There is a growing demand for natural sleep aids due to various side effects of long-term administration of pharmacological treatments for insomnia. Honey has been reported to exhibit numerous potential health benefits, and it is hypothesized that honey may favorably affect insomnia treatment. Therefore, this study was performed to investigate the possible hypnotic effect of clover honey (CH) and to determine its in vivo mechanism. The total flavonoid content (TFC) of CH and fractions extracted with ethylacetate (EtOAc) and H2O was measured. The pentobarbital-induced sleep test using GABAA-benzodiazepine (BZD) agonists and antagonists was conducted to evaluate the potential mechanism of action behind the sedative-hypnotic activity of CH in mice. The results showed that administration of 500 and 1,000 mg/kg of CH significantly (p<0.01) reduced the sleep latency to a level similar to that of diazepam (DZP, 2 mg/kg), and 1,000 mg/kg of CH significantly (p<0.01) prolonged the sleep duration, which was comparable to that of DZP (2 mg/kg). Administration of the EtOAc fraction with a higher TFC significantly reduced the sleep latency at 50 to 200 mg/kg and prolonged the sleep duration at 100 to 200 mg/kg, which were comparable to those after administration of DZP (2 mg/kg). However, co-administration of CH and EtOAc with flumazenil, a specific GABAA-BZD receptor antagonist, blocked the hypnotic effect. Our findings suggest that the hypnotic activity of CH may be attributed to allosteric modulation of GABAA-BZD receptors. The TFC of CH is expected to be a key factor that contributes to its hypnotic effect.

Among two different acetylcholinesterase (AmAChE1 and AmAChE2) of the western honey bee, the soluble AmAChE1might be related with a stress response as judged from its over-expression in honey bee workers when brood rearingwas suppressed. In this study, to ensure the nature of AmAChE1 responding to stress factors, the expression patternsof AmAChE1 were investigated following various treatments, including varroa mite infestation, bacterial challenge, broodrearing suppression, thermal stresses, chemical treatments, ultraviolet B irradiation, starvation, water restriction and crowdingstress. In addition, transcription profiles of four heat shock protein genes known as general stress markers and vitellogeningene, which is induced in several stress conditions, were tested as positive references. In every tested condition, onlybrood rearing suppression and heat shock were related with the expression of AmAChE1.

Perturbation of normal behaviors (e.g., nursing and foraging) in honey bee colonies by any external factors would immediately reduce the colony’s capacity for brood rearing, which can eventually lead to collapse of entire colony. To investigate the effects of brood rearing suppression in the biology of honey bee workers (nurse and forager), the gene-set enrichment analysis (GSEA) was performed for the transcriptomes of worker bees with or without their brood rearing being suppressed, from which functional profiles of pathways under influences by each condition were identified. Blocking of normal labor (i.e., nursing or foraging) induced the over-representation of pathways related with reshaping of worker bee physiology, suggesting that transition of labor is physiologically reversible. In addition, brood rearing suppression appeared to result in the reduction of neuronal excitability and aggressiveness in both forager and nurse, which would be necessary to manage the in-hive stress under unfavorable conditions

Honey bee swarming is a natural phenomenon that occurs by changes of colony (i.e. population size and queen condition) and environment conditions. As cuticular hydrocarbons (CHCs) are known to be involved in the communication between honey bee nest-mates, we investigated and compared the CHC profiles of worker bees from individual colonies of 9-days before swarming (PPSC), a day before swarming (PSC), swarming (SG) and remaining (non-swarming) (RG). A total of 53 CHCs were identified by GC-MS, among which 11 compounds showed significantly differential expression patterns between swarming states. Before swarming (between PPSC and PSC), detection levels of 4 CHCs were significantly different, suggesting that production of some CHCs changed prior to swarming for swarming preparation. Six CHCs were deferentially produced between PSC and RG. The differential profiles of CHCs with respect to different swarming states are currently under investigation.

