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.

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.

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.

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.

Feral honey bee populations have been reported to be in decline due to the spread of Varroa destructor, an ectoparasitic mite that when left uncontrolled leads to virus build-up and colony death. While pests and diseases are known causes of large-scale managed honey bee colony losses, no studies to date have considered the wider pathogen burden in feral colonies, primarily due to the difficulty in locating and sampling colonies, which often nest in inaccessible locations such as church spires and tree tops. In addition, little is known about the provenance of feral colonies and whether they represent a reservoir of Varroa tolerant material that could be used in apiculture. Samples of forager bees were collected from paired feral and managed honey bee colonies and screened for the presence of ten honey bee pathogens and pests using qPCR. Prevalence and quantity was similar between the two groups for the majority of pathogens, however feral honey bees contained a significantly higher level of deformed wing virus than managed honey bee colonies. An assessment of the honey bee race was completed for each colony using three measures of wing venation. There were no apparent differences in wing morphometry between feral and managed colonies, suggesting feral colonies could simply be escapees from the managed population. Interestingly, managed honey bee colonies not treated for Varroa showed similar, potentially lethal levels of deformed wing virus to that of feral colonies. The potential for such findings to explain the large fall in the feral population and the wider context of the importance of feral colonies as potential pathogen reservoirs is discussed.

Extreme temperature exposure can reduce stored sperm viability within queen honey bees; however, little is known about how thermal stress may directly impact queen performance or other maternal quality metrics. Here, in a blind field trial, we recorded laying pattern, queen mass, and average callow worker mass before and after exposing queens to a cold temperature (4°C, 2 h), hot temperature (42°C, 2 h), and hive temperature (33°C, control). We measured sperm viability at experiment termination, and investigated potential vertical effects of maternal temperature stress on embryos using proteomics. We found that cold stress, but not heat stress, reduced stored sperm viability; however, we found no significant effect of temperature stress on any other recorded metrics (queen mass, average callow worker mass, laying patterns, the egg proteome, and queen spermathecal fluid proteome). Previously determined candidate heat and cold stress biomarkers were not differentially expressed in stressed queens, indicating that these markers only have short-term post-stress diagnostic utility. Combined with variable sperm viability responses to temperature stress reported in different studies, these data also suggest that there is substantial variation in temperature tolerance, with respect to impacts on fertility, amongst queens. Future research should aim to quantify the variation and heritability of temperature tolerance, particularly heat, in different populations of queens in an effort to promote queen resilience.

In general, honey bees (Apis mellifera L.) feed on honey produced from collected nectar. In the absence of nectar, during certain times of the year or in monocultural landscapes, honey bees forage on honeydew. Honeydew is excreted by different herbivores of the order Hemiptera that consume phloem sap of plant species. In comparison to nectar, honeydew is composed of a higher variety of sugars and additional sugars with higher molecular weight, like the trisaccharide melezitose that can be a major constituent of honeydew. However, melezitose-containing honey is known to cause malnutrition in overwintering honey bees. Following the hypothesis that melezitose may be the cause for the so called 'honeydew flow disease', three independent feeding experiments with caged bees were conducted in consecutive years. Bees fed with melezitose showed increased food uptake, higher gut weights and elevated mortality compared to bees fed a control diet. Moreover, severe disease symptoms, such as swollen abdomen, abdomen tipping and impaired movement were observed in melezitose-fed bees. 16S-amplicon sequencing indicated that the melezitose diet changed the species composition of the lactic acid bacteria community within the gut microbiota. Based on these results, we conclude that melezitose cannot be easily digested by the host and may accumulate in the hindgut. Within cages or during winter, when there is no opportunity for excretion, the accumulated melezitose can cause severe intestinal symptoms and death of the bees, probably as result of poor melezitose metabolism capabilities in the intestinal microbiota. These findings confirm the causal relation between the trisaccharide melezitose and the honeydew flow disease and indicate a possible mechanism of pathogenesis.

Bumble bees and other wild bees are important pollinators of wild flowers and several cultivated crop plants, and have declined in diversity and abundance during the last decades. The main cause of the decline is believed to be habitat destruction and fragmentation associated with urbanization and agricultural intensification. Urbanization is a process that involves dramatic and persistent changes of the landscape, increasing the amount of built-up areas while decreasing the amount of green areas. However, urban green areas can also provide suitable alternative habitats for wild bees.

