Most traditional genome sequencing projects involving infectious viruses include culturing and purification of the virus. This can present difficulties as an analysis of multiple populations from multiple locations may be required to acquire sufficient amount of high-quality DNA for sequence analysis. The electrophoretic method provides a strategy whereby the genomic DNA sequences of the Korean isolate of Pieris rapae granulovirus (PiraGV-K) were analyzed by purifying it from host DNA by pulsed-field gel electrophoresis, thus simplifying sampling and labor time. The genomic DNA of infected P. rapae was embedded in agarose plugs, digested with a restriction nuclease and methylase, and pulsed-field gel electrophoresis (PFGE) was used to separate PiraGV-K DNA from the DNA of P. rapae, followed by mapping of fosmid clones of the separated viral DNA. The double-stranded circular genome of PiraGV-K encodes 120 open reading frames (ORFs), covering 92% of the sequenced genome. BLAST and ORF arrangement showed the presence of 78 homologs to other genes in the database. The mean overall amino acid identity of PiraGV-K ORFs was highest with the Chinese isolate of PiraGV (~99%), followed up with Choristoneura occidentalis ORFs at 58%. PiraGV-K ORFs were grouped, according to function, into 10 genes involved in transcription, 11 involved in replication, 25 structural protein genes, and 15 auxiliary genes. Genes for Chitinase (ORF 10) and cathepsin (ORF11), involved in the liquefaction of the host, were found in the genome. The recovery of PiraGV-K DNA genome by pulse-field electrophoretic separation from host genomic DNA had several advantages, compared with its isolation from particles harvested as virions or inclusions from the P. rapae host. We have sequenced and analyzed the 108,658 bp PiraGV-K genome purified by the pulsed field electrophoretic method. The method appears to be applicable to the analysis of genomes of large viruses. The chitinase, identified by PiraGV-K genome sequence, was functionally characterized by quantitative PCR, Western blot analysis, immunohistochemistry and transmission electron microscopy.

CD63, a member of tetraspanin membrane protein family, plays pivotal role in cell growth, motility, signal transduction, host-pathogen interactions and cancer. In this work, the cDNA encoding CD63 homologue (TmCD63) was cloned from larvae of coleopteran beetle, Tenebrio molitor. The cDNA is comprised of an open reading frame of 705 bp, encoding putative protein of 235 amino acid residues. In silico analysis shows that the protein has four putative transmembrane domains and one large extracellular loop. The characteristic ‘Cys-Cys-Gly’ motif and ‘Cys188’ residues are highly conserved in the large extracellular loop. Phylogenetic analysis of TmCD63 revealed that they belong to the insect cluster with 50-56% identity. Analysis of spatial expression patterns demonstrated that TmCD63 mRNA is mainly expressed in gut and Malphigian tubules of larvae and the testis of the adult. Developmental expression patterns of CD63 mRNA showed that TmCD63 transcripts are detected in late larval, pupal and adult stages. Interestingly, TmCD63 transcript was upregulated the maximum 4.5 fold in response to DAP-type peptidoglycan during the first 6 h, although other immune elicitors also made significant increase in the transcript level at later time-points. These results suggest that CD63 might contribute to T. molitor immune response against various microbial pathogens.

Apolipophorin III (apoLp-III) is a well-known hemolymph protein having a functional role in lipid transport and immune response of insects. We cloned full-length cDNA encoding putative apoLp-III from larvae of the coleopteran beetle, Tenebrio molitor (TmapoLp-III), by identification of clones corresponding to the partial sequence of TmapoLp-III, subsequently followed with full length sequencing by a clone-by-clone primer walking method. The complete cDNA consists of 890 nucleotides, including an ORF encoding 196 amino acid residues. Excluding a putative signal peptide of the first 20 amino acid residues, the 176-residue mature apoLp-III has a calculated molecular mass of 19,146 Da. Genomic sequence analysis with respect to its cDNA showed that TmapoLp-III was organized into four exons interrupted by three introns. Several immune-related transcription factor binding sites were discovered in the putative 5’-flanking region. BLAST and phylogenetic analysis reveals that TmapoLp-III has high sequence identity (88%) with Tribolium castaneum apoLp-III but shares little sequence homologies (<26%) with other apoLp-IIIs. Homology modeling of Tm apoLp-III shows a bundle of five amphipathic helices, including a short helix 3’. The ‘helix-short helix-helix’ motif was predicted to be implicated in lipid binding interactions, through reversible conformational changes and accommodating the hydrophobic residues to the exterior for stability. Highest level of TmapoLp-III mRNA was detected at late pupal stages, albeit it is expressed in the larval and adult stages at lower levels. The tissue specific expression of the transcripts showed significantly higher numbers in larval fat body and adult integument. In addition, TmapoLp-III mRNA was found to be highly up-regulated in late stages of L. monocytogenes or E. coli challenge. These results indicate that TmapoLp-III may play an important role in innate immune responses against bacterial pathogens in T. molitor.

