Biochemical characterization of Euphorbia resinifera floral cyathia.

Euphorbia resinifera O. Berg is a plant endemic to the Northern and Central regions of Morocco known since the ancient Roman and Greek times for secreting a poisonous latex containing resiniferatoxin. However, E. resinifera pseudo-inflorescences called cyathia are devoid of laticifers and, therefore, do not secrete latex. Instead, they exudate nectar that local honey bees collect and craft into honey. Honey and cyathium water extracts find a broad range of applications in the traditional medicine of Northern Africa as ointments and water decoctions. Moreover, E. resinifera monofloral honey has received the Protected Geographic Indication certification for its outstanding qualities. Given the relevance of E. resinifera cyathia for bee nutrition, honey production, and the health benefit of cyathium-derived products, this study aimed to screen metabolites synthesized and accumulated in its pseudo-inflorescences. Our analyses revealed that E. resinifera cyathia accumulate primary metabolites in considerable abundance, including hexoses, amino acids and vitamins that honey bees may collect from nectar and craft into honey. Cyathia also synthesize volatile organic compounds of the class of benzenoids and terpenes, which are emitted by flowers pollinated by honey bees and bumblebees. Many specialized metabolites, including carotenoids, flavonoids, and polyamines, were also detected, which, while protecting the reproductive organs against abiotic stresses, also confer antioxidant properties to water decoctions. In conclusion, our analyses revealed that E. resinifera cyathia are a great source of antioxidant molecules and a good food source for the local foraging honeybees, revealing the central role of the flowers from this species in mediating interactions with local pollinators and the conferral of medicinal properties to plant extracts.

Spatio-temporal expression of candidate genes for nectar spur development in Tropaeolum (Tropaeolaceae: Brassicales).

BACKGROUND AND AIMS: Tropaeolaceae (Brassicales) comprise ~100 species native to South and Central America. Tropaeolaceae flowers have a nectar spur, formed by a late expansion and evagination of the fused proximal region of the perianth (i.e. the floral tube). This spur is formed in the domain of the tube oriented towards the inflorescence axis, which corresponds to the adaxial floral region. However, little is known about the molecular mechanisms responsible for the evolution of spurs in Tropaeolaceae. METHODS: In this study, we examined the spatio-temporal expression of genes putatively responsible for differential patterns of cell division between the adaxial and abaxial floral regions in Tropaeolaceae. These genes include previously identified TCP and KNOX transcription factors and the cell division marker HISTONE H4 (HIS4). KEY RESULTS: We found a TCP4 homologue concomitantly expressed with spur initiation and elaboration. Tropaeolaceae possess two TCP4-like (TCP4L) copies, as a result of a Tropaeolaceae-specific duplication. The two copies (TCP4L1 and TCP4L2) in Tropaeolum longifolium show overlapping expression in the epidermis of reproductive apices (inflorescence meristems) and young floral buds, but only TlTCP4L2 shows differential expression in the floral tube at early stages of spur formation, restricted to the adaxial region. This adaxial expression of TlTCP4L2 overlaps with the expression of TlHIS4. Later in development, only TlTCP4L2 is expressed in the nectariferous tissue of the spur. CONCLUSIONS: Based on these results, we hypothesize that Tropaeolaceae TCP4L genes had a plesiomorphic role in epidermal development and that, after gene duplication, TCP4L2 acquired a new function in spur initiation and elaboration. To better understand spur evolution in Tropaeolaceae, it is critical to expand developmental genetic studies to their sister group, the Akaniaceae, which possess simultaneously an independent duplication of TCP4L genes and a spurless floral tube.

Evolutionary Adaptation of Genes Involved in Galactose Derivatives Metabolism in Oil-Tea Specialized Andrena Species.

Oil-tea (Camellia oleifera) is a woody oil crop whose nectar includes galactose derivatives that are toxic to honey bees. Interestingly, some mining bees of the genus Andrena can entirely live on the nectar (and pollen) of oil-tea and are able to metabolize these galactose derivatives. We present the first next-generation genomes for five and one Andrena species that are, respectively, specialized and non-specialized oil-tea pollinators and, combining these with the published genomes of six other Andrena species which did not visit oil-tea, we performed molecular evolution analyses on the genes involved in the metabolizing of galactose derivatives. The six genes (NAGA, NAGA-like, galM, galK, galT, and galE) involved in galactose derivatives metabolism were identified in the five oil-tea specialized species, but only five (with the exception of NAGA-like) were discovered in the other Andrena species. Molecular evolution analyses revealed that NAGA-like, galK, and galT in oil-tea specialized species appeared under positive selection. RNASeq analyses showed that NAGA-like, galK, and galT were significantly up-regulated in the specialized pollinator Andrena camellia compared to the non-specialized pollinator Andrena chekiangensis. Our study demonstrated that the genes NAGA-like, galK, and galT have played an important role in the evolutionary adaptation of the oil-tea specialized Andrena species.

