Occupancy modelling has received increasing attention as a tool for differentiating between true absence and non-detection in biodiversity data. This is thought to be particularly useful when a species of interest is spread out over a large area and sampling is constrained. We used occupancy modelling to estimate the probability of three phylogenetically independent pairs of native-introduced species [Megachile campanulae (Robertson)-Megachile rotundata (Fab.), Megachile pugnata Say-Megachile centuncularis (L.), Osmia pumila Cresson-Osmia caerulescens (L.)] (Apoidea: Megachilidae) being present when repeated sampling did not always find them. Our study occurred along a gradient of urbanization and used nest boxes (bee hotels) set up over three consecutive years. Occupancy modelling discovered different patterns to those obtained by species detection and abundance-based data alone. For example, it predicted that the species that was ranked 4th in terms of detection actually had the greatest occupancy among all six species. The native M. pugnata had decreased occupancy with increasing building footprint and a similar but not significant pattern was found for the native O. pumila. Two introduced bees (M. rotundata and M. centuncularis), and one native (M. campanulae) had modelled occupancy values that increased with increasing urbanization. Occupancy probability differed among urban green space types for three of six bee species, with values for two native species (M. campanulae and O. pumila) being highest in home gardens and that for the exotic O. caerulescens being highest in community gardens. The combination of occupancy modelling with analysis of habitat variables as an augmentation to detection and abundance-based sampling is suggested to be the best way to ensure that urban habitat management results in the desired outcomes.

A well-documented phenomenon among social insects is that brain changes occur prior to or at the onset of certain experiences, potentially serving to prime the brain for specific tasks. This insight comes almost exclusively from studies considering developmental maturation in females. As a result, it is unclear whether age-related brain plasticity is consistent across sexes, and to what extent developmental patterns differ. Using confocal microscopy and volumetric analyses, we investigated age-related brain changes coinciding with sexual maturation in the males of the facultatively eusocial sweat bee, Megalopta genalis, and the obligately eusocial bumble bee, Bombus impatiens. We compared volumetric measurements between newly eclosed and reproductively mature males kept isolated in the lab. We found expansion of the mushroom bodies-brain regions associated with learning and memory-with maturation, which were consistent across both species. This age-related plasticity may, therefore, play a functionally-relevant role in preparing male bees for mating, and suggests that developmentally-driven neural restructuring can occur in males, even in species where it is absent in females.

Gonadotropic hormones coordinate processes in diverse tissues regulating animal reproductive physiology and behavior. Juvenile hormone (JH) is the ancient and most common gonadotropin in insects, but not in advanced eusocial honey bees and some ants. To start probing the evolutionary basis of this change, we combined endocrine manipulations, transcriptomics, and behavioral analyses to study JH regulated processes in a bumble bee showing a relatively simple level of eusociality. We found that in worker fat body, more JH-regulated genes were up- rather than down-regulated, and enriched for metabolic and biosynthetic pathways. This transcriptomic pattern is consistent with earlier evidence that JH is the major gonadotropin in bumble bees. In the brain, more JH-regulated genes were down- rather than up-regulated and enriched for protein turnover pathways. Brain ribosomal protein gene expression shows a similar trend of downregulation in dominant workers, which naturally have high JH titers. In other species, similar downregulation of protein turnover is found in aging brains or under stress, associated with compromised long-term memory and health. These findings suggest a previously unknown gonadotropin-mediated tradeoff. Analysis of published data reveals no such downregulation of protein turnover pathways in the brain of honey bee workers, which exhibit more complex eusociality and in which JH is not a gonadotropin but rather regulates division of labor. These results suggest that the evolution of complex eusociality in honey bees was associated with modifications in hormonal signalling supporting extended and extremely high fertility while reducing the ancient costs of high gonadotropin titers to the brain.

