Pollen substitutes increase honey bee haemolymph protein levels as much as or more than does pollen

Adequate substitutes for pollen are necessary for maintaining healthy bee colonies during periods of pollen dearth, but testing them objectively is both time consuming and expensive. We compared two commercial diets with bee collected pollen and acacia pod flour (used by beekeepers in some parts of Brazil) by measuring their effect on haemolymph protein contents of young bees exclusively fed on these diets, which is a fast and inexpensive assay. The commercial diets included a new, non-soy-based, pollen substitute diet (named Feed-Bee (R)) and a soy-based diet, named Bee-Pro (R). The diets were each given in patty form to groups of 100 Africanized honey bees in hoarding cages, maintained and fed from emergence until six days of age. Sucrose, in the form of sugar syrup, was used as a protein free control. Feed-Bee (R), Bee-Pro (R), pollen and acacia pod flour diets increased protein titers in the haemolymph by factors of 2.65, 2.51, 1.76 and 1.69, respectively, over protein titers in bees fed only sucrose solution. The bees fed Feed-Bee (R) and Bee-Pro (R) had their haemolymph significantly enriched in protein compared to the controls and those fed acacia pod flour and to titers slightly higher than those fed pollen. All four proteinaceous diets were significantly superior to sucrose alone.

Representational Difference Analysis (RDA) reveals differential expression of conserved as well as novel genes during caste-specific development of the honey bee (Apis mellifera L.) ovary

In highly eusocial insects, such as the honey bee, Apis mellifera, the reproductive bias has become embedded in morphological caste differences. These are most expressively denoted in ovary size, with adult queens having large ovaries consisting of 150-200 ovarioles each, while workers typically have only 1-20 ovarioles per ovary. This morphological differentiation is a result of hormonal signals triggered by the diet change in the third larval instar, which eventually generate caste-specific gene expression patterns. To reveal these we produced differential gene expression libraries by Representational Difference Analysis (RDA) for queen and worker ovaries in a developmental stage when cell death is a prominent feature in the ovarioles of workers, whereas all ovarioles are maintained and extend in length in queens. In the queen library, 48% of the gene set represented homologs of known Drosophila genes, whereas in the worker ovary, the largest set (59%) were ESTs evidencing novel genes, not even computationally predicted in the honey bee genome. Differential expression was confirmed by quantitative RT-PCR for a selected gene set, denoting major differences for two queen and two worker library genes. These included two unpredicted genes located in chromosome 11 (Group11.35 and Group11.31, respectively) possibly representing long non-coding RNAs. Being candidates as modulators of ovary development, their expression and functional analysis should be a focal point for future studies. (C) 2011 Elsevier Ltd. All rights reserved.

Insulin-like peptide genes in honey bee fat body respond differently to manipulation of social behavioral physiology

Nutrient sensitive insulin-like peptides (ILPs) have profound effects on invertebrate metabolism, nutrient storage, fertility and aging. Many insects transcribe ILPs in specialized neurosecretory cells at changing levels correlated with life history. However, the major site of insect metabolism and nutrient storage is not the brain, but rather the fat body, where functions of ILP expression are rarely studied and poorly understood. Fat body is analogous to mammalian liver and adipose tissue, with nutrient stores that often correlate with behavior. We used the honey bee (Apis mellifera), an insect with complex behavior, to test whether ILP genes in fat body respond to experimentally induced changes of behavioral physiology. Honey bee fat body influences endocrine state and behavior by secreting the yolk protein precursor vitellogenin (Vg), which suppresses lipophilic juvenile hormone and social foraging behavior. In a two-factorial experiment, we used RNA interference (RNAi)-mediated vg gene knockdown and amino acid nutrient enrichment of hemolymph (blood) to perturb this regulatory module. We document factor-specific changes in fat body ilp1 and ilp2 mRNA, the bee`s ILP-encoding genes, and confirm that our protocol affects social behavior. We show that ilp1 and ilp2 are regulated independently and differently and diverge in their specific expression-localization between fat body oenocyte and trophocyte cells. Insect ilp functions may be better understood by broadening research to account for expression in fat body and not only brain.

