diethylether extract

Two entomopathogenic bacteria, Xenorhadus nematophila and Photorhabdus temperata temperata, are known to suppress immune responses of target insects by inhibiting eicosanoid biosynthesis. This study analyzed these bacterial metabolites in their effects on hemocyte-spreading behavior of the beet armyworm, Spodoptera exigua. Both bacterial culture broth significantly inhibited the hemocyte-spreading behavior, at which the culture broth derived from the stationary growth phase had the most potent effect. Three identified eicosanoid synthesis inhibitors (benzylideneacetone, PY and Ac-FGV) impaired the hemocyte-spreading behavior of S. xigua, at which benzylideneacetone was the most potent. These three compounds share a common chemical structure: a pentenebenzene ring. Alternation of this common structure resulted in significant loss of their inhibitory activity to the hemocyte-spreading behavior.

Two biogenic monoamines, octopamine and 5-hydroxytryptamine (5-HT), are known to play immune mediators in insects. They induce hemocyte behaviors by stimulating cytoskeleton rearrangement. However,it is not clear how they activate the hemocytes in terms of intracellular signal transduction. This study analyzed their interactions with signal pathways implicated in nodule formation via eicosanoids or hemocyte locomotory behavior via a small GTPase. Both octopamine and 5-HT increased hemocytic nodule formation in response to bacterial challenge in the beet armyworm, Spodoptera exigua. However, their immune mediation was inhibited by a treatment of dexamethasone (a specific inhibitor to phospholipase A2). In the presence of phentolamine (a specific antoganist to octopamine) or ketanserin (a specific antoganist to 5-HT), the inhibitory activity of dexamethasone was rescued by adding arachidonic acid (a precursor of eicosanoid biosynthesis). These results suggest that the mediation of nodule formation by the two monoamines is followed by eicosanoid signaling. Two monoamines also induced up-regulation of circulating hemocyte counts in S. exigua. This increase of hemocyte counts was not explained by de novo production of hemopoietic organ because even ligation between thorax and abdomenin order to block hemolymph circulation did not inhibit the increase of circulating hemocyte counts by octopamine.A small GTPase, Rac1, appeared to be involved in this hemocyte mobilization from a sessile compartment in S. exigua. Inhibition of Rac1 activity significantly suppressed hemocyte spreading behavior and the hemocyte mobilization. In summary, octopamine and 5-HT mediate cellular immune responses of S. exigua via eicosanoid signal or independently by activating Rac1 following increase of cAMP in the hemocytes.

Mitochondrial genome is inherited in maternal origin without recombination by mating and its specific regions have been used to monitor insect pest populations in agriculture. The oriental fruit moth, Grapholita molesta, is a serious pest on apple industry by its direct damage on fruits. This study reports a full sequence of mitochondrial genome of G. molesta. Sequence contigs were made by primary PCRs on conserved regions and subsequent PCRs to fill the gaps. Annotated genes were highly matched to the sequences of other lepidopteran species. However, a few positions of tRNA genes on the genome were different to other mitochondrial genomes.

Local and seasonal populations of the oriental fruit moth, Grapholita molesta, were monitored with sex pheromone trapping and RAPD (random amplified polymorphic DNA) molecular marker to analyze their movement in apple orchards. To detect their movements among farms, pheromone traps were placed at regions between apple farms ('outside-farms') as well as within-farms ('inside-farms'). Four seasonal adult peaks were evident in apple-cultivating fields from April to October in both trappings of inside- or outside-farms. After overwintering generation, populations of inside-farms were significantly reduced with frequent insecticide applications, compared to populations of outside-farms. Within apple farms, G. molesta tended to be unevenly distributed because of significant sublocal preference. Active movements of local and seasonal populations of G. molesta were supported by gene flow analysis using RAPD marker. Monitoring data using sex pheromone and seasonal reduction in initial genetic differentiation detected in the overwintering populations suggest that there must be significant movement of G. molesta among different orchards in apple-cultivating areas.

