Honeybee Venom Research Articles

Toxic and essential elements in honeybee venom from Slovakia: Potential health risk to humans

Honeybee venom is one of the natural substances produced by bees (Apis mellifera). Their venom gland produces venom which plays a defensive role. In this study a concentration of macro and trace elements (Ag, Al, As, Ba, Ca, Cd, Co, Cr, Cu, Fe, K, Li, Mg, Mn, Mo, Na, Ni, Sb, Se, Sr, Pb and Zn) in foragers′ and honeybees′ venom was analysed by axial inductively coupled plasma optical emission spectrometry (ICP OES) with good validation parameters to differentiate the element accumulation ability in honeybee venom. Cumulative ability for some elements (As, Al, Ba, Cr, Li, Mo, Pb, and Zn) in bee venom was clearly demonstrated. Oppositely, levels of macro elements (Ca, K, Mg and Na) in venom were several times lower compared to the levels detected in foragers. Moreover, PCA analysis of bee samples showed that Cr was associated with locality Košice, and Cd with locality Krompachy; both have rich industrial history. Since some of analysed elements are potentially toxic for humans, a risk assessment for bee-stung scenario was also calculated. A new way of exposure to potentially toxic elements via honeybee stung was showed in this study. Non-carcinogenic risk assessment for humans to selected toxic elements (As, Cd, Cr, Ni, and Pb) demonstrated acceptable risk and moreover the same we may conclude for potential carcinogenic risk for beekeepers exposed to As, Cd, Ni, and Pb via venom over their whole life.

Safety evaluation of the food enzyme triacylglycerol lipase from the non-genetically modified Limtongozyma cylindracea strain AE-LAYH (B).

The food enzyme, a triacylglycerol lipase (triacylglycerol acylhydrolase; EC 3.1.1.3), is produced with the non-genetically modified Limtongozyma cylindracea strain AE-LAYH (B) by Amano Enzyme Inc. It is intended to be used in six food manufacturing processes. Since residual amounts of food enzyme-total organic solids (TOS) are removed in one process, dietary exposure was calculated only for the remaining five food manufacturing processes. It was estimated to be up to 0.315 mg TOS/kg body weight (bw) per day in European populations. As the production strain qualifies for the quality presumption of safety (QPS) approach of safety assessment and no issue of concern arising from the production process of the food enzyme were identified, the Panel considered that no toxicological studies other than the assessment of allergenicity were necessary. A homology search for the amino acid sequence of the food enzyme to those of known allergens was made and one match with a honeybee venom allergen was found. The Panel considered that a risk of allergic reactions by dietary exposure, particularly in individuals allergic to honey, cannot be excluded, but is considered to be low. Based on the data provided, the QPS status of the production strain and the absence of issues of concern arising from the food enzyme manufacturing process, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Clinical complications in envenoming by Apis honeybee stings: insights into mechanisms, diagnosis, and pharmacological interventions.

Envenoming resulting from Apis honeybee stings pose a neglected public health concern, with clinical complications ranging from mild local reactions to severe systemic manifestations. This review explores the mechanisms underlying envenoming by honeybee sting, discusses diagnostic approaches, and reviews current pharmacological interventions. This section explores the diverse clinical presentations of honeybee envenoming, including allergic and non-allergic reactions, emphasizing the need for accurate diagnosis to guide appropriate medical management. Mechanistic insights into the honeybee venom's impact on physiological systems, including the immune and cardiovascular systems, are provided to enhance understanding of the complexities of honeybee sting envenoming. Additionally, the article evaluates emerging diagnostic technologies and therapeutic strategies, providing a critical analysis of their potential contributions to improved patient outcomes. This article aims to provide current knowledge for healthcare professionals to effectively manage honeybee sting envenoming, thereby improving patient care and treatment outcomes.

Venom immunotherapy in clinical practice: comparison of two ultra-rush protocols.