Organic volatiles produced by fruits can result in overestimation of sweetness by humans, but it is unknown if a comparable phenomenon occurs in other species. Honey bees collect nectar of varying sweetness at different flowering plants. Bees discriminate sugar concentration and generally prefer higher concentrations; they encounter floral volatiles as they collect nectar, suggesting that they, like humans, could be susceptible to sweetness enhancement by odorant. In this study, limonene, linalool, geraniol, and 6-methyl-5-hepten-2-ol were tested for their ability to alter behaviors related to perception of sweetness by honey bees. Honey bees were tested in the laboratory using proboscis extension response-based assays and in the field using feeder-based assays. In the laboratory assays, 6-methyl-5-hepten-2-ol and geraniol, but neither linalool nor limonene, significantly increased responses to low concentrations of sucrose compared with no odorant conditions in 15-day and 25-day-old adult worker honey bees, but not in 35-day-old bees. Limonene reduced responding in 15-day-old bees, but not in the older bees. There was no odorant-based difference in performance in field assays comparing geraniol and limonene with a no odorant control. The interaction of the tested plant volatiles with sucrose concentration revealed in laboratory testing is therefore unlikely to be a major determinant of nectar choice by honey bees foraging under natural conditions. Because geraniol is a component of honey bee Nasonov gland pheromone as well as a floral volatile, its impact on responses in the laboratory may reflect conveyance of different information than the other odorants tested.

A long-standing controversy in bee social evolution concerns whether highly eusocial behavior has evolved once or twice within the corbiculate Apidae. Corbiculate bees include the highly eusocial honey bees and stingless bees, the primitively eusocial bumble bees, and the predominantly solitary or communal orchid bees. Here we use a model-based approach to reconstruct the evolutionary history of eusociality and date the antiquity of eusocial behavior in apid bees, using a recent molecular phylogeny of the Apidae. We conclude that eusociality evolved once in the common ancestor of the corbiculate Apidae, advanced eusociality evolved independently in the honey and stingless bees, and that eusociality was lost in the orchid bees. Fossil-calibrated divergence time estimates reveal that eusociality first evolved at least 87 Mya (78 to 95 Mya) in the corbiculates, much earlier than in other groups of bees with less complex social behavior. These results provide a robust new evolutionary framework for studies of the organization and genetic basis of social behavior in honey bees and their relatives.

Given the role of infectious disease in global pollinator decline, there is a need to understand factors that shape pathogen susceptibility and transmission in bees. Here we ask how urbanization affects the immune response and pathogen load of feral and managed colonies of honey bees (Apis mellifera Linnaeus), the predominant economically important pollinator worldwide. Using quantitative real-time PCR, we measured expression of 4 immune genes and relative abundance of 10 honey bee pathogens. We also measured worker survival in a laboratory bioassay. We found that pathogen pressure on honey bees increased with urbanization and management, and the probability of worker survival declined 3-fold along our urbanization gradient. The effect of management on pathogens appears to be mediated by immunity, with feral bees expressing immune genes at nearly twice the levels of managed bees following an immune challenge. The effect of urbanization, however, was not linked with immunity; instead, urbanization may favor viability and transmission of some disease agents. Feral colonies, with lower disease burdens and stronger immune responses, may illuminate ways to improve honey bee management. The previously unexamined effects of urbanization on honey-bee disease are concerning, suggesting that urban areas may favor problematic diseases of pollinators.

Honeybees (Apis mellifera) discriminate multiple object features such as colour, pattern and 2D shape, but it remains unknown whether and how bees recover three-dimensional shape. Here we show that bees can recognize objects by their three-dimensional form, whereby they employ an active strategy to uncover the depth profiles. We trained individual, free flying honeybees to collect sugar water from small three-dimensional objects made of styrofoam (sphere, cylinder, cuboids) or folded paper (convex, concave, planar) and found that bees can easily discriminate between these stimuli. We also tested possible strategies employed by the bees to uncover the depth profiles. For the card stimuli, we excluded overall shape and pictorial features (shading, texture gradients) as cues for discrimination. Lacking sufficient stereo vision, bees are known to use speed gradients in optic flow to detect edges; could the bees apply this strategy also to recover the fine details of a surface depth profile? Analysing the bees' flight tracks in front of the stimuli revealed specific combinations of flight maneuvers (lateral translations in combination with yaw rotations), which are particularly suitable to extract depth cues from motion parallax. We modelled the generated optic flow and found characteristic patterns of angular displacement corresponding to the depth profiles of our stimuli: optic flow patterns from pure translations successfully recovered depth relations from the magnitude of angular displacements, additional rotation provided robust depth information based on the direction of the displacements; thus, the bees flight maneuvers may reflect an optimized visuo-motor strategy to extract depth structure from motion signals. The robustness and simplicity of this strategy offers an efficient solution for 3D-object-recognition without stereo vision, and could be employed by other flying insects, or mobile robots.

