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Urbanization and agricultural intensification continue to reshape landscapes, altering the habitat available to wildlife and threatening species of both economic and conservation concern. The honey bee, Apis mellifera, is a pollinator of economic importance to North American agriculture yet managed colonies are burdened by poor health and high annual mortality. Understanding the factors influencing this species is critical for improving colony health and supporting crop production. We used a nationwide cohort of 638 managed Canadian colonies to study the dominant drivers of colony health and overwintering mortality. We found that fall colony weight – a major predictor of overwintering survival – was strongly associated with landscape composition. Among four broadly defined land cover types, we discovered that urban and forested land covers were the least valuable sources of habitat for colonies, as inferred from fall colony weight measurements. Agricultural land appeared to provide habitat quality of slightly greater value, while herbaceous land cover was most strongly positively associated with fall colony weight. Herbaceous land cover also exhibited an associational effect size which was strongly statistically distinguishable from those of urban and forested land. Our research indicates that recent and ongoing land-use changes exacerbate modern apicultural challenges, and suggests variation in nutrition or floral resource availability plays a major role in modulating honey bee health. Our work highlights the need for additional research investigating whether land use change-associated alterations in floral resource availability increase the potential for resource competition between pollinator species.

The ideal-free distribution and central-place foraging are important ecological models that can explain the distribution of foraging organisms in their environment. However, this model ignores distance-based foraging costs from a central place (hive, nest), whereas central-place foraging ignores competition. Different foraging currencies and cooperation between foragers also create different optimal distributions of foragers, but are limited to a simple two-patch model. We present a hybrid model of the ideal-free distribution that uses realistic competitive effects although accounting for distance-based foraging, and test it using honey bees (Apis mellifera L.) foraging in canola fields (Brassica napus L.). Our simulations show that foragers maximizing efficiency (energy profits ÷losses) prioritize distance to their aggregation more than those maximizing net-rate (energy profits ÷time), and that social foragers move to more distant patches to maximize group benefits, meaning that social foragers do not approach an ideal-free distribution. Simulated efficiency-maximizers had a hump-shaped relationship of trip times with distance, spending shorter amounts of time in both nearby and far-away patches. Canola fields were far more attractive to simulated foragers than semi-natural areas, suggesting limited foraging on semi-natural lands during the bloom period of canola. Finally, we found that the observed distribution of honey bees in canola fields most closely resembled the optimal distribution of solitary efficiency-maximizers. Our model has both theoretical and practical uses, as it allows us to model central-place forager distributions in complex landscapes as well as providing information on appropriate hive stocking rates for agricultural pollination.

Nosema apis and N. ceranae are the two causative agents of Nosema disease in adult honey bees (Apis mellifera L.). Nosema apis has been a recognized parasite for over a century and its epizootiology is well known. In contrast, N. ceranae is an emerging parasite of honey bees, which is now globally prevalent and the dominant Nosema spp. in many parts of the world. Despite this, many gaps in our knowledge exist regarding this species. For example, we do not fully understand all of the routes of transmission of N. ceranae among bees, or how long this parasite is capable of surviving in honey bee colonies. Here we investigated the viability and infectivity of N. ceranae spores in water and 2 M sucrose over time after storage at 33, 20, −12 and −20°C. Spores in both 2 M sucrose and water maintained high viability, except in water at −20°C over the course of the 6-week experiment. Infectivity was variable for spores after storage at all four temperatures, but all were infective at the last time point. The results provide evidence for cold tolerance and suggest that both water and 2 M sucrose (fall bee feed) could act as routes of transmission for N. ceranae. This work also contains information that may help influence management recommendations for the parasite.

Nosema apis and N. ceranae are the two causative agents of Nosema disease in adult honey bees (Apis mellifera L.). Nosema apis has been a recognized parasite for over a century and its epizootiology is well known. In contrast, N. ceranae is an emerging parasite of honey bees, which is now globally prevalent and the dominant Nosema spp. in many parts of the world. Despite this, many gaps in our knowledge exist regarding this species. For example, we do not fully understand all of the routes of transmission of N. ceranae among bees, or how long this parasite is capable of surviving in honey bee colonies. Here we investigated the viability and infectivity of N. ceranae spores in water and 2 M sucrose over time after storage at 33, 20, −12 and −20°C. Spores in both 2 M sucrose and water maintained high viability, except in water at −20°C over the course of the 6-week experiment. Infectivity was variable for spores after storage at all four temperatures, but all were infective at the last time point. The results provide evidence for cold tolerance and suggest that both water and 2 M sucrose (fall bee feed) could act as routes of transmission for N. ceranae. This work also contains information that may help influence management recommendations for the parasite.

Honeybee veterinary medicine is a developing field in Canada and the United States. Veterinarians interested in working with honeybees should develop a comprehensive knowledge base on disease dynamics as it applies to the individual, colony, apiary, and broader honeybee populations. There are currently several governmental, academic, and industry organizations that are carrying out epidemiological-based surveys. Although honeybees face unique challenges in regard to biosecurity, the basic principles still apply. Veterinarians can use their expertise in the area of biosecurity to make improvements to current protocols within the apiary and beekeeping operations.

