The use of ETOH for the dilution of honey

Since the beginning of honey production, certain honey types are preferred because they taste better, are better for cooking, or do not rapidly crystallize. Pollen found in honey is used to determine the honey's type. Techniques used to extract pollen from honey vary in the amount of honey examined, the amount of water used to dilute the honey, and the time and speed of centrifugation. These variations address the disparity in pollen recovery that is due to the specific gravity and sinking rates of the different pollen types. Ethyl alcohol (ETOH) was used as a dilution liquid for honey and compared to two water-dilution techniques, one with a short centrifugation time (1?minute) and the other using a long centrifugation time (10?minutes). The ETOH samples were centrifuged for 3?minutes. All samples were centrifuged at 4000 r.p.m. Significantly higher pollen concentration values were found in the ETOH-diluted samples. Pollen concentration values of the ETOH-diluted samples were 5.26 times greater than water-diluted, short centrifugation samples, and 3.26 times greater than water-diluted, long centrifugation samples. ETOH-diluted samples produced 16% more taxa than the water-diluted short centrifugation technique, and 10% more taxa than the water-diluted long centrifugation technique. The pollen spectra were not consistent across the three techniques. Additional research is needed to determine the efficiency of other ETOH concentrations in recovering pollen from honey. We recommend that the ETOH-dilution technique become the standard technique for the extraction of pollen from honey for pollen analyses because of the improved pollen recovery.     

Pollen Contamination of Honey by Bees inside the Hive

This paper aims to assess to what degree and in what way the brood chamber affects the pollen content of the honey. Twenty-nine pieces of comb containing only honey were cut from different frames of hives. The percentage of cells in each frame occupied by the brood chamber and the distance between these cells and the cut piece were recorded. A honey sample was extracted from each comb piece, avoiding any contamination with pollen, its sediment examined under the microscope and its botanical constituents identified and counted. The results show that the pollen content of honey was higher in samples from frames containing brood or pollen cells; in these samples the pollen content was positively correlated with the amount of these cells in the frame and the proximity of the honey to them. The proportion of pollen grains from principally nectariferous plants was lower in honeys with a high pollen content.     

A preliminary palynological characterization of Spanish thyme honeys

Pollen was analysed in 25 thyme honey samples from Spain. The honey samples were provided by professional beekeepers, and pollen was obtained by centrifugation. A minimum of 8% of Thymus sp. pollen is considered sufficient to typify a honey as unifloral when considering pollen grains from anemophilous and nectarless plants. The quantitative analysis showed that nectar is the main honey source in the samples studied. The qualitative analysis of the samples revealed the presence of 53 taxa belonging to 27 families. The Spanish thyme honeys of the studied region are characterized by their medium to high numbers of grains of pollen (NGP; mean = 155 000) and their medium to high content of honeydew indicator elements (HDE; mean = 8300). Genista type (present in 100% of the samples), Echium plantagineum and Reseda luteola (90%), Eucalyptus type (80%), Calendula type and Olea europaea (75%), and Cistus monspeliensis (70%) may be mentioned among the characteristic accompanying species of this honey type.  © 2004 The Linnean Society of London, Botanical Journal of the Linnean Society , 2004, 146 , 323–330.     

Unifloral Honeys: Chemical Conversion and Pollen Reduction

Physico-chemical and pollen analyses were made of unifloral samples of nectar, crop content and honey. The samples were obtained by carrying out cage experiments exclusively with one plant species. The investigations of four plant species are presented: Centaurea montana, Coriandrum sativum. Euphorbia lathyris and Rubus idaeus. During the conversion of nectar to honey its chemical composition changes. The changes in carbohydrate composition cannot be explained by those enzymes described as specific for honey. During the period of time when the bees are working on gathered nectar, the chemical components of the honey, whose origin is the secretion from the bees, is increasing, while the amount of pollen is decreasing. There are negative correlations between the quantity of pollen grains and both enzymatic activity and the amount of proline. With respect to the botanical origin it is suggested that the absolute amount of pollen should be determined and compared with the chemical analysis of the honey.     