Cytoplasmic and nuclear localization of cadherin in honey bee (Apis mellifera L.) gonads

Cadherins are crucial molecules mediating cell-cell interactions between somatic and germline cells in insect and mammalian male and female gonads. We analysed the presence and localization of cadherins in ovaries of honeybee queens and in testes of drones. Transcripts representing two classical cadherins, E-cadherin (shotgun) and N-cadherin, as well as three protocadherins (Starry night, Fat and Fat-like) were detected in gonads of both sexes. Pan-cadherin antibodies, which most probably detect a honeybee N-cadherin, were used in immunolocalization analyses. In the germarium of ovarioles, cadherin-IR (cadherin immunoreactivity) was evidenced as homogeneously distributed in the cytoplasm and as nuclear foci, in both germline and somatic cells. It was also detected in polyfusomes and ring canals. In testiolar tubules, cadherin-IR showed a cytoplasmic and nuclear distributon alike in ovaries. The unexpected nuclear localization and cytoplasmic distribution in ovaries and testes were corroborated by immunogold electron microscopy, which revealed cadherin aggregates associated with electron-dense nuclear structures. With respect to cadherin localization, the honeybee differs from Drosophila, the model for gametogenesis in insects, raising the question as to how differences among solitary and social species may be built into and generated from the general architecture of polytrophic meroistic ovaries. It also indicates the possibility of divergent roles for cadherin in the functional architecture of insect gonads, in general, especially in taxa with high reproductive output.

In vitro study of the antimicrobial activity of Brazilian propolis against Paenibacillus larvae

The honey bee disease American foulbrood (AFB) is a serious problem since its causative agent (Paenibacillus larvae) has become increasingly resistant to conventional antibiotics. The objective of this study was to investigate the in vitro activity of propolis collected from various states of Brazil against P. larvae. Propolis is derived from plant resins collected by honey bees (Apis mellifera) and is globally known for its antimicrobial properties and particularly valued in tropical regions. Tests on the activity of propolis against P. larvae were conducted both in Brazil and Minnesota, USA using two resistance assay methods that measured zones of growth inhibition due to treatment exposure. The propolis extracts from the various states of Brazil showed significant inhibition of P. larvae. Clear dose responses were found for individual propolis extracts, particularly between the concentrations of 1.7 and 0.12 mg propolis/treatment disk, but the source of the propolis, rather than the concentration, may be more influential in determining overall activity. Two of the three tested antibiotics (tylosin and terramycin) exhibited a greater level of inhibition compared to most of the Brazilian samples, which could be due to the low concentrations of active compounds present in the propolis extracts. Additionally, the majority of the Brazilian propolis samples were more effective than the few collected in MN, USA. Due to the evolution of resistance of P. larvae to conventional antibiotic treatments, this research is an important first step in identifying possible new active compounds to treat AFB in honey bee colonies. (C) 2007 Elsevier Inc. All rights reserved.

Brood cell size of Apis mellifera modifies the reproductive behavior of Varroa destructor

We undertook a field study to determine whether comb cell size affects the reproductive behavior of Varroa destructor under natural conditions. We examined the effect of brood cell width on the reproductive behavior of V. destructor in honey bee colonies, under natural conditions. Drone and worker brood combs were sampled from 11 colonies of Apis mellifera. A Pearson correlation test and a Tukey test were used to determine whether mite reproduction rate varied with brood cell width. Generalized additive model analysis showed that infestation rate increased positively and linearly with the width of worker and drone cells. The reproduction rate for viable mother mites was 0.96 viable female descendants per original invading female. No significant correlation was observed between brood cell width and number of offspring of V. destructor. Infertile mother mites were more frequent in narrower brood cells.

Postembryonic Development of Rectal Pads in Bees (Hymenoptera, Apidae)

The morphology and development of the digestive tract of insects has been extensively studied, but little attention has been given to the development of the rectal pads. These organs are responsible for absorption of water and salts. In insects where they occur, there are usually six ovoid rectal pads located in the medial-anterior portion of the rectum. The rectal pad has three types of cells: principal, basal, and junctional. The arrangement of these three cell types delimits an intrapapillary lumen. The aim of the current study is to describe the development of the rectal pads during postembryonic development of Melipona quadrifasciata anthidioides and Melipona scutellaris. Specimens were analyzed at the following developmental stages: white-, pink-, brown-, and black-eyed pupae, and adult workers. The development of the rectal pad begins as a thickening of the epithelium in white-eyed pupae at 54 hr. At this stage, there is neither a basal cell layer nor intrapapillary lumen. The basal layers begin to form in the pink-eyed pupa and are completely formed at the end of the development of the brown-eyed pupa. In the brown-eyed pupal stage, the intrapapillary lumen is formed and the junctional cells are positioned and completely differentiated. Necrotic and apoptotic cell death were detected along with cell proliferation in the whole rectum during pupal development, suggesting that the development of the rectal pads involves cell proliferation, death, and differentiation. The rectal pads originate only from the ectoderm. Anat Rec, 292:1602-1611, 2009. (C) 2009 Wiley-Liss, Inc.