We have identified novel ricin-type (R-type) lectin by sequencing of random clones from cDNA library of the coleopteran beetle, T.molitor. The cDNA sequence is comprised of 495 bp encoding a protein of 164 amino acid residues and shows 49% identity with galectin of Tribolium castaneum. Bioinformatics analysis shows that the amino acid residues from 35 to 162 belong to ricin-type β-trefoil structure. The transcript was significantly upregulated after early hours of injection with peptidoglycans derived from Gram (+) and Gram (-) bacteria, beta-1, 3 glucan from fungi and an intracellular pathogen, L. monocytogenes suggesting putative function in innate immunity.

The aim of this study was to evaluate the biological mechanism of orally administered fly maggot ethanol extracts (EM) on hypocholesterolemic in rats fed a high-cholesterol diet. SDS male rats were divided into four groups (EM dose control=0, 5.0, 7.0, and 9.0 mg/100 g BW) and were treated for 6 weeks. EM groups revealed a significant reduction in serum triglyceride, total cholesterol, and low-density lipoprotein-cholesterol when compared with the control group. HMG-CoA reductase activity in EM groups were significantly lower than those of the control group, but total sterol, neutral sterol, and bile acid excretion were significantly increased in EM groups when compared with the control group. It was discovered that EM suppress the expression of SREBP-1⍺ and SREBP-2 mRNA in the liver tissues of high-cholesterol diet fed rats, while simultaneously increasing the expression of PPAR⍺ mRNA. This finding indicates that EM has a hypocholesterolemic effects in rats fed a high-cholesterol diet, by regulating cholesterol metabolism-related biochemical parameters and SREBP-1⍺ SREPB-2 and PPAR⍺ gene expression.

The chigger mite, Leptotrombidium pallidum, is widely distributed throughout South Korea and is a major vector for Orientia tsutsugamushi, the causative agent of scrub typhus. In this study, the genome size of the chigger mite was estimated to determine the necessary coverage level prior to whole genome sequencing. Cloning of EF1α and RpS3 as putative single copy reference genes were conducted and their partial sequences were determined. Using the serially diluted reference genes with known amount as standard templates, the weight of a single copy of the genome was predicted by a method based on quantitative real time PCR. The average genome length estimated from the weight using two methods was 191 ± 7 Mb. When the genome size of other arthropods (Drosophila melanogster, Apis mellifera and Tetranychus urticae), with their genome analysis completed, were estimated using the same method and compared with actual values, the estimation accuracy was 79.8-98.9%, suggesting our current estimation of L. pallidum genome size is reliable. The estimated L. pallidum genome size is in a similar range to other Acariform mites, such as the dust mite and scabie mite, but appoximately 10-fold smaller compared to the deer tick, which belongs to Parasitiform. Our finding provides key information for further genome sequencing and understanding of mite genome evolution.

For the investigation of the stability of purified bee venom(PBV) during the treatment in the temperature range of 50℃ to 120℃ for 24 hours, respectively, melittin contents, antibacterial effects, and cell regenerations were investigated. The changes in the melittin contents of PBV were not significantly different by treatment temperature below 70℃ for 24 houes and 80℃ for 4 hours. However the melttin contents is great decline after 24 hours above 80℃ for 24 hours. Antibacterial effects is not change below 80℃ for 4 houes but significantly decrease above 80℃ for 24 hours. Cell regenerations of PBV on human dermal fibroblast decreased at 80℃ for 24 houes, showing a significant difference from the below 80℃ for 4 houes. Through the temperature stability of PBV results of this study, it was treated that the melittin contents, antibacterial effects and cell regeneration effects of PBV could be maintained above 80℃ for 4 hours.

