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.