Botanical origin of honey from south of Calde´n district (Argentina)

We performed palynological analysis of 75 honey samples from the south of Caldén district, which is the part of the phytogeographical province del Espinal. Prosopis caldenia , "caldén" (Fabaceae) is the dominant arboreal species in this region. 79 pollen types that belong to 36 plant families were identified. Honeydew elements are absent or present in negligible amounts. Native flora is intensely utilized. Main nectar sources that characterize monofloral honey samples, are these natives Condalia microphylla (Rhamnaceae), Prosopis sp. and Vicia sp. (Fabaceae), Larrea divaricata (Zygophyllaceae) and Brassicaceae, which are foreign to the area. The pollen from these plants along with the pollen of Trichocline sp. (Asteraceae) and Prosopidastrum globosum (Fabaceae), Schinus fasciculatus (Anacardiaceae), Astereae and Lycium sp.(Solanaceae), characterize honey from Caldén.     

Pollen Analysis of Natural Honeys from the Central Region of Shanxi,North China

Based on qualitative and quantitative melissopalynological analyses, 19 Chinese honeys were classified by botanical origin to determine their floral sources. The honey samples were collected during 2010–2011 from the central region of Shanxi Province, North China. A diverse spectrum of 61 pollen types from 37 families was identified. Fourteen samples were classified as unifloral, whereas the remaining samples were multifloral. Bee-favoured families (occurring in more than 50% of the samples) included Caprifoliaceae (found in 10 samples), Laminaceae (10), Brassicaceae (12), Rosaceae (12), Moraceae (13), Rhamnaceae (15), Asteraceae (17), and Fabaceae (19). In the unifloral honeys, the predominant pollen types were Ziziphus jujuba (in 5 samples), Robinia pseudoacacia (3), Vitex negundo var. heterophylla (2), Sophora japonica (1), Ailanthus altissima (1), Asteraceae type (1), and Fabaceae type (1). The absolute pollen count (i.e., the number of pollen grains per 10 g honey sample) suggested that 13 samples belonged to Group I (<20,000 pollen grains), 4 to Group II (20,000–100,000), and 2 to Group III (100,000–500,000). The dominance of unifloral honeys without toxic pollen grains and the low value of the HDE/P ratio (i.e., honey dew elements/pollen grains from nectariferous plants) indicated that the honey samples are of good quality and suitable for human consumption.     

Palynological characteristics of different unifloral honeys from Greece

Three hundred and twenty-nine Greek honey samples of different botanical and geographical origin were collected and examined by organoleptic evaluation, melissopalynological analysis, measurement of electrical conductivity and colour. The results showed that 208 samples were unifloral with 178 of them representing the main types of unifloral honey produced in Greece; that is fir, pine, chestnut, cotton, orange and thyme honey. All honeys had the sensory characteristics typical of their origin and complied with the electrical conductivity standards set by Council Directive 2001/110/EC.

Fir and pine honeydew honey had a low honeydew element/pollen (HDE/P) ratio and belonged to Maurizio's Classes II or III. The pollen types identified in these honeys ranged from 11 to 45%. Chestnut nectar honey contained >90% chestnut pollen, had a total number of plant elements of >245,000/10?g, and low pollen diversity. Cotton honey contained 1.2 to 16.5% cotton pollen, belonged to Maurizio's Class II, and had 22 pollen types, with Castanea sativa L. present in all samples. Orange honey contained 2.9 to 26.5% Citrus spp. pollen, belonged to Maurizio's Class II, and was characterized by the presence of Brassicaceae, Fabaceae, Olea europea L., Quercus coccifera L. and Rosaceae. In thyme honeys Thymus capitatus Hoffm. &; Link. pollen was secondary or predominant ranging from 18.3 to 69.3%. These honeys belonged to Maurizio's Classes I or II and contained greater than 30 pollen types. Other Lamiaceae, Hypericum spp., Brassicaceae, Fabaceae, Rosaceae, and Cistus spp. pollen types appeared in the greatest number of thyme samples.     

Pollen analysis of honeys from the Gharb region (NW Morocco)

The pollen spectra in 18 honey samples from the Gharb region (Northwest of Morocco) have been studied. The samples were collected directly from the beekeepers, both professional and amateurs. The results show that nectar was the main honey source in this region, although one forest or mixed honey was detected. Seven samples belonging to the Class I, five to the Class II and six to the Class III were found. In all the samples some honeydew indicator elements were detected, with HDEN/NPGN= 0.005-1.38. By the qualitative results 58 pollen types belonging to 28 families were identified. Some species of Lythrum , Eucalyptus , Trifolium , Citrus , Mentha , Apiaceae and Scrophulariaceae are the main nectar sources,and some of Asteraceae, Quercus sp., Olea europaea , Plantago sp. and Fragaria 2 ananassa are the most important pollen sources. At least 14 unifloral honeys (c. 77%) from Citrus , Eucalyptus , Lythrum , Mentha and Teucrium were detected.     

