Decoding the Behavior of a Queenless Colony Using Sound Signals.

Honeybees are reported to be the most vital pollinators of agricultural and horticultural crops. However, their widespread decline has raised great attention to the need to monitor their activity in order to identify the causes and implement countermeasures. The recording and analysis of signals used by honeybees for their communication in their hive can be a very helpful tool to the beekeepers for the remote control of the hives. Thus, in the present study, we used a set of sound recording data taken inside the hives to automatically detect the sounds of the bees over a certain period, to distinguish between queenright and queenless states, and to find the gradual changes in the queenless state. Unlike what was commonly believed, noticeable changes in the sound signals of all experimental colonies were observed just one hour after the queens' removal from the hive, while the sound signals were intensified over a period of 5 h, after which the transmitted signal stabilized to the equivalent of a queenless state. The colonies seemed to return to their normal sounds 9-10 days after the reintroduction of the queens in the hives. Our study concluded that timely intervention of the queen's absence combined with the immediate intervention of the beekeeper may be a determining factor in mitigating the adverse effects that occur from the queen's loss.

Physicochemical Characterization and Biological Properties of Pine Honey Produced across Greece.

Pine honey is a honeydew honey produced in the East Mediterranean region (Greece and Turkey) from the secretions of the plant sucking insect Marchalina hellenica (Gennadius) (Coccoidea: Marchalini-dae) feeding on living parts of Pinus species. Nowadays, honeydew honey has attracted great attention due to its biological activities. The aim of this study was to study unifloral pine honey samples produced in Greece regarding their physicochemical parameters and antioxidant and antibacterial activity against five nosocomial and foodborne pathogens. These honeys showed physicochemical and microscopic characteristics within the legal limits, except for diastase activity, a parameter known to be highly variable, depending on various factors. Substantially higher levels of H2O2 were estimated compared to other types of honeydew honey, whereas protein content was similar. The total phenolic content was 451.38 ± 120.38 mg GAE/kg and antiradical activity ranged from 42.43 to 79.33%, while FRAP values (1.87 to 9.43 mmol Fe+2/kg) were in general higher than those reported in the literature. Various correlations could be identified among these parameters. This is the first attempt to investigate in depth the antibacterial activity of pine honey from Greece and correlate it with honey quality parameters. All tested honeys exerted variable but significant antibacterial activity, expressed as MIC and MBC values, comparable or even superior to manuka honey for some tested samples. Although honey antibacterial activity is mainly attributed to hydrogen peroxide and proteins in some cases (demonstrated by elevated MICs after catalase and Proteinase K treatment, respectively), no strong correlation between the antibacterial activity and hydrogen peroxide concentration or total protein content was demonstrated in this study. However, there was a statistically significant correlation of moisture, antioxidant and antibacterial activity against Klebsiella pneuomoniae, as well as antioxidant and antibacterial activity against Salmonella ser. Typhimurium. Interestingly, a statistically significant negative correlation has been observed between diastase activity and Staphylococcus aureus antibacterial activity. Overall, our data indicate multiple mechanisms of antibacterial activity exerted by pine honey.

The effect of high hydrostatic pressure on tetracycline hydrochloride and sulfathiazole residues in various food matrices - comparison with ultrasound and heat treatment.

Antibiotic residues in food pose serious direct and indirect risks for consumers. The aim of this study was to investigate the effect of High Hydrostatic Pressure (HHP) on tetracycline hydrochloride (TCH) and sulfathiazole (STZ) residues in honey, milk, and water. Three different pressures were tested for their efficiency and treatment at 580 MPa for 6 min was finally selected. Qualitative and quantitative determination of antibiotics were performed with HPLC and LC-MS. HHP treatment was compared to ultrasound and heat treatment. HHP treatment was found to be more effective than the other two methods for both antibiotics in water and milk. The reduction of STZ in honey was over 90%, while no reduction was observed for TCH. The highest TCH reduction was recorded after HHP treatment in water (76.4%) and the highest STZ reduction after ultrasound treatment in honey (94.3%). Reduction of the two antibiotics in different matrices did not follow a similar pattern. For the HHP treatment, the effect of the initial concentration of the two antibiotics was studied under two different storage conditions (refrigerated and frozen storage). The effectiveness of the method was found to be affected by the initial concentration, in both storage conditions for STZ, while for TCH significant differences were observed only for refrigerated storage.

