Bee venom contains a variety of peptide constituents, including low-molecular-weight protease inhibitors. While the putativelow-molecular-weight serine protease inhibitor Api m 6 containing a trypsin inhibitor-like cysteine-rich domain was identifiedfrom honeybee (Apis mellifera) venom, no anti-fibrinolytic or anti-microbial roles for this inhibitor have been elucidated.In this study, we identified an Asiatic honeybee (A. cerana) venom serine protease inhibitor (AcVSPI) that was shownto act as a microbial serine protease inhibitor and plasmin inhibitor. AcVSPI was found to consist of a trypsin inhibitor-likedomain that displays ten cysteine residues. Interestingly, the AcVSPI peptide sequence exhibited high similarity to the putativelow-molecular-weight serine protease inhibitor Api m 6, which suggests that AcVSPI is an allergen Api m 6-like peptide.Recombinant AcVSPI was expressed in baculovirus-infected insect cells, and it demonstrated inhibitory activity against trypsin,but not chymotrypsin. Additionally, AcVSPI has inhibitory effects against plasmin and microbial serine proteases; however,it does not have any detectable inhibitory effects on thrombin or elastase. Consistent with these inhibitory effects, AcVSPIinhibited the plasmin-mediated degradation of fibrin to fibrin degradation products. AcVSPI also bound to bacterial andfungal surfaces and exhibited anti-microbial activity against fungi as well as gram-positive and gram-negative bacteria. Thesefindings demonstrate the anti-fibrinolytic and anti-microbial roles of AcVSPI as a serine protease inhibitor.

Major royal jelly proteins (MRJPs) are important protein components of bee royal jelly (RJ) and exhibit various biologicaland pharmacological activities. The antimicrobial activities of royalisins and the jelleines contained within MRJP 1 andMRJP 2 in RJ have been elucidated. However, the antimicrobial effects of other bee RJ MRJPs remain largely unknown.In this study, we demonstrated that the Asiatic honeybee (Apis cerana) MRJP 4 (AcMRJP4) exhibits antimicrobial activitiesagainst bacteria, fungi, and yeast. Recombinant AcMRJP4 was expressed as a 63-kDa protein in baculovirus-infected insectcells. However, some of the recombinant AcMRJP4 proteins were cleaved into two fragments (i.e., 48-kDa (AcMRJP4-48)and 15-kDa (AcMRJP4-15) proteins) by the proteolytic cleavage of the C-terminus of the recombinant AcMRJP4. Interestingly,AcMRJP4, AcMRJP4-48, and AcMRJP4-15 exhibited antimicrobial activities, with AcMRJP4-15 exhibiting the highestantimicrobial activity, followed by AcMRJP4. AcMRJP4-15, which is a hydrophilic peptide with 88 amino acid residuesthat contains a high content of Asn and positively charged amino acids, induced structural damage in the cell walls ofthe assayed bacteria, fungi, and yeast. Altogether, our data demonstrated that AcMRJP4 functions as an antimicrobial agent.

RNA interference (RNAi) has been considered as an alternative strategy to control agricultural pests whereby double-strandedRNA triggers a potent and specific inhibition of its homologous mRNA. Since small double-stranded RNAs are requiredfor various RNAi applications, there is a need for cost-effective methods for producing large quantities of high-qualitydsRNA. Bacillus thuringiensis produces much insecticidal proteins with expression of their encoding genes being drivenby sporulation-dependent promoters. To develop dsRNA mass-production platform utilizing Bt, the pHT1K-EGFP plasmidvector which has cyt1Aa sporulation-dependent promoter was constructed. The transcriptional level of target gene (EGFP)is higher 113 times than Bt reference gene (ssPE). It was applied to protect honeybee from Sacbrood virus, so targetgene was replaced to SBV-vp1. By ingestion of Bt-derived dsRNA to honeybee shows positive effect on SBV suppression.

Due to their environmental and economical consequences, invasive species have become a major concern worldwide. Among them, the yellow-legged hornet, Vespa velutina, is a keen hunter of domestic honeybees. They use olfactory cues from the prey to assess food information. In this study, we investigated the attraction behavior of V. velutina to honeybee pheromone under outdoor conditions. Nine honeybee pheromones were tested and compared in order to find the best attractant. When testing specific compounds, the honeybee queen pheromone, homovanillyl alcohol, proved highly attractive.