Bee Pollen: Clinical Trials and Patent Applications.

Bee pollen is a natural cocktail of floral nectar, flower pollen, enzymes, and salivary secretions produced by honeybees. Bee pollen is one of the bee products most enriched in proteins, polysaccharides, polyphenols, lipids, minerals, and vitamins. It has a significant health and medicinal impact and provides protection against many diseases, including diabetes, cancer, infectious, and cardiovascular. Bee pollen is commonly promoted as a cost-effective functional food. In particular, bee pollen has been applied in clinical trials for allergies and prostate illnesses, with a few investigations on cancer and skin problems. However, it is involved in several patents and health recipes to combat chronic health problems. This review aimed to highlight the clinical trials and patents involving bee pollen for different cases and to present the role of bee pollen as a supplementary food and a potential product in cosmetic applications.

Convergent evolution of a blood-red nectar pigment in vertebrate-pollinated flowers.

Nearly 90% of flowering plants depend on animals for reproduction. One of the main rewards plants offer to pollinators for visitation is nectar. Nesocodon mauritianus (Campanulaceae) produces a blood-red nectar that has been proposed to serve as a visual attractant for pollinator visitation. Here, we show that the nectar's red color is derived from a previously undescribed alkaloid termed nesocodin. The first nectar produced is acidic and pale yellow in color, but slowly becomes alkaline before taking on its characteristic red color. Three enzymes secreted into the nectar are either necessary or sufficient for pigment production, including a carbonic anhydrase that increases nectar pH, an aryl-alcohol oxidase that produces a pigment precursor, and a ferritin-like catalase that protects the pigment from degradation by hydrogen peroxide. Our findings demonstrate how these three enzymatic activities allow for the condensation of sinapaldehyde and proline to form a pigment with a stable imine bond. We subsequently verified that synthetic nesocodin is indeed attractive to Phelsuma geckos, the most likely pollinators of Nesocodon We also identify nesocodin in the red nectar of the distantly related and hummingbird-visited Jaltomata herrerae and provide molecular evidence for convergent evolution of this trait. This work cumulatively identifies a convergently evolved trait in two vertebrate-pollinated species, suggesting that the red pigment is selectively favored and that only a limited number of compounds are likely to underlie this type of adaptation.

Correlation between octopaminergic signalling and foraging task specialisation in honeybees.

Regulation of pollen and nectar foraging in honeybees is linked to differences in the sensitivity to the reward. Octopamine (OA) participates in the processing of reward-related information in the bee brain, being a candidate to mediate and modulate the division of labour among pollen and nectar foragers. Here we tested the hypothesis that OA affects the resource preferences of foragers. We first investigated whether oral administration of OA is involved in the transition from nectar to pollen foraging. We quantified the percentage of OA-treated bees that switched from a sucrose solution to a pollen feeder when the sugar concentration was decreased experimentally. We also evaluated if feeding the colonies sucrose solution containing OA increases the rate of bees collecting pollen. Finally, we quantified OA and tyramine (TYR) receptor genes expression of pollen and nectar foragers in different parts of the brain, as a putative mechanism that affects the decision-making process regarding the resource type collected. Adding OA in the food modified the probability that foragers switch from nectar to pollen collection. The proportion of pollen foragers also increased after feeding colonies with OA-containing food. Furthermore, the expression level of the AmoctαR1 was upregulated in foragers arriving at pollen sources compared with those arriving at sugar-water feeders. Using age-matched pollen and nectar foragers that returned to the hive, we detected an upregulated expression of a TYR receptor gene in the suboesophageal ganglia. These findings support our prediction that OA signalling affects the decision in honeybee foragers to collect pollen or nectar.

Honeybee colonies provide foragers with costly fuel to promote pollen collection.