Background. Ultra-rush venom immunotherapy protocols have shown to be a safe and effective approach to prevent the occurrence of systemic reactions after hymenoptera stings. The aim was to describe our experience with two ultra-rush protocols - a five-step with 1 µg starting dose and a six-step with 0.1 µg starting dose, as well as to compare their safety profile. Methods. This is a retrospective study of all the patients who underwent VIT with honey bee or wasp venom between January 2008 and December 2021, in our department. Results. A total of 110 patients was included, with 109 patients (99%) completing the protocol. A total of 63 (57%) patients had no local or systemic reactions. Most systemic reactions occurred with 20 µg or higher doses (24, 83%). There were no documented grade IV systemic reactions (Mueller grading). No differences were found in local or systemic reactions regarding sex, atopy, β-blocker medication, the severity of the index reaction, ID test positivity, levels of total IgE, specific IgE and tryptase (all p > 0.05). Younger age, treatment with bee VIT or being a beekeeper were associated with more systemic reactions (p = 0.035, 0.006 and 0.047, respectively). No statistical differences in the number of local and systemic reactions were found when comparing both protocols (p = 1.000). Conclusions. Ultra-rush protocols are safe and effective, but systemic reactions are to be expected, especially with honeybee. Our data supports that ACE inhibitors do not compromise safety. Beginning with 1 µg is safe and can save time and resources.

Nasally delivered chitosan-coated poly(lactide-co-glycolide) encapsulating honeybee venom enhances T helper 1-related immunity against Salmonella Typhimurium infection in pigs.

Salmonella Typhimurium is a significant zoonotic concern for human food poisoning and a substantial economic burden in the swine industry. We previously reported that nasally delivered chitosan-coated poly(lactide-co-glycolide) (PLGA) encapsulating honeybee venom (CP-HBV) could enhance CD4+ T helper 1 (Th1)-related immune responses in healthy pigs. Building upon these findings, the current study aimed to investigate the protective immune enhancement by nasally delivered CP-HBV in pigs challenged with S Typhimurium. 36 healthy, 4-week-old, female, Landrace X Yorkshire X Duroc pigs. 36 pigs were allocated into 3 groups: CP-HBV (n = 16), control (n = 16), and healthy baseline control (n = 4). CP-HBV and control groups were challenged with S Typhimurium 7 days post-treatment. Pigs from the healthy control group were sacrificed at 0 days postinfection (DPI), and 4 pigs from each of the control and CP-HBV groups were sacrificed at 1, 2, 4, and 7 DPI. Salmonella shedding, immune cell frequencies, cytokines, and transcriptional factor expression levels were measured. The CP-HBV group exhibited lower bacterial shedding and an enhanced Th1-related immune response characterized by an upregulation of CD4+ T cells and CD4+ Interferon-γ+ T cells, accompanied by increased expression of Th1-related cytokines and reduced expression of regulatory T cells and immunosuppressive cytokines compared to the control group. CP-HBV is a promising strategy for controlling Salmonella infections in pigs and improving public health.

Ameliorative role of Honey and Bee venom of Apis mellifera against Methotrexate Induced Alterations On Biochemical Parameters of Liver and Kidney in Male Wistar Albino Rats

Ameliorative role of Honey and Bee venom of Apis mellifera against Methotrexate Induced Alterations On Biochemical Parameters of Liver and Kidney in Male Wistar Albino Rats

Venom Component Allergen IgE Measurement in the Diagnosis and Management of Insect Sting Allergy

Accurate identification of the allergy-eliciting stinging insect(s) is essential to insuring effective management of Hymenoptera venom-allergic individuals with venom-specific immunotherapy (VIT). Diagnostic testing using whole venom extracts with skin tests and serological-based analyses remains the first level of discrimination for honeybee versus vespid venom sensitization in clinical history-positive patients. As a second-level evaluation, serological testing using molecular venom allergens can further discriminate genuine sensitization (honeybee venom: Api m 1, 3, 4, and 10 versus yellow jacket venom/Polistes dominula venom Ves v 1/Pol d 1 and Ves v 5/Pol d 5) from inter-species cross-reactivity [hyaluronidases (Api m 2, Ves v 2, Pol d 2) and dipeptidyl peptidases IV (Api m 5, Ves v 3, Pol d 3)]. Clinical laboratories use a number of singleplex, oligoplex, and multiplex immunoassays that employ both extracted whole venom and molecular venom allergens (highlighted above) for confirmation of allergic venom sensitization. Established quantitative singleplex autoanalyzers have general governmental regulatory clearance worldwide for venom allergic patient testing with maximally achievable analytical sensitivity (0.1 kUA/L) and confirmed reproducibility (inter-assay CVs<10%). Emerging oligo- and multiplex (fixed panel) assays conserve on serum and are more cost-effective, but they need regulatory clearance in some countries and are prone to higher rates of detecting asymptomatic sensitization. Ultimately, the patient’s clinical history, combined with the proof of sensitization, is the final arbiter in the diagnosis of Hymenoptera venom allergy.