Vespa velutina nigrithorax prey on various insect and especially hunts domestic honeybees, such as the European honeybee, Apis melifera. For that reason, V. velutina predation has a direct economic impact on apiculture. In particular V. velutina queens are able to establish of new colonies, so they are suitable for V. velutina management. This study was conducted to investigate the attractant of V. velutina Queen. Four traps were tested and combined attractant such as honeybee comb extraction, honeybee extraction, pollen, rice wine and sugar syrup for attraction efficacy. We was able to observe V. velutina Queen in late April to May. And Honeybee extraction, rice wine and sugar syrup baits (5 : 3 : 2) was the best combination for trapping queen on spring.

Honeybees have faced many diseases which treaten bee colony including a serious population decline phenomenon called Colony Collapse Disorder (CCD). Nosema ceranae is a pathogen cause nosemosis, which is now wide spread around the world. According to the genome sequencing for N. ceranae, it has been identified that the presence of machinery for RNA silencing. Microsporidia N. ceranae that are obligated intracellular parasites depend on their host for energetic and metabolic needs. Here we selected the several genes from mitosome of N. ceranae to develop RNAi for the control of Nosema. Especially, TOM40, FNR1, FNR2 and Nar1 were chosen. After infection of N. ceranae, the Honeybee were treated with RNAi either by using only one or combining two or more. The infection rate and specific gene silecing in Nosema were analyzed.

We investigated pollination and foraging activities of honeybee (Apis mellifera L.) and bumblebee (Bombus terrestris L.) during flowering season of the asian pear (Pyrus pyrifolia N.) under different weather conditions. There was no significant statistical difference about the pollination activities of two species. However, the pollination activities of bumblebee were more active than those of honeybee under low temperature and rainfall period. The activities of honeybee and bumblebee were more influenced by temperature than other factors (i.e. illumination and wind velocity). Honeybee was more sensitive to temperature and illumination than bumblebee. At low temperatures (<20℃) on cloudy days (<30,000 lux) with a certain wind velocity (>4.0 m/s), the pollination activity of the honeybee was lower twice than that of bumblebee.

Varroa destructor is a devastating ectoparasitic mite which attacks Honeybee, Apis mellifera. V. destructor feeds on honeybee hemolymph, and often harbors small RNA viruses such as the deformed wing virus to transmit these viruses in the infested bee hive. To survey the genes of V. destructor, total RNA was subjected to high-throughput transcriptome sequencing to construct in silico cDNA library by using the Illumina HiSeq 2000 platform. Total of 2×107,748,792 paired-end short reads were obtained and quality filtered reads were subjected to Trinity de novo assembler followed by TransDecoder, and CD-HIT program to make a V. destructor reference cDNA library containing 28,023 of clustered contigs with protein coding capacity. These cDNA sequences will help us to understand the molecular biology of V. destructor.

Taste substances are recognized by gustatory sensory neurons that express putative seven transmembrane proteins in the gustatory receptor (Gr) family. However, the gustatory tuning of the molecular receptors encoded by these gustatory receptor genes remains unknown in honey bees. Here we first functionally characterize a gustatory receptor responding to umami taste L-amino acids in the western honey bee, Apis mellifera. Using Ca2+ imaging assay and two-voltage clamp recording, we first report that one of the gustatory receptors of honeybee, AmGr10, functions as a selectively tuned amino acid receptor in taste neurons. In addition, we report a floating electrode-based bioelectronic tongue mimicking honeybee taste systems for the detection and discrimination of umami substances. This floating electrode-based bioelectronic tongue mimicking insect taste systems can be a powerful platform for various applications such as food screening, and it also can provide valuable insights on insect taste systems.

Although it is believed that internal nutrient sensors play important roles in feeding behaviors, their molecular and neural mechanisms underlying of the modulation of physiological status and cell growth are poorly understood. Using a Ca2+ imaging experiments with heterologous expression systems, we show that one of the gustatory receptors in the western honey bee Apis mellifera is selectively tuned to amino acids. Remarkably, we report that this gustatory receptor of the honey bee is highly expressed in hypopharyngeal gland, which plays a role in caste differentiation as well as royal jelly production and secretion. Knocking down this gustatory receptor gene reduces cellular pathways responsible for nutritional sensing such as mTOR signals in hypopharageal gland. Furthermore, the interfering expression of this gustatory receptor gene not only alters morphological changes and developmental retardation of the hypopharyngeal gland, but it also blocks cellular growth signals to induce autophagy. This new report indicates that internal sensing and downstream signals detecting nutrients is essential for honey bee to maintain the cellular growth and development of internal organs essential for caste development and maintenance of social structure in the honey bee.

