Pine wood nematode, Bursaphelenchus xylophilus, is a causative organism to induce pine wilt disease in many varieties of pine trees. Until 2006, Monochamus alternatus had been known as the only insect vector of pine wood nematode in Korea which targeted on Pinus densiflora (Japanese red pine) and P. thunbergii (Japanese black pine). However, pine wilt disease was also reported from Korean pine tree (Pinus koraiensis) in 2006 and we found another insect vector, M. saltuarius, was involved to transmit pine wood nematode. Both Monochamus species were confirmed to transfer pine wood nematode to their hosts but, there is no detail information about other transmitted nematode. Especially Bursaphelenchus mucronatus is common species transmitted by Monochamus species which is morphologically closed to B. xylophilus. Moreover B. mucronatus have two genotypes; one is East Asian type and the other is European type. Both genotypes of B. mucronatus were found in Korea but, the host and vector information related to the genotypes of B. mucronatus was not clear. Monochamus saltuarius was collected from three different geographical locations and nematodes were extracted and identified. For the identification of the juveniles, nematode DNA was extracted and ITS-RFLP analysis was done by PCR and gel electrophoresis. The selected enzymes were Hinf I, Alu I, Msp I, Hae III, Rsa I. Most of Bursaphelenchus species carried by M. saltuarius, which collected from pine wilt disease-free area, was determined as European type of B. mucronatus. We will compare the nematode species and genotypes carried by M. alternatus and M. saltuarius. In addition the rate of nematode carrying insect and the average number of nematode per single insect will be counted and compared.

There are increasing interests in developing methods specifically detecting pathogenic Bursaphelenchus xylophilus. In order to develop a detecting method for B .xylophilus, at first we generated monoclonal antibodies (MAbs) specific to B. xylophilus, discriminating from other pine tree resident nematodes. Among 2304 hybridoma fusions screened. We finally selected a MAb clone, 9F10 and used for further study. To identify the antigenic target of MAb-9F10, we employed several biochemical methods such as SDS-PAGE, 2 dimensional electrophoresis, anion exchange chromatography, and immunoprecipitation to separate and isolate an antigenic target. Proteins from above methods were analyzed via nano-LC-ESI-Q-IT-MS. Peptides of GaLECtin were always detected from several proteomic analyses, suggesting that GaLECtin is the antigenic target of MAb-9F10.

The damage of citrus fruit by yellow tea thrips, Scirtothrips dorsalis Hood, was first confirmed in 2008, and has since been one of the serious pests of citrus in Jeju. This was reported on damage symptom on tangor cultivar, Setoka (Tangor Norin No. 8) fruits cultivated in a greenhouse and the characteristic of spatial disttribution of S. dorsalis caught on yellow sticky traps was conducted in three commercial citrus orchards. The feeding habits by S. dorsalis cause rind blemishes on the navel part of fruit. The damage is characterized by being scarped and figure like covered cloud on satsum mandarine fruit, but on by a brown ring of rough russeting that occurs on navel part of Setoka fruit. The season of damage occurrence was from middle of June to late of July. There was a highly significant relationship between the average number of thrips per trap and the maximum number caught on a trap. The overall differences between the linear regression models obtained from mean-variance relationship for each surveyed fields were tested by type Ⅲ error and contrast method. The characteristic of spatial distribution of S. dorsalis was better described by Taylor's power law than Iwao's patchiness regression, and the dispersion index which is the slope (=1.72) of linear regression model showed the aggregated distribution pattern. The sequential sampling stop lines at fixed precision level of 0.20, 0.25 and 0.30 were calculated.

Phospholipid-hydroperoxide glutathione peroxide (PHGPx) is an antioxidant enzyme that reduces lipid hydroperoxides in biomembranes. Here, we cloned and characterized cys-PHGPx from the bumblebee Bombus ignitus (Bi-PHGPx). The Bi-PHGPx gene consists of 4 exons, encoding 168 amino acid residues with a canonical cys-codon at residue 45 and active site residues Gln82 and Trp134. Recombinant Bi-PHGPx, expressed as a 19 kDa protein in baculovirus-infected insect cells, exhibited enzymatic activity against PLPC-OOH and H2O2 using glutathione as an electron donor. Tissue distribution analyses showed the presence of Bi-PHGPx in all tissues examined. Bi-PHGPx transcripts were upregulated by stresses, such as wounding, H2O2 exposure, external temperature shock, and starvation. Under H2O2 overload, the RNA interference (RNAi)-induced thioredoxin peroxidase (BiTPx1)-knock-down B. ignitus worker bees showed upregulated expression of Bi-PHGPx in the fat body. These results indicate that Bi-PHGPx is a stress-inducible antioxidant enzyme that acts on phospholipid hydroperoxide and H2O2.