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.

Upon oviposition, parasitoid wasps inject their eggs along with venom, teratocytes and polydnavirus (PDV) on the host. Among these parasitic factors, PDVs are known to suppress the host immune system and utilize the host translational mechanisms allowing the juvenile parasitoid to develop. Polydnavirusencoded genes can selectively inhibit host translation and still use the translation machinery of the host to synthesize their own proteins. In this study, we utilize a proteomic approach involving two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) that couples isoelectric focusing (IEF) and SDS-PAGE to resolve complex protein mixtures that results from the parastization of Cotesia plutellae on the lepidopteran host, Plutella xylostella. We specifically analyze the changes in protein synthesis using this technique after treatment of HTIFs that has been previously identified on C. plutellae. The difference in protein profile due to parasitization was confirmed by in vitro translation assay using rabbit reticulosyte lysate.

An entomopathogenic bacterium, Xenorhabdus nematophila, induces an immunosuppression by inhibiting phospholipase A2 (PLA2), which results in a fatal septicemia. PLA2 is an enzyme responsible for eicosanoid biosynthesis and the pathogenic molecular target of this bacterium. A PLA2 gene of the red flour beetle, Tribolium castaneum, was expressed in Escherichia coli. The recombinant T. castaneum PLA2 (TcPLA2) showed enzyme activity, which was specifically inhibited by bromophenacyl bromide (specific inhibitor to secretory PLA2) and ditheothreitol (reducing agent of disulfide bond). It was sensitive to pH (optimum at pH 7.0), temperature (optimum at 30°C), substrate specificity and calcium dependency. X. nematophila released compound(s) inhibiting TcPLA2during its stationary growth phase. The active compound (s) was resistant to heat treatment and could be extracted separately into both organic and aqueous phases. This PLA2 inhibitory fraction showed significant effect on immunosuppression of T. castaneum. These results suggest there may be several PLA2 inhibitors synthesized by X. nematophila and released into culture broth. The recombinant TcPLA2 was also used to screen potent PLA2 inhibitor compounds, which were designed based on a common chemical structure (a pentenebenzene ring) of two peptide inhibitors, proline-tyrosine (PY) and acetylated phenylalanine-glycine-valine (AcFGV). Alterations were made on amino acid sequence or specific functional groups on the pentenebenzene ring. Among 7 different peptides, AY and FGV showed the most potent effects on TcPLA2activity and also resulted in significant reductions in hemocyte spreading behavior of Plutella xylostella. The potent candidate molecules would be applied to control various insect pests to be developed into novel insecticides.

A monoterpenoid, benzylideneacetone (BZA), is synthesized by an entomopathogenic bacterium, Xenorhabdus nematophila K1, and known to suppress insect immune responses by inhibiting phospholipase A2(PLA2). This was designed to test its effect of insect digestion by oral administration. The beet armyworm, Spodoptera exigua, was tested by treating its artificial diet with different doses of BZA. The second instar larval were treated with the diets and monitored in their pupation, pupal weight, and adult emergence. BZA gave significant adverse effects on the larval development and subsequent adult metamorphosis. Digestive lumen of the fifth instar larval of S. exigua possessed activity, which was significant inhibited by BZA. These results support that BZA can be developed as a novel feeding deterrent.