There are two different types of acetylcholinesterase (AChE1 and AChE2) in the western honeybee as in most of insects. It is suggested that soluble AmAChE1 might be related with a stress response as judged from its elevated expression level in honey bee workers when brood rearing was suppressed. In this study, to ensure the nature of AmAChE1 responding to stress factors, the expression patterns of AmAChE1 following heat shock, brood rearing suppression and chemical treatments (Imidacloprid and fluvalinate) were investigated. Also, several heat shock protein (hsp) genes (hsp10, hsp60, hsp70 and hsp90) known as general stress markers were tested as positive references. Heat shock induced expression of every tested hsp along with AmAChE1. In brood rearing-suppressed worker bees, 7 days old bees showed much higher expression level of AmAChE1 and hsp90 compared to control honey bees. However, treatment of imidacloprid and fluvalinate did not induce any apparent overexpression of these genes. These results confirm that both HSP and AmAChE1 genes generally respond to temperature and brood rearing suppression and further suggest that AmAChE1 can serve as a potential biomarker along with hsps for the detection of stress in honey bee colonies.

In this study, we investigated and compared some chemical properties of Korean natural honeys and sugar-fed honeys for their quality characteristics. The natural honey samples were monofloral from chestnut and acacia flowers, and the sugar-fed honey samples were collected from honeybees feeding on sugar cane and sugar beet. The chemical properties of the honey samples, such as moisture, total protein, total lipids, ash, carbohydrate, minerals, vitamins, and free amino acids were determined. The moisture content was 18.5±0.9% in natural acacia honey, 17.2±0.9% in natural chestnut honey, 19.6±0.9% in sugar cane-fed honey, and 24.8±% in sugar beet-fed honey. The total protein and ash contents were the highest in natural chestnut honey. Maltose and sucrose were not detected in natural honeys but were detected at 2~7% in sugar-fed honeys. The vitamin, mineral, and free amino acids contents of natural honeys were higher than sugar-fed honeys. The natural chestnut honey is the highest in honeys. These results confirmed that the quality of natural honey was better than that of sugar-fed honey. Also, the vitamin, mineral, and free amino acids contents are potential characteristics for distinguishing between natural and sugar-fed honeys.

Two different types of acetylcholinesterae (AChE1 and AChE2) are present in majority of insects, including the Western honey bee. Out of the two honey bee AChEs (AmAChEs), the soluble AmAChE1 with little catalytic activity is widely distributed in both neuronal and non-neuronal tissues, including fat body. In this study, to identify stresss factors that can induce AmAChE expression, we tested various conditions that honey bees can encounter in natural setting, including heat shock, cold shock, bacterial challenge (Escherichia coli and Staphylococcus aureus) and Varroa mite infestations, and evaluated their effects on AmAChE expression. Among the stress factors tested, only heat shock condition induced AmAChE expression in a dose dependet manner. This finding suggests that one function of AmAChE1 is related with thermoregulations, especially against heat shock stress in honey bees.

Honey bee swarming is a natural phenomenon that occurs when the colony encounters changes in the in-hive (i.e. population size and queen condition) and environmental conditions. To better understand the molecular basis of swarming, we conducted the transcriptomic profiles of worker bees between before swarming [pre-swarming colony (PSC)] and after swarming [swarming group (SG) and remaining group (RG)]. Based on the gene set enrichment analysis (GSEA), we predicted the biological processes associated with swarming. In addition, we analyzed the composition of cuticular hydrocarbons (CHCs) by gas chromatography-mass spectrometry and compared their profiles between different bee groups. GSEA results showed that there were a little differences between PSC and RG while many of the pathways related with metabolism and protein processing were down regulated in SG relative to PSC and RG. CHCs profiling revealed a similar CHCs composition between PSC and RG but some differences in CHCs composition (i.e. heneicosane, octacosane, octacosanol) were detected between SG and RG. These differences in gene pathway and CHC composition were discussed with respect to physiological changes and social communication.

Colony collapse disorder (CCD), a phenomenon of honeybees disappearance, has been reported since 2006. Chronic exposure to neonicotinoid insecticides, particularly imidacloprid, has been suggested to impair forager’s ability for foraging and be a main cause of CCD. Recently, it has been reported that imidacloprid induces insulin resistance in animal cell line by blocking glucose uptake. Similarly to human insulin, insulin-like peptide (ILP) of insects is involved in maintaining blood glucose contents in hemolymph by regulating the concentration of trehalose and glycogen. Therefore, we have hypothesized that sublethal concentration of neonicotinoid may affect the metabolic pathway of honey bees as well. We investigated the transcription levels of the genes involved in the insulin/insulin-like signaling (IIS) pathway, such as AmILP and AmInR, following an acute or a chronic dietary exposure of sublethal concentrations of imidacloprid to foragers. In both experiments, honeybees showed increased expression levels of ILP and InR in a dose-dependent manner. Our results suggest that sublethal dose of imidacloprid likely upregulates IIS pathway, thereby rendering honey bees to become resistant to insulin.

