Malpighamoeba mellificae is a protozoan that infects the Malpighian tubules of honey bees. The amoebae, ingested as cysts, develop into trophozoites that feed upon tubule epithelia. The resulting damage of the Malpighian tubules can induce an imbalance of waste excretion and hemolymph exchange. This causes the so-called amoebiasis disease in adult bees, which may co-occur with Nosema infections. Most reports of this amoeba are from the 1960s and earlier, and knowledge of the disease and its spreading is very poor. The lack of any genetic marker for the species hampers its sensitive identification using molecular tools and gaining knowledge on its epidemiology. Here, we present a diagnostic RT-qPCR assay, consisting of two primers and one probe that were developed based on 18S rRNA sequences of the amoeba, generated with metagenomic sequencing of Malpighian tubules with and without M. mellificae cysts. The assay was initially tested and adjusted with samples microscopically tested for the presence of M. mellificae cysts. Later, it was validated and material with unknown infection status was tested. The sensitive diagnostic Malpighamoeba disease 18S assay is now ready to be applied for honey bee health monitoring purposes and to investigate the prevalence of M. mellificae in more detail.

Honey bee ecology demands they make both rapid and accurate assessments of which flowers are most likely to offer them nectar or pollen. To understand the mechanisms of honey bee decision-making, we examined their speed and accuracy of both flower acceptance and rejection decisions. We used a controlled flight arena that varied both the likelihood of a stimulus offering reward and punishment and the quality of evidence for stimuli. We found that the sophistication of honey bee decision-making rivalled that reported for primates. Their decisions were sensitive to both the quality and reliability of evidence. Acceptance responses had higher accuracy than rejection responses and were more sensitive to changes in available evidence and reward likelihood. Fast acceptances were more likely to be correct than slower acceptances; a phenomenon also seen in primates and indicative that the evidence threshold for a decision changes dynamically with sampling time. To investigate the minimally sufficient circuitry required for these decision-making capacities, we developed a novel model of decision-making. Our model can be mapped to known pathways in the insect brain and is neurobiologically plausible. Our model proposes a system for robust autonomous decision-making with potential application in robotics.

Honey bees, and pollinators in general, play a major role in the health of ecosystems. There is a consensus about the steady decrease in pollinator populations, which raises global ecological concern. Several drivers are implicated in this threat. Among them, honey bee pathogens are transmitted to other arthropods populations, including wild and managed pollinators. The western honey bee, Apis mellifera, is quasi-globally spread. This successful species acted as and, in some cases, became a maintenance host for pathogens. This systematic review collects and summarizes spillover cases having in common Apis mellifera as the mainteinance host and some of its pathogens. The reports are grouped by final host species and condition, year, and geographic area of detection and the co-occurrence in the same host. A total of eighty-one articles in the time frame 1960-2021 were included. The reported spillover cases cover a wide range of hymenopteran host species, generally living in close contact with or sharing the same environmental resources as the honey bees. They also involve non-hymenopteran arthropods, like spiders and roaches, which are either likely or unlikely to live in close proximity to honey bees. Specific studies should consider host-dependent pathogen modifications and effects on involved host species. Both the plasticity of bee pathogens and the ecological consequences of spillover suggest a holistic approach to bee health and the implementation of a One Health approach.

Bees play a key role in natural and agro-ecosystems and their diversity is worldwide threatened by anthropogenic causes. Despite this, there is little awareness of the existence of the numerous species of wild bees, and the common name "bee" is very often exclusively associated with Apis mellifera. Our aim was to create a citizen science project in Italy with the following objectives: (a) raising awareness of the importance and diversity of bees, (b) obtaining data on the biology, ecology and distribution of Italian species, and (c) launching the monitoring of alien bees. The first step of the project was to create a website platform with a section containing informative datasheets of the wild bee families and of the most common bee genera present in Italy, a form to send reports of observed bees and an interactive map with all citizen's reports. During the 2 years of the project 1086 reports were sent by 269 users, with 38 Apoidea genera reported on 190 plant genera; furthermore, 22 reports regarding the alien species Megachile sculpturalis arrived. The majority of bees (34 genera) were observed on spontaneous plants, including 115 genera native to Italy. Considering the increasing number of reports and data obtained in these first two years of the project, our objectives seem to be achieved. Future steps will be to outline the profile of beewatchers, to plan activities in a more targeted way, and also to start some sub-projects for conservation purposes.