Several Mites are currently the most serious threat to the world bee industry. The ectoparasitic honey bee mites was originally confined to the Asian honey bee(Apis cerana etc.). Varroa destructor and Tropilaelaps clareae has plagued European honey bees, Apis mellifera. Differences in mite tolerance are reported between two honey bee species A. mellifera and A. cerana. We were amplified antimicrobial peptide cDNA genes (Defencin, Abaecin, Royalisin, Apidaecin and Hymenoptaecin) by RT-PCR. We explored the transcriptional response to mite parasitism in A. mellifera 4th instars larvae which differ in susceptibility to V. destructor and T. clareae, comparing parasitized and non-parasitized 4th instars larvae (worker and Drone) from same hive. Differential gene expression of worker bees and Drone bees induced by mites (V. destructor and T. clareae) infection was investigated by northern blot. Mites (V. destructor and T. clareae) parasitism caused changes in the expression of genes related to sex distinction. Bees tolerant to mites (V. destructor and T. clareae) were mainly characterized by differences in the expression of genes regulating antimicrobial gene expression. It provides a first step toward better understanding molecular expression involved in this differential sex distinction host-parasite relationship. We were detected bee virus in A. mellifera, comparing parasitized and non-parasitized 4th instars larvae (worker and Drone). Therefore, this result was demonstrated that mites were another possible route of horizontal transmission, as several viruses were detected in mites and their hosts.

South Korea has over 38 millions of managed honey bee (Apis cerana) colonies before 2009 years ago, which produce the highest quantity of honey in the Korea; however, almost colony (99%) were collapsed by Korean Sacbrood Virus (KSBV) in South Korea. Korean Sacbrood Virus (KSBV) is the pathogen of A. cerana Sacbrood disease, which poses a serious threat to honeybee A. cerana, and tends to cause bee colony and even the whole apiary collapse. Colony collapse of A. cerana was first reported on the Pyeong-Chang of the South Korea in 2009. Symptoms of KSBV include the rapid transmission of larval stage honeybees (A. cerana), many dead larvae found in the bottom of hive and comb. Honeybees (A. cerana) are a very important species because they provide a number of pollination services for various ecosystems in some provinces (ex. jeon-nam, jeon-buk province). They are also extremely important organisms within human society, both agriculturally and economically. The fact that a direct cause has been determined suggests that colony collapse is a complex problem with a combination of natural and anthropogenic factors. Possible instigators of colony collapse include: wax moth, viral and fungal diseases, increased population, decreased genetic diversity, climate changing and a variety of other factors. The interaction among these potential causes may be resulting in immunity loss for honeybees and the increased likelihood of collapse.

Recently, the expression of acetylcholinesterase1 (AChE1) in honeybee worker has been found to be seasonally fluctuated. Seasonal investigation on the AChE1 expression profiles revealed that it is abundantly expressed in January but its expression was completely abolished in February in both head and abdomen. In an attempt to predict the physiological function of seasonally expressed AChE1, proteomic analysis of honeybee worker was conducted using the samples collected in January and February. Total protein samples separately extracted from the head and abdomen of honeybee forager were compared by 2-D electrophoresis (2-DE). More than 2-fold differences in expression patterns between the two different samples were observed in 50 and 85 protein spots in the head and abdomen, respectively. Among them, 20 protein spots showing >17-fold differences in expression between the two different samples of the head were identified by mass spectrometry. Most of the proteins were identified to be the major royal jelly protein (MRJP) families (e.g., MRJP, MRJP2 and MRJP3), which are known to be expressed in nurse bees during brooding season, and their expression was significantly higher in January than in February. This result was unexpected because brooding usually began in the study site apiary during February and the worker bees used for analysis were assumed to be foragers (old workers). Thus, current findings suggest, though speculative, that the workers collected in January may function as nurses despite their old ages in January or that MRJPs may have other not-yet-characterized functions, which is apart from the conventionally known roles. Finally, possible association of MRJPs with AChE1 was discussed.