Honey bees have evolved to use pollen, nectar, and water as their principal food sources. Their success is linked to the establishment of large colonies with one female reproductive member, three distinct social castes, a division of labor among workers, and genetically diverse subfamilies. Colonies also have the ability to recruit and communicate through complex mechanisms including dance language and pheromones. Pheromones produced by the queen maintain social order in the colony and ensure that she remains as the only female to lay eggs. Finally, honey bee colonies reproduce and disperse through a mechanism called swarming.

Honey bees fulfill a critical role as the principal managed pollinator for modern agricultural ecosystems, necessary for the production of many of the world's food crops. The beekeeper must be a knowledgeable manager of bee health, apicultural production systems, and food safety practices. Veterinarians play a vital role in apiculture in supporting beekeepers to treat current and emerging diseases and pests.

Social organisms combat pathogens through individual innate immune responses or through social immunity—behaviors among individuals that limit pathogen transmission within groups. Although we have a relatively detailed understanding of the genetics and evolution of the innate immune system of animals, we know little about social immunity. Addressing this knowledge gap is crucial for understanding how life-history traits influence immunity, and identifying if trade-offs exist between innate and social immunity. Hygienic behavior in the Western honey bee, Apis mellifera, provides an excellent model for investigating the genetics and evolution of social immunity in animals. This heritable, colony-level behavior is performed by nurse bees when they detect and remove infected or dead brood from the colony. We sequenced 125 haploid genomes from two artificially selected highly hygienic populations and a baseline unselected population. Genomic contrasts allowed us to identify a minimum of 73 genes tentatively associated with hygienic behavior. Many genes were within previously discovered QTLs associated with hygienic behavior and were predictive of hygienic behavior within the unselected population. These genes were often involved in neuronal development and sensory perception in solitary insects. We found that genes associated with hygienic behavior have evidence of positive selection within honey bees (Apis), supporting the hypothesis that social immunity contributes to fitness. Our results indicate that genes influencing developmental neurobiology and behavior in solitary insects may have been co-opted to give rise to a novel and adaptive social immune phenotype in honey bees.

Nosema disease is a prominent malady among adult honey bees [Apis mellifera L. (Hymenoptera: Apidae)], caused by the microsporidian parasites, Nosema apis Zander (Microspora: Nosematidae) and N. ceranae Fries et al. 1996. The biology of N. apis is well understood, as this parasite was first described over a century ago. As N. ceranae is an emerging parasite of the honey bee, we do not yet understand how long spores of this parasite survive in honey bee colonies, or all the potential modes of transmission among bees. We investigated the viability and infectivity of N. ceranae spores in honey and on beeswax over time after exposure to 33, 20, −12, and −20°C. Spores in honey maintained viability at freezing temperatures for up to 1 yr and remained viable considerably longer than those on beeswax. Based on this evidence, honey may act as an important reservoir for infective spores to initiate or perpetuate N. ceranae infections in honey bee colonies. This work provides information that may help enhance current management recommendations for apiculturalists.

The recent decline in honey bee (Hymenoptera: Apidae) colony health worldwide has had a significant impact on the beekeeping industry as well as on pollination-dependent crop sectors in North America and Europe. The pollinator crisis has been attributed to many environmental and anthropological factors including less nutrient rich agricultural monocultures, pesticide exposure, new parasite and pathogen infestations as well as beekeeper management and weather. Canadian beekeepers have indicated that issues with honey bee queens are the most significant factor affecting their colony health. In Canada, beekeepers manage colony losses by relying on the importation of foreign bees, particularly queens from warmer climates, to lead new replacement colonies. Unfortunately, the risks associated with imported queens include the introduction of new and potentially resistant pests and diseases, undesirable genetics including bees with limited adaptations to Canada’s unique climate and bees negatively affected by transportation. Importing a large proportion of our queens each year also creates an unsustainable dependency on foreign bee sources, putting our beekeeping and pollination sectors at an even greater risk in the case of border closures and restrictions. Increasing the domestic supply of queens is one mitigation strategy that could provide Canadian beekeepers, farmers and consumers with a greater level of agricultural stability through locally bred, healthier queens. Our study is the first rigorous analysis of the economic feasibility of Canadian queen production. We present the costs of queen production for three case study operations across Canada over two years as well as the profitability implications. Our results show that for a small to medium sized queen production operation in Canada, producing queen cells and mated queens can be profitable. Using a mated queen market price ranging from $30 to $50, a producer selling mated queens could earn a profit of between $2 and $40 per queen depending on price and the cost structure of his operation. If the producer chose to rear queens for his own operation, the cost savings would also be significant as imported queen prices continue to rise. Our case studies reveal that there is potential for both skilled labour acquisition over time in queen production as well as cost savings from economies of scale. Our queen producers also reduced their production costs by re-using materials year to year. Domestic queen production could be one viable strategy to help address the current pollinator crisis in Canada.

Dicyclohexylamine (DCH) is an excipient present in commercial formulations of fumagillin (Fumagilin-B® and Fumidil-B®), an antibiotic which is employed in apiculture for the control of nosema disease. DCH has been demonstrated to be stable in harvested honey; however, its fate in the beehive has not been investigated. In this study, DCH residues were determined in beeswax samples collected from brood chambers and honey supers from various apiaries throughout Alberta. The determination of DCH was performed using a simple extraction procedure followed by low-temperature cleanup and finally analysis with aqueous normal phase liquid chromatography-electrospray ionization tandem mass spectrometry. The method was found to yield high accuracy and precision (94.9 ± 1.7% to 110.8 ± 6.6%) for replicate beeswax samples spiked with 4, 80, 360 and 2,000 μg kg-1 of DCH. With a limit of quantification of 1 μg kg-1, 100% of the 61 beeswax samples analyzed were found to contain residues of DCH ranging from 15 to 6,410 μg kg-1. The results indicate that DCH can accumulate in beeswax when Fumagilin-B® or Fumidil-B® is applied in apiculture and that these concentrations can vary significantly among beekeeping operations.