A chemometric approach for the differentiation of 15 monofloral honeys based on physicochemical parameters.

BACKGROUND: Although the main method for authentication of monofloral honey is pollen analysis, other classification approaches have been also applied. However, the majority of the existing classification models so far have utilized a few honey types or a few honey samples of each honey type, which can lead to inaccurate results. Aiming at addressing this, the goal of the present study was to create a classification model by analysing in total 250 honey samples from 15 different monofloral honey types in ten physicochemical parameters and then, multivariate analysis [multivariate analysis of variance (MANOVA), principal component analysis (PCA) and multi-discriminant analysis (MDA)] was applied in an effort to distinguish and classify them. RESULTS: Electrical conductivity and colour were found to have the highest discriminative power, allowing the classification of monofloral honey types, such as oak, knotgrass and chestnut honey, as well as the differentiation between honeydew and nectar honeys. The classification model had a high predictive power, as the 84.4% of the group cases was correctly classified, while for the cases of chestnut, strawberry tree and sunflower honeys the respective prediction was correct by 91.3%, 95% and 100%, allowing further determination of unknown honey samples. CONCLUSION: It seems that the characterization of monofloral honeys based on their physicochemical parameters through the proposed model can be achieved and further applied on other honey types. The results could contribute to the development of methodologies for the determination of honey's botanical origin, based on simple techniques, so that these can be applied for routine analysis. © 2021 Society of Chemical Industry.

A targeted approach for studying the effect of sugar bee feeding on the metabolic profile of Royal Jelly.

Royal Jelly (RJ) constitutes one of the most popular beehive products and for this reason the use of inexpensive sweeteners during its production remains an important quality issue. In the present study we report results of metabolic profiling of RJ samples obtained after the application of artificial bee-feeding using different feeding protocols. The hydrophilic content of RJ samples was assessed by applying (HILIC)UPLC-MS/MS. In total 96 crude RJ samples were analysed with the developed method. Multivariate statistical analysis revealed clear differentiation between the RJ samples obtained from control (non-fed) bees and samples obtained after feeding. In total 27 out of 57 detected molecules were statistically found to be significantly altered in the different comparisons. Among them some amino acids (e.g. tryptophan, lysine), amino acid derivatives (pyroglutamic acid), amines (cadaverine, TMAO, etc.), carbohydrates and vitamins were found as potential markers. The results of the study could be further used for the development of an LC-MS based analytical tool for RJ quality control assessment.

Volatile compounds of Argentinean honeys: Correlation with floral and geographical origin.

The determination of the botanical/geographical origin of honey provides assurance of the product's quality. In the present work, honeys from different ecoregions of Argentina were analysed, and the possible link between the complete pollen profile of honey samples and their volatile composition was evaluated by multivariate statistical tools. A total of 110 volatile compounds were found and semiquantified in honey samples. Redundancy analysis showed significant correlations between the volatile profile of honeys and their production region (P = .0002). According to the present results, 3,8-p-menthatriene; cyclopropylidenemethylbenzene; 1,1,6-trimethyl-1,2-dihydronaphthalene; 1,2,4-trimethylbenzene; α-pinene; isopropyl 2-methylbutanoate; cymene; 2,6-dimethyl-1,6-octadiene; 3-methyloctane; 1-(1,4-dimethyl-3-cyclohexen-1-yl)ethanone; terpinolene; ethyl 2-phenylacetate; naphthalene and 7 unknown compounds could be used to classify Argentinean honeys according to their geographical origin with a prediction success of 96%. Moreover, it could be concluded that honeys with Eucalyptus sp., Aristotelia chilensis and T. Baccharis pollen types presented some characteristic volatile compounds which could be used as floral markers.

Targeted profiling of hydrophilic constituents of royal jelly by hydrophilic interaction liquid chromatography-tandem mass spectrometry.