The belief that honey bee venom (BV) can be used to treat certain immune-related diseases, such as arthritis and rheumatic conditions, goes back to antiquity. A growing number of reports have demonstrated that BV contains at least 18 pharmacologically active components, including phospholipase A2 (PLA2). Recent research has shown that bee venom PLA2 (bvPLA2) induces protective immune responses against several diseases including asthma, Parkinson’s disease, and drug-induced organ inflammation. However, the antiviral properties of bvPLA2 have not been well investigated. Hence, we examined the potential inhibitory effects of bvPLA2 and its possible mechanism of action against a broad panel of pathogenic viruses in vitro. Pre-treatment with bvPLA2 significantly inhibited the replication of vesicular stomatitis virus (VSV), coxsackie virus (H3), enterovirus-71 (EV-71), herpes simplex virus (HSV) and Adenovirus (AdV) dramatically. However, bvPLA2 did not show antiviral activity against Influenza A virus (PR8) and Newcastle disease virus (NDV). Such inhibitory effects were explained by blocking of the attachment of the virus to cells upon bvPLA2 treatment. Additionally, we observed that Heparan sulfate (HS) has an inhibitory effect on the attachment of HSV to the cell surface dose dependently, which was inconsistent with bvPLA2 treatment. These findings suggest that bvPLA2 has an inhibitory effect on the replication of diverse viruses by blocking their attachment to the cell surface and could be a promising source of natural antiviral agents.

HPLC-MS/MS를 이용하여 벌꿀의 동물용의약품 동시분석을 위한 분석법을 정립하고자 식품공전 중 벌꿀에 규격이 설정된 11종의 동물용의약품을 2개 그룹 Group 1 (streptomycine, dihydrostreptomycine, neomycine)과 Group 2 (oxytetracycline, enrofloxacin, ciprofloxacin, cymiazole, chloramphenicol, amitraz, coumaphos, fluvalinate)으로 나눠서 동시에 분석 할 수 있는 방법을 연구 하였다. 두 그룹모두 RT는 15분 이내였고, 검출한계는 0.0056~0.0643 μg/g, 정량한계는 0.0169~0.1948 μg/g으로 나타났으며 Group 1 (0.05~1.0 μg/g의 농도범위)과 Group 2 (0.01~1.0 μg/mL) 의 검량선을 작성한 결과 각 동물용의약품의 직선상관계수(R2)는 0.9917~0.9987, 0.9923~1.000으로 매우 양호한 상태를 보였고, 최종 농도가 0.25 μg/g, 1.0 μg/g에서의 회수율은 Group 1 65.1~80.6%, Group 2 64.2~90.3%를 나타났다, 또한 area 및 RT 에 대한 inter (n = 3), intra day (n = 6) RSD (%) 분석결과는 area는 10.92%이하, RT은 1.57% 이하로 나타나 양호한 수준을 보여 벌꿀 중 동물용의약품동시분석 방법으로 적합하다고 판단된다.

Mortality of honeybees(Apis cerana) is a serious problem that beekeepers have to face periodically in Korea. The presence of RNA viruses, in addition to other pathogens may be one of its possible causes. In this work, we were detected Black queen cell virus (BQCV), Kashmir bee virus (KBV), Korean Sacbrood virus (KSBV), Ascosphaera apis, and Nosema in samples of Apis cerana. Honey bee viruses was detected KSBV(58.5 %), KBV(6.5 %), BQCV(70 %) in 2015 by RT(Revers transcriptase)-PCR. Sacbrood virus (SBV) is an important disease of A. cerana. A. apis can cause chock brood disease to honey bee. It was detected 10.3 % of A. cerana colonies by PCR. Also, Nosema cerana was detected 50.5%. Conclusively, investigated disease of the A. cerana, and confirmed virus that lead to bee disease, this is thought by valuable thing as data for development of beekeeping industry such as Colony Collapse cause searching examination.