Research Progress in Detection of Bee Venom Allergens

Hymenopteran insect stings are a risk factor that cannot be ignored for the people allergic to hymenopteran venoms.In China,the current diagnostic tools cannot provide accurate information to identify sensitized insects,thus affecting clinical diagnosis and treatment.Honeybee is a common hymenopteran insect.Due to its wide distribution,large number,and complex venom composition,researchers have carried out recombination schemes for the main allergens of honeybee venom,laying a theoretical foundation for the detection of allergens.The development of diagnostic technologies for allergen components can accurately detect bee venom allergens,providing a new set of clinical diagnosis and treatment schemes for the population allergic to bee venom.

In vitro antibacterial and anticancer applications of chitosan nanoparticles loaded with Apis mellifera venom

In vitro antibacterial and anticancer applications of chitosan nanoparticles loaded with Apis mellifera venom

STRUCTURAL INSIGHTS AND ANTICANCER POTENTIAL OF MELITTIN IN CD147 INTERACTION

Objective: This study investigates the interaction between melittin (PDB ID: 2MLT), a bioactive peptide from honeybee venom, and CD147 (PDB ID: 5XF0), a glycosylated transmembrane protein implicated in tumor progression. Material and Method: Employing molecular docking and bioinformatics tools, our structural analysis reveals diverse binding features, including hydrogen bonds, salt bridges, and non-bonded contacts, between the CD147 complex and melittin. Result and Discussion: Non-bonded interactions between 2MLT and specific amino acids (Gly181 and Arg201) of CD147 are highlighted, resembling aspects of the CypA/CD147 binding mechanism (Pro180-Gly181 and Arg201). The elevated anticancer potential of 2MLT was substantiated by utilizing the AntiCP 2.0 server and the ENNAACT server, employing machine learning and artificial neural network algorithms. Additionally, hydrophobicity analysis aligns with characteristics associated with anticancer peptides. Notably, thermodynamic stability variations with temperature underscore the robust binding affinity of 2MLT to the 5XF0 receptor. While our study comprehensively explores molecular interactions and predictive analyses, further in vitro and in vivo investigations are crucial to validate these findings for potential therapeutic applications.

The Helix Ring Peptide U11 from the Venom of the Ant, Tetramorium bicarinatum, Acts as a Putative Pore-Forming Toxin.

An insect neuroactive helix ring peptide called U11-MYRTX-Tb1a (abbreviated as U11) from the venom of the ant, Tetramorium bicarinatum. U11 is a 34-amino-acid peptide that is claimed to be one of the most paralytic peptides ever reported from ant venoms acting against blowflies and honeybees. The peptide features a compact triangular ring helix structure stabilized by a single disulfide bond, which is a unique three-dimensional scaffold among animal venoms. Pharmacological assays using Drosophila S2 cells have demonstrated that U11 is not cytotoxic but instead suggest that it may modulate potassium channels via the presence of a functional dyad. In our work described here, we have tested this hypothesis by investigating the action of synthetically made U11 on a wide array of voltage-gated K and Na channels since it is well known that these channels play a crucial role in the phenomenon of paralysis. Using the Xenopus laevis oocyte heterologous expression system and voltage clamp, our results have not shown any modulatory effect of 1 μM U11 on the activity of Kv1.1, Kv1.3, Kv1.4, Kv1.5, Shaker IR, Kv4.2, Kv7.1, Kv10.1, Kv11.1 and KQT1, nor on DmNav and BgNav. Instead, 10 μM U11 caused a quick and irreversible cytolytic effect, identical to the cytotoxic effect caused by Apis mellifera venom, which indicates that U11 can act as a pore-forming peptide. Interestingly, the paralytic dose (PD50) on blowflies and honeybees corresponds with the concentration at which U11 displays clear pore-forming activity. In conclusion, our results indicate that the insecticidal and paralytic effects caused by U11 may be explained by the putative pore formation of the peptide.