Bee venom contains a variety of toxic enzymes and peptides. One of the major components of bumblebee venom is bombolitin, which is the most abundant venom constituent and biologically similar to melittin. Here, we first show the molecular cloning and antimicrobial activity of the venom bombolitin from the bumblebee Bombus ignitus. The B. ignitus venom bombolitin gene consists of 2 exons, encoding 56 amino acid residues. The bombolitin purified from B. ignitus venom was the 2104 Da mature peptide with 18 amino acid residues, which are created by cleavage of the probombolitin domain between Ala38 and Leu39. We examined the pattern of bombolitin expression to confirm that it is a component of bumblebee venom. B. igniutus venom bombolitin exhibits venom gland-specific expression. We also investigated the venom bombolitin for antimicrobial properties against bacteria and fungi. The venom bombolitin showed high antibacterial activity against both Gram-negative and Gram-positive bacteria. Most interestingly, the venom bombolitin showed high antifungal activity against Fulvia falva, a leaf mold, and Alternaria radicia, a black rot. These antimicrobial profiles of B. ignitus venom bombolitin reported herein will be useful in the application for potential antimicrobial agents.

We cloned and characterized two peroxiredoxins (Prxs), BiPrx1 (a 1-Cys Prx) and BiTPx1 (a 2-Cys Prx) from the bumblebee Bombus ignitus. The BiPrx1 gene consists of 5 exons, encoding 220 amino acid residues with one conserved cysteine residue. The BiTPx1 gene consists of three exons, encoding 195 amino acid residues with 2 conserved cysteine residues. Recombinant BiPrx1 (27 kDa) and BiTPx1 (25 kDa), expressed in baculovirus-infected insect Sf9 cells, reduced H2O2 in the presence of electrons donated by dithiothreitol. Unlike BiTPx1, however, BiPrx1 did not show reduction activity when thioredoxin was used as the electron donor. Both BiPrx1 and BiTPx1 protected super-coiled DNA from damage by metal-catalyzed oxidation (MCO) in vitro. Tissue distribution analyses showed the presence of BiPrx1 and BiTPx1 in the fat body, midgut, muscle and epidermis, but not in the hemolymph, suggesting that BiPrx1 and BiTPx1 are not secretable. When H2O2 was injected into B. ignitus bees, BiPrx1 and BiTPx1 transcripts were acutely up-regulated in the fat body tissues. We also demonstrated regulation of BiPrx1 and BiTPx1 expression via reduction of transcript levels in the fat body with RNA interference (RNAi). Under H2O2 overload, the RNAi-induced BiPrx1 knock-down B. ignitus worker bees showed up-regulated expression of BiTPx1. Reciprocally, BiTPx1 RNAi knockdowns showed up-regulated BiPrx1 expression in the fat body. These results indicate that loss of expression of BiPrx1 or BiTPx1 is compensated by up-regulation of expression of the other peroxidase in response to H2O2 overload.

House dust mites (HDMs) play an important role in the occurrence of allergic diseases such as asthma and atopic dermatitis. Dermatophagoides pteronyssinus (Der p) is one of the most prevalent HDMs. It mediates the activation of T cells and monocytes, and induces the elevation of immunoglobulin E levels in allergic diseases. However, the effects of Der p on human monocytes have not been fully understood. In the present study, we investigated whether or not Der p has a great effect on the chemotactic activity of the human monocytic cell line, THP-1 cells, as induced by CC chemokines. We also show that the Der p extract (DpE) increased the chemotactic activity of THP-1 cells in response to MCP-1, RANTES, MIP-1α, and TARC, but has no effect on the expressions of CC chemokine receptors (CCRs) binding to CC chemokines in THP-1 cells. Protease inhibitors, such as aprotinin and E64, blocked the increased chemotaxis, while cytoplasmic Ca2+ influx mediated by these chemokines was inhibited by DpE. These results indicate that DpE increases the chemotactic activity of THP-1 cells in response to CC chemokines by regulating the cells’ protease-dependent mechanism. This finding may be useful in identifying the pathogenesis of allergic diseases induced by Der p.