Honeybee pollen foragers departing the hive carry concentrated nectar to use as fuel for flight and glue for forming pollen loads. Since nectar is concentrated by in-hive bees at the cost of time and energy, using concentrated nectar increases the cost of foraging at the colony level. This experimental study explored the potential benefit to honeybees of using concentrated nectar for pollen collection by diluting nectar carried by pollen foragers from the hive. Mass feeding with 30% sugar solution successfully reduced the crop-load-sugar concentration in putative pollen foragers departing the hive, but while those bees tended to increase the crop-load volume, such increase did not fully compensate for the decreased amount of dissolved sugars in the crop load. Feeding 30% sugar solution reduced the pollen load dry weight by approx. 10-20% compared to the unfed control and to another test group fed 60% sugar solution. In addition, the pollen load size and sugar concentration of crop load remaining in returning pollen foragers was positively correlated. These results clearly show the advantage to honeybees of using concentrated nectar for pollen foraging.

Adjustment of fuel loads in stingless bees (Melipona subnitida).

Eusocial bee foragers leave their nest with nectar as flight fuel, therewith reducing the risk of starvation during a foraging trip. Yet, the extra mass results in an increase of energetic expenditure for flight. Thus, bees should tune their fuel loads to the respective foraging situation. In the present study, we investigated the fuel adjustment in the Brazilian stingless bee Melipona subnitida (Apidae, Meliponini). Specifically, we examined whether foragers of this species increase their fuel loads when they have low expectation for nectar collection during a foraging trip. Crop load measurements revealed that nectar foragers carried significantly less fuel on departing the nest than foragers collecting either pollen, clay, or resin. Surprisingly, 75% of nectar foragers left the nest without any detectable amount of nectar, which suggests that the majority of bees collected at nearby nectar sources and avoided an increase in foraging costs. Moreover, foragers increased their fuel loading when repeatedly experiencing empty food sources that had previously been rewarding. These results support our hypothesis and demonstrate that the capability of fuel adjustment is not restricted to honey bees.

A mathematical model of honey bee colony dynamics to predict the effect of pollen on colony failure.

The decline in colony populations of the honey bee, known as the Colony Collapse Disorder (CCD), is a global concern. Numerous studies have reported possible causes, including pesticides, parasites, and nutritional stress. Poor nutrition affects the immune system at both the individual and colony level, amplifying effects of other stress factors. Pollen is the only source of ten amino acids that are essential to honey bee development, brood rearing and reproduction. This paper presents a new mathematical model to explore the effect of pollen on honey bee colony dynamics. In this model, we considered pollen and nectar as the required food for the colony. The effect of pollen and nectar collected by foragers was evaluated at different mortality rates of pupa, pollen and nectar foragers.

Flagellum Removal by a Nectar Metabolite Inhibits Infectivity of a Bumblebee Parasite.

Plant phytochemicals can act as natural "medicines" for animals against parasites [1-3]. Some nectar metabolites, for example, reduce parasite infections in bees [4-7]. Declining plant diversity through anthropogenic landscape change [8-11] could reduce the availability of medicinal nectar plants for pollinators, exacerbating their decline [12]. Existing studies are, however, limited by (1) a lack of mechanistic insights into how phytochemicals affect pollinator diseases and (2) the restriction to few, commercially available chemicals, thereby potentially neglecting plants with the biggest antiparasitic effects. To rapidly identify plants with the greatest potential as natural bee medicines, we developed a bioactivity-directed fractionation assay for nectar metabolites. We evaluated 17 important nectar plants against the bumblebee pathogen Crithidia bombi (Trypanosomatidae) [13-17]. The most bioactive species was heather (Calluna vulgaris), the second most productive UK nectar plant [10]. We identified 4-(3-oxobut-1-enylidene)-3,5,5-trimethylcyclohex-2-en-1-one (callunene) from heather nectar as a potent inhibitor of C. bombi. Wild bumblebees (Bombus terrestris) foraging on heather ingest callunene at concentrations causing complete C. bombi inhibition. Feeding on callunene was prophylactic against infections. We show that C. bombi establishes infections by flagellar anchoring to the ileum epithelium. Short-term callunene exposure induced flagellum loss in C. bombi choanomastigotes, resulting in a loss of infectivity. We conclude that plant secondary metabolites can disrupt parasite flagellum attachment, revealing a mechanism behind their prophylactic effects. The decline of heathlands [18-21] reduces the availability of natural bee "medicine" and could exacerbate the contribution of diseases to pollinator declines. VIDEO ABSTRACT.

Flowers and climate change: a metabolic perspective.

