Bee venom contains a variety of peptides and enzymes, including serine proteases. While the presence of serine proteases in bee venom has been demonstrated, the role of these proteins in bee venom has not been elucidated. Furthermore, there is currently no information available regarding the melanization response or the fibrin(ogen)olytic activity of bee venom serine protease, and the molecular mechanism of its action remains unknown. Here we show that bee venom serine protease (Bi-VSP) is a multifunctional enzyme. In insects, Bi-VSP acts as an arthropod prophenoloxidase (proPO)-activating factor (PPAF), thereby triggering the phenoloxidase (PO) cascade. Bi-VSP injected through the stinger induces a lethal melanization response in target insects by modulating the innate immune response. In mammals, Bi-VSP acts similarly to snake venom serine protease, which exhibits fibrin(ogen)olytic activity. Bi-VSP activates prothrombin and directly degrades fibrinogen into fibrin degradation products, defining roles forBi-VSP as a prothrombin activator, a thrombin-like protease, and a plasmin-like protease. These findings provide a novel view of the mechanism of bee venom in which the bee venom serine protease kills target insects via a melanization strategy and exhibits fibrin(ogen)olytic activity.

Rice black-streaked dwarf virus (RBSDV), a member of the genus Fijivirus within the family Reoviridae, is the causative agent of maize rough dwarf and rice black-streaked dwarf diseases, both of which can lead to severe yield losses in east Asia. Although molecular approaches such as RT-PCR have potential for detection and diagnosis of this virus infections, their impact on high throughput certification is still limited. Therefore, the development of an antibody-based assay for rapid and effective diagnosis of RBSDV is preferable. In this study, we collected RBSDV from rice with rough dwarf disease and its complete nucleotide sequences of 10 genomic segments encoding 12 non-overlapping ORFs were determined. Among 12 ORFs, ORF1, 2 and 12 showed high level of similarities with the RdRp, major core protein and major outer shell protein, respectively. These ORFs were expressed as polyhedrin fusion protein or full-length soluble protein using baculovirus expression system for the preparation of specific antibody against RBSDV, which could be useful for the detection and diagnosis of this virus.

We isolated two baculoviruses, Spodoptera litura granulovirus (SlGV) and S. litura nucleopolyhedrovirus (SlNPV) in the dead larvae of S. litura. The granule of SlGV were ovoidal shape with an approximate measure of 240-340 nm×140-180 nm, and each granule contained one single rod-shape virion with a mean size of 180-200 nm×20-40 nm. Whereas, the polyhedra of SlNPV were irregular in shape with a approximate diameter of 1.0-1.5 ㎛, and numerous virions comprised of the multinucleocapsid were contained in each polyhedra. The major component of occlusion bodies produced by SlGV and SlNPV were about 29 and 30 kDa, respectively. When the phylogenic relationship between these viruses were analyzed using the nucleotide sequences of granulin gene from SlGV and polyhedrin gene from SlNPV, they were not closely related to each other. We also found that the two viruses showed similar insecticidal activity against 2nd instar larvae of Spodotera litura in terms of dose-response, but SlGV showed much longer LT50 than that of SlNPV. The two baculoviruses might be cooperatively be applied as biological control agent for the control of S. litura

Insects or insect remains found in beer are one of major issues in consumer claim. Accurate estimation of inflow time isa critical factor for the settlement of such claims related with beer-contaminating insects but no reliable methods have been developed. In an attempt to establish a molecular marker-based diagnostic method, the degradation rates of 18S rRNA genes in the insectssoaked in 500 ml beer were investigated by quantitative real-time PCR (qPCR) over one month period at room temperature. Among the six insect species tested, the house fly (Musca domestica) and honey bee (Apis mellifera) revealed high correlations (r2=0.974-0.990) between the degradation of 18S rRNA gene and inflow time. In these insects, statistically significant distinction was possible between the samples stored in beer less than 14 days and more than 14 days. Other insects, including the fruit fly, common house mosquito, German cockroach and Indian meal moth, displayed poor correlations, which appeared attributed to the inefficient genomic DNA extraction likely due to small sample size or disintegration of body parts during storage in beer. With proper improvement in DNA extraction, this 18S rRNA-based diagnostic method would be applicable for estimating the inflow time of beer-contaminating insects.