Surveys of 16S rDNA sequences from the honey bee, Apis mellifera, have revealed the presence of eight distinctive bacterial phylotypes in intestinal tracts of adult worker bees. Because previous studies have been limited to relatively few sequences from samples pooled from multiple hosts, the extent of variation in this microbiota among individuals within and between colonies and locations has been unclear. We surveyed the gut microbiota of 40 individual workers from two sites, Arizona and Maryland USA, sampling four colonies per site. Universal primers were used to amplify regions of 16S ribosomal RNA genes, and amplicons were sequenced using 454 pyrotag methods, enabling analysis of about 330,000 bacterial reads. Over 99% of these sequences belonged to clusters for which the first blastn hits in GenBank were members of the known bee phylotypes. Four phylotypes, one within Gammaproteobacteria (corresponding to "Candidatus Gilliamella apicola") one within Betaproteobacteria ("Candidatus Snodgrassella alvi"), and two within Lactobacillus, were present in every bee, though their frequencies varied. The same typical bacterial phylotypes were present in all colonies and at both sites. Community profiles differed significantly among colonies and between sites, mostly due to the presence in some Arizona colonies of two species of Enterobacteriaceae not retrieved previously from bees. Analysis of Sanger sequences of rRNA of the Snodgrassella and Gilliamella phylotypes revealed that single bees contain numerous distinct strains of each phylotype. Strains showed some differentiation between localities, especially for the Snodgrassella phylotype.

Eusociality represents a major transition in evolution and is typified by cooperative brood care and reproductive division of labor between generations. In bees, this division of labor allows queens and workers to phenotypically specialize. Worker traits associated with helping are thought to be crucial to the fitness of a eusocial lineage, and recent studies of honey bees (genus Apis) have found that adaptively evolving genes often have worker-biased expression patterns. It is unclear however if worker-biased genes are disproportionately acted on by strong positive selection in all eusocial insects. We undertook a comparative population genomics study of bumble bees (Bombus) and honey bees to quantify natural selection on queen- and worker-biased genes across two levels of social complexity. Despite sharing a common eusocial ancestor, genes, and gene groups with the highest levels of positive selection were often unique within each genus, indicating that life history and the environment, but not sociality per se, drives patterns of adaptive molecular evolution. We uncovered differences in the contribution of queen- and worker-biased genes to adaptive evolution in bumble bees versus honey bees. Unlike honey bees, where worker-biased genes are enriched for signs of adaptive evolution, genes experiencing positive selection in bumble bees were predominately expressed by reproductive foundresses during the initial solitary-founding stage of colonies. Our study suggests that solitary founding is a major selective pressure and that the loss of queen totipotency may cause a change in the architecture of selective pressures upon the social insect genome.

Increasingly invertebrates are being used to investigate the molecular and cellular effects of drugs of abuse to explore basic mechanisms of addiction. However, in mammals the principle factors contributing to addiction are long-term adaptive responses to repeated drug use. Here we examined whether adaptive responses to cocaine are also seen in invertebrates using the honey bee model system. Repeated topical treatment with a low dose of cocaine rendered bees resistant to the deleterious motor effects of a higher cocaine dose, indicating the development of physiological tolerance to cocaine in bees. Cocaine inhibits biogenic amine reuptake transporters, but neither acute nor repeated cocaine treatments caused measurable changes in levels of biogenic amines measured in whole bee brains. Our data show clear short and long-term behavioural responses of bees to cocaine administration, but caution that, despite the small size of the bee brain, measures of biogenic amines conducted at the whole-brain level may not reveal neurochemical effects of the drug.

The highly social (eusocial) corbiculate bees, comprising the honey bees, bumble bees, and stingless bees, are ubiquitous insect pollinators that fulfill critical roles in ecosystem services and human agriculture. Here, we conduct wide sampling across the phylogeny of these corbiculate bees and reveal a dynamic evolutionary history behind their microbiota, marked by multiple gains and losses of gut associates, the presence of generalist as well as host-specific strains, and patterns of diversification driven, in part, by host ecology (for example, colony size). Across four continents, we found that different host species have distinct gut communities, largely independent of geography or sympatry. Nonetheless, their microbiota has a shared heritage: The emergence of the eusocial corbiculate bees from solitary ancestors appears to coincide with the acquisition of five core gut bacterial lineages, supporting the hypothesis that host sociality facilitates the development and maintenance of specialized microbiomes.

Deformed wing virus (DWV) is a persistent pathogen of European honey bees and the major contributor to overwintering colony losses. The prevalence of DWV in honey bees has led to significant concerns about spillover of the virus to other pollinating species. Bumble bees are both a major group of wild and commercially-reared pollinators. Several studies have reported pathogen spillover of DWV from honey bees to bumble bees, but evidence of a sustained viral infection characterized by virus replication and accumulation has yet to be demonstrated. Here we investigate the infectivity and transmission of DWV in bumble bees using the buff-tailed bumble bee Bombus terrestris as a model. We apply a reverse genetics approach combined with controlled laboratory conditions to detect and monitor DWV infection. A novel reverse genetics system for three representative DWV variants, including the two master variants of DWV-type A and B-was used. Our results directly confirm DWV replication in bumble bees but also demonstrate striking resistance to infection by certain transmission routes. Bumble bees may support DWV replication but it is not clear how infection could occur under natural environmental conditions.