Insect pollination of flowers should change the within-season allocation of resources in plants. But the nature of this life-history response, particularly regarding allocation to roots, photosynthetic structures, and flowers, is empirically unresolved. This study uses a greenhouse experiment to investigate the effect of insect pollination on the reproductive output of 23 varieties of a globally important crop—canola (Brassica napus). Overall, insect pollination modified the functional characteristics (flower timing & effort, plant size & shape, seed packaging, root biomass) of canola, increasing seed production and quality, and pollinator dependence. Reproductive output and pollinator dependence were defined by strong trait trade-offs, which ranged from more pollinator-dependent plants favouring early reproductive effort, to less pollinator-dependent plants favouring a prolonged phenology with smaller plant size and lower seed quality. Seed production decreased with pollinator dependence in the absence of pollinators. The agricultural preference for hybrid varieties will increase seed production compared to open-pollinated varieties, but, even so, pollinators typically enhance seed production of both types. Our study elucidates how insect pollination alters the character and function of a globally important crop, supporting optimization of yield via intensification of insect pollination, and highlights the beneficial effects of insect pollination early in the season.

We present a novel way to select for highly polygenic traits. For millennia, humans have used observable phenotypes to selectively breed stronger or more productive livestock and crops. Selection on genotype, using single-nucleotide polymorphisms (SNPs) and genome profiling, is also now applied broadly in livestock breeding programs; however, selection on protein/peptide or mRNA expression markers has not yet been proven useful. Here we demonstrate the utility of protein markers to select for disease-resistant hygienic behavior in the European honey bee (Apis mellifera L.). Robust, mechanistically-linked protein expression markers, by integrating cis- and trans- effects from many genomic loci, may overcome limitations of genomic markers to allow for selection. After three generations of selection, the resulting marker-selected stock outperformed an unselected benchmark stock in terms of hygienic behavior, and had improved survival when challenged with a bacterial disease or a parasitic mite, similar to bees selected using a phenotype–based assessment for this trait. This is the first demonstration of the efficacy of protein markers for industrial selective breeding in any agricultural species, plant or animal.

A reversed phase (RP) liquid chromatography tandem mass spectrometric (LC-MS/MS) method was developed and validated to confirm and quantitate trace levels of fumagillin and dicyclohexylamine (DCH) residues in honey destined for human consumption. Chromatographic resolution of DCH and fumagillin, including the latter’s biologically active UV degradation products, was achieved using a C18 analytical column. A deuterium labelled d10-DCH internal standard was synthesized in a one-pot reaction from readily available starting materials and was used to compensate for observed matrix affects when quantitating DCH in honey from different floral origins. Sample extraction was achieved by using reversed phase polymeric solid phase extraction (SPE) with recoveries (±RSD%) at 10, 100 and 500 ng g−1 for 18 replicates at each respective concentration calculated as 105.7 ± 10.8, 100.8 ± 9.3 and 104.3 ± 8.7 for fumagillin and as 104.0 ± 5.9, 98.3 ± 6.9 and 104.0 ± 9.7 for DCH. Domestically produced honey samples (n = 16) were analyzed for fumagillin and DCH, and screened for UV-decomposed fumagillin, without having any prior knowledge of whether fumagillin was used in the bee colonies from which the honeys were produced. No UV-decomposed fumagillin could be detected in any of the samples. Fumagillin was detected in 11 of the 16 samples at levels below 10 ng g−1, and only in 2 samples at levels above 10 ng g−1 (11.9 and 11.6 ng g−1, respectively). DCH was detected in all of the tested samples with concentrations above 10 ng g−1 being observed for 15 out of the 16 samples, with concentrations ranging from 20.0 to 234.6 ng g−1.

Fumagillin is a potent fungal metabolite first isolated from Aspergillus fumigatus. It is widely used in apiculture and human medicine against a variety of microsporidian fungal infections. It has been the subject of research in cancer treatments by employing its angiogenesis inhibitory properties. The toxicity of fumagillin has limited its use for human applications and spurred the development of analogues using structure-activity relationships relating to its angiogenesis properties. These discoveries may hold the key to the development of alternative chemical treatments for use in apiculture. The toxicity of fumagillin to humans is important for beekeeping, because any residues remaining in hive products pose a direct risk to the consumer. The analytical methods published to date measure fumagillin and its decomposition products but overlook the dicyclohexylamine counterion of the salt form widely used in apiculture.

