Chemical Analyses and Antimicrobial Activity of Nine Kinds of Unifloral Chinese Honeys Compared to Manuka Honey (12+ and 20+).

Honey has good antimicrobial properties and can be used for medical treatment. The antimicrobial properties of unifloral honey varieties are different. In this study, we evaluated the antimicrobial and antioxidant activities of nine kinds of Chinese monofloral honeys. In addition, headspace gas chromatography-ion mobility spectrometry (HS-GC-IMS) technology was used to detect their volatile components. The relevant results are as follows: 1. The agar diffusion test showed that the diameter of inhibition zone against Staphylococcus aureus of Fennel honey (21.50 ± 0.41 mm), Agastache honey (20.74 ± 0.37 mm), and Pomegranate honey (18.16 ± 0.11 mm) was larger than that of Manuka 12+ honey (14.27 ± 0.10 mm) and Manuka 20+ honey (16.52 ± 0.12 mm). The antimicrobial activity of Chinese honey depends on hydrogen peroxide. 2. The total antioxidant capacity of Fennel honey, Agastache honey, and Pomegranate honey was higher than that of other Chinese honeys. There was a significant positive correlation between the total antioxidant capacity and the total phenol content of Chinese honey (r = 0.958). The correlation coefficient between the chroma value of Chinese honey and the total antioxidant and the diameter of inhibition zone was 0.940 and 0.746, respectively. The analyzed dark honeys had better antimicrobial and antioxidant activities. 3. There were significant differences in volatile components among Fennel honey, Agastache honey, Pomegranate honey, and Manuka honey. Hexanal-D and Heptanol were the characteristic components of Fennel honey and Pomegranate honey, respectively. Ethyl 2-methylbutyrate and 3-methylpentanoic acids were the unique compounds of Agastache honey. The flavor fingerprints of the honey samples from different plants can be successfully built using HS-GC-IMS and principal component analysis (PCA) based on their volatile compounds. Fennel honey, Agastache honey, and Pomegranate honey are Chinese honey varieties with excellent antimicrobial properties, and have the potential to be developed into medical grade honey.

Authentication of Apis cerana Honey and Apis mellifera Honey Based on Major Royal Jelly Protein 2 Gene.

In Asia, honey is mainly produced by Apis mellifera and Apis cerana. However, the price of A. cerana honey is usually much higher than A. mellifera honey. Seeing considerable profits, some dishonest companies and beekeepers mislabel A. mellifera honey as A. cerana honey or incorporate A. mellifera honey into A. cerana honey. In the present study, we developed methods to discriminate A. cerana honey from A. mellifera honey based on the MRJP2 (major royal jelly protein 2) gene. Two pairs of species-specific primers were designed. The amplification products of A. cerana and A. mellifera were 212 and 560 bp, respectively. As little as one percent incorporation of A. mellifera honey in the mixture can be detected by duplex PCR. Additionally, another method based on the melt curve analysis using the same primers was also developed, allowing a rapid discrimination of real-time PCR product of different species. Our study shows that the entomological authentication of honey samples can be identified by nuclear genes other than mitochondrial genes and this extends the possibility of gene selection in identification. The authentication system we proposed could be a useful tool for discriminating A. cerana honey from A. mellifera honey.

Israeli acute paralysis virus: epidemiology, pathogenesis and implications for honey bee health.

Israeli acute paralysis virus (IAPV) is a widespread RNA virus of honey bees that has been linked with colony losses. Here we describe the transmission, prevalence, and genetic traits of this virus, along with host transcriptional responses to infections. Further, we present RNAi-based strategies for limiting an important mechanism used by IAPV to subvert host defenses. Our study shows that IAPV is established as a persistent infection in honey bee populations, likely enabled by both horizontal and vertical transmission pathways. The phenotypic differences in pathology among different strains of IAPV found globally may be due to high levels of standing genetic variation. Microarray profiles of host responses to IAPV infection revealed that mitochondrial function is the most significantly affected biological process, suggesting that viral infection causes significant disturbance in energy-related host processes. The expression of genes involved in immune pathways in adult bees indicates that IAPV infection triggers active immune responses. The evidence that silencing an IAPV-encoded putative suppressor of RNAi reduces IAPV replication suggests a functional assignment for a particular genomic region of IAPV and closely related viruses from the Family Dicistroviridae, and indicates a novel therapeutic strategy for limiting multiple honey bee viruses simultaneously and reducing colony losses due to viral diseases. We believe that the knowledge and insights gained from this study will provide a new platform for continuing studies of the IAPV-host interactions and have positive implications for disease management that will lead to mitigation of escalating honey bee colony losses worldwide.