Adverse climatic conditions at the time of flowering severely hinder crop yields and threaten the interactions between plants and their pollinators. These features depend on a common trait: the metabolism of flowers. In this Viewpoint article, we aim to provide insight into the metabolic changes that occur in flowers in response to changes in climate and emphasize that these changes severely impact the fitness of autogamous and allogamous species, plant-pollinator interactions, and overall ecosystem health. We review the biochemical processes that lead to failure of gamete development and to alterations of color, scent and nectar secretion. Then, making use of open access expression data, we examine the expression of genes that may drive these changes in response to heat and drought. Finally, we present measurements of metabolites from flowers exposed to a heat wave and discuss how the results of this short-term experiment may give rise to misleading conclusions regarding the positive effect of heat on flower fitness. We hope this article draws attention to this often-neglected dynamic and its important consequences.

Insecticide dose and seasonal timing of trunk injection in apples influence efficacy and residues in nectar and plant parts.

BACKGROUND: Trunk injection is an established method for delivering pesticides in ornamental and shade trees, but further research is needed to determine efficacy and pollinator safety in tree fruit crops. Apple trees were injected in 2013 and 2014 with the insecticides emamectin benzoate, imidacloprid, dinotefuran, spinosad, chlorantraniliprole, or abamectin. Additional emamectin benzoate and imidacloprid injections were performed in the spring and fall of 2015. Nectar and pollen were sampled in the following spring to compare the effects of application timings on insecticide loading into flowers. RESULTS: Neonicotinoids reduced Empoasca fabae density in the field. Emamectin benzoate, chlorantraniliprole, and abamectin resulted in moderate to high mortality and reduced Choristoneura rosaceana feeding in bioassays. Imidacloprid was not detected in nectar or pollen when injected in the spring, and was detected at 0.39 ng g-1 in pollen when injected the previous fall. Emamectin benzoate was not detected in nectar or pollen when injected the previous fall, and was detected at 7.36 ng g-1 (nectar) and 1.15 ng g-1 (pollen) when injected in the spring. CONCLUSIONS: This study identified a broader list of possible trunk-injectable pesticides for apple trees. This study also shows that managing the seasonal timing of injection can reduce the risk of insecticide exposure to pollinators. © 2018 Society of Chemical Industry.

Variable pollen viability and effects of pollen load size on components of seed set in cultivars and feral populations of oilseed rape.

Pollination success is important for crop yield, but may be cultivar dependent. Less is known about which floral traits influence pollination success. Floral traits, e.g. traits related to attraction and reward, can also contribute to gene flow via pollen, the latter being of particular importance in oilseed rape (Brassica napus) where gene flow occurs between plants of crop, volunteer and feral origin as well as related taxa. We investigated the relationship between pollen load size and seed set in winter oilseed rape. We compared variability in pollen-viability traits, flower production (flowers from the main raceme times number of branches) and seed number and weight per siliqua among cultivars and feral populations (growing outside of agricultural fields) under controlled conditions. Both seed number and weight were saturated at relatively low pollen loads in the tested cultivar. Pollen viability and estimated flower production differed among cultivars, indicating that these traits could contribute to yield variability. Seed weight per siliqua, but not pollen traits or flower production, was lower in ferals compared to cultivars. Thus, while the probability of establishment may be reduced in ferals (due to lower seed weight per siliqua) this will not necessarily impact their contribution to gene flow via pollen. In oilseed rape a relatively low pollen load may be sufficient for full seed set in some cultivars, suggesting less dependence on insect pollination for high yield than generally expected. Our results also showed that previously less investigated floral traits, such as pollen viability, pollen tube growth rate and flower number, can differ between cultivars. Studies of these traits may provide targets for increasing crop yield and provide general knowledge about gene flow between cultivated, feral and related wild populations.

Storage conditions modulate the metabolomic profile of a black raspberry nectar with minimal impact on bioactivity.

Pre-clinical and clinical studies suggest black raspberries (BRBs) may inhibit the development of oral cancer. Lyophilized BRB powder is commonly used in these studies, but processed BRB products are more often consumed. The objective of this work was to understand how storage conditions influence the phytochemical profile and anti-proliferative activity of a BRB nectar beverage. Untargeted UHPLC-Q-TOF-MS based metabolomics analyses demonstrated that large chemical variation was introduced by storage above -20 °C over 60 days. However, minimal change in anti-proliferative activity was observed when stored nectar extracts were applied to SCC-83-01-82 premalignant oral epithelial cells. As proof of concept, cyanidin-3-O-rutinoside and its degradation product, protocatechuic acid, were administered in different ratios maintaining an equimolar dose, and anti-proliferative activity was maintained. This study shows the utility of metabolomics to profile global chemical changes in foods, while demonstrating that isolated phytochemicals do not explain the complete bioactivity of a complex food product.