Honey bee swarming is a naturally occurring phenomenon under the conditions of population increase, climate change and pollen deficit. However, unexpected swarming usually results in loss of bee colony, it poses a considerable trouble in bee keeping. In an attempt to search for molecular markers that can predict the swarming behavior, transcriptional profiling was conducted and compared between the heads of swarming group and the remaining group in the same honey bee colonies. A total of 25,551 transcripts were initially identified and 1,144 differentially expressed genes between the two groups were sorted by FC2 (fold change) cut-off value. Several transcripts, including 6 apidermin (structurally novel cuticular protein)-related, 16 cuticular and 3 odorant binding proteins, showed lower expression levels in the swarming group compared with the remaining group (FC range of –2.17 to –667.48, -2.04 to –54.34 and -2.08 to –21.34 respectively). Pathway analyses are currently in progress to understand the physiological and metabolic differences between swarming and remaining groups of honey bees.

Acetylcholinesterase 1 (AmAChE1) has low catalytic activity and is abundantly expressed in both neuronal and non-neuronal tissues. In previous experiments, we observed that AmAChE1 is rarely expressed in summer while highly expressed in winter. Through additional experiments, the expression of AmAChE1 was suggested to be associated with brood rearing status. Under the assumption that abnormal suppression of brood rearing activity may result in stressful condition in honey bee social community, it was further suggested that AmAChE1 is likely involved in stress management particularly during winter. We hypothesized that the increased docility usually observed in overwintering bees is likely an outcome of stress management in colony, which is mediated by AmAChE1 expression. To verify this, worker bees expressing abundant AmAChE1 were collected in early winter and injected with Amace1 dsRNA to knockdown Amace1. Then, the behavioral activity of the bees was investigated using the EthoVison video tracking system. Honey bees injected with Amace1 dsRNA showed significantly increased motility, which was strongly correlated with the suppressed expression level of AmAChE1 in the abdomen. No apparent reduced expression of AmAChE1 in the head was observed perhaps due to the limited efficacy of RNA interference in the blood-brain-barrier. Our finding suggests that behavioral activity can be regulated, at least, by AmAChE1 expression level in non-neuronal tissue (i.e., fatbody) perhaps via metabolic alteration.

Tropilaelaps mercedesae is an ectoparasite of immature honey bees belonging to the genus Tropilaelaps (Acari: Laelapidae). T. mercedesae has become a major threat to the Western honey bee Apis mellifera in Asia, including Korea, and is expanding its geographical range to northern regions due to global warming. To establish gene resources of T. mercedesae, the whole transcriptome was analyzed by RNA sequencing. An mRNA-focused library was generated from total RNA extracted from the mixed stages using the TruSeq RNA Library Preparation kit and sequenced using the HiSeq 2000 platform. A total of 6.0 Gb reads were obtained with 85% Q30 value. Trimmed sequence data were de novo assembled using the CLC Assembly Cell v 4.2. A total of 64,868 non-duplicate contigs were finally obtained and annotated by the Blast2GO using the NCBI nr database. The most abundant species in the resulting 14,336 Blast hits (22.1%) was Metaseiulus occidentalis, a predatory mite, followed by Ixodes scapularis and Tribolium castaneum, suggesting that the T. mercedesae transcriptome matches well with closely related other arthropod species, including mites and ticks. In order to provide basic information for efficient control and monitoring of potential resistance in T. mercedesae, acaricide target genes were annotated and characterized. One voltage-sensitive sodium channel gene encoding the molecular target of fluvalinate, a pyrethroid acaricide most widely used for the control of T. mercedesae, was identified and its molecular properties were investigated. In addition, other acaricide target genes, including acetylcholinesterase and glutamate (or GABA)-gated chloride channel, were identified and characterized.