14-3-3 proteins are known to play a pivotal role in a diverse array of cellular events such as cell survival, apoptosis, and signal transduction. Numerous 14-3-3 ζ have been cloned and characterized from a host of eukaryotic organisms including human, plants, yeast, fruit fly and silkworm. However, no study on Spodoptera exigua 14-3-3ζ in conjunction with virus infection has so far been reported in insects. It appears that expression of Se14-3-3ζ was decreased starting 24 h post-SeNPV infection as SeNPV titers seemed to increase as evidenced by intense bands of SeNPV IAP3. Interestingly, confocal microscopic analysis revealed that Se14-3-3ζ is expressed at the apical side of the NPV-uninfected gut cells, whereas it was detected mainly in the nucleus of the NPV-infected cells. Thus, despite the biological significance of Se14-3-3ζ in S. exigua in conjunction with molecular interactions between SeNPV and S. exigua is unclear now, our data suggest that Se14-3-3 ζ protein plays a role to protect S. exigua from the infection or inhibit replication of SeNPV.

Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway is known to play a pivotal role in various cellular events and antiviral responses in both vertebrates and insects. In an attempt to elucidate the potential involvement of STAT on S. exigua-SeNPV interactions, the full length cDNA of SeSTAT was cloned from S. exigua. Analysis of temporal expression patterns shows that SeSTAT is expressed in all stages of life cycle such as larvae, pupae, and adult. Spatial expression analysis shows that it is highly expressed in fat body and Malpighian tubule. Interestingly, SeSTAT is induced at 24 h in response to either laminarin or LTA injection in larvae. Electrophoretic mobility shift assay (EMSA) shows that the binding of nuclear extracts from fat body cells immune-challenged with LTA to STAT5 probe was observed. In addition, SeSTAT was nuclear-translocalized in both fat body and gut cells that were challenged with LTA and laminarin, respectively. Finally, gene silencing of SeSTAT shows that SeNPV number appears to be increased. It suggests that SeSTAT may act as a negative regulator against SeNPV in midgut.

Monochamus alternatus and M. saltuarius were reported as the vectors of Bursaphelenchus xylophilus, pine wood nematode in Korea. According to Kwon et al. (2006), each of 2 species has occupied their own regional distribution : M. saltuarius in southern part including Jeju island and M. alternatus in mid-northern part of Korean peninsula. We measured the supercooling point (SCP) of 2 species (laboratory-reared populations) by each of developmental stages. The SCPs of 2nd, 3rd and 5th instar larvae of M. saltuarius were -7.68±0.19℃, -7.02±0.69℃, -4.93±1.34℃ each of stages. On the other hand, the SCPs of 3rd, 4th, 5th instar larvae and pupae of M. alternatus. were -4.46±1.12℃, -5.94±1.33℃, -7.83±1.44℃, -9.53±1.78℃ each of stages. The SCPs of M. saltuarius larvae generally was lower than that of M. alternatus. The pupae of M. alternatus and 2nd instar larvae of M. saltuarius had the lowest SCP among measured samples. On the other hand, the highest SCP were recorded in 2nd and 5th instar larvae, each. This result shows that regional distribution of 2 beetles may be associated with the adaptation capacity to low temperature represented by the SCP as well as the developmental temperature. However, beetles experimented were not collected from pine forest fields. In further study, we are planning experiments with field populations and all developmental stages.

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.

This study was performed to analyze the sugar contents from six kinds of plant and investigate their effect on the life span of ggot-mae-mi, Lycorma delicatula. Part of plants were methanol extracted from host plants such as Ailanthus altissima, Vitis vinifera and non-host plants such as Malus pumila, Pyrus calleryana, Hibiscus syriacus, and Pinus densiflora, and concentrated the water layer and then analyzed the sugar contents using HPLC. Ailanthus altissima existed high in sugar contents and followed by Fructose>Glucose, Vitis vinifera was analyzed by an order of Glucose>Fructose>Maltose>Sucrose>Rhamnose; Malus pumila was as Glucose>Fructose; Pyrus calleryana was as Glucose>Unknown>Fructose; Hibiscus syriacus was as Sucrose>Glucose; Pinus densiflora was as Fructose>Glucose>Sucrose. A parafilm bioassay was used to investigate the longevity of L. delicatula nymphs to the sugar contents. Nymphs of L. delicatula was lived as long as 13.1 days in 5% Sucrose solution, but lived as short as 6.0 days in 5% Glucose solution. When provided with only water, L. delicatula lived for 5.4 days. Life span to the each sugar contents were longer lived in an order of Sucrose>Fructose>Rhamnose>Maltose>Glucose. As investigated the life span of L. delicatula nymph according to the combination of sugar contents founded in original plants were lived longer in 5% sugar combination solution of Ailanthus altissima. Analyzed original sugar contents from Malus pumila, Pyrus calleryana, L. delicatula was lived as 7.8, and 7.1 days, respectively, comparing to the Hibiscus syriacus, and Pinus densiflora lived as 6.0, and 4.7 days, respectively. This result were judged that sugar contents affected on choosing the host plants.