Propolis, bee glue is a sticky substances, which is used to prevent corruption of the ones who broke into the hive, and which is known as a substance that inhibits the growth of microorganisms within the hive. In order to use this propolis, is required to extract the active ingredients from the raw propolis. Mainly goes through the process of extraction with ethanol. Propolis is registered as health functional food for food and drug safety with a valid registration which is anti-oxidant effects and anti microbial effects of oralflora. Inthis study, in order to determine the antioxidant effect of propolis produced in various regions collected 58 regions. Antioxidant effects were tested by DPPH free radical scavenging effects method. Asa result, the propolis concentrations by 10, 50, 100, 500, 1000㎍ were 43, 73, 78, 82, and 73%, in Gangwon province, Gyeonggi Province, 57, 83, 83, 79, and 71%, Chungcheong Province, 55, 80, 80, 76, and 68%, Jeolla province 48, 75, 78, 78, and 71%, Gyeongsang province 47, 77, 81, 79, and 71%, respectively. More than a certain concentration was to determine antioxidant falling rather. These results can be said to suggest if you use the edible excess should be avoided.

Royal jelly (RJ) is one of the most attractive functional foods that have been a commercial product, especially in dietetics and cosmetics in many countries. However, RJ has been evoked with dermatitis, acute asthma and anaphylaxis because of major RJ proteins. Therefore, to access water soluble royal jelly (WSRJ) that removed allergy-induced proteins as an effective whitening agent for cosmetics and potential external treatment for topical use, we investigated its ability to inhibit melanin biosynthesis. B16F1 cells were treated with 10 nM α-melanocyte-stimulating hormone (α -MSH) for 48hr, and then were treated with various doses of WSRJ for 36hr. WSRJ (1-10ug/ml) inhibited direct tyrosinase activity and cellular tyrosinase activity, which lead to the decrease of melanin synthesis in α-MSH stimulated B16F1 melanoma cells. In addition, we examined RT-PCR and Western blotting for melanogenesis-related genes such as tyrosinase, tyrosinase related protein 1 (TRP-1) and 2. WSRJ suppressed mRNA and protein expression of tyrosinase, tyrosinase related protein (TRP)-1 and TRP-2 in α-MSH stimulated B16F1 cells, and similar to positive control, arbutin. Our findings suggest that WSRJ induced the downregulation of melanogenesis by inhibiting tyrosinase, TRP-1 and 2 activations. It may serve as a new candidate in the new skin-whitening agents.

Royal jelly (RJ) is exclusive food that is secreted from the hypopharyngeal and mandibular glands of worker honeybees, and it is well known to be a necessary for the growth of the queen honeybee Although fresh royal jelly have been demonstrated to enhance wound healing, the wound healing effects of water soluble royal jelly (WSRJ) have not been elucidated. We investigated whether WSRJ promotes the migration, attachment, and proliferation of human dermal fibroblasts (HDFs) during in vitro wound healing. HDFs were treated with 1-5ug/ml WSRJ and RJ for up to 24hr following wound formation. Cell migration was assessed by measuring recovery from wound margin, while cell attachment and proliferation were determined by MTT assay. By observing the numbers of cell attached, we confirmed that not only WSRJ but also RJ did not affect on the initial cell adhesion. WSRJ (5 ug/ml) enhanced cell migration rate approximately 84.3% in HDFs at 24hr, whereas RJ (5 ug/ml) increased cell migration rate 71.3% in HDFs at 24hr, which is similar to cell migration rate of WSRJ 1 ug/ml (73.7%). In cell proliferation assays, WSRJ induced an increase in the number of HDFs, compared with control and RJ. In conclusion, WSRJ promotes cell migration with increased cell proliferation in an in vitro wound healing model.

