The role of wax and resin in the nestmate recognition system of a stingless bee, Tetragonisca angustula

Recent research has shown that entrance guards of the stingless bee Tetragonisca angustula make less errors in distinguishing nestmates from non-nestmates than all other bee species studied to date, but how they achieve this is unknown. We performed four experiments to investigate nestmate recognition by entrance guards in T. angustula. We first investigated the effect of colony odours on acceptance. Nestmates that acquired odour from non-nestmate workers were 63% more likely to be rejected while the acceptance rate of non-nestmates treated with nestmate odour increased by only 7%. We further hypothesised that guards standing on the wax entrance tube might use the tube as an odour referent. However, our findings showed that there was no difference in the acceptance of non-nestmates by guards standing on their own colony's entrance tube versus the non-nestmate's entrance tube. Moreover, treatment of bees with nestmate and non-nestmate resin or wax had a negative effect on acceptance rates of up to 65%, regardless of the origin of the wax or resin. The role of resin as a source of recognition cues was further investigated by unidirectionally transferring resin stores between colonies. Acceptance rates of nestmates declined by 37% for hives that donated resin, contrasting with resin donor hives where acceptance of non-nestmates increased by 21%. Overall, our results confirm the accuracy of nestmate recognition in T. angustula and reject the hypothesis that this high level of accuracy is due to the use of the wax entrance tubes as a referent for colony odour. Our findings also suggest that odours directly acquired from resin serve no primary function as nestmate recognition cues. The lack of consistency among colonies plus the complex results of the third and fourth experiments highlight the need for further research on the role of nest materials and cuticular profiles in understanding nestmate recognition in T. angustula.

A method for harvesting unfermented pollen from stingless bees (Hymenoptera, Apidae, Meliponini)

Pollen traps used for harvesting pollen from Apis mellifera do not work for stingless bees, as most species have small entrances and rapidly deposit large quantities of propolis at any barrier in front of the nest. Some stingless beekeepers harvest pollen by removing it directly from pollen pots, but this pollen is normally fermented and unpalatable. The aim of this study was to test a new method for harvesting pollen from stingless bee colonies before it begins to ferment. Colonies of Scaptotrigona depilis were removed and replaced by empty hives, which were occupied by the returning foragers and used for storing pollen and nectar. After one week, the pollen and honey were harvested directly from the storing pots and weighed. On average, the colonies produced 8.7 g of honey and 54.2 g of unfermented pollen (n = 10). This method is a viable option for harvesting unfermented pollen from stingless bees, especially with species that harvest large amounts of pollen. The unfermented pollen of S. depilis was well received in taste tests, receiving higher scores than fermented pollen, and similar scores to A. mellifera pollen, so could have great commercial possibilities. It is also a good method for studying the foraging of stingless bees because the amount of harvested food can be easily and precisely quantified.

Anti-atherogenic and anti-angiogenic activities of polyphenols from propolis

Propolis is a polyphenol-rich resinous substance extensively used to improve health and prevent diseases. The effects of polyphenols from different sources of propolis on atherosclerotic lesions and inflammatory and angiogenic factors were investigated in LDL receptor gene (LDLr-/-) knockout mice. The animals received a cholesterol-enriched diet to induce the initial atherosclerotic lesions (IALs) or advanced atherosclerotic lesions (AALs). The IAL or AAL animals were divided into three groups, each receiving polyphenols from either the green, red or brown propolis (250 mg/kg per day) by gavage. After 4 weeks of polyphenol treatment, the animals were sacrificed and their blood was collected for lipid profile analysis. The atheromatous lesions at the aortic root were also analyzed for gene expression of inflammatory and angiogenic factors by quantitative real-time polymerase chain reaction and immunohistochemistry. All three polyphenol extracts improved the lipid profile and decreased the atherosclerotic lesion area in IAL animals. However, only polyphenols from the red propolis induced favorable changes in the lipid profiles and reduced the lesion areas in AAL mice. In IAL groups. VCAM, MCP-1, FGF, PDGF, VEGF, PECAM and MMP-9 gene expression was down-regulated, while the metalloproteinase inhibitor TIMP-1 gene was up-regulated by all polyphenol extracts. In contrast, for advanced lesions, only the polyphenols from red propolis induced the down-regulation of CD36 and the up-regulation of HO-1 and TIMP-1 when compared to polyphenols from the other two types of propolis. In conclusion, polyphenols from propolis, particularly red propolis, are able to reduce atherosclerotic lesions through mechanisms including the modulation of inflammatory and angiogenic factors. (C) 2012 Elsevier Inc. All rights reserved.