This study was performed to compare the host preference, survivorship and feeding behavior using EPG against ggot-mae-mi, Lycorma delicatula against seven plants such as Ailanthus altissima, Vitis vinifera, Malus pumila, Pyrus calleryana, Hibiscus syriacus, and Pinus densiflora. In host preference. L. delicatula was most preferred the Ailanthus altissima, Vitis vinifera and was not preferred the other plants. Survival rate of 3rd Nymph was recorded from Ailanthus altissima, Vitis vinifera as 15.0, and 15.4 days, respectively, it showed longest period. However, Malus pumila, Pyrus calleryana, Hibiscus syriacus, were survived within 6 days and Pinus densiflora was within 5 days. Moreover, L. delicatula was survived within 2 days to the three kinds of fruits. Ecdysis rate from 3rd to 4th nymph also high from Ailanthus altissima, Vitis vinifera as 63.3, 63.0 % and the order was followed as Malus pumila(17.7%) > Pyrus calleryana(9.3%) > Hibiscus syriacus(7.8%) > Pinus densiflora(5.9%). Especially, ecdysis rate was recorded 0% to the three kinds of fruits. Feeding behavior was analyzed using EPG and compared the differences their waveform from seven kinds of plants and three kinds of fruits. Non-probing time was short in host plants, reversely, Phloem-feeding time was recorded longer in Ailanthus altissima, Vitis vinifera as 45.7 and 13.7 min, respectively. And other plants and fruits were not showed feeding behavior.

This study was performed to investigate the correlation between changes of feeding behavior of green peach aphid, Myzus persicae and residual effect of an insecticide, Pyrifluzuinazon, using EPG technique. Pyrifluquinazon was showed the insecticidal activity until three days (72h) after treatment, and the activity was high in nymph than adult of GPA. There was no difference among treatment methods. Lethal sign was observed the slimed the abdomen of GPA after 3 days that rises the insecticidal activity, and dieㅇ as being sticked and/or pulled out the needle. Residual efficacy from 1, 3, 5, 7 to 20 days after treatment, insecticidal activity was showed 70% in 50 ppm, recommended concentration, until 5 days. And waveforms relating to non-probe time and phloem phase time using EPA, feeding inhibition efficacy was showed during 5 days after treatment, but showed similar level with control after 13 days. These results show that the change of feeding behavior of GPA is correlated with the change of residual effect of pyrifluzuinazon.

In order to understand the nature of population genetic structure of the notorious pest insect, Spodoptera litura (Lepidoptera: Noctuidae), a total of 159 individuals of the tobacco cutworm collected from six Korean and five Chinese localities were cloned and sequenced their mitochondrial A+T-rich region. A total of 90 haplotypes ranged 324 bp ~ 372 bp in length and 0.30% ~ 4.85% in sequence divergence. Compared to the preliminary mitochondrial COI gene sequence data that provided nearly identical sequences in all samples, the A+T-rich region provided substantially high sequence variation, validating the applicability of this molecule to the study of intraspecific variation. Overall, a very high per generation migration ratio (Nm = 5.91 ~ infinite), a very low level of genetic fixation (FST = 0 ~ 0.077796), and no discernable isolated population were noted to exist among populations of S. litura, although some significant genetic differentiations were found between several pairs of populations. These results collectively suggest that the S. litura populations in both Korea and China are very well interrelated to each other. The structural analysis revealed that the S. litura A+T-rich region harbored two stretches of the [TA(A)]n sequence, the poly-A stretch, and a stem-and-loop structure that are well found in other lepidopteran A+T-rich region. Furthermore, the region harbors the 13 ~ 19-bp long T stretch and 5-bp long motif that have been suggested to function as a possible recognition site for the initiation of replication of the minor strand of mtDNA also were found.