About 70% of total honey products produced by Korean bee keepers was acacia honey. The remaining 30% was chestnut honey, jujube honey, snowbell honey, and another honey. False acacia, went down in payability since the middle 2000s because of simultaneous blooming etiolation chlorsis decreases productivity after aging. Therefore, substitution honey plants were necessary. This study estimated nectar secretion amount of each flower and productivity per ha at 14 medical herbs. Each flower, Codonopsis lanceloata, estimated a majority nectar secretion amount at 176.08 ul for each of the 14 medical herbs. Astragalus membranaceus estimated majority nectar secretion amount at 1273.3 L per ha for each of the 14 medical herbs. Medical herbs were hypothesized with valuable honey plants.

Jujube trees, herbal medicine material, produce not only their fruit but also jujube honey for bee and human’ food sources. Although jujube is an important honey plant after acacia bloom, the research was done with 15-year-old jujube trees grown in ChungDo-Gun, which there was no information on jujube floral nectar. According to the research, jujube nectar secretion mostly happens during dawn and morning for two days. The average number of inflorescence per tree is 638.1 according to the research. And also, the average number of flowers per inflorescence is 64.4. The amount of nectar secretion is 11.6 ul on average per flower, and hypothesized nectar secretion from 15-year-old tree per tree is 476.682 ul. Also,a jujube tree has 545.5ul hypothesized nectar secretion per ha by the research.

This study was conducted to establish the optimized protocol for cytoplasm isolation of bee pollen. Data of biochemical parameters and amino acid profiles were obtained from acorn pollen grains treated with pulverization or lyophilization. Contents of moisture, ash, crude protein and crude fat of acorn pollen were 11.7%, 2.6%, 24.1% and 11.8%, respectively. After pulverizing, content of crude protein was decreased to 23.8% while crude fat was 22.5% which means 90% increase. Also content of crude protein was increased to 26.5% in case of the lyophilized pollen. Amino acids such as aspartic acid, glutamic acid, leucine and arginine were extensively found in acorn pollen while histidine, methionine and cystine were infrequent. The pulverized pollen was increased by 2.6% in the total amino acid percentage while the lyophilized pollen increased by 11.8% compared to the untreated pollen.

Most Korean beekeepers have moved from south to north of Korea to collect nectar from black locust (Robinia pseudoacacia) flowers for 2 months. This provided a valuable opportunity to sample bees originating from diverse areas in one location. We initiated a survey of honeybee (Apis mellifera) colonies on the blooming period of Acacia to determine the prevalence of Nosema apis and black queen cell virus (BQCV) in 2013. Nosema causes significant losses in population size of honeybees. Sixteenth hives were sampled for this study. Bees were collected on the 4th and 13th of May, 2013. Nosema spore counts ranged from zero to 1,948,333 spores per bee. The average number of nosema spores per bee was calculated to be 450,000. Approximately 94% of the apiaries examined were infected with nosema, based on the presence of spores in the flowering period of Acacia. Also nosema is thought to be associated with black queen cell virus. RT-PCR analysis shows that BQCV infection rate was 100%. This indicates that nosema and BQCV is the predominant species affecting honeybee colonies.

Virus infections of the honeybee(Apis mellifera) have been increasingly investigated during the last decade. In general, honeybee viruses are widespread and most of them persist as inapparent infections. We screened honeybee colonies for the presence of several bee viruses, including deformed wing virus(DWV), black queen virus(BQCV), Kashmir bee virus(KBV), Israeli acute paralysis virus (IAPV), sacbrood virus(SBV), acute bee paralysis virus(ABPV), using uniplex RT-PCR. Frequently simultaneous infections with different viruses are diagnosed in seemingly healthy bee colonies. Therefore we developed a multiplex RT-PCR assay for the simultaneous detection of multiple bee viruses.