This study presented a development of a phototactic chamber used for pest monitoring. The chamber was constructed by opaque acrylic body. Transparent acrylic wall of the chamber for light-exposure were fitted at both side end parts of the inside chamber. Side parts of the outside chamber were made of removable cover in combination with the air circulation system and light source such as LED or fluorescent. The insect entrance holes was positioned at the center part of the chamber to efficiently dispersed pests, and then nylon net was equipped inside the chamber to prevent the escape of inserted pests. Two opaque partition walls of the inside chamber were made of the movable plate, in order to the control of the light-exposure and the response termination. We also carried out behavioral experiment against various pest species by using the phototactic chamber. Consequently, the phototactic chamber was confirmed suitable result of behavioral experiment. Therefore, we believed that the test chamber help to understand the phototactic responses of various pests.

Classical swine fever virus (CSFV) envelope glycoprotein E2 is the main target for inducing neutralizing antibodies and protective immunity in swine. Here, we report a novel strategy forthe large-scale production of a CSFV E2 subunit vaccine that demonstrates a high immunogenic capability in the larvae of a baculovirus-infected silkworm, Bombyx mori. We constructed a recombinant B. mori nucleopolyhedrovirus (BmNPV) that expressed recombinant polyhedra together with the N-terminal 179 amino acids of CSFV E2 (CSFV E2ΔC). BmNPV-E2ΔC-infected silkworm larvae expressed an approximately 44-kDa fusion protein that was detected using both anti-polyhedrin and anti-CSFV E2 antibodies. Electron and confocal microscopy both demonstrated that the recombinant polyhedra were morphologically normal and contained CSFV E2ΔC. The CSFV E2ΔC antigen produced in BmNPV-E2ΔC-infected silkworm larvae reached 0.68 mg per ml of hemolymph and 0.53 mg per larva at 6 days post-infection. Mice that were immunized with the granule form of recombinant polyhedra or the soluble form of the fusion protein elicited CSFV E2 antibodies, which indicated that the recombinant polyhedra carrying CSFV E2ΔC were immunogenic. The virus neutralization test showed that the serum from mice that were treated with recombinant polyhedra or the soluble form of the fusion protein contained significant levels of virus neutralization activity. These results demonstrate that the present strategy can be used for the large-scale production of CSFV E2 antigen and that the recombinant polyhedra containing CSFV E2ΔC as a granule antigen can be used as a potential subunit vaccine against CSFV.

Glutathione S-transferases (GSTs) are multifunctional enzymes that are mainlyinvolved in the xenobiotic metabolism and protection against oxidative damage. Most studies of GSTs in insects have been focused on their role in detoxifying exogenous compounds in particular insecticides. Here, we show the expression profiles of GSTs of the bumblebee Bombus ignitus in response to oxidative stress. We identified a sigma-class GST from B. ignitus (BiGSTS). The BiGSTSgene consists of 4 exons that encode 201 amino acids. Comparative analysis indicates that the predicted amino acid sequence of BiGSTS shares a high identity with the sigma-class GSTs of hymenopteran insects such as Apis mellifera (70% protein sequence identity) and Solenopsis invicta (59% protein sequence identity). Tissue distribution analyses showed the presence of BiGSTS in all tissues examined, including the fat body, midgut, muscle and epidermis. The oxidative stress responses analyzed by quantitative real-time PCR showed that under H2O2 overload, BiGSTS and BiGSTD (identified in our previous study) were upregulated in all tissues examined, including the fat body and midgut of B. ignitus worker bees. Under uniform conditions of H2O2 overload, the expression profile of GSTs and other antioxidant enzyme genes, such as phospholipid-hydroperoxide glutathione peroxidase (Bi-PHGPx) and peroxiredoxins (BiPrx1 and BiTPx1), showed that other antioxidant enzyme genes are acutely induced at 3 h after H2O2 exposure, whereas BiGSTS and BiGSTD are highly induced at 9 h after H2O2 exposure in the fat body of B. ignitus worker bees. These findings indicate that GSTs and other antioxidant enzyme genes in B. ignitusare differentially expressed in response to oxidative stress. Taken together, our findings indicate that BiGSTS and BiGSTD are oxidative stress-inducible antioxidant enzymes that may play a role in oxidative stress response.

