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.     

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.     

Hierarchy of Attractants for Honey Bee Swarms

Chemical signals influence the selection of potential nest cavities by honey bee reproductive swarms. Attractants for swarms include the odors of old dark honey bee brood combs, odors from noncomb hive materials and propolis, and Nasonov pheromone, the odor released from the Nasonov glands of worker bees. Based on crossover and choice test experiments, swarms were shown to prefer, among otherwise identical cavities, those cavities containing Nasonov pheromone over cavities with only comb or other hive odors, cavities containing old comb over those with only noncomb odors or propolis, and cavities containing noncomb odors or propolis over those without bee or hive odor. Synergy between odors was not observed; that is, comb and/or noncomb hive odors did not enhance the attractiveness of Nasonov pheromone. The data support a model based on a hierarchy of olfactory attractants used by honey bee swarms, in order of highest to lowest: Nasonov pheromone, comb odor, noncomb and propolis odors, and, finally, absence of bee- or hive-produced odor.     

Ecologically appropriate xenobiotics induce cytochrome P450s in Apis mellifera

Background

Honey bees are exposed to phytochemicals through the nectar, pollen and propolis consumed to sustain the colony. They may also encounter mycotoxins produced by Aspergillus fungi infesting pollen in beebread. Moreover, bees are exposed to agricultural pesticides, particularly in-hive acaricides used against the parasite Varroa destructor. They cope with these and other xenobiotics primarily through enzymatic detoxificative processes, but the regulation of detoxificative enzymes in honey bees remains largely unexplored.

Methodology/Principal Findings

We used several approaches to ascertain effects of dietary toxins on bee susceptibility to synthetic and natural xenobiotics, including the acaricide tau-fluvalinate, the agricultural pesticide imidacloprid, and the naturally occurring mycotoxin aflatoxin. We administered potential inducers of cytochrome P450 enzymes, the principal biochemical system for Phase 1 detoxification in insects, to investigate how detoxification is regulated. The drug phenobarbital induces P450s in many insects, yet feeding bees with phenobarbital had no effect on the toxicity of tau-fluvalinate, a pesticide known to be detoxified by bee P450s. Similarly, no P450 induction, as measured by tau-fluvalinate tolerance, occurred in bees fed xanthotoxin, salicylic acid, or indole-3-carbinol, all of which induce P450s in other insects. Only quercetin, a common pollen and honey constituent, reduced tau-fluvalinate toxicity. In microarray comparisons no change in detoxificative gene expression was detected in phenobarbital-treated bees. However, northern blot analyses of guts of bees fed extracts of honey, pollen and propolis showed elevated expression of three CYP6AS P450 genes. Diet did not influence tau-fluvalinate or imidacloprid toxicity in bioassays; however, aflatoxin toxicity was higher in bees consuming sucrose or high-fructose corn syrup than in bees consuming honey.

Conclusions/Significance

These results suggest that regulation of honey bee P450s is tuned to chemicals occurring naturally in the hive environment and that, in terms of toxicological capacity, a diet of sugar is not equivalent to a diet of honey.     

Viral impacts on honey bee populations: A review

Honey bee is vital for pollination and ecological services, boosting crops productivity in terms of quality and quantity and production of colony products: wax, royal jelly, bee venom, honey, pollen and propolis. Honey bees are most important plant pollinators and almost one third of diet depends on bee’s pollination, worth billions of dollars. Hence the role that honey bees have in environment and their economic importance in food production, their health is of dominant significance. Honey bees can be infected by various pathogens like: viruses, bacteria, fungi, or infested by parasitic mites. At least more than 20 viruses have been identified to infect honey bees worldwide, generally from Dicistroviridae as well as Iflaviridae families, like ABPV (Acute Bee Paralysis Virus), BQCV (Black Queen Cell Virus), KBV (Kashmir Bee Virus), SBV (Sacbrood Virus), CBPV (Chronic bee paralysis virus), SBPV (Slow Bee Paralysis Virus) along with IAPV (Israeli acute paralysis virus), and DWV (Deformed Wing Virus) are prominent and cause infections harmful for honey bee colonies health. This issue about honey bee viruses demonstrates remarkably how diverse this field is, and considerable work has to be done to get a comprehensive interpretation of the bee virology.     