Descriptive Sensory Analysis and Acceptance of Stingless Bee Honey

Stingless bee honey samples were evaluated by sensory descriptive analysis using free choice profile methodology. Appearance, flavor, and aroma were described and the data were treated with Generalized Procrustes Analysis. Individual descriptive terms ranged from 8 to 20. Plotting the samples in a bidimensional plan indicated that appearance attributes (color and viscosity) and sweet, sour and acid flavor were strongly correlated with x axis (Dimension 1) while coconut, wood, acid, sour, and sweet flavor aroma attributes were correlated with y axis (Dimension 2). The affective test was also performed and with the exception of the Melipona scutellaris honey, all the other samples showed good acceptance. Honeys that were described as sweeter and less acid were preferred by nontrained assessors, indicating that the regular consumer recognizes honey produced by Apis mellifera bee as a standard.

A molecular phylogeny of the stingless bee genus Melipona (Hymenoptera: Apidae)

Stingless bees (Meliponini) constitute a diverse group of highly eusocial insects that occur throughout tropical regions around the world. The meliponine genus Melipona is restricted to the New World tropics and has over 50 described species. Melipona, like Apis, possesses the remarkable ability to use representational communication to indicate the location of foraging patches. Although Melipona has been the subject of numerous behavioral, ecological, and genetic studies, the evolutionary history of this genus remains largely unexplored. Here, we implement a multigene phylogenetic approach based on nuclear, mitochondrial, and ribosomal loci, coupled with molecular clock methods, to elucidate the phylogenetic relationships and antiquity of subgenera and species of Melipona. Our phylogenetic analysis resolves the relationship among subgenera and tends to agree with morphology-based classification hypotheses. Our molecular clock analysis indicates that the genus Melipona shared a most recent common ancestor at least similar to 14-17 million years (My) ago. These results provide the groundwork for future comparative analyses aimed at understanding the evolution of complex communication mechanisms in eusocial Apidae. (C) 2010 Elsevier Inc. All rights reserved.

Ecological niche similarities of Peponapis bees and non-domesticated Cucurbita species

Peponapis bees are considered specialized pollinators of Cucurbita flowers, a genus that presents several species of economic value (squashes and pumpkins). Both genera originated in the Americas, and their diversity dispersion center is in Mexico. Ten species of Peponapis and ten species of Cucurbita (only non-domesticated species) were analyzed considering the similarity of their ecological niche characteristics with respect to climatic conditions of their occurrence areas (abiotic variables) and interactions between species (biotic variables). The similarity of climatic conditions (temperature and precipitation) was estimated through cluster analyses. The areas of potential occurrence of the most similar species were obtained through ecological niche modeling and summed with geographic information system tools. Three main clusters were obtained: one with species that shared potential occurrence areas mainly in deserts (P. pruinosa, P. timberlakei, C. digitata, C. palmata, C. foetidissima), another in moist forests (P. limitaris, P. atrata, C. lundelliana, C. o. martinezii) and a third mainly in dry forests (C. a. sororia, C. radicans, C. pedatifolia, P. azteca, P. smithi, P. crassidentata, P. utahensis). Some species with similar ecological niche presented potential shared areas that are also similar to their geographical distribution, like those occurring predominantly on deserts. However, some clustered species presented larger geographical areas, such as P. pruinosa and C. foetidissima suggesting other drivers than climatic conditions to shape their distributions. The domestication of Cucurbita and also the natural history of both genera were considered also as important factors. (C) 2011 Elsevier B.V. All rights reserved.

Genetic structure of Partamona helleri (Apidae, Meliponini) from Neotropical Atlantic rainforest

Stingless bees of the genus Partamona are distributed from southern Mexico to southern Brazil. This genus has been subject to different approaches to solve questions concerning general biology, taxonomy, systematics and biogeography, but population studies applying molecular techniques are inexistent. We analyzed the genetic structure of P. helleri across its geographic distribution along the coastal Atlantic tropical rainforest in Brazil. Ten mtDNA haplotypes were observed in 47 colonies of P. helleri of which some were exclusive and others shared among geographic sub-groups. Statistical analysis showed high genetic differentiation between geographic areas sampled. Fragmentation of the Atlantic forest during Pleistocene glaciations is discussed as a possible cause of the present haplotype distribution and frequency.

Determination of the maximum inhibitory dilution of cetylpyridinium chloride-based mouthwashes against staphylococcus aureus: an in vitro study