Autographa californica nucleopolyhedrovirus (AcMNPV) has a large doublestrand DNA genome of approximately 134 kbp and harbors 156 open reading frames (ORFs). To elucidate DNA replication cascade of AcMNPV, we developed a novel baculovirus genome that can be maintained in Escherichia coli as a plasmid and can infect susceptible lepidopteran insect cells. This genome, named bAc-MK, contains a mini-F replicon and a kanamycin resistance marker. Using a convenient Tn7 transposon-based system, pPCS-S, 55 single ORF-truncated mutants were generated by random insertion into bAc-MK genome. These single ORF-truncated mutants were independently transfected into Sf9 cells, 16 of them were found affecting viral replication since they defected in producing polyhedra. Furthermore, to verify the pathogenicity of the single ORF-truncated mutants, the remaining 39 mutants were subjected to bioassay to Spodoptera exigua 3rd instar larvae. Among them, ac9-, ac49-, ac103- and ac105-knockout mutants showed higher mortality compared to that of bAc-MK. These results suggested that these ORFs could be related to pathogenicity of AcMNPV.

The Bacillus thuringiensis strain K4 was isolated from fallen leaves, sampled in a forest region of the city of Mungyeong, Korea. The flagellated vegetative cells of B. thuringiensis strain K4 were agglutinated with the H3 reference antiserum and further, agglutinated with 3b and 3d monospecific antisera but non-reactive for 3c and 3e factor sera. These results create a new serogroup with flagellar antigenic structure of 3a3b3d, designated serovar mogi. The strain K4 showed high activity against dipteran larvae, Anopheles sinensis and Culex pipiens pallens while no lepidopteran toxicity. It produced a single ovoidal-shaped parasporal crystal whose SDS-PAGE protein profile consisted of several bands ranging from 75 to 30 kDa. Through the protein identification by nano-LC-ESI-IT MS analysis, the putative peptides of Cry19Ba, Cry40ORF2, Cry27Aa and Cry20Aa were detected. In contrast to the plasmid profile of B. thuringiensis H3 serotype strains, the strain K4 contained only a large plasmid (~100 kb) and we cloned partial cry27Aa, cry19Ba and cry40ORF2 genes from it by thermal asymmetric interlaced PCR. Sequencing analysis showed 87%, 88% and 88% homologous with known cry27Aa, cry19Ba and cry40ORF2 genes, respectively. The new type strain, B. thuringiensis subsp. mogi (H3a3b3d) will be a good resource for new mosquitocidal cry genes.

This study was performed to investigate the antifeeding activity of Perilla frutescens extracts against diamondback moth, Plutella xylostella larvae and to confirm the electrophysiological responses of two sensilla (LST=lateral styloconic sensillum, MST=medial styloconic sensillum) in maxilla galea, a chemoreceptor. Crude extract of P. frutescens in methanol was showed antifeeding activity approximately 70%, and subsequently separated into four fractions - n-hexane(H), chloroform(C), ethylacetate(E), and water(W). Antifeeding activity was only showed in n-Hexane fraction around 99%. H1, H2, H3, H4, H5 were isolated from n-Hexane fraction using an open column chromatography, and the bioassay showed strongest antifeeding activity in H1 fraction. Using an oscilloscope, electrophysiological responses of two sensilla showed more seven times activity in MST. H11, H12, H13, H14 were separated from H1 fraction and antifeeding activity was showed highest in H11 fraction. H11 fraction was examined electrophysiological responses at doses of 100, 10, and 1 ppm, and MST of P. xylostella responded at a dose of 100 ppm. H11 fraction was separated using HPLC and identified using GC/MS and NMR. Finally, the structure of active compound proved to be farnesene with molecular weight of 204, and a formula of C15H24