Micromorphological and histochemical attributes of flowers and floral reward in Linaria vulgaris (Plantaginaceae).

The self-incompatible flowers of Linaria vulgaris have developed a range of mechanisms for attraction of insect visitors/pollinators and deterrence of ineffective pollinators and herbivores. These adaptive traits include the flower size and symmetry, the presence of a spur as a "secondary nectar presenter," olfactory (secondary metabolites) and sensual (scent, flower color, nectar guide-contrasting palate) signals, and floral rewards, i.e. pollen and nectar. Histochemical tests revealed that the floral glandular trichomes produced essential oils and flavonoids, and pollen grains contained flavonoids, terpenoids, and steroids, which play a role of olfactory attractants/repellents. The nectary gland is disc-shaped and located at the base of the ovary. Nectar is secreted through numerous modified stomata. Nectar secretion began in the bud stage and lasted to the end of anthesis. The amount of produced nectar depended on the flower age and ranged from 0.21 to 3.95 mg/flower (mean = 1.51 mg). The concentration of sugars in the nectar reached up to 57.0%. Both the nectar amount and sugar concentration demonstrated a significant year and population effect. Pollen production was variable between the years of the study. On average, a single flower of L. vulgaris produced 0.31 mg of pollen. The spectrum of insect visitors in the flowers of L. vulgaris differed significantly between populations. In the urban site, Bombus terrestris and Apis mellifera were the most common visitors, while a considerable number of visits of wasps and syrphid flies were noted in the rural site.

Impact of pre- and post-pollination barriers on pollen transfer and reproductive isolation among three sympatric Pedicularis (Orobanchaceae) species.

Pedicularis is the largest genus in the Orobanchaceae (>300) with many species co-occurring and co-blooming in subalpine to alpine meadows in the Himalayas. Although it is well known that different Pedicularis species place pollen on different parts of the same bumblebee's body, thus reducing interspecific pollen transfer, it is not known whether post-pollination components also contribute to reproductive isolation (RI). In this study, we quantified the individual strengths and absolute contributions of six pre- and post-pollination components of RI between three sympatric species in two pairs; Pedicularis gruina × Pedicularis tenuisecta (gru × ten) and Pedicularis comptoniifolia × Pedicularis tenuisecta (com × ten). All three Pedicularis species shared the same Bombus species. Individual foragers showed a high, but incomplete, floral constancy for each species. Therefore, pre-pollination barriers were potentially 'leaky' as Bombus species showed a low but consistent frequency of interspecific visitation. The RI strength of pre-pollination was lower in com × ten than in gru × ten. In contrast, post-pollination barriers completely blocked gene flow between both sets of species pairs. Two post-pollination recognition sites were identified. Late acting rejection of interspecific pollen tube growth occurred in com♀ × ten♂, while seeds produced in bi-directional crosses of gru × ten failed to germinate. We propose that, although floral isolation based on pollen placement on pollinators in the genus Pedicularis is crucial to avoid interspecific pollen transfer, the importance of this mode of interspecific isolation may be exaggerated. Post-pollination barriers may play even larger roles for currently established populations of co-blooming and sympatric species in this huge genus in the Himalayas.

Evolutionary and structural analyses uncover a role for solvent interactions in the diversification of cocoonases in butterflies.

Multi-omic approaches promise to supply the power to detect genes underlying disease and fitness-related phenotypes. Optimal use of the resulting profusion of data requires detailed investigation of individual candidate genes, a challenging proposition. Here, we combine transcriptomic and genomic data with molecular modelling of candidate enzymes to characterize the evolutionary history and function of the serine protease cocoonase. Heliconius butterflies possess the unique ability to feed on pollen; recent work has identified cocoonase as a candidate gene in pollen digestion. Cocoonase was first described in moths, where it aids in eclosure from the cocoon and is present as a single copy gene. In heliconiine butterflies it is duplicated and highly expressed in the mouthparts of adults. At least six copies of cocoonase are present in Heliconius melpomene and copy number varies across H. melpomene sub-populations. Most cocoonase genes are under purifying selection, however branch-site analyses suggest cocoonase 3 genes may have evolved under episodic diversifying selection. Molecular modelling of cocoonase proteins and examination of their predicted structures revealed that the active site region of each type has a similar structure to trypsin, with the same predicted substrate specificity across types. Variation among heliconiine cocoonases instead lies in the outward-facing residues involved in solvent interaction. Thus, the neofunctionalization of cocoonase duplicates appears to have resulted from the need for these serine proteases to operate in diverse biochemical environments. We suggest that cocoonase may have played a buffering role in feeding during the diversification of Heliconius across the neotropics by enabling these butterflies to digest protein from a range of biochemical milieux.