The recent global decline in insect populations is of particular concern for pollinators. Wild and managed bees (Hymenoptera, Apoidea) are of primary environmental and economic importance because of their role in pollinating cultivated and wild plants, and synthetic pesticides are among the major factors contributing to their decline. Botanical biopesticides may be a viable alternative to synthetic pesticides in plant defence due to their high selectivity and short environmental persistence. In recent years, scientific progress has been made to improve the development and effectiveness of these products. However, knowledge regarding their adverse effects on the environment and non-target species is still scarce, especially when compared to that of synthetic products. Here, we summarize the studies concerning the toxicity of botanical biopesticides on the different groups of social and solitary bees. We highlight the lethal and sublethal effects of these products on bees, the lack of a uniform protocol to assess the risks of biopesticides on pollinators, and the scarcity of studies on specific groups of bees, such as the large and diverse group of solitary bees. Results show that botanical biopesticides cause lethal effects and a large number of sublethal effects on bees. However, the toxicity is limited when comparing the effects of these compounds with those of synthetic compounds.

Glyphosate, the primary herbicide used globally for weed control, targets the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) enzyme in the shikimate pathway found in plants and some microorganisms. Thus, glyphosate may affect bacterial symbionts of animals living near agricultural sites, including pollinators such as bees. The honey bee gut microbiota is dominated by eight bacterial species that promote weight gain and reduce pathogen susceptibility. The gene encoding EPSPS is present in almost all sequenced genomes of bee gut bacteria, indicating that they are potentially susceptible to glyphosate. We demonstrated that the relative and absolute abundances of dominant gut microbiota species are decreased in bees exposed to glyphosate at concentrations documented in the environment. Glyphosate exposure of young workers increased mortality of bees subsequently exposed to the opportunistic pathogen Serratia marcescens Members of the bee gut microbiota varied in susceptibility to glyphosate, largely corresponding to whether they possessed an EPSPS of class I (sensitive to glyphosate) or class II (insensitive to glyphosate). This basis for differences in sensitivity was confirmed using in vitro experiments in which the EPSPS gene from bee gut bacteria was cloned into Escherichia coli All strains of the core bee gut species, Snodgrassella alvi, encode a sensitive class I EPSPS, and reduction in S. alvi levels was a consistent experimental result. However, some S. alvi strains appear to possess an alternative mechanism of glyphosate resistance. Thus, exposure of bees to glyphosate can perturb their beneficial gut microbiota, potentially affecting bee health and their effectiveness as pollinators.

Varroa destructor and its associated viruses, in particular deformed wing virus (DWV), have been identified as probable causes of honey bee (Apis mellif era L.) colony losses. Evidence suggests that elevated DWV titres in bees could compromise sensory and communication abilities resulting in negative consequences for hygienic behaviour. As antennae play a central role in this behaviour, we compared antennal ultrastructure in DWV-symptomatic and asymptomatic bees. The results show that virus capsids accumulate in the basal regions of the antennal epithelium, close to the haemolymph. No virus particles were detected at the level of sensory sensilla, such as pore plates, nor within the sensory cell dendrites associated with these sensilla. However, membranous structures appeared to be more prevalent in supporting cells surrounding the dendrites of DWV-symptomatic bees. Para-crystalline arrays containing large numbers of virus particles were detected in the antennae of DWV-symptomatic bees but not in asymptomatic bees.