Spray adjuvants are often applied to crops in conjunction with agricultural pesticides in order to boost the efficacy of the active ingredient(s). The adjuvants themselves are largely assumed to be biologically inert and are therefore subject to minimal scrutiny and toxicological testing by regulatory agencies. Honey bees are exposed to a wide array of pesticides as they conduct normal foraging operations, meaning that they are likely exposed to spray adjuvants as well. It was previously unknown whether these agrochemicals have any deleterious effects on honey bee behavior.

Brain structure and learning capacities both vary with experience, but the mechanistic link between them is unclear. Here, we investigated whether experience-dependent variability in learning performance can be explained by neuroplasticity in foraging honey bees. The mushroom bodies (MBs) are a brain center necessary for ambiguous olfactory learning tasks such as reversal learning. Using radio frequency identification technology, we assessed the effects of natural variation in foraging activity, and the age when first foraging, on both performance in reversal learning and on synaptic connectivity in the MBs. We found that reversal learning performance improved at foraging onset and could decline with greater foraging experience. If bees started foraging before the normal age, as a result of a stress applied to the colony, the decline in learning performance with foraging experience was more severe. Analyses of brain structure in the same bees showed that the total number of synaptic boutons at the MB input decreased when bees started foraging, and then increased with greater foraging intensity. At foraging onset MB structure is therefore optimized for bees to update learned information, but optimization of MB connectivity deteriorates with foraging effort. In a computational model of the MBs sparser coding of information at the MB input improved reversal learning performance. We propose, therefore, a plausible mechanistic relationship between experience, neuroplasticity, and cognitive performance in a natural and ecological context.

In this study, the first complete mitogenome of Andrenidae, namely Andrena camellia, is newly sequenced. It includes 13 protein-coding (PCG) genes, 22 transfer RNA (rRNA) genes, two ribosomal RNA (tRNA) genes, and a control region. Among PCGs, high conservation is observed in cytochrome oxidase genes with cox1 exhibits the highest conservation. Conversely, NADH dehydrogenase and ATPase subunit genes are more variable with atp8 presents the maximal variation. Comparison of the gene order indicates complex rearrangement in bees. Most of the rearranged events are located in the tRNA clusters of trnI-trnQ-trnM, trnW-trnC-trnY, and trnA-trnR-trnN-trnS1-trnE-trnF. Furthermore, we present the most comprehensive mitochondrial phylogeny of bee families. The monophyly of each family and the long-tongued bees is highly supported. However, short-tongued bees are inferred as paraphyletic relative to the sister relationship between Melittidae and other bee families. Furthermore, to improve the resolution of phylogeny, various datasets and analytical approaches are performed. It is indicated that datasets including third codons of PCGs facilitate to produce identical topology and higher nodal support. The tRNA genes that have typical cloverleaf secondary structures also exhibit similar positive effects. However, rRNAs present poor sequence alignment and distinct substitution saturation, which result in negative effects on both tree topology and nodal support. In addition, Gblocks treatment can increase the congruence of topologies, but has opposite effects on nodal support between the two inference methods of maximum likelihood and Bayesian inference.

Evidence of inter-species pathogen transmission from managed to wild bees has sparked concern that emerging diseases could be causing or exacerbating wild bee declines. While some pathogens, like RNA viruses, have been found in pollen and wild bees, the threat these viruses pose to wild bees is largely unknown. Here, we tested 169 bees, representing 4 families and 8 genera, for five common honey bee (Apis mellifera) viruses, finding that more than 80% of wild bees harbored at least one virus. We also quantified virus titers in these bees, providing, for the first time, an assessment of viral load in a broad spectrum of wild bees. Although virus detection was very common, virus levels in the wild bees were minimal-similar to or lower than foraging honey bees and substantially lower than honey bees collected from hives. Furthermore, when we experimentally inoculated adults of two different bee species (Megachile rotundata and Colletes inaequalis) with a mixture of common viruses that is lethal to honey bees, we saw no effect on short term survival. Overall, we found that honey bee RNA viruses can be commonly detected at low levels in many wild bee species, but we found no evidence that these pathogens cause elevated short-term mortality effects. However, more work on these viruses is greatly needed to assess effects on additional bee species and life stages.