Honey bee (<Emphasis Type="Italic">Apis mellifera capensis/A. m. scutellata</Emphasis> hybrid) nesting behavior in the Eastern Cape,South Africa

Little is known about the natural history of wild honey bee (Apis mellifera) colonies in the Eastern Cape Province of South Africa. The goal of this research was to examine nest site characteristics of honey bee (A. m. capensis/A. m. scutellata hybrid) colonies sampled from a variety of habitats (nature reserves, livestock farms, and an urban setting) in the Eastern Cape. We also determined how nest site location related to various colony strength parameters. In general, colonies not nesting in ground cavities tended to nest in locations >6 m high when nesting in cliffs and buildings and >2 m high when nesting in trees. Colonies typically nested in cavities whose entrances faced a southeasterly direction and were ~40 L in volume. We sampled a subset of colonies to determine the relationship between nest type and the following colony strength parameters: total area of comb in the colony, the volume of stored honey, pollen, and brood, adult bee population, the weight per adult bee, and the bee/nest cavity volume ratio. In general, colonies nesting in cliffs tended to be stronger than those nesting in the ground or trees. Our findings provide new insights into the nesting biology of honey bees in the Eastern Cape, South Africa, perhaps leading to the formation of conservation recommendations for honey bees in this region.     

Behavioral responses of the small hive beetle,Aethina tumida,to odors of three meliponine bee species and honey bees,Apis mellifera scutellata

Recent studies have shown that honey bees, bumble bees, and some meliponine bee species of the genera Trigona, Meliponula, and Dactylurina are hosts of the small hive beetle (SHB) Aethina tumidaMurray (Coleoptera: Nitidulidae), a pest of honey bee colonies in various regions of the world. Olfaction has been implicated in SHB infestations of honey bee and bumble bee colonies. We used olfactometer bioassays to investigate responses of adult male and female SHBs to odors from intact colonies and separate hive components (pot honey, pot pollen, cerumen, and propolis) of three African meliponine bee species, Meliponula ferruginea (Lepeletier) (black morphospecies), M. ferruginea (reddish brown morphospecies), and Meliponula bocandei (Spinola) (Hymenoptera: Apidae). Although both sexes of the beetle strongly preferred intact colony, pot honey, and pot pollen odors, there was no evidence of attraction to propolis and cerumen odors from the three meliponine bee species. Both sexes of SHB also strongly preferred odors from honey bees, Apis mellifera L. (Hymenoptera: Apidae), over odors from the three meliponine bee species. Our results provide substantial evidence of the host potential of African meliponine bees for the SHB, and we discuss this complex association of the SHB with species within the Apidae family.     

PVC nest boxes are less at risk of occupancy by feral honey bees than timber nest boxes and natural hollows

Feral European Honey Bee (Apis mellifera) has been identified as a potential nest competitor for Australian hollow nesting species, but few studies have investigated the impact of feral honey bee competition on Threatened species. Our study used data from Glossy Black‐cockatoo (Calyptorhynchus lathami halmaturinus) nests on Kangaroo Island, monitored and managed over an 11‐year period, and found 12% of nests became occupied by feral honey bees during that period. Our results indicate that feral honey bees were less likely to occupy nest boxes made of PVC (5%) compared with wooden nest boxes (24%) or natural hollows in Eucalyptus trees (14%). The removal of feral honey bee hives from nests is a priority for long‐term conservation of glossy black‐cockatoos on Kangaroo Island. We recommend that PVC nest boxes are chosen for future nesting habitat restoration, due to the more frequent use of wooden nest boxes by feral honey bees.     

Unique Honey Bee (Apis mellifera) Hive Component-Based Communities as Detected by a Hybrid of Phospholipid Fatty-Acid and Fatty-Acid Methyl Ester Analyses

Microbial communities (microbiomes) are associated with almost all metazoans, including the honey bee Apis mellifera. Honey bees are social insects, maintaining complex hive systems composed of a variety of integral components including bees, comb, propolis, honey, and stored pollen. Given that the different components within hives can be physically separated and are nutritionally variable, we hypothesize that unique microbial communities may occur within the different microenvironments of honey bee colonies. To explore this hypothesis and to provide further insights into the microbiome of honey bees, we use a hybrid of fatty acid methyl ester (FAME) and phospholipid-derived fatty acid (PLFA) analysis to produce broad, lipid-based microbial community profiles of stored pollen, adults, pupae, honey, empty comb, and propolis for 11 honey bee hives. Averaging component lipid profiles by hive, we show that, in decreasing order, lipid markers representing fungi, Gram-negative bacteria, and Gram-positive bacteria have the highest relative abundances within honey bee colonies. Our lipid profiles reveal the presence of viable microbial communities in each of the six hive components sampled, with overall microbial community richness varying from lowest to highest in honey, comb, pupae, pollen, adults and propolis, respectively. Finally, microbial community lipid profiles were more similar when compared by component than by hive, location, or sampling year. Specifically, we found that individual hive components typically exhibited several dominant lipids and that these dominant lipids differ between components. Principal component and two-way clustering analyses both support significant grouping of lipids by hive component. Our findings indicate that in addition to the microbial communities present in individual workers, honey bee hives have resident microbial communities associated with different colony components.     