The bumblebee, Bombus ignitus (Hymenoptera: Apidae), is a valuable natural resource that is one of the most notably utilized for greenhouse pollination in Korea. In order to understand the nature of genetic relationships, gene flow, and population structure of the species we sequenced a partial COI gene of mitochondrial DNA (mtDNA) corresponding to “animal barcode” region and the complete internal transcribed spacer 2 (ITS2) of the nuclear ribosomal DNA (nrDNA) with the individuals collected from Korean localities. Although the 658-bp long mtDNA sequence provided only six haplotypes with the maximum sequence divergence of 0.456% (3 bp, from 91 individuals), but the ITS sequences provided 33 sequence types with the maximum sequence divergence of 0.78% (16 bp, from 35 individuals), confirming better applicability of the ITS sequences to the study of intraspecific variation. The ITS2 sequences of B. ignitus were shown to be the longest among known insects, ranging in size from 2,034 bp ~ 2,045 bp and harbored two duplicated repeats, indicating unusual structure of B. ignitus ITS2 sequences compared with other insect ITS sequences. Overall, a very high per generation migration ratio, a very low level of genetic fixation, and no discernable hierarchical population/population group were noted to exist among populations of B. ignitus on the basis of both molecules, thus suggesting that the B. ignitus populations on the Korean peninsula are panmictic, which is consistent with our understanding of the dispersal capability.

Eumenis autonoe, a member of the lepidopteran family Nymphalidae (superfamily Papilionoidea) is an endangered species, and is found only on one isolated remote island, Jeju in South Korea, on Halla Mt., at altitudes higher than 1,400 meters. In this study, we report the complete mitochondrial genome (mitogenome) of E. autonoe. The 15,489-bp long E. autonoe genome evidenced the typical gene content found in animal mitogenomes, and harbors the gene arrangement identical to all other sequenced lepidopteran insects, which differs from the most common type found in insects, due to the movement of tRNAMet to a position 5’-upstream of tRNAIle. As has been observed in many other lepidopteran insects, no typical ATN codon for the COI gene is available. Thus, we also designated the CGA (arginine) found at the beginning of the COI gene as a lepidopteran COI starter, in accordance with previous suggestions. The 678-bp long A+T-rich region, which is second longest in sequenced lepidopteran insects, harbors 10 identical 27-bp long tandem repeats plus one 13-bp long incomplete final repeat. Such a repeat sequence has been, thus far, only rarely detected in lepidopteran mitogenomes. The E. autonoe A+T-rich region harbors a poly-T stretch of 19 bp and a conserved ATAGA motif located at the end of the region, which have been suggested to function as structural signals for minor-strand mtDNA replication.

Deciduous tree fruits such as pears and apples are widely grown in the Pacific Northwest of the United States and are well adapted to the seasonal environment in that region. Extended cold periods provide adequate chilling to break dormancy and reinitiate growth in the spring. Cold exposure synchronizes the physiological processes and makes sure that bloom is uniform and that fruit matures in a uniform manner. As a result of global warming, some fruit-growing areas may experience inadequate cold exposure during the winter months, gradually shifting the southern boundary for production of deciduous fruits further north. However, climate change will affect not only growth and fruiting habits of fruit trees but also the insect and mite pests which feed on them. There is general agreement that in temperate regions a trend towards warmer summers and milder winters will generally benefit insect and mite pests and increase their injuriousness. Temperature changes in particular will impact the development, mortality, phenology, and voltinism of fruit pests. Here we discuss how climate change may affect pests and control practices on deciduous tree fruits in the Hood River Valley of northern Oregon. This small valley extends in a north-south direction from the Columbia River to the foothills of Mt. Hood and is characterized by a varied topography and large altitudinal differences (sea level to 600 m). The major pest of pears and apples in that area is codling moth, Cydia pomonella L., a cosmopolitan pest which is present in most deciduous fruit-growing areas of the world. Like its host trees, the codling moth is well adapted to a seasonal environment. Diapause is the principal mechanism which synchronizes its phenology with the tree and the presence of fruit, the larval food source. Diapausing overwintering larvae require cold exposure (chilling) to terminate diapause in late winter or early spring. At the lower elevations close to the Columbia River the codling moth is bivoltine but gradually becomes univoltine at the higher elevations where the growing season is shorter and fewer heat units (above 10oC) are available for development. Long-term temperature records from the lower Hood River Valley indicate that the 25 years since 1985 have been considerable warmer than the 25 years prior to 1985. For instance, the average heat units available for codling moth development over a season have increased by more than 10% over the last 25 years. The codling moth is adapting to this warming trend by gradually increasing its voltinism (number of generations). As a result, the severity of codling moth as a pest can be expected to increase. Therefore, fruit growers will have to adjust and intensify control practices to keep fruit free from codling moth damage. We will also explore how other fruit and foliage feeding pests which are part of the pest complex of pears and apples in northern Oregon fare under different global warming scenarios compared to codling moth.