We present a novel way to select for highly polygenic traits. For millennia, humans have used observable phenotypes to selectively breed stronger or more productive livestock and crops. Selection on genotype, using single-nucleotide polymorphisms (SNPs) and quantitative trait loci (QTLs), is also now applied broadly in livestock breeding programs; however, selection on protein or mRNA expression markers have not been proved useful yet. Here we demonstrate the utility of protein markers to select for disease-resistant behaviour in the European honey bees (Apis melliferaL.). Robust, mechanistically-linked protein expression markers, by integrating cis and trans effects from many genomic loci, may overcome limitations of genomic markers to allow for selection. After three generations of selection, the resulting stock performed as well or better than bees selected using phenotype–based assessment of this trait, when challenged with disease. This is the first demonstration of the efficacy of protein markers for selective breeding in any agricultural species, plant or animal.Significance statementThe honey bee has been in the news a lot recently, largely because of world-wide die-offs due to the parasitic Varroa mite, which is becoming resistant to the chemical controls the bee industry uses. In this study, we show that robust expression biomarkers of a disease-resistance trait can be used, in an out-bred population, to select for that trait. After three generations of selection, the resulting stock performed as well or better than bees selected using the phenotypic best method for assessing this trait when challenged with disease. This is the first demonstration of an expression marker for selective breeding in any agricultural species, plant or animal. This also represents a completely novel way to select for highly polygenic traits.

International audience; AbstractFumagilin-B® is the only currently registered chemical treatment available to combat nosema disease in apiculture. Fumagillol, the basic hydrolysis product of fumagillin, two semisynthetic fumagillin analogues, and four in-house purely synthetic compounds which were designed to mimic the mode of action of fumagillin against the methionine aminopeptidase type 2 (MetAP-2) enzyme, was observed to exhibit statistically significant biological activity against Nosema ceranae-infected caged bees. None of these compounds were, however, as effective as Fumagilin-B®. The commercially available thymol and enilconazole also exhibited activity against N. ceranae, with thymol being the most promising chemical treatment other than Fumagilin-B®. High cumulative bee mortality was associated with the therapeutic dosage of Fumagilin-B® during our study, suggesting the need for continued investigation.

International audience; AbstractBoth commercially available fumagillin-based treatments for honey bees (Apis mellifera), Fumagilin-B® as well as Fumidil-B®, contain the reportedly genotoxic and tumorigenic compound dicyclohexylamine (DCH) as the counter ion in the fumagillin-DCH salt. The effect of DCH, purified fumagillin (containing no DCH), and the commercial formulation Fumagilin-B® (containing both fumagillin as well as DCH) on the mortality of caged Nosema ceranae-infected honey bees was investigated. A statistically significant risk of bee mortality associated with oral exposure to DCH was observed. DCH is also known to be significantly more stable than fumagillin in honey under a variety of temperature conditions, both in the presence and absence of light. The presence of DCH in the hive is therefore a potential concern for bee health and also for food safety.

Chalkbrood, caused by Ascosphaera apis (Maassen and Claussen) Olive and Spiltor, is a cosmopolitan fungal disease of honey bee larvae (Apis mellifera L.) for which there is no chemotherapeutic control. We evaluated the efficacy of lysozyme-HCl, an inexpensive food-grade antimicrobial extracted from hen egg white, for the treatment of chalkbrood disease in honey bee colonies. Our study compared three doses of lysozyme-HCl in sugar syrup (600, 3,000, and 6,000 mg) administered weekly for 3 wk among chalkbrood-inoculated colonies, colonies that were inoculated but remained untreated, and colonies neither inoculated or treated. Lysozyme-HCl at the highest dose evaluated was found to suppress development of chalkbrood disease in inoculated colonies to levels observed in uninoculated, untreated colonies, and did not adversely affect adult bee survival or brood production. Honey production was significantly negatively correlated with increased disease severity but there were no significant differences in winter survival among treatment groups. Based on our results, lysozyme-HCl appears to be a promising, safe therapeutic agent for the control of chalkbrood in honey bee colonies.

In 2008 the COLOSS network was formed by honey bee experts from Europe and the USA. The primary objectives set by this scientific network were to explain and to prevent large scale losses of honey bee (Apis mellifera) colonies. In June 2008 COLOSS obtained four years support from the European Union from COST and was designated as COST Action FA0803 – COLOSS (Prevention of honey bee COlony Journal of Apicultural Research 51(1): 100-114 (2012) © IBRA 2012 DOI 10.3896/IBRA.1.51.1.12 Honey bee colony losses in Canada, China, Europe, Israel and Turkey in 2008-10 101 LOSSes). To enable the comparison of loss data between participating countries, a standardized COLOSS questionnaire was developed. Using this questionnaire information on honey bee losses has been collected over two years. Survey data presented in this study were gathered in 2009 from 12 countries and in 2010 from 24 countries. Mean honey bee losses in Europe varied widely, between 7-22% over the 2008-9 winter and between 7-30% over the 2009-10 winter. An important finding is that for all countries which participated in 2008- 9, winter losses in 2009-10 were found to be substantially higher. In 2009-10, winter losses in South East Europe were at such a low level that the factors causing the losses in other parts of Europe were absent, or at a level which did not affect colony survival. The five provinces of China, which were included in 2009-10, showed very low mean (4%) A. mellifera winter losses. In six Canadian provinces, mean winter losses in 2010 varied between 16-25%, losses in Nova Scotia (40%) being exceptionally high. In most countries and in both monitoring years, hobbyist beekeepers (1-50 colonies) experienced higher losses than practitioners with intermediate beekeeping operations (51-500 colonies). This relationship between scale of beekeeping and extent of losses effect was also observed in 2009- 10, but was less pronounced. In Belgium, Italy, the Netherlands and Poland, 2008-9 mean winter losses for beekeepers who reported ‘disappeared’ colonies were significantly higher compared to mean winter losses of beekeepers who did not report ‘disappeared’ colonies. Mean 2008-9 winter losses for those beekeepers in the Netherlands who reported symptoms similar to “Colony Collapse Disorder” (CCD), namely: 1. no dead bees in or surrounding the hive while ; 2. capped brood was present, were significantly higher than mean winter losses for those beekeepers who reported ‘disappeared’ colonies without the presence of capped brood in the empty hives. In the winter of 2009-10 in the majority of participating countries, beekeepers who reported ‘disappeared’ colonies experienced higher winter losses compared with beekeepers, who experienced winter losses but did not report ‘disappeared’ colonies.

