Insensitive acetylcholinesterase (AChE) was determined to be basically involved in a EPN-resistant (ER) strain and ER contaminated susceptible (CS) strain of diamondback moth (DBM, Plutella xylostella L.), as estimated by the AChE inhibition assay using DDVP as a inhibitor in nondenaturing electrophoresis gel. ER strain exhibited very high insensitivity revel, high resistance ratio and two point mutations (G227A, A201S) in ace1. CS strain showed intermediate insensitivity level, low resistance ratio and a point mutation (G227A). This finding suggests that the A201S mutation along with reported G227A mutation (Baek et al, 2005) can be main player to develop the organophosphate resistance. Additionally, surveyed seven local population DBMs saturated G227A mutation and A201S mutation mixed in some population. Also A441G mutation was detected in some population, but there was no significant correlation. These results suggest that A201S mutation could be one of the good candidate for molecular diagnosis marker of resistance monitoring.

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.

Two entomopathogenic bacteria, Xenorhabdus nematophila and Photorhabdus temperata subsp. temperata, are known to be potent against the diamondback moth, Plutella xylostella, when the bacteria are injected into the hemocoel. This study investigated any pathogenic effect of their culture broth on P. xylostella by oral administration. Only culture broth of both bacterial species did not give enough pathogenic effects by the oral administration. However, when the culture broth was orally treated together with Bacillus thuringiensis (Bt), both cell-free culture broth significantly enhanced Bt pathogenicity against the 3rd instar larvae of P. xylostella. The culture broth was then fractionated into hexane, ethyl acetate, and aqueous extracts. Most synergistic effect on Bt pathogenicity was found in ethyl acetate extracts of both bacterial species. Thin layer chromatography of these extracts clearly showed that ethyl acetate extracts of both bacterial culture broths possessed metabolites that were different to those of hexane and aqueous extracts. These results suggest that the both entomopathogenic bacteria produce and secrete different factors to give significant synergistic effect on Bt pathogenicity.

Insensitive acetylcholinesterase (AChE) was determined to be basically involved in a EPN-resistant (ER) strain of diamondback moth (DBM, Plutella xylostella L.), as estimated by the AChE inhibition assay using DDVP and thiodicarb as inhibitors in nondenaturing electrophoresis gel. AChEs were clearly separated into four different bands (a major band and three minor bands) in susceptible strain (CS) and all bands inhibited by used inhibitors almost same level, however, only two bands (a major band and a minor band) showed in that of ER strain and major band showed higher insensitivity. Moreover, ER strain showed cross resistance against used inhibitors and DDVP highly inhibited esterase in both strains. About 2kb of ace1,2 cloned and point mutations were detected in ER strain.

This study was conducted to develop the economic threshold for the diamondback moth (Plutella xylostella (L.) on Leaf Broccoli (leaf vegetable) in 2007. To investigate the relationship between initial density of diamondback moth larvae and broccoli leaf yield, experimental plots with five treatments (0, 0.5, 1.0, 2.0, 4.0 larvae per plant) as initial density were established. We inoculated larvae in chesses cloth covering to survey larvae density change. When grown to eat, leaves of broccoli were harvested by periods. 60-70% of larvae were removed whenever we picked the leaves. High levels of larvae were associated with significant reductions in leaf yield. There were 85%, 64%, 58%, and 56% yield reductions from the diamond back moth larvae density in 0, 0.5, 1,0, 1.5, 2.0 per plant, respectively 25days after larvae inoculation. The regression equation used to predict leaf yield based on the number of initial larvae density per a plant was y=1635-393x(R2=0.79***). The economic injury level of diamondback moth on leaf broccoli was 2-3 larvae per 10 plants for a damage level of 5%. The economic thresholds was 1-2 larvae per 10 plants. Thus, the diamond back moth management should be initiated 1-2 larvae occurrence per plant.

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.

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 toxicity of perilla-chinese Basil, Perilla frutescens whole plant-derived materials to third-instar of larva Plutella xylostella was examined using that of four insecticides and 5 constituents of P. frutescens from other research materials. The active principle of P. frutescens was identified as the sesquiterpenoids α-farnesene by spectroscopic analysis. In leaf-dipping bioassay, α-farnesene (LD50, 36.9) was 3.9 times more toxic than β-farnesene (LD50, 145.2) against P. xylostella larva, based on 48h LD50 values. This compound was less toxic than insecticides. Naturally occurring α-farnesene merit further study as potential diamond back moth control agent.

Cytochrome P450s (P450s) are known to oxidize a variety of insecticides including pyrethroids, thereby conferring metabolic resistance in diamondback moth (DBM), Plutella xylostella. Synergism assay with piperonyl butoxide indicated that the enhanced activity of P450 is associated with pyrethroid resistance in a cypermethrin-resistant (CR) strain. However, there were little differences in the basal transcription levels of all the P450s examined between susceptible (Sus) and CR strains, suggesting that constitutive overexpression of P450 is not likely involved in the cypermethrin resistance but induction of P450 by cypermethrin is rather associated with metabolic resistance. To determine the conditions resulting in maximum levels of P450 induction, several factors including the way of adminstration (topical application vs. leaf dipping), exposure dose and exposure duration were examined. In general, leaf dipping method resulted in greater levels of induction in a wider array of P450s. The conditions of ‘low dose (sublethal dose or concentration) and short exposure (less than 3 hr)' to cypermethrin were more efficient in P450 induction than those of ‘high dose (around LD50 or LC50) and long exposure (more than overnight)’, which have been employed in many other studies to date. Cross-strain comparison revealed that 9 of 11 P450s were induced 1.4-2.2 fold in CR whereas only 3 P450s in Sus under the optimal induction conditions, demonstrating that metabolic resistance in CR strain is actually conferred by the mechanism of selective P450 induction when exposed to cypermethrin.