The Asian honeybee, Apis cerana, is a native honeybee species in Korea which is important in agriculture for pollination and honey production. For better understanding of the physiology of A. cerana, high-throughput Illumina transcriptome sequencing was performed to analyze the gene expression profiles of queen, worker and larva. A total of 219,799,682 clean reads corresponding to 22.2Gb of nucleotide sequences was obtained from the whole body total RNA samples. The Apis mellifera reference mRNA sequence database was used to measure the gene expression level with Bowtie2 and eXpress software and the Illumina short reads were mapped to 11,459 out of 11,736 A. mellifera reference genes. Total of 9,221 genes with FPKM value greater than 5 of each sample group were subjected to evolutionary genealogy of genes: Non-supervised Orthologous Groups (eggNOG) with BLASTX for gene ontology analysis. The differential gene expression between queen and worker, and worker and larva were analyzed to screen the overexpressed genes in each sample group. in the queen and worker sample group, total of 1,766 genes were differentially expressed with 887 and 879 genes overexpressed over two folds in queen and worker, respectively. In the worker and larva sample group, total of 1,410 genes were differentially expressed with 1,009 and 401 genes overexpressed over two folds in worker and larva, respectively.

Nosema disease caused by microsporidia which belongs to fungi is a major cause of honey production loss and is worldwide in distribution. The most common method of nosema disease controlling is the application of the fumagillin. It is an antibiotic derived from the fungus Aspergillus fumigates. It has been used for many years to treat microsporidiosis caused by Nosema in honeybees. However, Fumagillin does not kill Nosema spores, and has rapidly deteriorating potency after application, resulting in only partial and temporary anti-Nosema effect, since new bees emerge constantly in a colony, and re-application is required several times a year. Also it is no longer practised for the associated risk of honey contamination. In this study we compare the effect in spore reduction of Cordyceps militaris extract(CMX) and of fumagillin. Three weekly administrations of CMX showed a noticeable decrease in the number of spores infecting the honey bee intestines, without negative effects for adults and comb health. Therefore this product could play an important role in prevention and reduction of nosema disease without risks of honey with antibiotics.

Sacbrood virus (SBV) is one of the most fatal pathogens against Asian honeybee, Apis cerana. This virus cause failure of the insect larvae to pupate and death of the adult insects. This study has analyzed the host genes affected by viral infection, by comparing the expression level of host transcripts infected with or without SBV. As a first step, we sequenced the cDNA libraries of Asian honeybee by using illumina RNA sequencing. The sequences were de novo assembled to acquire honeybee transcriptome sequences. The transcriptome was annotated by the sequence comparison to known protein sequences by BLASTX and evolutionary genealogy of genes: Non-supervised Orthologous Groups (eggNOG) database with functional categories and description. By mapping the RNA-seq data to de novo assembled transcripts, we characterized the differentially expressed transcripts between SBV-infected and non-infected Asian honeybee.

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.

Sacbrood virus (SBV), a causative agent of larval death in honeybees, is one of the most devastating diseases in bee industry throughout the world. Lately the Korean Sacbrood virus (KSBV) induced great losses in Korean honeybee (Apis cerana) colonies. However, there is no culture system available for honeybee viruses, including SBV, therefore, the research on honeybee viruses is practically limited until present. In this study, we investigated the growth and replication of KSBV in cell cultures. The growth of KSBV was demonstrated by RT-PCR, quantitative real-time PCR, TEM and nucleotide sequence analysis. The results demonstrated that SBVshowed the replication signals in mammalian cell lines, including Vero cells without any signs of cytopathic effect (CPE). The results of RT-PCR, quantitative real-time PCR and in vivo infection with KSBV were also indicated the replication. Phylogenetic tree analysis shows our sequence included in distinct group with other SBV strains from China and Korea. It clearly showed the differenciation between field strain and attenuated strain through cell culture. The results of present study demonstrated for the first time that SBV like other animal viruses could be adapted and attenuated in cells through the sequential passages. The sequential adaptation through cell culture could result in discrepancy of pathogenicity of virus and morphological characterization. For this reason, the present results indicated that the cell adapted SBV could be a valuable tool to study the general properties of this emerging virus, including pathogenicity in the future.

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).