Nosematosis is currently a frequently discussed honey bee disease caused by two types of Microsporidia: Nosema apis and Nosema ceranae. Nosematosis as an intestinal disease caused by these species is one of the main factors associated with the weakening and loss of hives, with none of the stressors acting in isolation and all having an important synergistic or additive effect on the occurrence of parasitic infection. The most important factors are exposure to pesticides and nutritional stress, both worsening the immune response. Honey bees Apis mellifera become more susceptible to parasites and subsequently the disease manifests itself. Choosing the right laboratory diagnostics is important to determine the prevalence of both species. Our review summarizes the most commonly used methodologies, especially polymerase chain reaction (PCR), which is a reliable method for detecting nosematosis, as well as for distinguishing between the two species causing the disease.

When it comes to the brain, bigger is generally considered better in terms of cognitive performance. While this notion is supported by studies of birds and primates showing that larger brains improve learning capacity, similar evidence is surprisingly lacking for invertebrates. Although the brain of invertebrates is smaller and simpler than that of vertebrates, recent work in insects has revealed enormous variation in size across species. Here, we ask whether bee species that have larger brains also have higher learning abilities. We conducted an experiment in which field-collected individuals had to associate an unconditioned stimulus (sucrose) with a conditioned stimulus (coloured strip). We found that most species can learn to associate a colour with a reward, yet some do so better than others. These differences in learning were related to brain size: species with larger brains-both absolute and relative to body size-exhibited enhanced performance to learn the reward-colour association. Our finding highlights the functional significance of brain size in insects, filling a major gap in our understanding of brain evolution and opening new opportunities for future research.

Bees are essential pollinators of many plants in natural ecosystems and agricultural crops alike. In recent years the decline and disappearance of bee species in the wild and the collapse of honey bee colonies have concerned ecologists and apiculturalists, who search for causes and solutions to this problem. Whilst biological factors such as viral diseases, mite and parasite infections are undoubtedly involved, it is also evident that pesticides applied to agricultural crops have a negative impact on bees. Most risk assessments have focused on direct acute exposure of bees to agrochemicals from spray drift. However, the large number of pesticide residues found in pollen and honey demand a thorough evaluation of all residual compounds so as to identify those of highest risk to bees. Using data from recent residue surveys and toxicity of pesticides to honey and bumble bees, a comprehensive evaluation of risks under current exposure conditions is presented here. Standard risk assessments are complemented with new approaches that take into account time-cumulative effects over time, especially with dietary exposures. Whilst overall risks appear to be low, our analysis indicates that residues of pyrethroid and neonicotinoid insecticides pose the highest risk by contact exposure of bees with contaminated pollen. However, the synergism of ergosterol inhibiting fungicides with those two classes of insecticides results in much higher risks in spite of the low prevalence of their combined residues. Risks by ingestion of contaminated pollen and honey are of some concern for systemic insecticides, particularly imidacloprid and thiamethoxam, chlorpyrifos and the mixtures of cyhalothrin and ergosterol inhibiting fungicides. More attention should be paid to specific residue mixtures that may result in synergistic toxicity to bees.

Evolutionary transitions to a social lifestyle in insects are associated with lineage-specific changes in gene expression, but the key nodes that drive these regulatory changes are unknown. We examined the relationship between social organization and lineage-specific microRNAs (miRNAs). Genome scans across 12 bee species showed that miRNA copy-number is mostly conserved and not associated with sociality. However, deep sequencing of small RNAs in six bee species revealed a substantial proportion (20-35%) of detected miRNAs had lineage-specific expression in the brain, 24-72% of which did not have homologues in other species. Lineage-specific miRNAs disproportionately target lineage-specific genes, and have lower expression levels than shared miRNAs. The predicted targets of lineage-specific miRNAs are not enriched for genes with caste-biased expression or genes under positive selection in social species. Together, these results suggest that novel miRNAs may coevolve with novel genes, and thus contribute to lineage-specific patterns of evolution in bees, but do not appear to have significant influence on social evolution. Our analyses also support the hypothesis that many new miRNAs are purged by selection due to deleterious effects on mRNA targets, and suggest genome structure is not as influential in regulating bee miRNA evolution as has been shown for mammalian miRNAs.