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 cDNA of PBAN receptor (Plx-PBANR) isolated from female pheromone gland of the diamondback moth (DBM, Plutella xylostella (L.) encodes 338 amino acids. Plx-PBANR includes 7 transmembranes, indicating it belongs to G-protein coupled receptor family. Plx-PBANR showed high similarities with other moth PBANRs and its expression was only found in female pheromone gland, demonstrating that pheromone gland is the only molecular target of Plx-PBAN. To accomplish the funcional expression of Plx-PBANR, Human uterus carcinoma was stably transfected with Plx-PBANR gene and Plx-PBANR expression was confirmed by RT-PCR analysis. Plx-PBANR expressing cells increased level of Ca2+ influx when challenged with Plx-PBAN and Hez-PBAN from Heliothis zea, as ionomycin as a positive control does. To inhibit Plx-PBNAR expression in vivo, RNAi fragment for Plx-PBANR was injected into pupae. Suppression of PBANR expression was confirmed by RT-PCR and also induced inhibition of mating behavior in adults, revealing that reproductive organ of the female has no spermatocyte and that there are no successful reproductive behaviors. RNAi-treated adults showed reduced pheromone production. These results suggests that inhibition of PBANR expression affects the molecular biological events of PBAN and eventually suppresses mating behavior.

The diamondback moth, Plutella xylostella parasitized by its endoparasitoid Cotesia plutellae undergoes various physiological alterations which involves immunosuppression and developmental arrest. Its symbiotic virus, C. plutellae bracovirus (CpBV) is highly essential for their successful parasitization which possesses more than 136 genes encoded. CpBV15βunique in CpBV genome is expressed at low levels in early and at higher levels during late parasitization period. This gene product alters the hemocyte-spreading behavior through inhibition of protein synthesis under in vitro conditions. In the current study, we investigated its specific involvement in physiological functions in the host by transient expression and RNA interference techniques. The open reading frame of CpBV15βwas cloned into a eukaryotic expression vector and this recombinant CpBV15β was transfected into healthy non-parasitized 3rd instar P. xylostella by microinjection. CpBV15βwas expressed as early as 24 h and was consistent up to 72 h. Due to the expression of this gene, the hemolymph storage protein levels were significantly reduced and the ability of the hemocytes to adhere and spread on extracellular matrix was altered or reduced, wherein CpBV15βwas detectable in the cytoplasm of hemocytes based on indirect immunofluorescence assay. To confirm the role of CpBV15β, its double stranded RNA could efficiently recover the functional efficacy of hemocytes towards non-self and synthesis of storage proteins. Thus these results clearly demonstrate the role of CpBV15βin altering the host physiology by involving in cellular immune response and host protein synthesis.

Phospholipase A2 (PLA2) is the committed catalytic step of eicosanoid biosynthesis, which has been a common molecular target of several entomopathogens to induce insect immunosuppression. Despite critical importance of PLA2 in insect immunity, its gene structure was not known. This study identified insect PLA2 gene associated with immune reactions in the red flour beetle, Tribolium castaneum. Based on a previous study that an immune-associated PLA2 in insect is secretory type of PLA2 (sPLA2), five highly matched cDNA sequences were obtained from T. castaneum genome database using an sPLA2 sequence probe encoded in Drosophila melanogaster. The expressions of these five putative PLA2 were confirmed by reverse transcriptase-polymerase chain reaction. Out of five genes, one PLA2 gene called TcPLA2B was chosen because it showed specific expression in hemocyte and fat body. TcPLA2B was cloned and expressed in Escherichia coli and its protein was purified. The purified TcPLA2B showed PLA2enzyme activity, which was specifically inhibited by bromophenacyl bromide (a specific sPLA2inhibitor) and dithiothreitol (reducing agent of disulfide bond). It was sensitive to pH (optimum at pH 6.0) and reaction temperature (optimum at 10-30°C), and calcium dependency. An immunofluorescence assay indicated that TcPLA2B was localized near to cellular membrane of the cytosol in the hemocytes of T. castaneum at immune chanlenge. Double-stranded RNA (dsRNA) of TcPLA2B-treated larvae showed knockdown of its mRNA expression and did not form hemocyte nodule formation, while control larvae could exhibit time- and bacterial dose-dependent nodule formation in response to bacterial challenge. Addition of arachidonic acid (the catalytic product of PLA2) to the dsRNA-treated larvae rescued the inhibition of nodule formation. These results suggest that TcPLA2B gene is associated with insect immune reaction.