International audience; Gains in hygienic behaviour based on maternal selection of queens were evaluated among four commercial beekeeping operations in northern Alberta, Canada over four generations. While the proportion of breeding colonies expressing the trait increased with each subsequent generation, levels of hygienic behaviour among progeny remained relatively unchanged. Estimated breeding values for the final generation of progeny confirmed only a slight effect of selection; the expected average increase in the removal of frozen brood in the next generation was determined to be 0.17% at 24 h and 0.34% at 48 h. This modest increase may be explained by the low (24 h, h 2 = 0.17) to moderate (48 h, h 2 = 0.25) heritability of hygienic behaviour in our population. Our work implies that year-to-year comparisons of breeding colonies may not be an accurate predictor of selection gains within honeybee populations and that honey producers in this region may need to focus selection within elite breeding populations.

Native to sub-Saharan Africa, Aethina tumida Murray (Coleoptera: Nitidulidae) is now an invasive pest of honey bee, Apis mellifera L., colonies in Australia and North America. Knowledge about the introduction (s) of this beetle from Africa into and among the current ranges will elucidate pest populations and invasion pathways and contribute to knowledge of how a parasite expands in new populations. We examined genetic variation in adult beetle samples from the United States, Australia, Canada, and Africa by sequencing a 912-base pair region of the mitochondrial DNA cytochrome c oxidase subunit I gene and screening 10 informative microsatellite loci. One Canadian introduction of small hive beetles can be traced to Australia, whereas the second introduction seems to have come from the United States. Beetles now resident in Australia were of a different African origin than were beetles in North America. North American beetles did not show covariance between two mitochondrial haplotypes and their microsatellite frequencies, suggesting that these beetles have a shared source despite having initial genetic structure within their introduced range. Excellent dispersal of beetles, aided in some cases by migratory beekeeping and the bee trade, seems to lead to panmixis in the introduced populations as well as in Africa.

We report on the first proficiency test in the context of honey bee disease testing on a broad international scale. Honey samples were distributed to 12 participating laboratories for isolation and identification of spores of the etiological agent of American foulbrood, Paenibacillus larvae. Of the 11 laboratories responding to this proficiency testing event, only 6 (54%) provided data that were in full agreement with the previously determined status of the honey samples; eight (72%) laboratories fell within the threshold for qualification as defined a priori in this proficiency test (=level of agreement of at least 90%). Some lessons can now be drawn from the organizational point of view and will certainly improve new initiatives to be taken.

Background The Western honey bee (Apis mellifera L.) is a critical component of human agriculture through its pollination activities. For years, beekeepers have controlled deadly pathogens such as Paenibacillus larvae, Nosema spp. and Varroa destructor with antibiotics and pesticides but widespread chemical resistance is appearing and most beekeepers would prefer to eliminate or reduce the use of in-hive chemicals. While such treatments are likely to still be needed, an alternate management strategy is to identify and select bees with heritable traits that allow them to resist mites and diseases. Breeding such bees is difficult as the tests involved to identify disease-resistance are complicated, time-consuming, expensive and can misidentify desirable genotypes. Additionally, we do not yet fully understand the mechanisms behind social immunity. Here we have set out to discover the molecular mechanism behind hygienic behavior (HB), a trait known to confer disease resistance in bees. Results After confirming that HB could be selectively bred for, we correlated measurements of this behavior with protein expression over a period of three years, at two geographically distinct sites, using several hundred bee colonies. By correlating the expression patterns of individual proteins with HB scores, we identified seven putative biomarkers of HB that survived stringent control for multiple hypothesis testing. Intriguingly, these proteins were all involved in semiochemical sensing (odorant binding proteins), nerve signal transmission or signal decay, indicative of the series of events required to respond to an olfactory signal from dead or diseased larvae. We then used recombinant versions of two odorant-binding proteins to identify the classes of ligands that these proteins might be helping bees detect. Conclusions Our data suggest that neurosensory detection of odors emitted by dead or diseased larvae is the likely mechanism behind a complex and important social immunity behavior that allows bees to co-exist with pathogens. Electronic supplementary material The online version of this article (doi:10.1186/s12864-014-1193-6) contains supplementary material, which is available to authorized users.

An analytical method for the determination of residues of the antibiotic drugs lincomycin and tylosin in honey was developed. The procedure employed a solid-phase extraction for the isolation of lincomycin and tylosin from diluted honey samples. The antibiotic residues were subsequently analyzed by reversed-phase HPLC with atmospheric pressure chemical ionization mass spectrometric detection. Average analyte recoveries for lincomycin and tylosin ranged from 84 to 107% in replicate sets of honey samples fortified with drug concentrations of 0.01, 0.5, and 10 microg/g. The method detection limits were determined to be 0.007 and 0.01 microg/g for lincomycin and tylosin, respectively.