A morphologically specialized soldier caste improves colony defense in a neotropical eusocial bee

Division of labor among workers is common in insect societies and is thought to be important in their ecological success. In most species, division of labor is based on age (temporal castes), but workers in some ants and termites show morphological specialization for particular tasks (physical castes). Large-headed soldier ants and termites are well-known examples of this specialization. However, until now there has been no equivalent example of physical worker subcastes in social bees or wasps. Here we provide evidence for a physical soldier subcaste in a bee. In the neotropical stingless bee Tetragonisca angustula, nest defense is performed by two groups of guards, one hovering near the nest entrance and the other standing on the wax entrance tube. We show that both types of guards are 30% heavier than foragers and of different shape; foragers have relatively larger heads, whereas guards have larger legs. Low variation within each subcaste results in negligible size overlap between guards and foragers, further indicating that they are distinct physical castes. In addition, workers that remove garbage from the nest are of intermediate size, suggesting that they might represent another unrecognized caste. Guards or soldiers are reared in low but sufficient numbers (1-2% of emerging workers), considering that <1% usually perform this task. When challenged by the obligate robber bee Lestrimelitta limao, an important natural enemy, larger workers were able to fight for longer before being defeated by the much larger robber. This discovery opens up opportunities for the comparative study of physical castes in social insects, including the question of why soldiers appear to be so much rarer in bees than in ants or termites.

Geopropolis from Melipona scutellaris decreases the mechanical inflammatory hypernociception by inhibiting the production of IL-1 beta and TNF-alpha

Ethnopharmacological relevance: The pharmacological activity of geopropolis collected by stingless bees (important and threatened pollinators), a product widely used in folk medicine by several communities in Brazil, especially in the Northeast Region, needs to be studied. Objective: The aim of this study was to evaluate the antinociceptive activity of Melipona scutellaris geopropolis (stingless bee) using different models of nociception. Material and methods: The antinociceptive activity of the ethanolic extract of geopropolis (EEGP) and fractions was evaluated using writhing induced by acetic acid, formalin test, carrageenan-induced hypernociception, and quantification of IL-1 beta and TNF-alpha. The chemical composition was assessed by quantification of total flavonoids and phenolic compounds. Results: EEGP and its hexane and aqueous fractions showed antinociceptive activity. Both EEGP and its aqueous fraction presented activity in the mechanical inflammatory hypernociception induced by the carrageenan model, an effect mediated by the inhibition of IL-1 beta and TNF-alpha. The chemical composition of EEGP and its hexane and aqueous fractions showed a significant presence of phenolic compounds and absence of flavonoids. Conclusion: Our data indicate that geopropolis is a natural source of bioactive substances with promising antinociceptive activity. (C) 2012 Elsevier Ireland Ltd. All rights reserved.