Basophil activation in insect venom allergy: comparison of an established test using liquid reagents with a test using 5-color tubes with dried antibody reagents

BackgroundFlow cytometry-based basophil activation tests (BAT) have been performed with various modifications, differing in the use of distinct identification and activation markers. Established tests use liquid reagents while a new development involves the use of tubes with dried antibody reagents. The aim of this pilot study was to compare these two techniques in patients with insect venom allergy.MethodsSeventeen patients with an insect venom allergy were included in the study. The established “BAT 1” utilizes conventional antibody solutions of anti-CCR3 for basophil identification and anti-CD63 to assess basophil activation, whereas “BAT 2” uses dried anti-CD45, anti-CD3, anti-CRTH2, anti-203c and anti-CD63 for identification and activation measurement of basophils. Negative and positive controls as well as incubations with honey bee venom and yellow jacket venom at three concentrations were performed.ResultsSeven patients had to be excluded due to low basophil counts, high values in negative controls or negative positive controls. For the remaining 10 patients the overall mean (± SD) difference in activated basophils between the two tests was 0.2 (± 12.2) %P. In a Bland-Altman plot, the limit of agreement (LoA) ranged from 24.0 to -23.7. In the qualitative evaluation (value below/above cut-off) Cohen’s kappa was 0.77 indicating substantial agreement. BAT 2 took longer to perform than BAT 1 and was more expensive.ConclusionThe BAT 2 technique represents an interesting innovation, however, it was found to be less suitable compared to an established BAT for the routine diagnosis of insect venom allergies.

Formulation and Evaluation of Poly Herbal Body Scrub

The research aimed to produce an Polyherbal body scrub. These days, the demand for skin care products and treatments has increased to a larger extend.Having a proper appearance and a beauty standard has gotten a lot of importance. A typical skin care routine consists of a cleanser, a serum, a moisturizer and a sunscreen.Among these, it has been seen that the serums are the new go to when it comes to building an excellent skin routine. Serums come in various types of formulation be it for oily, dry or anything in between type of skin. Facial wrinkles and skin aging are undesirable outcome of photo damage and ultraviolet (UV) rays. Currently no effective strategies are available to delay skin aging process. Aloe Vera, glycerin and honeybee venom face serum is a highly concentrated cosmetic product. When using aloe Vera we get not only a quick cosmetic effects but also psychological satisfactions, serum has a property of rapid absorption and ability to penetrate into deeper layer of the skin. In the present work we have formulated the poly herbal body scrub by using Orange peel powder, SCI powder, SLS powder, Honey, Glycerine, Bees wax, Aloe vera. These formulations are safe to use for skin and found to be satisfied with all required characterizations. The scrub was evaluated by using the parameters like Appearance, Viscosity, pH, Spreadibility, Washability, Grittiness, Foamability, Moisture content, Consistancytest and Irritation.

Melittin—The principal toxin of honeybee venom—Is also produced in the honeybee fat body

Melittin is a powerful toxin present in honeybee venom that is active in a wide range of animals, from insects to humans. Melittin exerts numerous biological, toxicological, and pharmacological effects, the most important of which is destruction of the cell membrane. The phospholipase activity of melittin and its ability to activate phospholipases in the venom contribute to these actions. Using analytical methods, we discovered that the honeybee Apis mellifera produces melittin not only in the venom gland but also in its fat body cells, which remain resistant to this toxin's effects. We suggest that melittin acts as an anti-bacterial agent, since its gene expression is significantly upregulated when honeybees are infected with Escherichia coli and Listeria monocytogenes bacteria; additionally, melittin effectively kills these bacteria in the disc diffusion test. We hypothesize that the chemical and physicochemical properties of the melittin molecule (hydrophilicity, lipophilicity, and capacity to form tetramers) in combination with reactive conditions (melittin concentration, salt concentration, pH, and temperature) are responsible for the targeted destruction of bacterial cells and apparent tolerance towards own tissue cells. Considering that melittin is an important current and, importantly, potential broad-spectrum medication, a thorough understanding of the observed phenomena may significantly increase its use in clinical practice.

Antioxidant Activity and Mechanism of Action of Amwaprin: A Protein in Honeybee (Apis mellifera) Venom.