In the present work a Hydrophilic Interaction Liquid Chromatography-tandem Mass Spectrometry (HILIC-MS/MS) method was developed for the efficient separation and quantification of a large number of small polar bioactive molecules in Royal Jelly. The method was validated and provided satisfactory detection sensitivity for 88 components. Quantification was proven to be precise for 64 components exhibiting good linearity, recoveries R% >90% for the majority of analytes and intra- and inter-day precision from 0.14 to 20% RSD. Analysis of 125 fresh royal jelly samples of Greek origin provided useful information on royal jelly's hydrophilic bioactive components revealing lysine, ribose, proline, melezitose and glutamic acid to be in high abundance. In addition the occurrence of 18 hydrophilic nutrients which have not been reported previously as royal jelly constituents is shown.

The determination of the botanical origin in honeys with over-represented pollen: combination of melissopalynological, sensory and physicochemical analysis.

BACKGROUND: Pollen analysis of honey is the basic method for the determination of its botanical origin. However, the presence of over-represented pollen in honeys may lead the analysis to false results. This can be more severe if this pollen is present in unifloral under-represented honeys of commercial importance (e.g. thyme honey). In the present study, we investigated the abundance of over-represented pollen grains on several quality characteristics in honey samples. In particular, we mixed honeys characterised as over-represented, specifically chestnut and eucalyptus, with thyme honeys in different analogies, and we also analysed the melissopalynological, organoleptic, physicochemical (water content, electrical conductivity, colour) and volatile characteristics of the blends. RESULTS: The most sensitive parameters were the microscopic characteristics, followed by the organoleptic ones. Blends of thyme honey with an originally low percentage of thyme pollen were the most influenced and could not be characterised as unifloral regarding their melissopalynological characteristics, even when they were mixed with small quantities of honeys with over-represented pollen (i.e. 5%). CONCLUSION: The present study confirms that, in the case of presence of over-represented pollen in honeys, pollen analysis alone cannot give trustworthy results for the determination of the botanical origin, even though their exclusion during pollen analysis, when they are present in percentages of up to 30%, could provide more accurate results. Consequently, pollen analysis should also be combined with the other analyses, especially in honeys with under-represented and over-represented pollens, to give safer results for the botanical characterisation of honeys. © 2017 Society of Chemical Industry.

A suggestion for royal jelly specifications.

This article proposes guidelines for quality standards of royal jelly. The proposals are based on two sets of data; the first from our study of the factors that may affect the royal jelly's chemical composition (protein and sugar supplementation of beehives) and the second on the analysis of a great number of samples from across Greece to establish natural variability of this product. We compared our findings with the adopted national limits, the proposals of the working group of the International Honey Commission (IHC), and the draft proposal of the International Organization of Standardization (ISO). The studied parameters included moisture, total proteins, sugars (fructose, glucose, sucrose, total sugars), and 10-hydroxy- 2-decenoic acid (10-HDA). Our results indicate that the limits for royal jelly in some countries should be amended and the proposals of the IHC and the ISO reviewed in view of recent data on variability. We believe that our proposals could be considered for setting global standards for royal jelly, as they incorporate national legislations, proposals of scientific groups, experimental data, and updated information.

Nontarget effects of aerial mosquito adulticiding with water-based unsynergized pyrethroids on honey bees and other beneficial insects in an agricultural ecosystem of north Greece.

We assessed the nontarget effects of ultra-low-volume (ULV) aerial adulticiding with two new water-based, unsynergized pyrethroid formulations, Aqua-K-Othrine (FFAST antievaporant technology, 2% deltamethrin) and Pesguard S102 (10% d-phenothrin). A helicopter with GPS navigation technology was used. One application rate was tested per formulation that corresponded to 1.00 g (AI)/ha of deltamethrin and 7.50 g (AI)/ha of d-phenothrin. Three beneficial nontarget organisms were used: honey bees (domesticated hives), family Apidae (Apis mellifera L.); mealybug destroyers, family Coccinellidae (Cryptolaemus montrouzieri Mulsant); and green lacewings, family Chrysopidae (Chrysoperla carnea (Stephens)). No significant nontarget mortalities were observed. No bees exhibited signs of sublethal exposure to insecticides. Beehives exposed to the insecticidal applications remained healthy and productive, performed as well as the control hives and increased in weight (25-30%), in adult bee population (14-18%), and in brood population (15-19%).