Bee pollination is an indispensable component of global food production and plays a crucial role in sustainable agriculture. The worldwide decline of bee populations, including wild pollinators, poses a threat to this system. However, most studies to date are situated in temperate regions where Apini and Bombini are very abundant pollinators. Tropical and subtropical regions where stingless bees (Apidae: Meliponini) are generally very common, are often overlooked. These bees also face pressure due to deforestation and agricultural intensification as well as the growing use and spread of exotic pollinators as Apis mellifera and Bombus species. The loss or decline of this important bee tribe would have a large impact on their provided ecosystem services, in both wild and agricultural landscapes. The importance of pollinator diseases, which can contribute to decline, has not been investigated so far in this bee tribe. Here we report on the first large pathogen screening of Meliponini species in southern Brazil. Remarkably we observed that there was an absence of Leishmaniinae and Nosematidae, and a very low occurrence of Apicystis bombi. Our data on disease prevalence in both understudied areas and species, can greatly improve our knowledge on the distribution of pathogens among bee species.

Hemolymph plays key roles in honey bee molecule transport, immune defense, and in monitoring the physiological condition. There is a lack of knowledge regarding how the proteome achieves these biological missions for both the western and eastern honey bees (Apis mellifera and Apis cerana). A time-resolved proteome was compared using two-dimensional electrophoresis-based proteomics to reveal the mechanistic differences by analysis of hemolymph proteome changes between the worker bees of two bee species during the larval to pupal stages.

The rapid conversion of natural habitats to anthropogenic landscapes is threatening insect pollinators worldwide, raising concern regarding the negative consequences on their fundamental role as plant pollinators. However, not all pollinators are negatively affected by habitat conversion, as certain species find appropriate resources in anthropogenic landscapes to persist and proliferate. The reason why some species tolerate anthropogenic environments while most find them inhospitable remains poorly understood. The cognitive buffer hypothesis, widely supported in vertebrates but untested in insects, offers a potential explanation. This theory suggests that species with larger brains have enhanced behavioural plasticity, enabling them to confront and adapt to novel challenges. To investigate this hypothesis in insects, we measured brain size for 89 bee species, and evaluated their association with the degree of habitat occupancy. Our analyses revealed that bee species mainly found in urban habitats had larger brains relative to their body size than those that tend to occur in forested or agricultural habitats. Additionally, urban bees exhibited larger body sizes and, consequently, larger absolute brain sizes. Our results provide the first empirical support for the cognitive buffer hypothesis in invertebrates, suggesting that a large brain in bees could confer behavioural advantages to tolerate urban environments.

In temperate climates, honey bees rely on stored carbohydrates to sustain them throughout the winter. In nature, honey serves as the bees' source of carbohydrates, but when managed, beekeepers often harvest honey and replace it with cheaper, artificial feed. The effects of alternative carbohydrate sources on colony survival, strength, and individual bee metabolic health are poorly understood. We assessed the impacts of carbohydrate diets (honey, sucrose syrup, high-fructose corn syrup, and invert syrup) on colony winter survival, population size, and worker bee nutritional state (i.e., fat content and gene expression of overwintered bees and emerging callow bees). We observed a nonsignificant trend for greater survival and larger adult population size among colonies overwintered on honey compared to the artificial feeds, with colonies fed high-fructose corn syrup performing particularly poorly. These trends were mirrored in individual bee physiology, with bees from colonies fed honey having significantly larger fat bodies than those from colonies fed high-fructose corn syrup. For bees fed honey or sucrose, we also observed gene expression profiles consistent with a higher nutritional state, associated with physiologically younger individuals. That is, there was significantly higher expression of vitellogenin and insulin-like peptide 2 and lower expression of insulin-like peptide 1 and juvenile hormone acid methyltransferase in the brains of bees that consumed honey or sucrose syrup relative to those that consumed invert syrup or high-fructose corn syrup. These findings further our understanding of the physiological implications of carbohydrate nutrition in honey bees and have applied implications for colony management.

Honey bees (Apis mellifera) can be infected by many viruses, some of which pose a major threat to their health and well-being. A critical step in the dynamics of a viral infection is its mode of transmission. Here, we compared for the first time the effect of mode of horizontal transmission of Black queen cell virus (BQCV), a ubiquitous and highly prevalent virus of A. mellifera, on viral virulence in individual adult honey bees. Hosts were exposed to BQCV either by feeding (representing direct transmission) or by injection into hemolymph (analogous to indirect or vector-mediated transmission) through a controlled laboratory experimental design. Mortality, viral titer and expression of three key innate immune-related genes were then quantified. Injecting BQCV directly into hemolymph in the hemocoel resulted in far higher mortality as well as increased viral titer and significant change in the expression of key components of the RNAi pathway compared to feeding honey bees BQCV. Our results support the hypothesis that mode of horizontal transmission determines BQCV virulence in honey bees. BQCV is currently considered a benign viral pathogen of adult honey bees, possibly because its mode of horizontal transmission is primarily direct, per os. We anticipate adverse health effects on honey bees if BQCV transmission becomes vector-mediated.