This study was estimated for cabbage aphid, Brevicoryne brassicae in Brassica campestris L. var. rapa (L.) Hartm. in order to institute of Economic injury levels(EILs). B. brassicae was innoculated on April 29, in differently 0, 5, 10, 20 and 40 adults per ten plant, respectively. After inoculated of B. brassicae, the density was increased until harvest ing gradually in all plots except non innoculate plot. and Higher inoculation density were increased higher than lower inoculation density. Percentage of damage leaf was higher in plots with higher initial aphid density than in plots with lower initial aphid density. And the leaf weight of commodity were decreased in higher initial aphid density. The decreasing rates of leaf weight of commodity was increased with increasing initial aphid density. The relationship between initial B. brassicae densities and the decreasing rates of leaf weight of commodity was well described by a linear regression, Y=0.8416X-3.5147, R2=0.94. Based on the relationship, the number of adults per 10 plant which can cause 5% loss of yield was estimated to be approximately 10.1. And EILs was estimated to be approximately 1.0 adults/plant in late April.

To response evaluation of high power light emitting diodes (HPLEDs) as potential attractants to the Spodoptera exigua adults, we investigated the attractiveness of specific wavelength, illuminance intensity and light-exposure time, and compared them to the fluorescent. The all light treatments with the 40 lux intensity attracted the significantly highest number of S. exigua. The optimal light-exposure time exhibited the highest attraction rate at the 60 min. When the attraction and repellent rate in the optimal conditions to the S. exigua was surveyed, the white HPLED exhibited the highest attraction rate (91.1%), whereas the red HPLED exhibited the most repellent rate (33.3%). When evatuated of illuminance efficiency with fluorescent as control, white and red HPLED were found to be 9.14 and 10.34 times more efficient than fluorescent. These data clearly show that both the 40 lux intensity and the 60 min light-exposure time by using the white HPLED was the most suitable for attraction of the S. exigua.

In the previous study, the plant essential oils such as clove bud , thyme white and garlic oils gave potent toxicity with the range of 1.5 to 4.5×10-3 ul cm-3 of LC50 values against Bemisia tabaci. Based on the bio-assay results, three plant essential oils(clove bud, thyme white and garlic) were applied to 30% emulsion type. The 100, 200 and 400 fold dilution of these emulsions was showed mild toxicity under port scale treatment against Q biotype. At each fold dilution, clove bud showed 44, 26 and 14%, and thyme white produced 44, 39 and 27%, respectively. Of garlic emulsion, its toxicity was evaluated with 27, 26 and 11%, respectively. But, in case of clove bud and thyme white mixture, their toxicity was more good under the mixture than under the single treatment. Mixture with clove bud 200 fold dilution adding to thyme white 200 fold dilution showed potent activity with 86% mortality. And also 200 adding to 400, and 400 to 400 exhibited 61%, 76%, respectively. These essential oil mixtures showed similar strong toxicity against B biotype under port scale test. In particular, the mixture with clove bud 200 fold dilution adding to thyme white 200 fold dilution showed the highest synergistic effect. Toxicity to plants, except the 100 fold dilution of these emulsions was not observed.

The pinewood nematode (PWN, Bursaphelenchus xylophilus) is known to be a major pathogen of the pine wilt disease (PWD). However molecular pathology of B. xylophilus is not completely understood, the pathogenecity of PWD is related to cell wall-degrading enzymes such as endoglucanases, expansins and pectate lyases (PELs). Recently, we developed stage-specific expressed tag library of B. xylophilus and identified a novel PEL, Bx-PEL3. We cloned Bx-PEL3 gene with RT-PCR, which showed high similarity to previously reported Bx-PELs. Phylogenetic analysis revealed that PEL3 was much closer to PELs of B. xylophilus than any other PELs. PEL3 has a conserved intron site as found in Bx-PEL2 in the genomic DNA analysis. Quantitative real-time PCR analysis revealed that Bx-PEL1 and Bx-PEL2 were more predominantly expressed than the Bx-PEL3 in B. xylophilus. The difference of expression level among Bx-PELs according to growth condition suggests that each Bx-PEL plays different biochemical role in the pathogenesis of the PWD.