Pollen analyses of honey from Finland

The present study reports results of qualitative melissopalynological analyses of Finnish honey between the years 2000–2007 and changes in its pollen content from the period 1960–2007. Altogether the pollen content of 734 honey samples was analysed with an average of 415 pollen grains counted from a sample. Pollen of Trifolium repens type, Rubus spp., Salix spp. and the Brassicaceae family were present in more than 90% of the samples, and these pollen types were also found in the highest proportions. Annual variation in the relative amounts of the most numerous pollen types could be as high as 10%. On the basis of the pollen spectra of the honey samples, four regions of forage plants for bees could be identified in Finland. In the period between 1960 and 2007, the most marked change observed was that the percentage of the Trifolium spp. pollen type had decreased from 70% to 10%, while the proportions of Brassicaceae and Rosaceae pollen types showed a corresponding increase.     

A study of variation in the pollen spectra of honeys sampled from the Baixa Limia‐Serra do Xurés Nature Reserve in north‐west Spain

The pollen content of eleven honey samples from ten different apiaries in the Baixa Limia – Serra do Xurés Nature Reserve and other honey commercialised by the cooperative as “Mel do Xurés” (north‐west Spain) was subjected to quantitative and qualitative melissopalynological analysis. The quantitative analysis found that ten samples belonged to Maurizio's Class III and one to Class IV. According to the qualitative analysis, four samples were classified as unifloral honey with Erica, four samples as multifloral honey with Erica pollen as the principal component and three samples as multifloral honey with Cytisus‐type pollen and Erica as the principal component pollen. The pollen spectra differ between the diverse honeys analysed, with a common denominator being Erica and Cytisus‐type pollen being abundant in all. For the rest of the samples, the pollen spectra were mainly the same, but with different relative percentages among secondary elements. Thus, either as a secondary or an important element, 91% of the honeys contained Quercus, 82% Castanea sativa Miller, 45% Rubus, 36% Cistus and 27% Lithodora prostrate (Loisel) Griseb,. In particular, we record for the first time the presence of Ribes and Ilex aquifolium L. pollen in Spanish honeys as an important minor or minor pollen component.     

Honey Pollen: Using Melissopalynology to Understand Foraging Preferences of Bees in Tropical South India

The aim of the study was to use melissopalynology to delineate the foraging preferences of bees in tropical environs. This was done by comparing pollen spectra obtained from the same hives every three months for three years at four sampling locations (in two sites) within a confined landscape mosaic. If melissopalynology is highly replicable, the spatial variation of the pollen spectrum from the honey samples would be much more than the temporal (inter-annual) variations. In other words, given the three factors, Month, Year and Location, honey pollen from different Locations, in a given Year and Month, would be much less similar than samples from different Years, in a given Location and Month. We then determined how the factors, Month, Year and Location, influenced the pollen influx of honey. The pollen analyses of the 42 honey samples collected during the three years yielded 80 pollen taxa/types: 72 dicotyledonous and 8 monocotyledonous, encompassing 41 botanical families spread into seven life forms namely, trees, shrubs, epiphytes, herbs, climbers, grasses, and sedges. Our results showed that pollen spectra were equally comparable between Locations and between Months and Years; the importance of this result is that it helped to demonstrate the complexity of ecological/environmental phenomena involved in the process of foraging by bees in a heterogeneous and complex landscape.     

The first find in southern Georgia of fossil honey from the Bronze Age,based on palynological data

The results of a palynological analysis of the organic content of earthenware pots from the Kodiani burial mound (27th–25th centuries b.c.) are reported. The character of the palynological spectrum differs significantly from that of a buried soil within the same burial mound. In the samples taken from the pots, pollen concentration is very high, pollen grains are perfectly preserved and an abundance of pollen from insect-pollinated plants is recorded. It is well known that these features are peculiar to honey palynospectra. In all three pots the pollen of Rosaceae, a family of plants that produce good honey, is dominant. However, the second, third and fourth most dominant pollen types in all three samples are different. For example, Tilia pollen is the second dominant in only one pot. In the second pot, Apiaceae and Poaceae are predominant, and in the third pot, Poaceae, both wild and cultural, is the second dominant. It is clear that the different pots contained different types of honey. The variety of honey types indicates highly developed bee-keeping in the Early Bronze Age. In the same regions of Georgia, agriculture was also highly developed. Wheat cultivation was very important. According to the palynospectra, the landscape and climate of this period were probably quite different to those of today.     