The habitat disruption induces immune-suppression and oxidative stress in honey bees

The honey bee is a major insect used for pollination of many commercial crops worldwide. Although the use of honey bees for pollination can disrupt the habitat, the effects on their physiology have never been determined. Recently, honey bee colonies have often collapsed when introduced in greenhouses for pollination in Japan. Thus, suppressing colony collapses and maintaining the number of worker bees in the colonies is essential for successful long-term pollination in greenhouses and recycling of honey bee colonies. To understand the physiological states of honey bees used for long-term pollination in greenhouses, we characterized their gene expression profiles by microarray. We found that the greenhouse environment changes the gene expression profiles and induces immune-suppression and oxidative stress in honey bees. In fact, the increase of the number of Nosema microsporidia and protein carbonyl content was observed in honey bees during pollination in greenhouses. Thus, honey bee colonies are likely to collapse during pollination in greenhouses when heavily infested with pathogens. Degradation of honey bee habitat by changing the outside environment of the colony, during pollination services for example, imposes negative impacts on honey bees. Thus, worldwide use of honey bees for crop pollination in general could be one of reasons for the decline of managed honey bee colonies.     

Survival and health improvement of Nosema infected Apis florea (Hymenoptera: Apidae) bees after treatment with propolis extract

Nosema ceranae is now considered to be an emerging infectious disease of the European honey bee Apis mellifera. Only one antibiotic, Fumagillin, is commercially available to combat Nosema infections. This antibiotic treatment is banned from use in Europe and elsewhere there is a high probability for antibiotic resistance to develop. We are therefore interested in investigating the effects of a natural propolis extract on its ability to reduce N. ceranae infection loads in the dwarf honey bee, Apis florea, a native honey bee with a range that overlaps with Apis cerana and Apis mellifera that is at risk of infection. Experimentally infected caged bees were fed a treatment consisting of 0%, 50%, or 70% propolis extract. All 50% and 70% propolis treated bees had significantly lower infection loads, and the 50% treated bees had higher survival in comparison to untreated bees. In addition, propolis treated bees had significantly higher haemolymph trehalose levels and hypopharyngeal gland protein content similar to levels of uninfected bees. Propolis ethanolic extract treatment could therefore be considered as a possible viable alternative to Fumagillin to improve bee health. This natural treatment deserves further exploration to develop it as a possible alternative to combat N. ceranae infections distributed around the world.     

The scent of the waggle dance

The waggle dance of honey bee (Apis mellifera L.) foragers communicates to nest mates the location of a profitable food source. We used solid-phase microextraction and gas chromatography coupled with mass spectrometry to show that waggle-dancing bees produce and release two alkanes, tricosane and pentacosane, and two alkenes, Z-(9)-tricosene and Z-(9)-pentacosene, onto their abdomens and into the air. Nondancing foragers returning from the same food source produce these substances in only minute quantities. Injection of the scent significantly affects worker behavior by increasing the number of bees that exit the hive. The results of this study suggest that these compounds are semiochemicals involved in worker recruitment. By showing that honey bee waggle dancers produce and release behaviorally active chemicals, this study reveals a new dimension in the organization of honey bee foraging.     

The immunological dependence of plant-feeding animals on their host’s medical properties may explain part of honey bee colony losses

The honey bee (Apis mellifera) is an important pollinator of agricultural and horticultural crops, but also of wild flowers. The species has been facing declines in many areas of the world, the causes being identified as multifactorial. Recently, it has been theorised that some plant-dwelling animals may develop a dependence on the medicinal properties of their hots plant’s secondary metabolites. Here, the question of honey bee self-medication using organic materials, namely propolis, nectar, honey, honeydew, pollen, wood, and algae for self-medication is addressed. Self-medication in honey bees is a largely unexplored area and thus a comprehensive overview of the field is provided. Prior studies suggest that recent honey bee colony declines are driven by decreased forage plant availability. The problem is expanded and it is suggested, that if honey bees developed a dependence on medical properties of some disappearing plants or materials, this could explain a part of the colony losses observed around the world. To date, convincing evidence points towards self-medication with honey and propolis. Bees also contact plant secondary metabolites, fatty acids, essential oils, and microorganisms that are active against the causative agents of American foulbrood, European foulbrood, nosemosis, chalkbrood, stonebrood, and varroasis. In the future, selected taxa of plants with medicinal properties may be planted to boost honey bee health without chemotherapy. Future directions of research are discussed.     