We evaluated the influence of pollen-based cues on the foraging decisions made by honeybees using a series of two-choice bioassays, performed within a highly controlled indoor environment. We examined behaviours related to the choice and collection of pollen by foragers among six floral species and three artificial substrates (pollen analogues). First, we evaluated the responses of honeybees to the odours produced by different pollens (or pollen analogues) and pollen lipid extracts. Honeybees displayed similar levels of preference to the odours produced by all pollen species over those of pollen analogues, with a similar pattern of response shown to their extracts. We then evaluated behaviours of foragers in response to variation in particle size, using soybean meal that was ground and sifted to create a hierarchy of particle size classes. Bees preferred particle sizes below 150 μm, but the greatest response was shown for those particle sizes below 45 μm. We also assayed the effect of varying protein content on the foraging decisions made by bees by mixing soy flour with different proportions of cellulose powder. Foragers, however, were incapable of discriminating protein content. We determined changes in the response of foragers to different levels of handling time using different sized screens through which bees were forced to crawl to reach an attractive pollen odour source. In these tests, pollen-seeking behaviours were seen to decrease with increases in handling time. When odour was presented simultaneously with other stimuli, it was the primary and overriding cue used by bees to select pollen. These results suggest that individual honeybee foragers do not discriminate among pollen sources based on intrinsic differences in quality, but instead evaluate cues that may increase their efficiency of collection and recruitment to such a food resource.

Fumagillin is extensively used to control nosema disease in apiculture. In the commercial formulation, fumagillin is present as a salt in an equimolar quantity with dicyclohexylamine (DCH). In this study DCH was observed to be significantly more resistant to degradation in honey than fumagillin using LC-MS/MS analysis. Observed half-lives for DCH ranged from a minimum of 368 days when kept at 34 °C in darkness, to a maximum of 852 days when stored at 21 °C in darkness. A maximum half-life of 246 days was observed for fumagillin in samples kept in darkness at a temperature of 21 °C. The observed half-life of fumagillin was estimated to be 3 days when exposed to light at 21 °C, and complete decomposition was observed after 30 days under the same conditions. The stability of DCH, combined with its genotoxicity and tumorigenic properties make it an important potential contaminant in honey destined for human consumption.

Individual and colony-level foraging behaviors were evaluated in response to changes in the quantity or nutritional quality of pollen stored within honeybee (Apis mellifera L.) colonies. Colonies were housed in vertical, three-frame observation hives situated inside a building, with entrances leading to the exterior. Before receiving treatments, all colonies were deprived of pollen for 5 days and pollen foragers were marked. In one treatment group, colony pollen reserves were quantitatively manipulated to a low or high level, either by starving colonies of pollen or by providing them with a fully provisioned frame of pollen composed of mixed species. In another treatment group, pollen reserves were qualitatively manipulated by removing pollen stores from colonies and replacing them with low- or high-protein pollen supplements. After applying treatments, foraging rates were measured four times per day and pollen pellets were collected from experienced and inexperienced foragers to determine their weight, species composition, and protein content. Honeybee colonies responded to decreases in the quantity or quality of pollen reserves by increasing the proportion of pollen foragers in their foraging populations, without increasing the overall foraging rate. Manipulation of pollen stores had no effect on the breadth of floral species collected by colonies, or their preferences for the size or protein content of pollen grains. In addition, treatments had no effect on the weight of pollen loads collected by individual foragers or the number of floral species collected per foraging trip. However, significant changes in foraging behavior were detected in relation to the experience level of foragers. Irrespective of treatment group, inexperienced foragers exerted greater effort by collecting heavier pollen loads and also sampled their floral environment more extensively than experienced foragers. Overall, our results indicate that honeybees respond to deficiencies in the quantity or quality of their pollen reserves by increasing the gross amount of pollen returned to the colony, rather than by specializing in collecting pollen with a greater protein content. Individual pollen foragers appear to be insensitive to the quality of pollen they collect, indicating that colony-level feedback is necessary to regulate the flow of protein to and within the colony. Colonies may respond to changes in the quality of their pollen stores by adjusting the numbers of inexperienced to experienced foragers within their foraging populations.

(2010). Honey bee colony losses in Canada. Journal of Apicultural Research: Vol. 49, Honey bee colony losses, pp. 104-106.

SUMMARYA bioassay was developed to test various aspects of the orientation behaviour of the honey bee ectoparasite, Varroa jacobsoni The bioassay arena consisted of a petri dish, 60 mm in diameter, in which live honey bee larvae and previously frozen adults were used as hosts. Bioassays were conducted in a dark incubator at 32°C for 60 min. Greater numbers of mites parasitized worker larvae than drone larvae, but this trend was not significant. Mites of various ages given a choice between nurse bees and fifth instar worker larvae preferred nurse bees at all ages of mites tested, excluding newly emerged mites. When given a choice between a nurse bee and a pollen forager, V. jacobsoni did not show a preference, and similarly when given a choice between a nurse bee and an adult drone no preference was observed. Finally, when given a choice between a nurse bee extracted with hexane and a non—extracted nurse bee, V. jacobsoni preferred the non-extracted host.