Nectar and oleiferous trichomes as floral attractants in Bulbophyllum saltatorium Lindl. (Orchidaceae).

Although many Orchidaceae have deceit flowers that produce no reward, the most common reward, when present, is nectar. Bulbophyllum, however, is unusual in that the labellar secretions of most species investigated to date lack sugars, and, therefore, cannot be considered true nectar. The African species Bulbophyllum saltatorium is an exception in that it produces not only nectar but also possesses specialized, capitate oleiferous trichomes. The nectary of B. saltatorium is borne on the labellum and is represented by a deep, narrow, median longitudinal groove, having a small aperture, and flanked by trichomes. Isodiametric epidermal cells lining this groove secrete nectar which collects both in the groove and on the surface of the labellum. As well as a nectary, the labellum of B. saltatorium also bears three types of unicellular trichomes: the longest trichomes are borne distally and abaxially; the marginal ones form a rim around the entire labellum, and finally, massive, capitate trichomes occur proximally and adaxially. These are oleiferous, containing large quantities of oil which might function as precursors of volatile components of fragrance or provide a food-reward. To the best of our knowledge, this is the first time for such oleiferous trichomes to be described for Bulbophyllum. Therefore, apart from their color and markings, flowers of this species are able to attract pollinators in at least two, possibly three ways: food-reward in the form of nectar; fragrance; and possibly food-rewards in the form of food-hairs.

Resilience of honeybee colonies via common stomach: A model of self-regulation of foraging.

We propose a new regulation mechanism based on the idea of the "common stomach" to explain several aspects of the resilience and homeostatic regulation of honeybee colonies. This mechanism exploits shared pools of substances (pollen, nectar, workers, brood) that modulate recruitment, abandonment and allocation patterns at the colony-level and enable bees to perform several survival strategies to cope with difficult circumstances: Lack of proteins leads to reduced feeding of young brood, to early capping of old brood and to regaining of already spent proteins through brood cannibalism. We modeled this system by linear interaction terms and mass-action law. To test the predictive power of the model of this regulatory mechanism we compared our model predictions to experimental data of several studies. These comparisons show that the proposed regulation mechanism can explain a variety of colony level behaviors. Detailed analysis of the model revealed that these mechanisms could explain the resilience, stability and self-regulation observed in honeybee colonies. We found that manipulation of material flow and applying sudden perturbations to colony stocks are quickly compensated by a resulting counter-acting shift in task selection. Selective analysis of feedback loops allowed us to discriminate the importance of different feedback loops in self-regulation of honeybee colonies. We stress that a network of simple proximate mechanisms can explain significant colony-level abilities that can also be seen as ultimate reasoning of the evolutionary trajectory of honeybees.

Microbial characterization of bee pollen from the Vesuvius area collected by using three different traps.

Flower pollen is collected by honeybee foragers, adhered on their rear legs and transported into the hives in the form of pellets. Once in the hives, bee pollen is moisturised with nectar and bee mouth secretions and due to enzymatically modifications it becomes the so-called bee-bread, the protein reservoir of young bees. Bee pollen can be artificially removed from bee legs and collected by using specific systems, the bee pollen traps. Bee pollen is commercialized for human consumption as fresh product and after freezing or drying. Although bee pollen is nowadays largely consumed in developed countries, as food or food supplement according to local legislation, little is known on its safety related to microbiological hazards. In this work, we aimed to characterize for the first time the microbiological profile of Italian bee pollen in fresh, frozen and dried form collected along an entire harvesting season. Moreover, monthly microbiological analyses were performed on frozen (storage at -18°C) and dried (storage at room temperature) bee pollen over a 4 months period. Further aim of this work was the evaluation of the possible impact on production level of three different traps used for pollen collection. Our results on microbial contamination of fresh and frozen bee pollen show that a more comprehensive microbiological risk assessment of bee pollen is required. On the other side, dried pollen showed very low microbial contamination and no pathogen survived after the drying process and during storage.