Division of Labor Associated with Brood Rearing in the Honey Bee: How Does It Translate to Colony Fitness?

Division of labor is a striking feature observed in honey bees and many other social insects. Division of labor has been claimed to benefit fitness. In honey bees, the adult work force may be viewed as divided between non-foraging hive bees that rear brood and maintain the nest, and foragers that collect food outside the nest. Honey bee brood pheromone is a larval pheromone that serves as an excellent empirical tool to manipulate foraging behaviors and thus division of labor in the honey bee. Here we use two different doses of brood pheromone to alter the foraging stimulus environment, thus changing demographics of colony division of labor, to demonstrate how division of labor associated with brood rearing affects colony growth rate. We examine the effects of these different doses of brood pheromone on individual foraging ontogeny and specialization, colony level foraging behavior, and individual glandular protein synthesis. Low brood pheromone treatment colonies exhibited significantly higher foraging population, decreased age of first foraging and greater foraging effort, resulting in greater colony growth compared to other treatments. This study demonstrates how division of labor associated with brood rearing affects honey bee colony growth rate, a token of fitness.     

Antibacterial Immune Competence of Honey Bees (Apis mellifera) Is Adapted to Different Life Stages and Environmental Risks

The development of all honey bee castes proceeds through three different life stages all of which encounter microbial infections to a various extent. We have examined the immune strength of honey bees across all developmental stages with emphasis on the temporal expression of cellular and humoral immune responses upon artificial challenge with viable Escherichia coli bacteria. We employed a broad array of methods to investigate defence strategies of infected individuals: (a) fate of bacteria in the haemocoel; (b) nodule formation and (c) induction of antimicrobial peptides (AMPs). Newly emerged adult worker bees and drones were able to activate efficiently all examined immune reactions. The number of viable bacteria circulating in the haemocoel of infected bees declined rapidly by more than two orders of magnitude within the first 4–6 h post-injection (p.i.), coinciding with the occurrence of melanised nodules. Antimicrobial activity, on the other hand, became detectable only after the initial bacterial clearance. These two temporal patterns of defence reactions very likely represent the constitutive cellular and the induced humoral immune response. A unique feature of honey bees is that a fraction of worker bees survives the winter season in a cluster mostly engaged in thermoregulation. We show here that the overall immune strength of winter bees matches that of young summer bees although nodulation reactions are not initiated at all. As expected, high doses of injected viable E.coli bacteria caused no mortality in larvae or adults of each age. However, drone and worker pupae succumbed to challenge with E.coli even at low doses, accompanied by a premature darkening of the pupal body. In contrast to larvae and adults, we observed no fast clearance of viable bacteria and no induction of AMPs but a rapid proliferation of E.coli bacteria in the haemocoel of bee pupae ultimately leading to their death.     

Experience-dependent plasticity in the mushroom bodies of the solitary bee Osmia lignaria (Megachilidae)

All members of the solitary bee species Osmia lignaria (the orchard bee) forage upon emergence from their natal nest cell. Conversely, in the honey bee, days-to-weeks of socially regulated behavioral development precede the onset of foraging. The social honey bee's behavioral transition to foraging is accompanied by neuroanatomical changes in the mushroom bodies, a region of the insect brain implicated in learning. If these changes were general adaptations to foraging, they should also occur in the solitary orchard bee. Using unbiased stereological methods, we estimated the volume of the major compartments of the mushroom bodies, the neuropil and Kenyon cell body region, in adult orchard bees. We compared the mushroom bodies of recently emerged bees with mature bees that had extensive foraging experience. To separate effects of general maturation from field foraging, some orchard bees were confined to a cage indoors. The mushroom body neuropil of experienced field foragers was significantly greater than that of both recently emerged and mature caged orchard bees, suggesting that, like the honey bee, this increase is driven by outdoor foraging experience. Unlike the honey bee, where increases in the ratio of neuropil to Kenyon cell region occur in the worker after emerging from the hive cell, the orchard bee emerged from the natal nest cell with a ratio that did not change with maturation and was comparable to honey-bee foragers. These results suggest that a common developmental endpoint may be reached via different development paths in social and solitary species of foraging bees.     

Increased Resin Collection after Parasite Challenge: A Case of Self-Medication in Honey Bees?