Evidence of Apis cerana Sacbrood virus Infection in Apis mellifera.

Sacbrood virus(SBV) is one of the most destructive viruses in the Asian honeybee Apis cerana but is much less destructive in Apis mellifera In previous studies, SBV isolates infecting A. cerana(AcSBV) and SBV isolates infecting A. mellifera(AmSBV) were identified as different serotypes, suggesting a species barrier in SBV infection. In order to investigate this species isolation, we examined the presence of SBV infection in 318A. mellifera colonies and 64A. cerana colonies, and we identified the genotypes of SBV isolates. We also performed artificial infection experiments under both laboratory and field conditions. The results showed that 38A. mellifera colonies and 37A. cerana colonies were positive for SBV infection. Phylogenetic analysis based on RNA-dependent RNA polymerase (RdRp) gene sequences indicated that A. cerana isolates and most A. mellifera isolates formed two distinct clades but two strains isolated fromA. mellifera were clustered with theA. cerana isolates. In the artificial-infection experiments, AcSBV negative-strand RNA could be detected in both adult bees and larvae ofA. mellifera, although there were no obvious signs of the disease, demonstrating the replication of AcSBV inA. mellifera Our results suggest that AcSBV is able to infectA. melliferacolonies with low prevalence (0.63% in this study) and pathogenicity. This work will help explain the different susceptibilities ofA. cerana and A. melliferato sacbrood disease and is potentially useful for guiding beekeeping practices.

High Royal Jelly-Producing Honeybees (Apis mellifera ligustica) (Hymenoptera: Apidae) in China.

China is the largest producer and exporter of royal jelly (RJ) in the world, supplying >90% of the global market. The high production of RJ in China is principally owing to the high RJ-producing lineage of honeybees (Apis mellifera ligustica Spinola, 1806) established by beekeepers in the 1980s. We describe the development of high royal jelly-producing honeybees and the management of this lineage today. Previous research and recent advances in the genetic characterization of this lineage, and the molecular markers and mechanisms associated with high RJ production are summarized. The gaps in our knowledge and prospects for future research are also highlighted.

Detection of Spiroplasma melliferum in honey bee colonies in the US.

Spiroplasma infections in honey bees have been reported in Europe and Asia quite recently, due to intensive studies on the epidemiology of honey bee diseases. The situation in the US is less well analyzed. Here, we examined the honey bee colonies in Beltsville, MD, where Spiroplasmamelliferum was originally reported and found S. melliferum infection in honey bees. Our data showed high variation of S. melliferum infection in honey bees with a peak prevalence in May during the course of one-year study period. The colony prevalence increased from 5% in February to 68% in May and then decreased to 25% in June and 22% in July. Despite that pathogenicity of spiroplasmas in honey bee colonies remains to be determined, our results indicated that spiroplasma infections need to be included for the consideration of the impacts on honey bee health.

Spore Loads May Not be Used Alone as a Direct Indicator of the Severity of Nosema ceranae Infection in Honey Bees Apis mellifera (Hymenoptera:Apidae).

Nosema ceranae Fries et al., 1996, a microsporidian parasite recently transferred from Asian honey bees Apis cerana F., 1793, to European honey bees Apis mellifera L., 1758, has been suspected as one of the major culprits of the worldwide honey bee colony losses. Spore load is a commonly used criterion to describe the intensity of Nosema infection. In this study, by providing Nosema-infected bees with sterilized pollen, we confirmed that pollen feeding increased the spore loads of honey bees by several times either in the presence or absence of a queen. By changing the amount of pollen consumed by bees in cages, we showed that spore loads increased with an increase in pollen consumption. Nosema infections decrease honey bee longevity and transcription of vitellogenin, either with or without pollen feeding. However, the reduction of pollen consumption had a greater impact on honey bee longevity and vitellogenin level than the increase of spore counts caused by pollen feeding. These results indicate that spore loads may not be used alone as a direct indicator of the severity of N. ceranae infection in honey bees.