No abstract available

Systemic RNAi, initiated by double-stranded RNA (dsRNA) ingestion, has been reported in diverse invertebrates, including honey bees, demonstrating environmental RNA uptake that undermines homologous gene expression. However, the question why any organism would take up RNA from the environment has remained largely unanswered. Here, we report on horizontal RNA flow among honey bees mediated by secretion and ingestion of worker and royal jelly diets. We demonstrate that transmission of jelly-secreted dsRNA to larvae is biologically active and triggers gene knockdown that lasts into adulthood. Worker and royal jellies harbor differential naturally occurring RNA populations. Jelly RNAs corresponded to honey bee protein-coding genes, transposable elements, and non-coding RNA, as well as bacteria, fungi, and viruses. These results reveal an inherent property of honey bees to share RNA among individuals and generations. Our findings suggest a transmissible RNA pathway, playing a role in social immunity and signaling between members of the hive.

Similar to many other animals, the honey bee Apis mellifera relies on a beneficial gut microbiota for regulation of immune homeostasis. Honey bees exposed to agrochemicals, such as the herbicide glyphosate or antibiotics, usually exhibit dysbiosis and increased susceptibility to bacterial infection. Considering the relevance of the microbiota-immunity axis for host health, we hypothesized that glyphosate exposure could potentially affect other components of the honey bee physiology, such as the immune system.

Fumagillin is the only antibiotic approved for control of nosema disease in honey bees and has been extensively used in United States apiculture for more than 50 years for control of Nosema apis. It is toxic to mammals and must be applied seasonally and with caution to avoid residues in honey. Fumagillin degrades or is diluted in hives over the foraging season, exposing bees and the microsporidia to declining concentrations of the drug. We showed that spore production by Nosema ceranae, an emerging microsporidian pathogen in honey bees, increased in response to declining fumagillin concentrations, up to 100% higher than that of infected bees that have not been exposed to fumagillin. N. apis spore production was also higher, although not significantly so. Fumagillin inhibits the enzyme methionine aminopeptidase2 (MetAP2) in eukaryotic cells and interferes with protein modifications necessary for normal cell function. We sequenced the MetAP2 gene for apid Nosema species and determined that, although susceptibility to fumagillin differs among species, there are no apparent differences in fumagillin binding sites. Protein assays of uninfected bees showed that fumagillin altered structural and metabolic proteins in honey bee midgut tissues at concentrations that do not suppress microsporidia reproduction. The microsporidia, particularly N. ceranae, are apparently released from the suppressive effects of fumagillin at concentrations that continue to impact honey bee physiology. The current application protocol for fumagillin may exacerbate N. ceranae infection rather than suppress it.

Although few honey bee diseases are known to be caused by bacteria, pathogens of adult worker bees may be underrecognized due to social immunity mechanisms. Specifically, infected adult bees typically abandon the hive or are removed by guards. Serratia marcescens, an opportunistic pathogen of many plants and animals, is often present at low abundance in the guts of honey bee workers and has recently been isolated from Varroa mites and from the hemolymph of dead and dying honey bees. However, the severity and prevalence of S. marcescens pathogenicity in honey bees have not been fully investigated. Here we characterized three S. marcescens strains isolated from the guts of honey bees and one previously isolated from hemolymph. In vivo tests confirmed that S. marcescens is pathogenic in workers. All strains caused mortality when a few cells were injected into the hemocoel, and the gut-isolated strains caused mortality when administered orally. In vitro assays and comparative genomics identified possible mechanisms of virulence of gut-associated strains. Expression of antimicrobial peptide and phenoloxidase genes was not elevated following infection, suggesting that these S. marcescens strains derived from honey bees can evade the immune response in their hosts. Finally, surveys from four locations in the United States indicated the presence of S. marcescens in the guts of over 60% of the worker bees evaluated. Taken together, these results suggest that S. marcescens is a widespread opportunistic pathogen of adult honey bees and that it may be highly virulent under some conditions such as perturbation of the normal gut microbiota or the presence of Varroa mites that puncture the integument, thereby enabling entry of bacterial cells.IMPORTANCE Recently, it has become apparent that multiple factors are responsible for honey bee decline, including climate change, pests and pathogens, pesticides, and loss of foraging habitat. Of the large number of pathogens known to infect honey bees, very few are bacteria. Because adult workers abandon hives when diseased, many of their pathogens may go unnoticed. Here we characterized the virulence of Serratia marcescens strains isolated from honey bee guts and hemolymph. Our results indicate that S. marcescens, an opportunistic pathogen of many plants and animals, including humans, is a virulent opportunistic pathogen of honey bees, which could contribute to bee decline. Aside from the implications for honey bee health, the discovery of pathogenic S. marcescens strains in honey bees presents an opportunity to better understand how opportunistic pathogens infect and invade hosts.