Evaluation of the impact of Exomite Pro on Varroa mite (Varroa destructor) populations and honeybee (Apis mellifera) colonies: efficacy, side effects and residues.

In this research, we examined the application of thymol-based powder, directly over the top of the brood frames in colonies with different population in a 2-year study. The efficacy against mites, the side effects on bees and the contamination of honey were studied comparably to thymol-based gel treatment. In one-store colonies, thymol-based powder treatment gave average efficacy 64.5% and did not differ significantly from thymol-based gel treatment (65.4%). The natural mortality in control colonies was 41.4% and the corrected efficacy (E T) during 2 years was 39.4 and 40.9%, respectively. In two-store bee colonies, the application of thymol-based powder on top of each hive body gave higher E T (45,4%) than on top of the double body hive (40.4%), without statistically significant differences. The average concentration of thymol residues in honey 24 days after the application was 368 and 1,119 µg kg(-1) for the honey of colonies treated with thymol-based powder and thymol-base gel, respectively.

Residue distribution of the acaricide coumaphos in honey following application of a new slow-release formulation.

Acaricide used in beehives for the control of varroa often leaves residues in bee products. The behaviour and distribution of the acaricide coumaphos in honey following the application of a new slow-release strip formulation (CheckMite+) was assessed. The bee colonies were allowed to build new combs without foundation, and two strips were hung in the brood chamber of each colony for a period of 42 days. The distribution of coumaphos residues in honey in relation to the position of the frame and the duration of treatment was examined by collecting samples from each comb at various time intervals up to 145 days after treatment. In the brood chamber, coumaphos was incorporated into honey from the first day of application, and residues accumulated mainly in combs placed next to strips. In the adjacent combs, residues remained at low concentrations with slight variations. In the honey chamber, residue concentrations on the day of strip removal ranged between 0.006 and 0.020 mg kg(-1), while 79 days after application the concentration of coumaphos residues was below 0.020 mg kg(-1). Residues above the EC fixed maximum residue limit (MRL) of 0.1 mg kg(-1) were measured only in brood chamber honey obtained from those combs placed next to strips. In these samples, 0.060-0.111 mg kg(-1) of coumaphos was detected up to 103 days after strip removal. Coumaphos residues in honey extracted from combs that were placed at the edge of the brood chamber were found below the MRL value, even during the 42 day period of CheckMite+ strip treatment.

Glutathione S-transferase in the developmental stages of the insect Apis mellifera macedonica.

We investigated the pattern of glutathione S-transferase (GST) activity in the course of the development of Apis mellifera macedonica. GST activity is present in all developmental stages of A. mellifera macedonica. The highest activity towards the substrate 1-chloro-2,4-dinitrobenzene (CDNB) is found in the adult stage and the lowest in the egg. The kinetic characteristics of the whole enzyme change as the insect develops. Significant changes are observed in substrate specificity, inhibitor sensitivity and V(max). The number of isoenzymes and their rate of expression vary as the insect develops. However, two main isoenzymes are present in all developmental stages, one in the alkaline area and the other in the acidic. While in the larval stage the acidic isoenzyme is expressed at a slightly higher rate (52.2% over 47.8% for the alkaline isoenzyme), in the adult stage, the rate is reversed dramatically (13.24% and 84.2%, respectively).

Glutathione S-transferase in the insect Apis mellifera macedonica kinetic characteristics and effect of stress on the expression of GST isoenzymes in the adult worker bee.

The glutathione S-transferase present in the adult worker bee Apis mellifera macedonica was purified and analyzed for its physicochemical and kinetic properties. The enzyme is heterodimeric with subunit molecular masses of 29 and 25 kDa, respectively. Two main isoenzymes with distinct kinetic properties are present, with isoelectric points of 7.40 for the alkaline and 4.58 for the acidic forms, respectively. The two enzymes are induced independently by factors such as insecticide treatments and environmental conditions, including low temperatures or starvation.