This study aimed to evaluate the antioxidant activity of chestnut honey which were harvested at various areas in South Korea. First at all, we measured the total phenols content through a spectrophotometric determination with a modified Folin-Ciocalteu method and total flavonoids content determined with aluminium chloride. Total phenolic compounds was highest in Sunchang of Chestnut honey(2.21mg/ml)and flavonoids contents was also the highest in Sunchang of Chestnut honey(1.02mg/ml) than other samples. For measured the antioxidant activity of chestnut honey, we performed DPPH(2,2-diphenyl-1-picrylhydrazyl) test and FRAP(ferric reducing-antioxidant assay)test. DPPH scavenging activity highest in Sunchang of Chestnut honey more than 50% DPPH scavenging activitywhile other samples (Gong-ju, Yechen, Chung-ju, Imsil, Ha-dong) showed more than 25% DPPH scavenging activity. The ferric reducing-antioxidant assay (FRAP) is based on the reduction of ferric 2,4,6-tris(2-pyridyl)-1,3,5-triazine [Fe(III)-TPTZ] by spectrophotometric analysis. Sunchang were found to have more than 532μM FRAP activity while other samples (Gong-ju, Yechen, Chung-ju, Imsil, Ha-dong) showed more than 300μM FRAP activity. The results suggested that chestnut honey strong antioxidant activity and it could be utilized as a source of natural antioxidant.

We report for the first time the occurrence of DWV-infected bumble bees (Bombus ignitus). For the present study, the detection of DWV virus from the female and male bumble bee was investigated in the same colony. The Deformed wing virus (DWV) of honeybee (Apis mellifera) is closely associated with characteristic wing deformities, abdominal bloating, paralysis, and rapid mortality of emerging adult bees. Using specific RT-PCR protocols for the detection of DWV followed by sequencing of the PCR products we could demonstrate that the bumble bees were indeed infected with DWV. The virus was detected from Bombus ignitus, and its partial DWV gene was cloned and sequenced. The partial DWV gene encoding the polyprotein is 711-nt of 235 amino acid residues. The deduced nucleotide sequence of the polyprotein partial gene of DWV showed 96.9%, 96.2%, 96.8%, and 96.5% homology to other structure polyprotein partial gene of DWV from insects, respectively. Phylogenetic analysis further conformed that the deduced nucleotide sequence of the polyprotein partial gene of DWV divided to the outside tree. We describe the first time that presence of Deformed wing virus(DWV) from bumble bee(Bombus terrestris) in korea using RT-PCR.

The traditional use of insects as food continues to be widespread in tropical and subtropical countries and to provide significant nutritional, economic and ecological benefits for rural communities. Specially, Bee brood serves as a food source to humans in many countries although limited data exists concerning its nutrient composition. Bee brood (pupa and larvae) were analyzed for Carbohydrate, Saturated fatty acid, Cholesterol, protein, fat, fiber, minerals, and vitamins. Bee brood was high in protein(46.4%～46.73%), fat(18.84%～ 20.75%),carbohydrate(24.66 %～35.79 %), Folic acid(222.30 ㎍/100g), and vitamins. Differentially, folic acid had been contained by high density in pupa of drone. While low in iron, bee brood was a good source of folic acid, and carbohydrate. The fat was composed mostly of saturated and mono-unsaturated fatty acids. The present data suggest bee brood to be an excellent source of many valuable nutrients including energy, amino acids, many essential minerals, and B-vitamins. These data suggest bee brood could be a valuable source of nutrients to various populations.

Bacillus thuringiensis 1-3 (Bt 1-3), belonging to subsp. aizawai (H7), showed different characteristics in plasmid profiles from type strain and had cry2A gene in addition to cry1Aa, cry1Ab, cry1C and cry1D. To clone its plasmids and construct E.coli-Bt shuttle vector, we constructed the plasmid capture system (PCS) by inserting attB sites including lacZ between transposable elements (designated as pPCS-Troy). Through in vitro transposition with total plasmids DNA of Bt 1-3, 53 clones were acquired and their sizes were approximately 10 kb. Based on the sequence analysis, they were classified in four groups showing similarities with four known Bt plasmids, pGI3, pBMB175, pGI1 and pGI2, respectively. One of pGI3-like clones, named as pBt1-3, was fully sequenced and its putative open reading frames (ORFs), Rep-protein, double-strand origin of replication (dso), single-strand origin of replication (sso), have been identified. The structure of pBt1-3 showed high similarity with pGI3 which is one of rolling-circle replication (RCR) group VI family. As a donor for construction of shuttle vector, pDonr-attPEm vector harboring erythromycin resistant gene between attP sites was constructed. Through BP recombination with pPCS-Troy-cloned Bt plasmids and pDonr-attPEm, erythromycin resistant gene was transposed to Bt plasmids. This scheme proposes that in vitro transposition using pPCS-Troy and BP recombination using pDonr-attPEm can easily clone Bt plasmids and construct novel shuttle vectors.