Propolis Standardized Extract (EPP-AF (R)), an Innovative Chemically and Biologically Reproducible Pharmaceutical Compound for Treating Wounds

The aim of this study was to develop a formulation, containing the propolis standardized extract (EPP-AF (R)), which can assist in the healing of skin lesions. To achieve this objective the antimicrobial activity and chemical composition of the propolis extract was determined. The final product was subjected to in vitro and in vivo pre-clinical evaluation. The broth macrodi-lution method was used to determine the antimicrobial activity of the extracts and formulations against the microorganisms most commonly found in burns, Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, Staphylococcus aureus and Staphylococcus epidermidis. Wistar rats with puncture wounded skin were used to evaluate the wound healing properties of propolis. The results of chemical and biological characterization demonstrated the batch-to-batch reproducibility of the standardized extract which is an unprecedented result. The antimicrobial and wound healing activity of the pharmaceutical studied showed the best results when samples contain 3.6% propolis, suggesting that this is the most promising composition.

Habitat Fragmentation Effects on the Orchid Bee Communities in Remnant Forests of Southeastern Brazil

The effect of habitat fragmentation on the structure of orchid bee communities was analyzed by the investigation of the existence of a spatial structure in the richness and abundance of Euglossini species and by determining the relationship between these data and environmental factors. The surveys were carried out in four different forest fragments and one university campus. Richness, abundance, and diversity of species were analyzed in relation to abiotic (size of the area, extent of the perimeter, perimeter/area ratio, and shape index) and biotic characteristics (vegetation index of the fragment and of the matrix of each of the locations studied). We observed a highly significant positive correlation between the diversity index and the vegetation index of the fragment, landscape and shape index. Our analysis demonstrated that the observed variation could be explained mainly by the vegetation index and the size of the fragment. Variations in relative abundance showed a tendency toward an aggregated spatial distribution between the fragments studied, as well as between the sampling stations within the same habitat, demonstrating the existence of a spatial structure on a small scale in the populations of Euglossini. This distribution will determine the composition of species that coexist in the area after fragmentation. These data help in understanding the differences and similarities in the structure of communities of Euglossini resulting from forest fragmentation.

Pollination services at risk: Bee habitats will decrease owing to climate change in Brazil

Native bees are important providers of pollination services, but there are cumulative evidences of their decline. Global changes such as habitat losses, invasions of exotic species and climate change have been suggested as the main causes of the decline of pollinators. In this study, the influence of climate change on the distribution of 10 species of Brazilian bees was estimated with species distribution modelling. We used Maxent algorithm (maximum entropy) and two different scenarios, an optimistic and a pessimistic, to the years 2050 and 2080. We also evaluated the percentage reduction of species habitat based on the future scenarios of climate change through Geographic Information System (GIS). Results showed that the total area of suitable habitats decreased for all species but one under the different future scenarios. The greatest reductions in habitat area were found for Melipona bicolor bicolor and Melipona scutellaris, which occur predominantly in areas related originally to Atlantic Moist Forest. The species analysed have been reported to be pollinators of some regional crops and the consequence of their decrease for these crops needs further clarification. (C) 2012 Elsevier B.V. All rights reserved.

PLGA microspheres containing bee venom proteins for preventive immunotherapy

Bee venom (BV) allergy is potentially dangerous for allergic individuals because a single bee sting may induce an anaphylactic reaction, eventually leading to death. Currently, venom immunotherapy (VIT) is the only treatment with long-lasting effect for this kind of allergy and its efficiency has been recognized worldwide. This therapy consists of subcutaneous injections of gradually increasing doses of the allergen. This causes patient lack of compliance due to a long time of treatment with a total of 30-80 injections administered over years. In this article we deal with the characterization of different MS-PLGA formulations containing BV proteins for VIT. The PLGA microspheres containing BV represent a strategy to replace the multiple injections, because they can control the solute release. Physical and biochemical methods were used to analyze and characterize their preparation. Microspheres with encapsulation efficiencies of 49-75% were obtained with a BV triphasic release profile. Among them, the MS-PLGA 34 kDa-COOH showed to be best for VIT because they presented a low initial burst (20%) and a slow BV release during lag phase. Furthermore, few conformational changes were observed in the released BV. Above all, the BV remained immunologically recognizable, which means that they could continuously stimulate the immune system. Those microspheres containing BV could replace sequential injections of traditional VIT with the remarkable advantage of reduced number of injections. (C) 2011 Elsevier B.V. All rights reserved.