Pollen collection is necessary for bee survival and important for flowering plant reproduction, yet if and how pollen extraction motor routines are modified with experience is largely unknown. Here, we used an automated reward and monitoring system to evaluate modification in a common pollen-extraction routine, floral sonication. Through a series of laboratory experiments with the bumblebee, Bombus impatiens, we examined whether variation in sonication frequency and acceleration is due to instrumental learning based on rewards, a fixed behavioral response to rewards, and/or a mechanical constraint. We first investigated whether bees could learn to adjust their sonication frequency in response to pollen rewards given only for specified frequency ranges and found no evidence of instrumental learning. However, we found that absence versus receipt of a pollen reward did lead to a predictable behavioral response, which depended on bee size. Finally, we found some evidence of mechanical constraints, in that flower mass affected sonication acceleration (but not frequency) through an interaction with bee size. In general, larger bees showed more flexibility in sonication frequency and acceleration, potentially reflecting a size-based constraint on the range over which smaller bees can modify frequency and acceleration. Overall, our results show that although bees did not display instrumental learning of sonication frequency, their sonication motor routine is nevertheless flexible.

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.

Caffeine has long been used as a stimulant by humans. Although this secondary metabolite is produced by some plants as a mechanism of defense against herbivores, beneficial or detrimental effects of such consumption are usually associated with dose. The Western honey bee, Apis mellifera, can also be exposed to caffeine when foraging at Coffea and Citrus plants, and low doses as are found in the nectar of these plants seem to boost memory learning and ameliorate parasite infection in bees. In this study, we investigated the effects of caffeine consumption on the gut microbiota of honey bees and on susceptibility to bacterial infection. We performed in vivo experiments in which honey bees, deprived of or colonized with their native microbiota, were exposed to nectar-relevant concentrations of caffeine for a week, then challenged with the bacterial pathogen Serratia marcescens. We found that caffeine consumption did not impact the gut microbiota or survival rates of honey bees. Moreover, microbiota-colonized bees exposed to caffeine were more resistant to infection and exhibited increased survival rates compared to microbiota-colonized or microbiota-deprived bees only exposed to the pathogen. Our findings point to an additional benefit of caffeine consumption in honey bee health by protecting against bacterial infections. IMPORTANCE The consumption of caffeine is a remarkable feature of the human diet. Common drinks, such as coffee and tea, contain caffeine as a stimulant. Interestingly, honey bees also seem to like caffeine. They are usually attracted to the low concentrations of caffeine found in nectar and pollen of Coffea plants, and consumption improves learning and memory retention, as well as protects against viruses and fungal parasites. In this study, we expanded these findings by demonstrating that caffeine can improve survival rates of honey bees infected with Serratia marcescens, a bacterial pathogen known to cause sepsis in animals. However, this beneficial effect was only observed when bees were colonized with their native gut microbiota, and caffeine seemed not to directly affect the gut microbiota or survival rates of bees. Our findings suggest a potential synergism between caffeine and gut microbial communities in protection against bacterial pathogens.

Solitary bees and honey bees from a neighbouring apiary were screened for a broad set of putative pathogens including protists, fungi, spiroplasmas and viruses. Most sampled bees appeared to be infected with multiple parasites. Interestingly, viruses exclusively known from honey bees such as Apis mellifera Filamentous Virus and Varroa destructor Macula-like Virus were also discovered in solitary bees. A microsporidium found in Andrena vaga showed most resemblance to Nosema thomsoni. Our results suggest that bee hives represent a putative source of pathogens for other pollinators. Similarly, solitary bees may act as a reservoir of honey bee pathogens.