The aim of this in vitro study was to determine the maximum inhibitory dilution (MID) of four cetylpyridinium chloride (CPC)-based mouthwashes: CPC+Propolis, CPC+Malva, CPC+Eucaliptol+Juá+Romã+Propolis (Natural Honey®) and CPC (Cepacol®), against 28 Staphylococcus aureus field strains, using the agar dilution method. Decimal dilutions ranging from 1/10 to 1/655,360 were prepared and added to Mueller Hinton Agar. Strains were inoculated using Steers multipoint inoculator. The inocula were seeded onto the surface of the culture medium in Petri dishes containing different dilutions of the mouthwashes. The dishes were incubated at 37ºC for 24 h. For readings, the MID was considered as the maximum dilution of mouthwash still capable of inhibiting microbial growth. The obtained data showed that CPC+Propolis had antimicrobial activity against 27 strains at 1/320 dilution and against all 28 strains at 1/160 dilution, CPC+Malva inhibited the growth of all 28 strains at 1/320 dilution, CPC+Eucaliptol+Juá+Romã+Propolis inhibited the growth of 2 strains at 1/640 dilution and all 28 strains at 1/320 dilution, and Cepacol® showed antimicrobial activity against 3 strains at 1/320 dilution and against all 28 strains at 1/160 dilution. Data were submitted to Kruskal-Wallis test, showing that the MID of Cepacol® was lower than that determined for the other products (p<0.05). In conclusion, CPC-mouthwashes showed antimicrobial activity against S. aureus and the addition of other substances to CPC improved its antimicrobial effect.

Age polyethism in Plebeia emerina (Friese) (Hymenoptera: Apidae) colonies related to propolis handling

Stingless bees collect plant resins and make it into propolis, although they have a wider range of use for this material than do honey bees (Apis spp.). Plebeia spp. workers employ propolis mixed with wax (cerumen) for constructing and sealing nest structures, while they use viscous (sticky) propolis for defense by applying it onto their enemies. Isolated viscous propolis deposits are permanently maintained at the interior of their colonies, as also seen in other Meliponini species. Newly-emerged Plebeia emerina (Friese) workers were observed stuck to and unable to escape these viscous propolis stores. We examined the division of labor involved in propolis manipulation, by observing marked bees of known age in four colonies of P. emerina from southern Brazil. Activities on brood combs, the nest involucrum and food pots were observed from the first day of life of the marked bees. However, work on viscous propolis deposits did not begin until the 13th day of age and continued until the 56th day (maximum lifespan in our sample). Although worker bees begin to manipulate cerumen early, they seem to be unable to handle viscous propolis till they become older.

Cuticular hydrocarbons in the stingless bee Schwarziana quadripunctata (Hymenoptera, Apidae, Meliponini): differences between colonies, castes and age

Chemical communication is of fundamental importance to maintain the integration of insect colonies. In honey bees, cuticular lipids differ in their composition between queens, workers and drones. Little is known, however, about cuticular hydrocarbons in stingless bees. We investigated chemical differences in cuticular hydrocarbons between different colonies, castes and individuals of different ages in Schwarziana quadripunctata. The epicuticle of the bees was extracted using the nonpolar solvent hexane, and was analyzed by means of a gas chromatograph coupled with a mass spectrometer. The identified compounds were alkanes, branched-alkanes and alkenes with chains of 19 to 33 carbon atoms. Discriminant analyses showed clear differences between all the groups analyzed. There were significant differences between bees from different colonies, workers of different age and between workers and virgin queens.

The four hexamerin genes in the honey bee: structure, molecular evolution and function deduced from expression patterns in queens, workers and drones

Background: Hexamerins are hemocyanin-derived proteins that have lost the ability to bind copper ions and transport oxygen; instead, they became storage proteins. The current study aimed to broaden our knowledge on the hexamerin genes found in the honey bee genome by exploring their structural characteristics, expression profiles, evolution, and functions in the life cycle of workers, drones and queens. Results: The hexamerin genes of the honey bee (hex 70a, hex 70b, hex 70c and hex 110) diverge considerably in structure, so that the overall amino acid identity shared among their deduced protein subunits varies from 30 to 42%. Bioinformatics search for motifs in the respective upstream control regions (UCRs) revealed six overrepresented motifs including a potential binding site for Ultraspiracle (Usp), a target of juvenile hormone (JH). The expression of these genes was induced by topical application of JH on worker larvae. The four genes are highly transcribed by the larval fat body, although with significant differences in transcript levels, but only hex 110 and hex 70a are re-induced in the adult fat body in a caste-and sex-specific fashion, workers showing the highest expression. Transcripts for hex 110, hex 70a and hex70b were detected in developing ovaries and testes, and hex 110 was highly transcribed in the ovaries of egg-laying queens. A phylogenetic analysis revealed that HEX 110 is located at the most basal position among the holometabola hexamerins, and like HEX 70a and HEX 70c, it shares potential orthology relationship with hexamerins from other hymenopteran species. Conclusions: Striking differences were found in the structure and developmental expression of the four hexamerin genes in the honey bee. The presence of a potential binding site for Usp in the respective 5' UCRs, and the results of experiments on JH level manipulation in vivo support the hypothesis of regulation by JH. Transcript levels and patterns in the fat body and gonads suggest that, in addition to their primary role in supplying amino acids for metamorphosis, hexamerins serve as storage proteins for gonad development, egg production, and to support foraging activity. A phylogenetic analysis including the four deduced hexamerins and related proteins revealed a complex pattern of evolution, with independent radiation in insect orders.