The acetylcholinesterase 1 (AmAChE1) of the honey bee is known to be abundantly expressed both in the central and peripheral nervous systems. AmAChE1 exists mostly in the soluble form with little catalytic activity and has non-neuronal functions. Our preliminary observation showed that AmAChE1 expression fluctuated between the forages and nurses. A more systematic expression profiling of AmAChE1 over a year cycle on a monthly basis revealed that AmAChE1 was predominantly expressed during the winter months with being moderately expressed during the rainy summer time. However, no significant difference in AmAChE1 expression was noticed between the nurse and forager workers. Interestingly, AmAChE1 expression was inhibited when bees were allowed for brooding by placing overwintering bee hives in strawberry green houses with the supplement of pollen diets whereas it was resumed when the bee hives were removed from the green houses, thereby suppressed brooding. To confirm whether brooding status is a main determining factor for the suppression of AmAChE1 expression, active bee hives were placed in a screen tent, thereby hindering foraging, until brooding was completely suppressed, and then allowed to restore brooding by removing the screen. The AmAChE1 expression in the head was up-regulated when brooding was suppressed whereas its expression was down-regulated when brooding was resumed. These finding demonstrates that AmAChE1 expression in the central nervous system (i.e., head) is related with brooding status of honey bee. To understand the connection between the AmAChE1 expression and other pathways related with brooding, currently in progress are the analyses of head transcriptomes of honey bee workers with or without their brooding suppressed.

Varroa destructor and Tropilaelaps mercedesae mites are ectoparasitic to honey bee having similar life cycle and damage symptoms. Both invade into the last instar larval cell and reproduce during capped brood period of honey bee development. Female adult mites escape from the comb cell on the back of the emerging adult bee (phoretic period) and invade another cell for reproduction. Objective of this study was to study the effect of competitive interaction on each parasitic mite species population. We assessed population monitoring of host and parasitic mites. Honey bee population was monitored by approximating sealed brood and adult bees based on the coverage of the combs. Parasitic mites were monitored by detection technique like sugar shake, stick board, and sealed brood. This monitoring continued at weekly interval during 2008, 2014, and 2015. Additionally Invasion distribution of each species was checked. We calculated carrying capacity, population growth rate, and competition parameter from population monitoring data. Single parasitic mite, Varroa occurred and infestation increased continuously throughout the year in 2008. Co-occurrence of Varroa and Tropilaelaps in honey bee colonies was studied in 2014 and 2015. Carrying capacity was higher in single parasite infesting honeybee than parasites in co-occurrence. While using sugar method, carrying capacity of Varroa alone was found higher than in its co-occurrence with Tropilaelaps. Population growth rate of Varroa when tested alone was higher than its co-occurrence with Tropilaelaps in sugar method. Population growth rate of Varroa and Tropilaepas was higher in sticky method than sugar methods when they were tested in co-occurrence. Population growth rate is higher in Tropilaelaps (0.09) than Varroa (0.05) when both are tested in co-occurrence. We calculated competition parameter of Varroa and Tropilaelaps which was 1.9 and 0.53, respectively. Negative effect on regulation of carrying capacity and population growth rate is due to interspecies competition. Varroa population was higher than Tropilaelaps because there was high intraspecies competition among Tropilaelaps. Single Varroa or its co-occurrence with Tropilaelaps both can destroy honeybee colonies.

In order to use as a new functional food material, we analyzed the chemical components including the organic compounds, minerals and Vitamin C of sorbus and acacia honey which were produced in South Korea. The condensed rate of methanol extraction in honey was 77% of sorbus honey and it was 93.06% of acacia honey. In the case of sorbus honey, main organic compounds that extract by organic solvents in GC-MS analysis were trichloromethane, propylcarbinol, cyclopentane, acetoxyethane, tetrasol, aziridine etc. and main aromatic compounds that extract by organic solvents in SPME analysis were Skelly solve, Benzaldehyde, Hyacinthin, Dodecanal, Lauraldehyde etc. Also, in occasion of acacia honey, main organic compounds were trichloromethan, Acetoxyethane, Hexanaphthene, acetidin etc. and main aromatic compounds were Hydrazomethan, Azulene, Cyclotrisiloxane, Hydrazine etc. Proximate composition was crude protein 0.54%, crude fat 0.44%, crude ash 0.25% in sorbus honey and crude protein 0.10%, crude fat 0.29%, crude ash 0.06% in acacia honey. Free sugar that analyze by HPLC was fructose 26.81%, glucose 20.42%, total sugars 47.23% in sorbus honey and fructose 48.52%, glucose 24.29%, total sugars 72.81% in acacia honey. Vitamin C was not detected in two sample honeys. Minerals by ICP analysis were detected total 18 kinds in sorbus honey, K 11.715 ppm>Na 7.857 ppm>Si 4.407 ppm>Ca 3.673 ppm etc. and total 22 kinds in acacia honey, Na 4.527 ppm>Si 3.420 ppm>K 3.091 ppm>Zn 1.482 ppm etc.