Host-associated microbiomes, particularly gut microbiomes, often harbor related but distinct microbial lineages, but how this diversity arises and is maintained is not well understood. A prerequisite for lineage diversification is reproductive isolation imposed by barriers to gene flow. In host-associated microbes, genetic recombination can be disrupted by confinement to different hosts, for example following host speciation, or by niche partitioning within the same host. Taking advantage of the simple gut microbiome of social bees, we explore the diversification of two groups of gut-associated bacteria, Gilliamella and Snodgrassella, which have evolved for 80 million y with honey bees and bumble bees. Our analyses of sequenced genomes show that these lineages have diversified into discrete populations with limited gene flow. Divergence has occurred between symbionts of different host species and, in some cases, between symbiont lineages within a single host individual. Populations have acquired genes to adapt to specific hosts and ecological niches; for example, Gilliamella lineages differ markedly in abilities to degrade dietary polysaccharides and to use the resulting sugar components. Using engineered fluorescent bacteria in vivo, we show that Gilliamella lineages localize to different hindgut regions, corresponding to differences in their abilities to use spatially concentrated nitrogenous wastes of hosts. Our findings show that bee gut bacteria can diversify due to isolation in different host species and also due to spatial niche partitioning within individual hosts, leading to barriers to gene flow.

The microbial communities in animal digestive systems are critical for host development and health. They stimulate the immune system during development, synthesize important chemical compounds like hormones, aid in digestion, competitively exclude pathogens, etc. Compared to the bacterial and fungal components of the microbiome, we know little about the temporal and spatial dynamics of bacteriophage communities in animal digestive systems. Recently, the bacteriophages of the honey bee gut were characterized in two European bee populations. Most of the bacteriophages described in these two reports were novel, harbored many metabolic genes in their genomes, and had a community structure that suggests coevolution with their bacterial hosts. To describe the conservation of bacteriophages in bees and begin to understand their role in the bee microbiome, we sequenced the virome of Apis mellifera from Austin, TX, and compared bacteriophage compositions among three locations around the world. We found that most bacteriophages from Austin are novel, sharing no sequence similarity with anything in public repositories. However, many bacteriophages are shared among the three bee viromes, indicating specialization of bacteriophages in the bee gut. Our study, along with the two previous bee virome studies, shows that the bee gut bacteriophage community is simple compared to that of many animals, consisting of several hundred types of bacteriophages that primarily infect four of the dominant bacterial phylotypes in the bee gut. IMPORTANCE Viruses that infect bacteria (bacteriophages) are abundant in the microbial communities that live on and in plants and animals. However, our knowledge of the structure, dynamics, and function of these viral communities lags far behind our knowledge of their bacterial hosts. We sequenced the first bacteriophage community of honey bees from the United States and compared the U.S. honey bee bacteriophage community to those of samples from Europe. Our work is an important characterization of an economically critical insect species and shows how bacteriophage communities can contain highly conserved individuals and be highly variable in composition across a wide geographic range.

"Ecological intelligence" hypotheses posit that animal learning and memory evolve to meet the demands posed by foraging and, together with social intelligence and cognitive buffer hypotheses, provide a key framework for understanding cognitive evolution.1-5 However, identifying the critical environments where cognitive investment reaps significant benefits has proved challenging.6-8 Here, we capitalize upon seasonal variation in forage availability for a social insect model (Bombus terrestris audax) to establish how the benefits of short-term memory, assayed using a radial arm maze (RAM), vary with resource availability. Following a staggered design over 2 years, whereby bees from standardized colonies at identical life-history stages underwent cognitive testing before foraging in the wild, we found that RAM performance predicts foraging efficiency-a key determinant of colony fitness-in plentiful spring foraging conditions but that this relationship is reversed during the summer floral dearth. Our results suggest that the selection for enhanced cognitive abilities is unlikely to be limited to harsh environments where food is hard to find or extract,5,9-11 highlighting instead that the challenges of rich and plentiful environments, which present multiple options in short succession, could be a broad driver in the evolution of certain cognitive traits. VIDEO ABSTRACT.