The use of fossilized honey for paleoecological reconstruction: A palynological study of archeological material from Georgia

Palynological analysis of the organic contents of ceramic pots from the Kodiani burial mound, which is dated as 27th-25th centuries B.C., revealed that they contained honey. The samples are extremely rich in excellently preserved pollen grains, including numerous pollen grains of insect-pollinated plants. Such characteristics are typical of palynological assemblages from honey. The palynological assemblages from three pot fragments studied are dominated by pollen grains of Rosaceae; however, they differ from one another in the subdominants. The discovery of several kinds of honey testifies to the presence of well-developed beekeeping in the time of the Early Kurgans. Agriculture, with a significant role of wheat, was also developed in the region of Georgia under study. According to the composition of the palynospectra, the ecological conditions that existed during the epoch studied differ significantly from the present day.     

Pollen characterization of multifloral honeys from La Palma (Canary Islands)

The pollen content of twenty-five honey samples from twelve different apiaries on La Palma (Canary Islands) was subjected to qualitative and quantitative melissopalynological analysis. The quantitative analysis showed that 4% of the honey belonged to Maurizio Class II, 88% to Class III, 4% to Class IV and 4% to Class V. In the qualitative analysis, 60 pollen types were identified from 40 different families. The number of pollen types per honey sample ranges between 16 and 37 (mean of 25.8). Foeniculum vulgare-type and Rumex sp. pollen were present in all the samples. Castanea sativa and Echium plantagineum pollen were found in 96% of them and the combination Aspalathium bituminosum, Bidens pilosa-type and Erica arborea in 92%.     

Mapping of key bee flora of upper Gangetic region of India – a palynological assessment through regional honeys

Fifty‐one regional honey samples, collected mainly from Uttarakhand and Uttar Pradesh districts, were studied to determine the pollen composition. Pollen spectra of the local honeys varied according to the vegetation type utilized by the bees within this floristically diverse region. Eight pollen types from Uttarakhand and seven from Uttar Pradesh were the predominant pollen types in honey and included both local naturalized flora as well as cultivated crops. The investigation revealed that in addition to already known bee forage (e.g. Brassica, Coriandrum, and Litchi) some other species including Eucalyptus, Callistemon that are planted for social forestry programs, are also heavily utilized as pollen and nectar sources by honey bees in urban and semi urban areas. Naturalized flora including Myrica, Rumex, Erigeron are also utilized and therefore are important for apiculture in these regions.     

Overview of pesticide residues in stored pollen and their potential effect on bee colony (Apis mellifera) losses in Spain

In the last decade, an increase in honey bee (Apis mellifera L.) colony losses has been reported in several countries. The causes of this decline are still not clear. This study was set out to evaluate the pesticide residues in stored pollen from honey bee colonies and their possible impact on honey bee losses in Spain. In total, 1,021 professional apiaries were randomly selected. All pollen samples were subjected to multiresidue analysis by gas chromatography-mass spectrometry (MS) and liquid chromatography-MS; moreover, specific methods were applied for neonicotinoids and fipronil. A palynological analysis also was carried out to confirm the type of foraging crop. Pesticide residues were detected in 42% of samples collected in spring, and only in 31% of samples collected in autumn. Fluvalinate and chlorfenvinphos were the most frequently detected pesticides in the analyzed samples. Fipronil was detected in 3.7% of all the spring samples but never in autumn samples, and neonicotinoid residues were not detected. More than 47.8% of stored pollen samples belonged to wild vegetation, and sunflower (Heliantus spp.) pollen was only detected in 10.4% of the samples. A direct relation between pesticide residues found in stored pollen samples and colony losses was not evident accordingly to the obtained results. Further studies are necessary to determine the possible role of the most frequent and abundant pesticides (such as acaricides) and the synergism among them and with other pathogens more prevalent in Spain.     