Recently, the genome of Spodoptera litura granulovirus (SlGV) which encodes 133 putative open reading frames (ORFs) was completely sequenced. In this study, to screen novel insecticidal genes of SlGV, we first constructed an advanced plasmid capture system, pPCS-TPI, which contains not only pUC19 ori and ampicillin resistance gene but also Autographa californica nucleopolyhedrovirus (AcMNPV) ORF603 and ORF1629 homologous region between Tn7L and Tn7R. In order to introduce genomic segments of SlGV into the genome of AcMNPV, genomic DNA of SlGV was digested with EcoRI and self-ligated. These self-ligated segments were in vitro transposed with the pPCS-TPI donor by the help of TnsABC* transposase. By this, 10 EcoRI-digested genomic segments of the SlGV were cloned, and these clones were co-transfected with the bApGOZA DNA into sf9 cells to generate corresponding recombinant virus, respectively. The resulting recombinant viruses harboring genomic segments of the SlGV could be used to investigate the insecticidal activity and/or other functions originated from the introduced genomic segments of the SlGV.

The Classical Swine Fever Virus (CSFV) is a member of the Pestivirus genus of the Flaviviridae. The genome of CSFV is a positive single-stranded RNA molecule 12.3 kb and contains a single large open reading frame (ORF). The polyprotein composed of eight nonstructural and four structural proteins (nucleocapsid protein C and three envelope glycoprotein E0, E1 and E2). E2, the most immunogenic of the CSFV glycoproteins, induces a protective immune response in swine. To determine the characteristics of the CSFV, LOM strain, we investigated the nucleotide sequence of the glycoprotein E0, E1 and E2. Comparison of the LOM with the other strains revealed nucleotide sequence identity ranging from 97 to 98%. Expression of the glycoprotein E2 was identified by SDS-PAGE and Western blot analysis using anti-CSFV E2 monoclonal antibodies in Sf21 cells. The expression levels of glycoprotein E2 were observed from day 3 and 5 days maximum. In addition, its expression efficiency by media and cell line was investigated. The result showed that High-Five cells and Grace’s insect media for Sf21 were the best conditions for the expression of the glycoprotein E2.

Because mealybugs are one of the most economically damaging groups of insects on food crops and ornamental plants, some are regulated-species in quarantine with the foreign trade of agricultural products. However, the absence of morphological characteristics enabling the discrimination of early life stages often causes a significant delay or the rejection of a shipment when fruit is discovered containing them, causing much economic loss. A PCR-based method for species identification was developed for six mealybug species known from Korean pears including two regulated insects, Planococcus kraunhiae (Kuwana) and Crisicoccus matsumotoi (Siraiwa). Six sets of species-specific primers were designed based on the sequence comparison of the internal transcribed spacer 1 and 2 regions. Efficiency tests against many mealybug samples showed that this method could effectively discriminate different mealybug species regardless of their developmental stages. Blind tests against 11 field collected mealybug nymph samples indicated that a single PCR is enough to discriminate unidentified mealybugs collected on Korean pears. This new method will be useful in quarantine as well as pest monitoring by providing an easy, accurate way of identifying any life stage of these mealybugs.

The baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV), a large circular double-stranded DNA virus whose genome encodes at least 155 open reading frames (ORFs), is highly pathogenic to a number of lepidopteran insects and widely used to transduce various cells for exogenous gene expression. Although many genes of AcMNPV have been identified, the genome-wide study related to viral replication has not been well announced. In this study, to elucidate DNA replication cascade of AcMNPV, we firstly 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, which contains an ampicillin resistance gene, ORF knock-out mutants were generated by random insertion into bAc-MK genome. These mutants will be suffered DNA microarray to elucidate AcMNPV replication cascade.

Plasmid capture systems (PCS) facilitate cloning and manipulation of circular double-stranded DNA. We recently developed an improved PCS (PCS-LZ) to clone relatively large DNA molecules of 30-150 kb. The PCS-LZ donor consists of a mini-F replicon and a kanamycin resistance marker between Tn7 left and Tn7 right ends. Both the replicon and marker gene of the PCS-LZ donor are transferred into target plasmid DNAs by in vitro transposition, followed by replication in E. coli. Colonies are tested for lacZ expression by blue/white screening. Circular DNAs were obtained from plasmids of Bacillus thuringiensis, genome segments of Cotesia glomerata bracovirus and polymorphic genomes of Autographa californica nucleopolyhedrovirus. PCS-LZ is a powerful tool for use in genomic analysis and mutagenesis in invertebrate pathology, and we are extending its application to include vertebrate research.