Nectar sugar composition and temporal patterns of nectar sugar production were examined in oilseed summer rape (Brassica napus L. ssp. oleifera (Metzg.)) from six open-pollinated, eight pol cytoplasmic male-sterile (CMS) hybrid and seven dominant self-incompatible (SI) hybrid cultivars at three field plot sites in Manitoba. The total sugar content of nectar samples was measured by the Dreywood anthrone reaction for total carbohydrate, and simple sugar composition was determined using an enzymatic bioanalysis for D-glucose, D-fructose and sucrose. Hybrid and open-pollinated cultivar flowers had similar sugar content. Mean total sugar content per flower also did not vary among hybrid breeding systems when compared within individual weeks of the bloom period or within daily sampling periods. However, for all cultivars, total nectar sugar content per flower was lower during the 08:00 and 11:00 h sampling periods and increased to maximum levels during the 14:00 and 16:00 h sampling periods. Significant differences in nectar sugar content were also found in relation to the bloom phenology of the cultivars. Cultivars produced the greatest amount of sugar per flower during the first 2 wk of the bloom period, then sugar production decreased in the third and fourth weeks. Nectar sugar ratios from all cultivars averaged approximately 1:1 glucose:fructose. Nectar glucose content among cultivars was similar but, among breeding systems, CMS cultivars tended to have lower amounts of glucose than SI or open-pollinated cultivars. Selecting for higher total sugar content may produce nectars more attractive to foraging honeybees (Apis mellifera L.), thereby ensuring adequate pollination of hybrid parental lines and F1 hybrid plants. Selecting for lower nectar glucose will produce honeys with more desirable granulation characteristics. Overall, the production and quality of nectar sugar in oilseed rape hybrids are similar to those of open-pollinated cultivars, and are not likely to adversely affect the pollinating activities of honeybees or their potential for honey production. Key words: oilseed summer rape, Brassica napus, honeybees, Apis mellifera, nectar, simple sugars

Background: Disease is a major factor driving the evolution of many organisms. In honey bees, selection for social behavioral responses is the primary adaptive process facilitating disease resistance. One such process, hygienic behavior, enables bees to resist multiple diseases, including the damaging parasitic mite Varroa destructor. The genetic elements and biochemical factors that drive the expression of these adaptations are currently unknown. Proteomics provides a tool to identify proteins that control behavioral processes, and these proteins can be used as biomarkers to aid identification of disease tolerant colonies. Results We sampled a large cohort of commercial queen lineages, recording overall mite infestation, hygiene, and the specific hygienic response to V. destructor. We performed proteome-wide correlation analyses in larval integument and adult antennae, identifying several proteins highly predictive of behavior and reduced hive infestation. In the larva, response to wounding was identified as a key adaptive process leading to reduced infestation, and chitin biosynthesis and immune responses appear to represent important disease resistant adaptations. The speed of hygienic behavior may be underpinned by changes in the antenna proteome, and chemosensory and neurological processes could also provide specificity for detection of V. destructor in antennae. Conclusions Our results provide, for the first time, some insight into how complex behavioural adaptations manifest in the proteome of honey bees. The most important biochemical correlations provide clues as to the underlying molecular mechanisms of social and innate immunity of honey bees. Such changes are indicative of potential divergence in processes controlling the hive-worker maturation.

Background There is a major paradox in our understanding of honey bee immunity: the high population density in a bee colony implies a high rate of disease transmission among individuals, yet bees are predicted to express only two-thirds as many immunity genes as solitary insects, e.g., mosquito or fruit fly. This suggests that the immune response in bees is subdued in favor of social immunity, yet some specific immune factors are up-regulated in response to infection. To explore the response to infection more broadly, we employ mass spectrometry-based proteomics in a quantitative analysis of honey bee larvae infected with the bacterium Paenibacillus larvae. Newly-eclosed bee larvae, in the second stage of their life cycle, are susceptible to this infection, but become progressively more resistant with age. We used this host-pathogen system to probe not only the role of the immune system in responding to a highly evolved infection, but also what other mechanisms might be employed in response to infection. Results Using quantitative proteomics, we compared the hemolymph (insect blood) of five-day old healthy and infected honey bee larvae and found a strong up-regulation of some metabolic enzymes and chaperones, while royal jelly (food) and energy storage proteins were down-regulated. We also observed increased levels of the immune factors prophenoloxidase (proPO), lysozyme and the antimicrobial peptide hymenoptaecin. Furthermore, mass spectrometry evidence suggests that healthy larvae have significant levels of catalytically inactive proPO in the hemolymph that is proteolytically activated upon infection. Phenoloxidase (PO) enzyme activity was undetectable in one or two-day-old larvae and increased dramatically thereafter, paralleling very closely the age-related ability of larvae to resist infection. Conclusion We propose a model for the host response to infection where energy stores and metabolic enzymes are regulated in concert with direct defensive measures, such as the massive enhancement of PO activity.

As a result of the application of tylosin to honey bee colonies for the control of American foulbrood disease, antibiotic residues may exist in honey destined for human consumption. It has been recognized that the parent compound, tylosin A, degrades in acidic media such as honey to yield the antimicrobially active degradation product, desmycosin. Data is presented documenting levels of incurred tylosin and desmycosin in honey resulting from simulated therapeutic applications of a commercial formulation of tylosin during the fall. It is demonstrated that honey destined for human consumption should be analyzed for both tylosin A and desmycosin (tylosin B) rather than the parent antibiotic alone. An analytical method that permits the simultaneous determination of tylosin A and desmycosin in honey using liquid chromatography-tandem mass spectrometry is also presented.