Geographical influences on content of 10-hydroxy-trans-2-decenoic acid in royal jelly in China.

The content of 10-hydroxy-trans-2-decenoic acid (10-HDA), a marker compound in royal jelly (RJ), is the most important criterion in grading RJ for commercial trade and varies with its origin. To identify the effect of geographical origin on 10-HDA content in RJ, 138 samples were collected from 19 provinces of China (divided into three groups) produced by either Apis mellifera ligustica Spinola, 1806 or a hybrid of A. m. ligustica and Apis mellifera carnica Pollman, 1879 and analyzed for moisture, sugar, crude protein, ash, acid, and 10-HDA concentration. The results show that RJ from western China has a significantly higher 10-HDA level (2.01 +/- 0.05%) than those from northeastern (1.87 +/- 0.05%) and eastern (1.75 +/- 0.03%) China. RJ secreted by hybrid bees contained more 10-HDA (1.89 +/- 0.03%) than that secreted by A. m. ligustica (1.78 +/- 0.03%). The 10-HDA content of RJ produced during flowering of rape (Brassica campestris L.), lime (Tilia amurensis Ruprecht), and vitex (Vitex negundo L. variety heterophylla (Franch.) Rehder) was 1.92, 1.80, and 1.68%, respectively. The results would be helpful during the process of price determination of RJ by providing some basis of geographical, bee strain, and botanical information for commercial trade.

Investigation of the Maturity Evaluation Indicator of Honey in Natural Ripening Process: The Case of Rape Honey.

Honey maturity, a critical factor for quality evaluation, is difficult to detect in the current industry research. The objective of this study was to explore the changes in the composition and find potential maturity indicators of rape honey at different maturity stages through evaluating physicochemical parameters (moisture, sugars, pH, electrical conductivity, total protein, total phenols, total flavonoids, proline, and enzyme activity), the antioxidant capacity, and volatile components. The relevant results are as follows: 1. As the maturity increased, the moisture, sucrose, and maltose content of rape honey gradually decreased, while the glucose, fructose, and total protein content gradually increased. The activities of diastase, invertase, and ß-glucosidase showed a significant increase with the elevation of ripening days, and the activity of glucose oxidase reached the highest before completely capping. 2. The antioxidant capacity of honey increased with the increase in honey maturity. There is a significant and strong correlation between the bioactive components of rape honey and antioxidant capacity (p < 0.01, |r| > 0.857). 3. Thirty-five volatile components have been identified. Nonanal, benzaldehyde monomer, and benzaldehyde dimer can be used as potential indicators for the identification of honey maturity stages. Principal component analysis (PCA) based on antioxidant parameters and volatile components can identify the maturity of honey.

Unique dynamic mode between Artepillin C and human serum albumin implies the characteristics of Brazilian green propolis representative bioactive component.

As a representative bioactive component in Brazil green propolis, Artepillin C (ArtC; 3, 5-diprenyl-4-hydroxycinnamic acid) has been reported a wide variety of physiological activities including anti-tumor, anti-inflammatory, and antimicrobial activity etc. However, it seems incompatible that ArtC in vivo was characterized as low absorption efficiency and low bioavailability. In order to obtain the elucidation, we further investigated the physicochemical basis of ArtC interacting with human serum albumin (HSA) in vitro. We found a unique dynamic mode interaction between ArtC and HSA, which is completely different from other reported propolis bioactive components. Thermodynamic analysis showed that hydrophobic interactions and electrostatic forces are the main driving force. The competitive assay indicates that the binding site of ArtC with HSA is close to the Sudlow's site I. The findings of this study reveal the unique physicochemical transport mechanism of ArtC in the human body, which helps to further understand the uniqueness of the representative functional components of Brazilian green propolis in the human body.

Discrimination of the entomological origin of honey according to the secretions of the bee (Apis cerana or Apis mellifera).