To determine differential gene expression profiles in a cypermethrin-resistant strain (CR) of diamondback moth, Plutella xylostella Linnaeus (1758), a subtractive cDNA library was constructed by suppression subtractive hybridization. A total of 1196 expressed sequence tags (EST) were clustered and assembled into 579 contigs (100 multiplets and 479 singletons). About 46% (267) of 579 contigs had the matched BLASTx hits (E ≤ 10-5). Among these, 143 contigs had similarity to proteins with assigned molecular function in the Functional Catalogue database, and most of them (86%) were homologous to the genes from insects, particularly to Lepidoptera (56%). The contigs encoding carboxylesterase and cytochrome P450 known to be involved in the insecticide resistance were found in the library. They were identified as pxest3, pxest4, and CYP9G2 gene by 5' and 3' RACE. Among these, pxest4 was determined to be 2-fold over-transcribed in the CR strain by qrtPCR. Several contigs encoding enzymes including cytochrome oxidase subunit I that are likely involved in the insecticide resistance were also identified from the library. Discovery of the genes specific to cypermethrin resistance should promote further studies on the molecular mechanisms of insecticide resistance in P. xylostella.

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.

Hemolin is a member of the immunoglobulin (Ig) superfamily and contains four Ig domainsthat are similar to neural cell adhesion molecules. It has been regarded as a recognition molecule at immune challenge in insects. This study showed that hemolin of Plutella xylostella was expressed during pupal and adult stages but absent in all larval instars without any immune challenge. It is, however, strongly induced by the injection of Escherichia coli or its lipopolysaccharide in hemocytes, fat body and gut. A double-stranded RNA (dsRNA) interference experiment revealedits role in activation of prophenoloxidase (PPO) in the hemolymph during bacterial infection. Also its involvement in cellular defense was investigated in its mediation of the adherence of hemocytes to rat blood erythrocytes which was knocked down by its dsRNA. Finally, its physiological significance in pupal stage was confirmed by using dsRNA, which significantly prevented adult development. Therefore, it is concluded that hemolin plays roles in both immune and adult development in P. xylostella.

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.

Many female moths produce and release sex pheromones to mate successfully with a conspecific male. Sex pheromone production in lepidopteran moth is known to be under the regulation of a pheromone biosynthesis activating neuropeptide (PBAN). A PBAN polypeptide is processed into five neuropeptides (NPs) after post-translational modification, resulting in diapause-like NP, PBAN, α-, β- and γ-NP. All of the peptides are amidated in their C-termini and shared a conserved motif, FXPR(or K)L structure. PBAN (Plx-PBAN) from Plutella xylostella consists of 30 amino acids, the shortest PBAN so far reported. When female adults were injected with synthetic Plx-PBAN, pheromone production showed a maximal increase 1 h post-injection. RT-PCR screening revealed that Plx-PBAN cDNA was expressed in both sexes, with the highest expression level in the head of female adults. Plx-PBAN binds to its receptor on pheromone gland cells. PBAN receptor has seven transmembranes, indicating G-protein coupled receptor family and transduces its signal via G-protein mediated signal transduction. Subsequently, calcium channels remain activated and stimulate biochemical reactions for sex pheromone production in the pheromone gland.

Five strains of Korean entomopathogenic nematodes (EPN), steinernematids and heter-orhabditids were evaluated and tried in laboratory, pot, and vegetable greenhouses for environmentally friendly control of diamondback moth (DBM), Plutella xylostella, from 2002 to 2005. LC₅₀ values of five EPN strains against DBM were different depending on nematode strain and DBM instar. LC₅₀ value of Steinernema carpocapsae GSN1 (GSN1) was the lowest representing 2.6~3.9 infective juveniles (Ijs, 3rd stage) to 2nd to 4th instars of DBM. Pathogenicity of five effective strains against DBM was different depending on nematode strain, concentration, application times, and vegetable in pot. The most effective nematode was GSN1. Steinernema spp. was more effective than Heterorhabditis spp. against DBM. Two or three times of applications of EPN were effective regardless of nematode strain and concentration in pot. Efficacy of EPN was different depending on vegetable species. Efficacy was higher on Chinese cabbage, red mustard, and Ssamchoo than that on cabbage, kale, and leaf broccoli. Efficacy of GSN1, Steinernema GSNUS-10, Steinernema GSNUS-14, and Heterorhabditis GSNUH-l was variable depending on nematode strain, concentration, application times, and vegetable in greenhouse experiments. GSN1 was the most effective and 100,000 infective juveniles per ㎡ (=1 × 10⁹ Ijs/ha) resulted in higher efficacy. Three times of application of nematodes led to higher control efficacy than one or two applications. Efficacy of nematodes was higher on Chinese cabbage than cabbage or kale in greenhouse.

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.