The use of Open Reading frame ESTs (ORESTES) for analysis of the honey bee transcriptome

Abstract Background The ongoing efforts to sequence the honey bee genome require additional initiatives to define its transcriptome. Towards this end, we employed the Open Reading frame ESTs (ORESTES) strategy to generate profiles for the life cycle of Apis mellifera workers. Results Of the 5,021 ORESTES, 35.2% matched with previously deposited Apis ESTs. The analysis of the remaining sequences defined a set of putative orthologs whose majority had their best-match hits with Anopheles and Drosophila genes. CAP3 assembly of the Apis ORESTES with the already existing 15,500 Apis ESTs generated 3,408 contigs. BLASTX comparison of these contigs with protein sets of organisms representing distinct phylogenetic clades revealed a total of 1,629 contigs that Apis mellifera shares with different taxa. Most (41%) represent genes that are in common to all taxa, another 21% are shared between metazoans (Bilateria), and 16% are shared only within the Insecta clade. A set of 23 putative genes presented a best match with human genes, many of which encode factors related to cell signaling/signal transduction. 1,779 contigs (52%) did not match any known sequence. Applying a correction factor deduced from a parallel analysis performed with Drosophila melanogaster ORESTES, we estimate that approximately half of these no-match ESTs contigs (22%) should represent Apis-specific genes. Conclusions The versatile and cost-efficient ORESTES approach produced minilibraries for honey bee life cycle stages. Such information on central gene regions contributes to genome annotation and also lends itself to cross-transcriptome comparisons to reveal evolutionary trends in insect genomes.

Bioassay guided purification of the antimicrobial fraction of a Brazilian propolis from Bahia state

Abstract Background Brazilian propolis type 6 (Atlantic forest, Bahia) is distinct from the other types of propolis especially due to absence of flavonoids and presence of other non-polar, long chain compounds, but presenting good in vitro and in vivo antimicrobial activity. Several authors have suggested that fatty acids found in this propolis might be responsible for its antimicrobial activity; however, so far no evidence concerning this finding has been reported in the literature. The goals of this study were to evaluate the antibacterial activity of the main pure fatty acids in the ethanolic extract and fractions and elucidate the chemical nature of the bioactive compounds isolated from Brazilian propolis type 6. Methods Brazilian propolis type 6 ethanolic extract (EEP), hexane fraction (H-Fr), major fatty acids, and isolated sub-fractions were analyzed using high performance liquid chromatography (HPLC), high resolution gas chromatography with flame ionization detection (HRGC-FID), and gas chromatography-mass spectrometry (GC-MS). Three sub-fractions of H-Fr were obtained through preparative HPLC. Antimicrobial activity of EEP, H-Fr, sub-fractions, and fatty acids were tested against Staphyloccus aureus ATCC 25923 and Streptococcus mutans Ingbritt 1600 using minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Results EEP and H-Fr inhibited the growth of the microorganisms tested; nevertheless, no antimicrobial activity was found for the major fatty acids. The three sub-fractions (1, 2, and 3) were isolated from H-Fr by preparative HPLC and only sub-fraction 1 showed antimicrobial activity. Conclusion a) The major fatty acids tested were not responsible for the antimicrobial activity of propolis type 6; b) Sub-fraction 1, belonging to the benzophenone class, was responsible for the antimicrobial activity observed in the present study. The identification of the bioactive compound will improve the development of more efficient uses of this natural product.