The eastern honeybee Apis cerana and the western honeybee Apis mellifera are the two most economically valuable honeybee species used in apiculture. In market, the price of Apis cerana honey (ACH) is usually several times higher than that of Apis mellifera honey (AMH) due to the production limit, resulting in wide adulteration and counterfeiting of ACH by AMH. In the present study, we compared honeybee secretions in these two kinds of honey, and found significant differences in protein profiles and hydrocarbon components. The SDS-PAGE pattern showed three species-specific bands with molecular weights between 15.0 and 29.4 KDa in ACH, and six species-specific bands in AMH with molecular weights between 13.8 and 33.1 KDa. The GC-MS-MS detection of the petroleum ether extracts of the two kinds of honey showed that 17-Pentatriacontene and Hentriacontane were the characteristic constituents of ACH and AMH, respectively. These two methods constitute a system to satisfy different needs for entomological authentication of honey samples.

Ethanol Extract of Chinese Propolis Attenuates Early Diabetic Retinopathy by Protecting the Blood-Retinal Barrier in Streptozotocin-Induced Diabetic Rats.

Propolis has been shown to reduce the level of blood glucose and suppress the histopathological changes in diabetics. However, it still remains unknown if propolis has a similar effect on diabetic retinopathy (DR). Our study aimed to evaluate the effect of the ethanol extract of Chinese propolis (EECP) on early DR in streptozotocin (STZ)-induced diabetic rats. EECP was given to diabetic rats by oral intubation for 12 weeks. The concentrations of fasting blood glucose (FBG), glycated hemoglobin (HbA1c), malondialdehyde (MDA), reactive oxygen species (ROS), and reactive nitrogen species (RNS) were measured. Pathological examinations, including hematoxylin and eosin (HE) staining, transmission electron microscopy (TEM), and immunofluorescence, were also conducted to provide further evidence of EECP's effect on early DR. EECP was able to attenuate diabetes via directly decreasing the levels of FBG and HbA1c, which also resulted in the reduction of MDA, ROS, and RNS. Furthermore, EECP could protect against the damages of photoreceptor cells, as well as retinal thickening. And the inhibition of blood-retinal barrier (BRB) leakage was also observed in EECP-treated diabetic rats, along with the inhibition the loss of tight junction proteins (occludin, ZO-1). These results suggest that EECP has an ameliorating effect on early DR by inhibition of blood-retinal barrier breakdown. PRACTICAL APPLICATION: This study sheds light on the protective effect of the ethanol extract of Chinese propolis on early diabetic retinopathy and the molecular actions underlying the inhibition of blood-retinal barrier breakdown. Our study suggests that ethanol extract of Chinese propolis can be considered as a potential therapeutic agent in the treatment of early diabetic retinopathy.

The effects of clove oil on the enzyme activity of Varroa destructor Anderson and Trueman (Arachnida: Acari: Varroidae).

Varroa destructor, a key biotic threat to the Western honey bee, has played a major role in colony losses over the past few years worldwide. Overuse of traditional acaricides, such as tau-fluvalinate and flumethrin, on V. destructor has only increased its tolerance to them. Therefore, the application of essential oils in place of traditional pesticides is an attractive alternative, as demonstrated by its high efficiency, lack of residue and tolerance resistance. To study the acaricidal activity of essential oils, we used clove oil (Syzygium aromaticum L.), a typical essential oil with a wide range of field applications, and examined its effects on the enzyme activities of Ca2+-Mg2+-ATPase, glutathione-S-transferase (GST) and superoxide dismutase (SOD) and its effects on the water-soluble protein content of V. destructor body extracts after exposure to 0.1 µl and 1.0 µl of clove oil for 30 min. Our results showed that the water-soluble protein content significantly decreased after the treatments, indicating that the metabolism of the mites was adversely affected. The bioactivity of GSTs increased significantly after a low dosage (0.1 µl) exposure but decreased at a higher dosage (1.0 µl), while the activities of SOD and Ca2+-Mg2+-ATPase were significantly elevated after treatments. These results suggest that the protective enzyme SOD and detoxifying enzymes Ca2+-Mg2+-ATPase and GST contributed to the stress reaction of V. destructor to the essential oils and that the detoxification ability of V. destructor via GST was inhibited at higher dosages. Our findings are conducive to understanding the physiological reactions of V. destructor to treatment with essential oils and the underlying mechanisms behind the acaricidal activities of these natural products.

Evidence of the synergistic interaction of honey bee pathogens Nosema ceranae and Deformed wing virus.