Chungbuk province has number of management honey bee(Apis cerana) colonies. A. cerana produce honey, and are pollinators with in agricultural crops and natural environmental ecosystems. Korean Sacbrood Virus(SBV) causes colony collapses of A. cerana Feb. in Korean apiaries from 2009 years. It causes a fatal disease(sacbrood) of honeybee larvae, which fail to pupate, change color and shape, and finally die. We thus conducted a molecular survey of honey bee RNA viruses, Nosema microsporidia, Foul broods and fungal disease(Chalk brood and Stone brood) associated with environmental conditions in apiaries and differential type(Traditional and Modern) of A. cerana hives in Chungbuk province. We found the presence of Israel Acute Paralysis Virus(IAPV), Nosema apis, and Sacbrood virus(SBV) was detected in the tested samples. IAPV were detected from mordern hive. Nosema apis, and Sacbrood virus(SBV) was detected from traditional hives. Our results demonstrate that honey bee RNA viruse(SBV) and N. apis are present in Traditional hives. It can suggest SBV and other disease might be related environment conditions(Type of hives).

This is the first record of endoparasitic Hymenoptera Apanteles galleriae recorded from Apis cerana colonies in Korea. A simple rearing protocol was established to allow the morphology, mating behavior and infestation rate of A galleriae. In total, 55 lesser wax moth fresh cocoons were kept in the tissue culture test plate at room temperature (25.6 ± 1.5˚C, RH 21 ± 3.7%). The females were 3.4 ± 0.3 longer than male 3.3 ± 0.2. The male antenna was longer than females. The copulation lasts 24.4 ± 2.4 seconds. The larvae of A. galleriae were pupated inside the cocoons of lesser wax moth. Ninety percent of adults A. galleriae was successfully emerged from the lesser wax moth cocoons. A. galleriae can be used as bio-logical control in store and in live colonies to control lesser wax moth.

We investigated the duration of laying worker oviposition and egg-laying behavior in three queenless colonies of A. cerana by in situ video recording. Egg load was determined by dissecting laying workers in September 2012. Egg size, length and breadth, shape index and egg elongation were calculated. To determine the number of eggs laid by laying workers per cell at 24, 53, 74, 120 and 171 hours was also monitored. To estimate the number of eggs per cell per week, a small comb was squeezed between two frames. The combs were collected at given hours and weekly to count the number of eggs, respectively. The results showed that the duration of oviposition of laying workers on average was 109.2 ± 67.5 seconds per cell. During oviposition, egg-laying workers showed two types of behaviors, viz; a still phase, where the egg-laying workers did not move, and a recovery phase, where the egg-laying workers vigorously wagged their abdomens after oviposition. The results showed that on average, 4.0 ± 5.1 of worker eggs per cell per week was recorded. The highest number of eggs was recorded at 120 hours compare to at 24, 53, 74 and 171 hours. Three different shapes of the eggs namely oval, elongated long and elongated curve shaped was laid by workers. The results showed that the laying worker carried 1 to 4 mature eggs in her ovaries and may lay from one to four per oviposition. In conclusion, the laying worker shows a still and a recovery phase during and after laying the eggs. The laying workers retain 1 to 4 eggs in their ovaries. The breadth of eggs is strong positive relationships with length. One worker cell can accumulate up to 33 eggs in queenless colonies.

Ovarioles are smooth, gradually widening white tubes with different stages of eggs. The ovarioles were gently removed, and the right and left ovarioles were separated and counted the ovarioles. We observed that the ovaries of laying queens were extended from second abdominal up to fourth abdominal segments. Each ovariole is supplied with tracheae. The tracheae are auriferous types characterized by coating spiracle tubules with permeable cuticle, which may bring the tracheal air into close contact with haemolymph.

RNA interference(RNAi) is the method which controls phenotypes of gene in live cells. Chitinase is the enzyme helping digestion and absorption of old cuticles during the ecdysis of insects. In order to investigate molting-inhibition effect with the chitinase related gene in Spodoptera litura, RNA was extracted from the 5th instars. cDNA was synthesized and then we obtained about 645bp size chitinase. After PCR products were cloned into a pGEM T-easy vector, colonies were picked. DNA was extracted from the colony cultures. EcoR I enzyme was used to check whether PCR products were inserted or not. To synthesize the dsRNA, each DNA was cut with Spe I and Nco I enzymes. After synthesis, of dsRNA, approximately 5ul dsRNA was injected into the 3rd abdominal segment of S. litura 4th larvae. We confirmed insects that were phenotypically abnormal - for instance molting inhibition, change of integument color, malformation.