The honey bee has been an important model organism for studying learning and memory. More recently, the honey bee has become a valuable model to understand perception and cognition. However, the techniques used to explore psychological phenomena in honey bees have been limited to only a few primary methodologies such as the proboscis extension reflex, sting extension reflex, and free flying target discrimination-tasks. Methods to explore operant conditioning in bees and other invertebrates are not as varied as with vertebrates. This may be due to the availability of a suitable response requirement. In this manuscript we offer a new method to explore operant conditioning in honey bees: the cap pushing response (CPR). We used the CPR to test for difference in learning curves between novel auto-shaping and more traditional explicit-shaping. The CPR protocol requires bees to exhibit a novel behavior by pushing a cap to uncover a food source. Using the CPR protocol we tested the effects of both explicit-shaping and auto-shaping techniques on operant conditioning. The goodness of fit and lack of fit of these data to the Rescorla-Wagner learning-curve model, widely used in classical conditioning studies, was tested. The model fit well to both control and explicit-shaping results, but only for a limited number of trials. Learning ceased rather than continuing to asymptotically approach the physiological most accurate possible. Rate of learning differed between shaped and control bee treatments. Learning rate was about 3 times faster for shaped bees, but for all measures of proficiency control and shaped bees reached the same level. Auto-shaped bees showed one-trial learning rather than the asymptotic approach to a maximal efficiency. However, in terms of return-time, the auto-shaped bees' learning did not carry over to the covered-well test treatments.

Honey bee colonies are subject to numerous pathogens and parasites. Interaction among multiple pathogens and parasites is the proposed cause for Colony Collapse Disorder (CCD), a syndrome characterized by worker bees abandoning their hive. Here we provide the first documentation that the phorid fly Apocephalus borealis, previously known to parasitize bumble bees, also infects and eventually kills honey bees and may pose an emerging threat to North American apiculture. Parasitized honey bees show hive abandonment behavior, leaving their hives at night and dying shortly thereafter. On average, seven days later up to 13 phorid larvae emerge from each dead bee and pupate away from the bee. Using DNA barcoding, we confirmed that phorids that emerged from honey bees and bumble bees were the same species. Microarray analyses of honey bees from infected hives revealed that these bees are often infected with deformed wing virus and Nosema ceranae. Larvae and adult phorids also tested positive for these pathogens, implicating the fly as a potential vector or reservoir of these honey bee pathogens. Phorid parasitism may affect hive viability since 77% of sites sampled in the San Francisco Bay Area were infected by the fly and microarray analyses detected phorids in commercial hives in South Dakota and California's Central Valley. Understanding details of phorid infection may shed light on similar hive abandonment behaviors seen in CCD.

Ongoing losses of pollinators are of significant international concern because of the essential role they have in our ecosystem, agriculture, and economy. Both chemical and non-chemical stressors have been implicated as possible contributors to their decline, but the increasing use of neonicotinoid insecticides has recently emerged as particularly concerning. In this study, honey bees were exposed orally to sublethal doses of the neonicotinoid clothianidin in the field in order to assess its effects on the foraging behavior, homing success, and dance communication. The foraging span and foraging activity at the contaminated feeder decreased significantly due to chronic exposure at field-realistic concentrations. Electrostatic field of dancing bees was measured and it was revealed that the number of waggle runs, the fanning time and the number of stop signals were significantly lower in the exposed colony. No difference was found in the homing success and the flight duration between control and treated bees released at a novel location within the explored area. However, a negative effect of the ambient temperature, and an influence of the location of the trained feeder was found. Finally, the residues of clothianidin accumulated in the abdomens of exposed foraging bees over time. These results show the adverse effects of a chronic exposure to sublethal doses of clothianidin on foraging and dance communication in honey bees.

Varroa destructor, the introduced parasite of European honey bees associated with massive colony deaths, spreads readily through populations of honey bee colonies, both managed colonies living crowded together in apiaries and wild colonies living widely dispersed in natural settings. Mites are hypothesized to spread between most managed colonies via phoretically riding forager bees when they engage in robbing colonies or they drift between hives. However, widely spaced wild colonies show Varroa infestation despite limited opportunities for robbing and little or no drifting of bees between colonies. Both wild and managed colonies may also exchange mites via another mechanism that has received remarkably little attention or study: floral transmission. The present study tested the ability of mites to infest foragers at feeders or flowers. We show that Varroa destructor mites are highly capable of phoretically infesting foraging honey bees, detail the mechanisms and maneuvers by which they do so, and describe mite behaviors post-infestation.