Foraging behaviour of the stingless bee Melipona orbignyi (Hymenoptera: Apidae: Meliponini) in a dry forest assessed by multivariate analysis from palynological data

Pollen analysis was applied to study the botanical composition of honey and pollen stored by Melipona orbignyi in its southernmost distributional range. Many studies have revealed that robust buzzing bees of this genus are associated with diverse plant families, some of which are different to those exploited by the remaining genera of Meliponini. Cluster analysis was performed to assess similarity in botanical composition between pot-pollen and pot-honey provisions from different colonies. The results indicated that each colony was capable to access to different plant species for obtaining nectar and pollen, a fact observed when discarding the contaminated honey samples. As honey samples were not clustered together with other ones but with pollen samples indicating a similar botanical composition, it could be assumed that a same plant species provided nectar and pollen to bees. Resource partitioning was not clearly observed among a subset of late spring colonies, as two families of high richness of species and abundant in this dry forest (the mimosoid clade in Fabaceae and Capparaceae) were foraged. A higher number of synchronously sampled colonies would be necessary to detect this ecological aspect. The botanical composition of samples of M. orbignyi was governed by both random factors such as local and temporary flower availability plus preferences for particular plant species such as those from Solanaceae and Ximeniaceae. The Capparaceae and Ximeniaceae are here reported for the first time as intensively foraged pollen resources by the genus Melipona.     

Secondary pollen types characteristic of Louisiana honeys

Nine secondary pollen types (those comprising 16–45% of the pollen sample) were recovered from 54 honey samples (10 g) collected from the major beekeeping areas of Louisiana. The pollen types in order of decreasing importance are:Berchemia scandens, rattan vine;Rubus spp., blackberry and dewberry;Salix spp., willow;Trifolium repens, white clover;Glycine max, soybean;Parthenocissus quinquefolia, Virginia creeper; Rosaceae, rose family;Cephalanthus occidentalis, buttonbush; andCornus spp., dogwood. The first four secondary types also appeared as dominant pollen (over 45%) in some of the Louisiana honey samples investigated and were previously reported. The remaining five pollen types and the “bee plants” associated with each pollen type are identified and described.     

Crop Pollination Exposes Honey Bees to Pesticides Which Alters Their Susceptibility to the Gut Pathogen Nosema ceranae

Recent declines in honey bee populations and increasing demand for insect-pollinated crops raise concerns about pollinator shortages. Pesticide exposure and pathogens may interact to have strong negative effects on managed honey bee colonies. Such findings are of great concern given the large numbers and high levels of pesticides found in honey bee colonies. Thus it is crucial to determine how field-relevant combinations and loads of pesticides affect bee health. We collected pollen from bee hives in seven major crops to determine 1) what types of pesticides bees are exposed to when rented for pollination of various crops and 2) how field-relevant pesticide blends affect bees’ susceptibility to the gut parasite Nosema ceranae. Our samples represent pollen collected by foragers for use by the colony, and do not necessarily indicate foragers’ roles as pollinators. In blueberry, cranberry, cucumber, pumpkin and watermelon bees collected pollen almost exclusively from weeds and wildflowers during our sampling. Thus more attention must be paid to how honey bees are exposed to pesticides outside of the field in which they are placed. We detected 35 different pesticides in the sampled pollen, and found high fungicide loads. The insecticides esfenvalerate and phosmet were at a concentration higher than their median lethal dose in at least one pollen sample. While fungicides are typically seen as fairly safe for honey bees, we found an increased probability of Nosema infection in bees that consumed pollen with a higher fungicide load. Our results highlight a need for research on sub-lethal effects of fungicides and other chemicals that bees placed in an agricultural setting are exposed to.     

Maize pollen foraging by honey bees in relation to crop area and landscape context

The increasing demand for insect pollinated crops and high recent losses of honey bee colonies raise concerns about food security. Systemic insecticides are recognized as one of the drivers of worldwide honey and wild bee declines. Particularly honey bees in agricultural environments are exposed to pesticides when they collect crop pollen and nectar. However, landscape scale studies which analyze pollen use and foraging distances of honey bees on mass-flowering crops like maize to evaluate potential exposure risks are currently lacking. In an experimental approach on a landscape scale we took advantage of intra-colonial dance communication to gather information about the location of utilized pollen resources. During maize flowering, four observation hives were placed in and rotated between 11 different landscapes which covered a gradient from low to high maize acreage. A higher frequency of dances for foraging locations on maize fields compared to other land use types shows that maize is an intensively used pollen resource for honey bee colonies. Mean foraging distances were significantly shorter for maize pollen than for other pollen origins. The percentage of maize pollen foragers did not increase with maize acreage in the landscape. The proportion of grassland area providing alternative pollen sources did not reduce the percentage of maize pollen foragers. Our findings allow estimating the distance-related exposure risk of honey bee colonies to pollen from surrounding maize fields treated with systemic insecticides. Similarly, the results can be used to estimate the exposure to transgenic maize pollen, which is relevant for honey production in European countries. Provision of alternative pollen resources within agri-environmental schemes could potentially reduce exposure risk to pesticide contaminated crop pollen.