The constant pressure posed by parasites has caused species throughout the animal kingdom to evolve suites of mechanisms to resist infection. Individual barriers and physiological defenses are considered the main barriers against parasites in invertebrate species. However, behavioral traits and other non-immunological defenses can also effectively reduce parasite transmission and infection intensity. In social insects, behaviors that reduce colony-level parasite loads are termed "social immunity." One example of a behavioral defense is resin collection. Honey bees forage for plant-produced resins and incorporate them into their nest architecture. This use of resins can reduce chronic elevation of an individual bee's immune response. Since high activation of individual immunity can impose colony-level fitness costs, collection of resins may benefit both the individual and colony fitness. However the use of resins as a more direct defense against pathogens is unclear. Here we present evidence that honey bee colonies may self-medicate with plant resins in response to a fungal infection. Self-medication is generally defined as an individual responding to infection by ingesting or harvesting non-nutritive compounds or plant materials. Our results show that colonies increase resin foraging rates after a challenge with a fungal parasite (Ascophaera apis: chalkbrood or CB). Additionally, colonies experimentally enriched with resin had decreased infection intensities of this fungal parasite. If considered self-medication, this is a particularly unique example because it operates at the colony level. Most instances of self-medication involve pharmacophagy, whereby individuals change their diet in response to direct infection with a parasite. In this case with honey bees, resins are not ingested but used within the hive by adult bees exposed to fungal spores. Thus the colony, as the unit of selection, may be responding to infection through self-medication by increasing the number of individuals that forage for resin.     

Resource overlap and possible competition between honey bees and wild bees in central Europe

Evidence for interspecific competition between honey bees and wild bees was studied on 15 calcareous grasslands with respect to: (1) foraging radius of honey bees, (2) overlap in resource use, and (3) possible honey bee effects on species richness and abundance of flower-visiting, ground-nesting and trap-nesting wild bees. The grasslands greatly differed in the number of honey bee colonies within a radius of 2 km and were surrounded by agricultural habitats. The number of flower-visiting honey bees on both potted mustard plants and small grassland patches declined with increasing distance from the nearest apiary and was almost zero at a distance of 1.5–2.0 km. Wild bees were observed visiting 57 plant species, whereas honey bees visited only 24 plant species. Percentage resource overlap between honey bees and wild bees was 45.5%, and Hurlbert’s index of niche overlap was 3.1. In total, 1849 wild bees from 98 species were recorded on the calcareous grasslands. Neither species richness nor abundance of wild bees were negatively correlated with the density of honey bee colonies (within a radius of 2 km) or the density of flower-visiting honey bees per site. Abundance of flower- visiting wild bees was correlated only with the percentage cover of flowering plants. In 240 trap nests, 1292 bee nests with 6066 brood cells were found. Neither the number of bee species nor the number of brood cells per grassland was significantly correlated with the density of honey bees. Significant correlations were found only between the number of brood cells and the percentage cover of shrubs. The number of nest entrances of ground-nesting bees per square metre was not correlated with the density of honey bees but was negatively correlated with the cover of vegetation. Interspecific competition by honey bees for food resources was not shown to be a significant factor determining abundance and species richness of wild bees. Received: 22 March 1999 / Accepted: 24 September 1999     

Chronic Nosema ceranae infection inflicts comprehensive and persistent immunosuppression and accelerated lipid loss in host Apis mellifera honey bees

Nosema ceranae is an intracellular microsporidian parasite of the Asian honey bee Apis cerana and the European honey bee Apis mellifera. Until relatively recently, A. mellifera honey bees were naïve to N. ceranae infection. Symptoms of nosemosis, or Nosema disease, in the infected hosts include immunosuppression, damage to gut epithelium, nutrient and energetic stress, precocious foraging and reduced longevity of infected bees. Links remain unclear between immunosuppression, the symptoms of nutrient and energetic stress, and precocious foraging behavior of hosts. To clarify physiological connections, we inoculated newly emerged A. mellifera adult workers with N. ceranae spores, and over 21?days post inoculation (21?days?pi), gauged infection intensity and quantified expression of genes representing two innate immune pathways, Toll and Imd. Additionally, we measured each host’s whole-body protein, lipids, carbohydrates and quantified respirometric and activity levels. Results show sustained suppression of genes of both humorally regulated immune response pathways after 6?days?pi. At 7?days?pi, elevated protein levels of infected bees may reflect synthesis of antimicrobial peptides from an initial immune response, but the lack of protein gain compared with uninfected bees at 14?days?pi may represent low de novo protein synthesis. Carbohydrate data do not indicate that hosts experience severe metabolic stress related to this nutrient. At 14?days?pi infected honey bees show high respirometric and activity levels, and corresponding lipid loss, suggesting lipids may be used as fuel for increased metabolic demands resulting from infection. Accelerated lipid loss during nurse honey bee behavioral development can have cascading effects on downstream physiology that may lead to precocious foraging, which is a major factor driving colony collapse.