Nosema ceranae and Deformed wing virus (DWV) are two of the most prevalent pathogens currently attacking Western honey bees, Apis mellifera, and often simultaneously infect the same hosts. Here we investigated the effect of N. ceranae and Deformed wing virus (DWV) interactions on infected honey bees under lab conditions and at different nutrition statuses. Our results showed that Nosema could accelerate DWV replication in infected bees in a dose-dependent manner at the early stages of DWV infection. When bees were restricted from pollen nutrition, inoculation with 1×10(4) and 1×10(5) spores/bee could cause a significant increase in DWV titer, while inoculation with 1×10(3) spores/bee did not show any significant effect on the DWV titer. When bees were provided with pollen, only inoculation with 1×10(5) spores/bee showed significant effect on DWV titer. However, our results also showed that the two pathogens did not act synergistically when the titer of DWV reached a plateau. This study suggests that the synergistic effect of N. ceranae and DWV is dosage- and nutrition-dependent and that the synergistic interactions between the two pathogens could have implications on honey bee colony losses.

Fast determination of royal jelly freshness by a chromogenic reaction.

UNLABELLED: Royal jelly is one of the most important products of honeybees. Given its role in development of bee brood into fertile individuals of the royal caste it is also used in health products for human consumption. Royal jelly spoils and loses its health-promoting properties depending on storage duration and conditions. To ensure product quality before selling, it is therefore necessary to assess royal jelly freshness. Many indexes of freshness have been suggested, but they all lack reliability or require complex and time-consuming analyses. Here we describe a method to detect royal jelly freshness based on a chromogenic reaction between royal jelly and HCl. We demonstrate that analyses based on color parameters allow for the discrimination of royal jelly samples based on the duration of their storage. Color parameters of royal jelly stored at -18 and 4 °C for 28 d remained comparable to that of fresh samples, which supports the reliability of the method. The method of freshness determination described is practical, cheap, and fast and can thus be used in real-time when trading royal jelly. PRACTICAL APPLICATION: The method developed can be used to assess royal jelly freshness. It is practical, cheap, and fast and can thus be used in real-time when trading royal jelly.

Extraction, partial characterization, and storage stability of ß-glucosidase from propolis.

Extraction and assay conditions for ß-glucosidase from propolis were optimized. Highest enzyme activity was obtained in a citric acid-disodium hydrogen phosphate buffer at pH 6.0 with 2.5% insoluble polyvinylpyrrolidone at incubation temperature of 57 °C. ß-Glucosidase activities were found in all freshly harvested propolis while ß-glucosidase activities were scarcely present in the randomly bought propolis. Propolis was stored at -20 °C and 4 °C for 3 mo with almost no loss of ß-glucosidase activity, but at room temperature the activity decreased exponentially with the increase of storage time. These results indicated that the activity of ß-glucosidase could be a candidate for propolis-freshness index. ß-Glucosidase from propolis was capable of hydrolyzing p-nitrophenyl-ß-D-glucoside and p-nitrophenyl-ß-D-galactoside, but lacked activity toward p-nitrophenyl-ß-D-glucuronide, p-nitrophenyl-ß-D-cellobioside, amygdalin, cellobiose, and gentiobiose. These results were consistent with the hypothesis that flavonoid glucosides were hydrolyzed by ß-glucosidase during propolis collection and processing and provided a possible explanation for why some flavonoid biosides (that is, rutin and isorhamnetin-3-O-rutinoside) exist in propolis. Practical Application: ß-Glucosidase activity was detected and partial characterization of the enzyme was determined in propolis. The enzyme activity decreased exponentially with the increase of storage time at room temperature, which suggested that the activity of ß-glucosidase could be regarded as a freshness index of propolis. The research will be useful for studying the chemical constituents of propolis.

Self assessment in insects: honeybee queens know their own strength.

Contests mediate access to reproductive opportunities in almost all species of animals. An important aspect of the evolution of contests is the reduction of the costs incurred during intra-specific encounters to a minimum. However, escalated fights are commonly lethal in some species like the honeybee, Apis mellifera. By experimentally reducing honeybee queens' fighting abilities, we demonstrate that they refrain from engaging in lethal contests that typically characterize their reproductive dominance behavior and coexist peacefully within a colony. This suggests that weak queens exploit an alternative reproductive strategy and provides an explanation for rare occurrences of queen cohabitation in nature. Our results further indicate that self-assessment, but not mutual assessment of fighting ability occurs prior to and during the agonistic encounters.