Transcriptional profiling of Saccharomyces cerevisiae exposed to propolis

Abstract Background Propolis is a natural product of plant resins collected by honeybees (Apis mellifera) from various plant sources. Our previous studies indicated that propolis sensitivity is dependent on the mitochondrial function and that vacuolar acidification and autophagy are important for yeast cell death caused by propolis. Here, we extended our understanding of propolis-mediated cell death in the yeast Saccharomyces cerevisiae by applying systems biology tools to analyze the transcriptional profiling of cells exposed to propolis. Methods We have used transcriptional profiling of S. cerevisiae exposed to propolis. We validated our findings by using real-time PCR of selected genes. Systems biology tools (physical protein-protein interaction [PPPI] network) were applied to analyse the propolis-induced transcriptional bevavior, aiming to identify which pathways are modulated by propolis in S. cerevisiae and potentially influencing cell death. Results We were able to observe 1,339 genes modulated in at least one time point when compared to the reference time (propolis untreated samples) (t-test, p-value 0.01). Enrichment analysis performed by Gene Ontology (GO) Term finder tool showed enrichment for several biological categories among the genes up-regulated in the microarray hybridization such as transport and transmembrane transport and response to stress. Real-time RT-PCR analysis of selected genes showed by our microarray hybridization approach was capable of providing information about S. cerevisiae gene expression modulation with a considerably high level of confidence. Finally, a physical protein-protein (PPPI) network design and global topological analysis stressed the importance of these pathways in response of S. cerevisiae to propolis and were correlated with the transcriptional data obtained thorough the microarray analysis. Conclusions In summary, our data indicate that propolis is largely affecting several pathways in the eukaryotic cell. However, the most prominent pathways are related to oxidative stress, mitochondrial electron transport chain, vacuolar acidification, regulation of macroautophagy associated with protein target to vacuole, cellular response to starvation, and negative regulation of transcription from RNA polymerase II promoter. Our work emphasizes again the importance of S. cerevisiae as a model system to understand at molecular level the mechanism whereby propolis causes cell death in this organism at the concentration herein tested. Our study is the first one that investigates systematically by using functional genomics how propolis influences and modulates the mRNA abundance of an organism and may stimulate further work on the propolis-mediated cell death mechanisms in fungi.

Topical Brazilian propolis improves corneal wound healing and inflammation in rats following alkali burns

Abstract Background The objective of this study was to investigate the effects of the Brazilian Scaptotrigona sp propolis, a widely used folk medicine, in corneal wound healing and inflammation. Methods Corneal epithelial defects of 1 mm in diameter were made in the right eyes of Wistar male adult rats by cauterization with silver nitrate sticks. Subsequently, they were divided in two groups (n = 40 rats/group): Brazilian propolis (BP) group was topically treated with a microemulsion containing 1% Brazilian propolis; vehicle (VH) group received the same formulation without propolis. The epithelial defect area was photographed and measured at t = 0 (wound induction), and after 12, 24, 48 and 120 h of treatment. The inflammatory response was evaluated based on counting of neutrophils. Epithelial regeneration rates were determined based on Ki-67 expression in basal epithelial cells. Comparisons were made using the Kruskal-Wallis and the Mann–Whitney U test. Results The BP group presented both smaller epithelial defect areas at 12, 24 and 48 h and fewer corneal infiltrating neutrophils at 24 and 48 h (P < 0.01) than the VH group. These effects were associated with more pervasive Ki-67 staining in the BP group at 12 and 24 h (P < 0.05). Conclusions Topically applied BP accelerated wound healing and reduced the inflammatory response to silver nitrate-induced corneal alkali burns in rats.

Description of an ancient social bee trapped in amber using diagnostic radioentomology

The application of non-invasive imaging technologies using X-radiation (diagnostic radioentomology, ‘DR’) is demonstrated for the study of amber-entombed social bees. Here, we examine the external and internal morphology of an Early Miocene (Burdigalian) stingless bee (Apinae: Meliponini) from the Dominican Republic using non-destructive X-ray microtomography analysis. The study permits the accurate reconstruction of features otherwise obscured or impossible to visualize without destroying the sample and allows diagnosis of the specimen as a new species, Proplebeia adbita Greco and Engel.