Bee venom contains several bioactive components, including enzymatic and non-enzymatic proteins. There is increasing interest in the bioactive components of bee venom since they have exhibited various pharmacological effects. Recently, Apis mellifera waprin (Amwaprin) was identified as a novel protein in Apis mellifera (honeybee) venom and characterized as an antimicrobial agent. Herein, the novel biological function of Amwaprin as an antioxidant is described. In addition, the antioxidant effects of Amwaprin in mammalian cells were investigated. Amwaprin inhibited the growth of, oxidative stress-induced cytotoxicity, and inflammatory response in mammalian NIH-3T3 cells. Amwaprin decreased caspase-3 activity during oxidative stress and exhibited protective activity against oxidative stress-induced cell apoptosis in NIH-3T3 and insect Sf9 cells. The mechanism underlying the cell protective effect of Amwaprin against oxidative stress is due to its direct binding to the cell membrane. Furthermore, Amwaprin demonstrated radical-scavenging activity and protected against oxidative DNA damage. These results suggest that the antioxidant capacity of Amwaprin is attributed to the synergistic effects of its radical-scavenging action and cell shielding, indicating its novel role as an antioxidant agent.

Exploring the potential of melittin peptide: Expression, purification, anti-pathogenic properties, and promising applications as a bio-preservative for beef slices

Melittin peptide is a short cationic linear peptide derived from honey bee venom with 26 amino acids, demonstrates potential in controlling and eliminating pathogens. In this study, the genetic sequence of melittin was inserted into the pcDNA3.1+ plasmid and expressed in HEK-293 cells with a His-tag. Purification was done through affinity chromatography and a nickel column. In this study, the antimicrobial effect of melittin in beef sirloin was first investigated, and the minimum inhibitory concentration (MIC) was determined. Then, the effect of combining it with nanochitosan (n-CH) coating, melittin (0.1% and 0.2%v/v), and nanochitosan alone on the physicochemical, microbiological, and sensory properties of beef was examined over a 9-day at 4 °C. At the end of the storage period, the coated samples effectively prevented an increase in pH (5.75 ± 0.06) and showed a significant decrease in the peroxide value (PV) (4.50 ± 0.07). The most favorable result was observed when using the n-CH+0.2%M, which led to malondialdehyde level of 0.44 ± 0.0.05 mg/kg in the amount of thiobarbituric acid reactant. Additionally, the total volatile base nitrogen content in the treatment involving 0.2%M and n-CH+0.2% and 0.1%M was deemed acceptable. Although the total viable count (TVC) of microorganisms in samples covered with melittin increased over time (7.88 ± 0.12 CFU/g), the microbial growth rate in these samples was lower than the control sample (12.10 ± 0.13 CFU/g). Therefore, the use of nanochitosan based edible coatings in combination with melittin in meat packaging reduces microbial population and increases its shelf life.

Melittin-Phospholipase A2 Synergism Is Mediated by Liquid-Liquid Miscibility Phase Transition in Giant Unilamellar Vesicles.

The primary constituents of honeybee venom, melittin and phospholipase A2 (PLA2), display toxin synergism in which the PLA2 activity is significantly enhanced by the presence of melittin. It has been shown previously that this is accomplished by the disruption in lipid packing, which allows PLA2 to become processive on the membrane surface. In this work, we show that melittin is capable of driving miscibility phase transition in giant unilamellar vesicles (GUVs) and that it raises the miscibility transition temperature (Tmisc) in a concentration-dependent manner. The induced phase separation enhances the processivity of PLA2, particularly at its boundaries, where a substantial difference in domain thickness creates a membrane discontinuity. The catalytic action of PLA2, in response, induces changes in the membrane, rendering it more conducive to melittin binding. This, in turn, facilitates further lipid phase separation and eventual vesicle lysis. Overall, our results show that melittin has powerful membrane-altering capabilities that activate PLA2 in various membrane contexts. More broadly, they exemplify how this biochemical system actively modulates and capitalizes on the spatial distribution of membrane lipids to efficiently achieve its objectives.