Worldwide, American foulbrood (AFB) is the most devastating bacterial disease of the honey bee (Apis mellifera). Because the distinction between AFB and powdery scale disease is no longer considered valid, the pathogenic agent has recently been reclassified as one species Paenibacillus larvae, eliminating the subspecies designations Paenibacillus larvae subsp. larvae and Paenibacillus larvae subsp. pulvifaciens. The creamy or dark brown, glue-like larval remains of infected larvae continue to provide the most obvious clinical symptom of AFB, although it is not conclusive. Several sensitive and selective culture media are available for isolation of this spore-forming bacterium, with the type of samples that may be utilized for detection of the organism being further expanded. PCR methods for identification and genotyping of the pathogen have now been extensively developed. Nevertheless, biochemical profiling, bacteriophage sensitivity, immunotechniques and microscopy of suspect bacterial strains are entirely adequate for routine identification purposes., Facultad de Ciencias Agrarias y Forestales, Centro de Investigaciones de Fitopatología

A simple and rapid analytical method was developed for the determination of lincomycin and tylosin residues in honey as part of field studies examining the efficacy and target animal safety of these antibiotics to control American foulbrood disease in honey bees. Residues of the antibiotics were determined using liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS). Honey samples were diluted and injected directly into the LC/MS/MS system without additional cleanup by solid-phase extraction or liquid-liquid partitioning. A six-port valve system was utilized to selectively route eluant from the LC column into the mass spectrometer only during a relatively short portion of the chromatographic run corresponding to the elution of the analytes of interest. Minimal contamination of the MS source chamber was observed despite the analysis of large numbers of samples. Using internal standard quantitation, excellent accuracy and precision were obtained with no apparent matrix-to-matrix variation. Based on the analysis of fortified replicates, the mean percent deviation from the theoretical concentration and the percent relative standard deviation were both less than 10% for tylosin over an analytical range of 10–1000 μg/kg. Slightly higher mean percent deviations and relative standard deviations were observed for the analysis of lincomycin in fortified replicate samples. The method detection limits were determined to be 5 and 2 μg/kg for lincomycin and tylosin, respectively. Copyright © 2005 John Wiley & Sons, Ltd.

International audience; Newly-emerged honey bees were placed in cages and fed sucrose syrup and one of the following single-pollen diets: Malus domestica Borkh., Brassica campestris L., Phacelia tanacetifolia L., Melilotus officinalis (L.) Pall., Helianthus annuus L., Pinus banksiana (Lamb.), artificial supplement (Bee-Pro$^{\circledR}$) or nothing. Hypopharyngeal gland protein was determined at intervals of 0, 3, 8 and 14 days and ovary development was visually scored on day 14. The development of hypopharyngeal glands and ovaries varied with diet and, collectively, proved to be sensitive measures of protein utilization and pollen quality. For workers fed 1-year-old Phacelia pollen, protein was utilized in a differential fashion, promoting the development of ovaries over that of hypopharyngeal glands. Development of glands and ovaries was strongly correlated with the amount of protein workers consumed from pollen diets, and to a lesser extent, the crude protein content of diets. Storing pollen for 1 year by freezing did not affect gland or ovary development.; Qualité de différents pollens monofloraux, frais et d'un an, comme nourriture pour les ouvrières d'abeilles domestiques (Apis mellifera L.). Des groupes de 150 abeilles naissantes ont été placées dans des cages en bois, nourries avec une solution de saccharose à 2 M et avec l'un des six pollens suivants : Malus domestica Borkh., Brassica campestris L., Phacelia tanacetifolia L., Melilotus officinalis (L.) Pall., Helianthus annuus L. et Pinus banksiana (Lamb.). Un succédané commercial de pollen, Bee-Pro$^{\circledR}$, et un régime sans pollen ont également été testés. Le pollen fourni aux abeilles encagées était soit du pollen fraîchement récolté, soit du pollen congelé à $-30~^\circ$C pendant un an et conservé dans des récipients en verre sous atmosphère réduite en oxygène. Au début de l'expérience, puis aux jours J3, J8 et J14, dix abeilles ont été prélevées dans chaque cage et leurs glandes hypopharyngiennes extraites. La teneur totale en protéines des glandes a été déterminée par le test de Bradford et utilisée comme mesure du développement et de la taille des glandes. A la fin de l'expérience (J14), 25 abeilles de chaque cage ont été disséquées et leur développement ovarien classé visuellement de 0 à 4. Le développement des glandes hypopharyngiennes et des ovaires a varié en fonction du régime et s'est comporté, dans l'ensemble, comme une mesure fiable et sensible de l'utilisation des protéines et de la qualité du pollen. Pour les ouvrières nourries avec du pollen de phacélie d'un an, les protéines ont été utilisées différemment : elles ont plus favorisé le développement des ovaires que celui des glandes hypopharyngiennes. Le développement des ovaires et des glandes était fortement corrélé avec la quantité de protéines consommées issues du pollen et, à un moindre degré, avec la teneur brute en protéines des pollens. La conservation du pollen par congélation durant un an n'a eu aucun effet sur le développement des glandes ni des ovaires. Le pollen de tournesol et le succédané Bee-Pro$^{\circledR}$ devraient être complétés par d'autres espèces de pollen lorsqu'ils sont donnés en nourrissement ; le pollen de pin devrait être évité.