Eusocial insects offer a unique opportunity to analyze the evolution of body size differences between sexes in relation to social environment. The workers, being sterile females, are not subject to selection for reproductive function providing a natural control for parsing the effects of selection on reproductive function (i.e., sexual and fecundity selection) from other kinds of natural selection. Patterns of sexual size dimorphism (SSD) and testing of Rensch's rule controlling for phylogenetic effects were analyzed in the Meliponini or stingless bees. Theory predicts that queens may exhibit higher selection for fecundity in eusocial taxa, but contrary to this, we found mixed patterns of SSD in Meliponini. Non-Melipona species generally have a female-biased SSD, while all analyzed species of Melipona showed a male-biased SSD, indicating that the direction and magnitude of the selective pressures do not operate in the same way for all members of this taxon. The phylogenetic regressions revealed that the rate of divergence has not differed between the two castes of females and the males, that is, stingless bees do not seem to follow Rensch's rule (a slope >1), adding this highly eusocial taxon to the various solitary insect taxa not conforming with it. Noteworthy, when Melipona was removed from the analysis, the phylogenetic regressions for the thorax width of males on queens had a slope significantly smaller than 1, suggesting that the evolutionary divergence has been larger in queens than males, and could be explained by stronger selection on female fecundity only in non-Melipona species. Our results in the stingless bees question the classical explanation of female-biased SSD via fecundity and provide a first evidence of a more complex determination of SSD in highly eusocial species. We suggest that in highly eusocial taxa, additional selection mechanisms, possibly related to individual and colonial interests, could influence the evolution of environmentally determined traits such as body size.

Honey bees perform robustly in different conditioning paradigms. This makes them excellent candidates for studying mechanisms of learning and memory at both an individual and a population level. Here we introduce a novel method of honey bee conditioning: APIS, the Automatic Performance Index System. In an enclosed walking arena where the interior is covered with an electric grid, presentation of odors from either end can be combined with weak electric shocks to form aversive associations. To quantify behavioral responses, we continuously monitor the movement of the bee by an automatic tracking system. We found that escapes from one side to the other, changes in velocity as well as distance and time spent away from the punished odor are suitable parameters to describe the bee's learning capabilities. Our data show that in a short-term memory test the response rate for the conditioned stimulus (CS) in APIS correlates well with response rate obtained from conventional Proboscis Extension Response (PER)-conditioning. Additionally, we discovered that bees modulate their behavior to aversively learned odors by reducing their rate, speed and magnitude of escapes and that both generalization and extinction seem to be different between appetitive and aversive stimuli. The advantages of this automatic system make it ideal for assessing learning rates in a standardized and convenient way, and its flexibility adds to the toolbox for studying honey bee behavior.

Although the honeybee is a crucial agricultural agent and a prominent scientific model organism, crucial aspects of its reproductive behaviour are still unknown. During the mating season, honeybee males, the drones, gather in congregations 10-40 m above ground. Converging evidence suggests that drones emit a pheromone that can attract other drones, thereby increasing the size of the congregation. Virgin queens join the vicinity of the congregation after it has formed, and mate with as many as 20 males in mid-air. It is still unclear which sensory cues help virgin queens find drone congregations in the first place. Beside visual cues for long-range orientation, queens may use olfactory cues. We thus tested virgin queens' olfactory orientation on a walking simulator in which they have full control over odour stimulation. We show that sexually-mature virgin queens are attracted to the odour bouquet from a group of living drones. They are not attracted to the bouquet from a group of workers. In addition, non-sexually receptive females (workers) of the same age are not attracted to the drone odour bouquet. Interpreted in the context of mating, these results may suggest that virgin queens use volatile olfactory cues from the drones to find the congregations.