Scirtothrips dorsalis Hood is a newly advent pest on citrus in Jeju. Both larvae and adult attack the new shoots and young and matured fruits of citrus. S. dorsalis is highly polyphagous with over 100 recorded species, but not known in Jeju. As a result from investigating the host plants in and/or around citrus orchards in Jeju during 2009 to 2010, they were 25 families 39 species. The total thrips examined on those plants were 13 species, and the richness thrips among them was S. dorsalis, Frankliniella occidentalis, Thrips tabaci and Thrips hawaiiensis. The widespread host plants of S. dorsalis were Mallotus japonicus, Paederia scandens, Hedera rhombea, Cayratia japonica and Clematis apiifolia and they can be also used for monitoring plants. On early May, the larvae of S. dorsalis were first investigated on the shoots of Mallotus japonicus, Lonicera japonica, Paederia scandens, Viburnum awabukira, Fraxinus rhynchophylla and Celtis sinensis. The other thrips species except S. dorsalis and T. tabaci, especially F. intonsa, T. hawaiiensis, T. flavus and T. coloratus were just found on flower stalks during the blooming season.

Blossom midge, Contarinia maculipennis Felt (Diptera: Cecidomyiidae) is originated in Southeast Asia and has been present in Japan, USA and Hawaii. Recently, C. maculipennis was intercepted in Japan, USA and Netherlands with orchid flowers imported from Southeast Asian countries. This pest is designated as quarantine pest in Korea because of its potential damages to vegetables and ornamental plants in case when this pest introduced in Korea. C. maculipennis has known to feed inside unopened flower buds, causing deformed, discolored buds and blossoms. And, in severe infestations, cause premature bud or blossom drop. In Korea, similar damage symptoms have been reported in western orchids which mighty be caused by C. maculipennis from Mid-2000s, especially on D. phalaenopsis. An official recognition to C. maculipennis was given in 2007. And, the occurrences of C. maculipennis and its damages were investigated mainly on D. phalaenopsis in 2008~2009. We emphasize caution about recognizing the possibilities that C. maculipennis could infest flower buds of an orchids and carry out management strategies for this pest in the future.

The black soldier fly (BSF), Hermetia illucens, is known as a beneficial insect and feeds on organic materials derived from animals and human, resulting in reduction of food waste and conversion of organic materials. Despite of many studies on the BSF, there have been no reports of cloned genes encoding serine proteases in the BSF. Thus, the primary objective of this study is to clone and to investigate expression pattern of genes encoding serine proteases released from the midgut of the BSF larvae in order to gain a better understanding of expression mechanism of serine proteases. We cloned two serine proteases from the BSF larva. Based on phylogenetic tree analysis, one was chymotrypsin, the other was trypsin. The open reading frame (ORF) of chymotrypsin was 804bp, which encoded a polypeptide of 267 amino acids. In case of trypsin, the ORF was 744bp, which encoded a polypeptide of 247 amino acids. To investigate expression pattern of two serine proteases, we conducted semi-quantitative RT-PCR at different tissues and different developmental stages. A chymotrypsin and trypsin transcripts were revealed strongly in mid gut. Especially, a chymotrypsin was detected largely at feeding stage more than molting stage, while trypsin was expressed similarly between feeding stage and molting stage

Two acetylcholinesterases (AChEs; BgAChE1 and BgAChE2) from Blattella germanica were functionally expressed using the baculovirus system. Kinetic analysis demonstrated that BgAChE2 had higher catalytic efficiency but lower substrate specificity than BgAChE1. Except paraoxon, BgAChE1 was generally less sensitive to inhibitors than BgAChE2. Western blot analysis using anti-BgAChE antibodies revealed that BgAChE1 was far more abundant in all examined tissues compared to BgAChE2, which is only present in the central nervous system. Both BgAChEs existed in dimeric form, covalently connected via a disulfide bridge under native conditions. Most fractions of BgAChE1 had a glycophosphatidylinositol (GPI) anchor, but a small fraction comprised a collagenlike tail. BgAChE2 appeared to have a collagen-GPI-fused tail. Based on the kinetic and molecular properties, tissue distribution and abundance, BgAChE1 was confirmed to play a major role in postsynaptic transmission.