Comparative study of the commonly used protein quantitation assays on different Hymenoptera venoms: A fundamental aspect of Hymenoptera venom proteome analysis

Determination of protein concentration in Hymenoptera venoms requires an accurate and reproducible assay as the results will be used to support subsequent proteomic techniques employed in their analyses. However, all protein assay techniques have inherent strengths and weaknesses, demanding their assessment before selecting the most suitable platform for sample analysis. In this study, protein profiles of ant, honeybee, and wasp venoms, and bovine serum albumin (BSA) and hyaluronidase standards were qualitatively assessed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Their amino acid and protein concentration were quantitatively determined via Amino Acid Analysis (AAA). Amino acid concentration was determined via hydrolysis, derivatization, and chromatographic quantification. Protein concentration was estimated using four different protein concentration assays. The ratios of protein concentration in venom samples to protein standards were calculated, and the accuracy of the protein concentration assays was analysed relative to the concentration determined from AAA. SDS-PAGE analysis showed that BSA contained several protein bands, while hyaluronidase contained a mixture of peptide and protein bands. Ant and honeybee venoms contained a higher proportion of peptide bands, while wasp venom contained more protein bands. As determined by AAA, the ratio of protein concentration in Hymenoptera venoms varied between 1.01 and 1.11 to BSA, and between 0.96 and 1.06 to hyaluronidase. Overall, the Bradford assay was found to be the least accurate and the BCA assay was the most accurate in estimating protein concentration in Hymenoptera venoms. There was no significant advantage in using hyaluronidase as a standard or increasing incubation temperature of BCA assay when analysing Hymenoptera venoms. Diluent solutions containing phenol and human serum albumin interfered with Lowry-based assays.

Investigation of imaging the somatostatin receptor by opening the blood-brain barrier with melittin – A feasibility study using positron emission tomography and [64Cu]Cu-DOTATATE

DOTATATE is a somatostatin peptide analog used in the clinic to detect somatostatin receptors which are highly expressed on neuroendocrine tumors. Somatostatin receptors are found naturally in the intestines, pancreas, lungs, and brain (mainly cortex). In vivo measurement of the somatostatin receptors in the cortex has been challenging because available tracers cannot cross the blood-brain barrier (BBB) due to their intrinsic polarity. A peptide called melittin, a main component of honeybee venom, has been shown to disrupt plasma membranes and increase the permeability of biological membranes. In this study, we assessed the feasibility of using melittin to facilitate the passage of [64Cu]Cu-DOTATATE through the BBB and its binding to somatostatin receptors in the cortex. Evaluation included in vitro autoradiography on Long Evans rat brains to estimate the binding affinity of [64Cu]Cu-DOTATATE to the somatostatin receptors in the cortex and an in vivo evaluation of [64Cu]Cu-DOTATATE binding in NMRI mice after injection of melittin. This study found an in vitro Bmax = 89 ± 4 nM and KD = 4.5 ± 0.6 nM in the cortex, resulting in a theoretical binding potential (BP) calculated as Bmax/KD ≈ 20, which is believed suitable for in vivo brain PET imaging. However, the in vivo results showed no significant difference between the control and melittin injected mice, indicating that the honeybee venom failed to open the BBB. Additional experiments, potentially involving faster injection rates are required to verify that melittin can increase brain uptake of non-BBB permeable PET tracers. Furthermore, an evaluation of whether a venom with a narrow therapeutic range can be used for clinical purposes needs to be considered.

Factors affecting the quantity and quality of honey bee venom collected from entrance and inside the beehive under Egyptian conditions

Modern medicine has rediscovered the therapeutic properties of honey bee venom (HBV), a crucial byproduct of beehives. Since HBV has the potential to treat several diseases, a thorough evaluation of its quality and production practices is necessary through the measurement of chemical composition. The aim of this work is to comprehend how the season and beehive harvesting location (inside or entrance) impact the quality and production. The changes in the components, when HBV is collected from the entrance or within the hives and during different seasons, were studied using each set of 20 colonies. Since samples were measured every month from April to September 2021. The findings revealed that the collection site on the beehives, and season affect insignificantly on the amounts of the components that had been evaluated. While, seasons were found to differ significantly in the amounts of collected bee venom between inside and outside beehives which scored 0.030 ± 0.003 and 0.072 ± 0.002g in spring with outside and inside bee hives, respectively, while reached 0.107 ± 0.001 and 0.194 ± 0.011g in summer with outside and inside bee hives, respectively. However, the most important factors that affect the quality of HBV may be the incorrect harvesting and storage methods used by beekeepers.