Previously, we found that expression by translational fusion of the polyhedrin (Polh)-green fluorescence protein (GFP) led to the formation of granular structures and these fluorescent granules were easily precipitated by high-speed centrifugation. Here, we developed an easy, fast, and mass purification system using this baculovirus expression system (BES). An enhanced GFP (EGFP) fused with Polh gene at the N-terminus including an adaptor and enterokinase (EK) site between Polh and EGFP was expressed in Sf9 cells. The cells infected by AcPolhEKA-EGFP produced fluorescent granules. The EGFP fusion protein was purified from granule-containing cells according to three steps; cell harvest, sonication and EK digestion. Through the final enterokinase digestion, EGFP was presented mainly in the supernatant (93.3%) and the supernatant also showed a pure EGFP band. These results suggest that the combined procedure of Polh fusion expression and enterokinase digestion can used for the rapid and easy purification of other proteins.

Aujeszky’s disease (AD), also called pseudorabies, is an infectious viral disease, caused by an alpha herpes virus and has domestic and wild pigs, as well as a wide range of domestic and wild animals, as the natural host. AD affects many countries and regions in the world, causing important economic losses, mainly due to international trade restrictions. In this study, to determine the characteristics of the Aujeszky’s disease virus (ADV), NYJ strain, which was isolated from the serum of an infected pig in 1987, we investigated the nucleotide sequence and expression of the glycoproteins gB, gC, and gD using the bBpGOZA system. We found that the glycoproteins gB, gC, and gD of NYJ consisted of 2751 bp, 1443 bp, and 1203 bp, respectively. Comparison of the NYJ with the other strains revealed nucleotide sequence identity ranging from 91.tito 99.0%. To better understand the genetic relationships between other strains, phylogenetic analyses were performed. The NYJ strain was formed a distinct branch with high bootstrap support. The expression of glycoprotein gD in insect cells was characterized by SDS-PAGE and Western blotting with an anti-ADV polyclonal antibody. Glycoprotein gD of approximately 45 kDa was detected. The results of this study have implications for both the taxonomy of ADV and vaccine development.

We investigated the effects of cadmium exposure and various stress on the transcription of heat shock protein 70 and 82 (HSP70 and HSP82) from Pardosa astrigera wolf spider. To do this, P. astrigera HSP70 and HSP82 genes were cloned and its full-length sequence determined. Female spiders were long-term exposed to cadmium or to polychlorinated biphenyl (PCB) for 2, 4 and 6 weeks and short-term exposed to endosulfan by dietary uptake. Female spiders were also exposed to various temperatures. HSP82 did not show a clear tendency of transcription induction following exposure to cadmium. On the contrary, HSP70 transcription gradually increased during the exposure to 2, 20 and 40 mM of cadmium for 2, 4 and 6 weeks. Transcript level of HSP70 was not significantly changed by endosulfan and PCB exposure. In the short-term (3 hr) temperature exposure, an increased expression of HSP70 was observed under the heat shock to 30°C and then slightly decreased at 35°C. However, induction of HSP70 transcription was not observed during the long-term (7 days) temperature exposure. Taken together, HSP70 gene appears to be up-regulated by cadmium in a time-dependent manner but little affected by other potential contaminants. Analysis of HSP70 transcript levels in P. astrigera collected from various fields revealed that levels of cadmium concentration were well correlated with HSP70 transcript levels (r2 = 0.76). Taken together, it was suggested that transcript level of HSP70 could be useful as a biomarker for the long-term cadmium exposure of P. astrigera.

Diamondback moth (Plutella xylostella L.) belonging to genus Lepidoptera is a notorious pest of cruciferous crops worldwide. We evaluated the bioinsecticidal activity of the liquid cultures (LB and NB) of a bacterial strain, Serratia sp. EML-SE1, isolated from a diseased diamondback moth. The pathogenicity of a bacterial strain to diamondback moth was confirmed by the following procedures: treatment of liquid culture on cabbage leaves, ingestion of inoculated cabbage and mortality response. For the test, twenty 3rd instar larvae of diamondback moth were placed on the Chinese cabbage leaf in a round plastic cage (Ø 10 × 6 cm) and sprayed with the liquid cultures. After 72 hours, insecticidal activity of LB and NB cultures of Serratia sp. against P. xylostella larvae showed 91.7% and 88.3%, respectively. In addition, the bioinsecticidal activity on potted cabbage with 14 leaves in a growth cage (165 × 83 × 124 cm) also was similar to that of plastic cage experiment. Summarized, the Serratia sp. EML-SE1 may be a potent candidate as a bioinsecticidal agent to control diamondbac kmoth.

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.