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.

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.

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 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.

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.