From pollen to putrid: Comparative metagenomics reveals how microbiomes support dietary specialization in vulture bees.

For most animals, the microbiome is key for nutrition and pathogen defence, and is often shaped by diet. Corbiculate bees, including honey bees, bumble bees, and stingless bees, share a core microbiome that has been shaped, at least in part, by the challenges associated with pollen digestion. However, three species of stingless bees deviate from the general rule of bees obtaining their protein exclusively from pollen (obligate pollinivores) and instead consume carrion as their sole protein source (obligate necrophages) or consume both pollen and carrion (facultative necrophages). These three life histories can provide missing insights into microbiome evolution associated with extreme dietary transitions. Here, we investigate, via shotgun metagenomics, the functionality of the microbiome across three bee diet types: obligate pollinivory, obligate necrophagy, and facultative necrophagy. We find distinct differences in microbiome composition and gene functional profiles between the diet types. Obligate necrophages and pollinivores have more specialized microbes, whereas facultative necrophages have a diversity of environmental microbes associated with several dietary niches. Our study suggests that necrophagous bee microbiomes may have evolved to overcome cellular stress and microbial competition associated with carrion. We hypothesize that the microbiome evolved social phenotypes, such as biofilms, that protect the bees from opportunistic pathogens present on carcasses, allowing them to overcome novel nutritional challenges. Whether specific microbes enabled diet shifts or diet shifts occurred first and microbial evolution followed requires further research to disentangle. Nonetheless, we find that necrophagous microbiomes, vertebrate and invertebrate alike, have functional commonalities regardless of their taxonomy.

Plant-pollinator network architecture does not impact intraspecific microbiome variability.

Variation in how individuals interact with food resources can directly impact, and be affected by, their microbial interactions due to the potential for transmission. The degree to which this transmission occurs, however, may depend on the structure of forager networks, which determine the community-scale transmission opportunities. In particular, how the community-scale opportunity for transfer balances individual-scale barriers to transmission is unclear. Examining the bee-flower and bee-microbial interactions of over 1000 individual bees, we tested (1) the degree to which individual floral visits predicted microbiome composition and (2) whether plant-bee networks with increased opportunity for microbial transmission homogenized the microbiomes of bees within that network. The pollen community composition carried by bees was associated with microbiome composition at some sites, suggesting that microbial transmission at flowers occurred. Contrary to our predictions, however, microbiome variability did not differ based on transfer opportunity: bee microbiomes in asymmetric networks with high opportunity for microbial transfer were similarly variable compared to microbiomes in networks with more evenly distributed links. These findings suggest that microbial transmission at flowers is frequent enough to be observed at the community level, but that community network structure did not substantially change the dynamics of this transmission, perhaps due to filtering processes in host guts.

Pollen Diet Diversity does not Affect Gut Bacterial Communities or Melanization in a Social and Solitary Bee Species.

Pollinators face many stressors, including reduced floral diversity. A low-diversity diet can impair organisms' ability to cope with additional stressors, such as pathogens, by altering the gut microbiome and/or immune function, but these effects are understudied for most pollinators. We investigated the impact of pollen diet diversity on two ecologically and economically important generalist pollinators, the social bumble bee (Bombus impatiens) and the solitary alfalfa leafcutter bee (Megachile rotundata). We experimentally tested the effect of one-, two-, or three-species pollen diets on gut bacterial communities in both species, and the melanization immune response in B. impatiens. Pollen diets included dandelion (Taraxacum officinale), staghorn sumac (Rhus typhina), and hawthorn (Crataegus sp.) alone, each pair-wise combination, or a mix of all three species. We fed bees their diet for 7 days and then dissected out guts and sequenced 16S rRNA gene amplicons to characterize gut bacterial communities. To assess melanization in B. impatiens, we inserted microfilament implants into the bee abdomen and measured melanin deposition on the implant. We found that pollen diet did not influence gut bacterial communities in M. rotundata. In B. impatiens, pollen diet composition, but not diversity, affected gut bacterial richness in older, but not newly-emerged bees. Pollen diet did not affect the melanization response in B. impatiens. Our results suggest that even a monofloral, low-quality pollen diet such as dandelion can support diverse gut bacterial communities in captive-reared adults of these bee species. These findings shed light on the effects of reduced diet diversity on bee health.

Sunflower plantings reduce a common gut pathogen and increase queen production in common eastern bumblebee colonies.

Community diversity can reduce the prevalence and spread of disease, but certain species may play a disproportionate role in diluting or amplifying pathogens. Flowers act as both sources of nutrition and sites of pathogen transmission, but the effects of specific plant species in shaping bee disease dynamics are not well understood. We evaluated whether plantings of sunflower (Helianthus annuus), whose pollen reduces infection by some pathogens when fed to bees in captivity, lowered pathogen levels and increased reproduction in free-foraging bumblebee colonies (Bombus impatiens). Sunflower abundance reduced the prevalence of a common gut pathogen, Crithidia bombi, and reduced infection intensity, with an order of magnitude lower infection intensity at high sunflower sites compared with sites with little to no sunflower. Sunflower abundance was also positively associated with greater queen production in colonies. Sunflower did not affect prevalence of other detected pathogens. This work demonstrates that a single plant species can drive disease dynamics in foraging B. impatiens, and that sunflower plantings can be used as a tool for mitigating a prevalent pathogen while also increasing reproduction of an agriculturally important bee species.

Individual Dietary Specialization in a Generalist Bee Varies across Populations but Has No Effect on the Richness of Associated Microbial Communities.

AbstractDespite the increasingly documented occurrence of individual specialization, the relationship between individual consumer interactions and diet-related microbial communities in wild populations is still unclear. Using data from nests of Ceratina australensis from three different wild bee populations, we combine metabarcoding and network approaches to explore the existence of individual variation in resource use within and across populations and whether dietary specialization affects the richness of pollen-associated microbes. We reveal the existence of marked dietary specialization. In the most specialized population, we also show that individuals' diet breadth was positively related to the richness of fungi but not bacteria. Overall, individual specialization appeared to have a weak or negligible effect on the microbial richness of nests, suggesting that different mechanisms beyond environmental transmission may be at play regarding microbial acquisition in wild bees.

Floral resources shape parasite and pathogen dynamics in bees facing urbanization.

Urbanization is associated with increases in impervious land cover, which alters the distribution of resources available to wildlife and concentrates activity in unbuilt spaces such as parks and gardens. How resource shifts alter the dynamics of parasite and pathogen transmission has not been addressed for many important species in urban systems. We focus on urban gardens, resource-rich "islands" within the urban matrix, to examine how the availability of floral resources at local and landscape scales influences the prevalence of six RNA viruses and three parasites in honey bees and bumble bees. Because parasites and pathogens are transmitted at flowers between visitors, we expected that floral abundance would concentrate bees within gardens, amplifying infection rates in pollinators, unless increases in floral resources would enhance bee diversity enough to dilute transmission. We found that garden size and flowering perennial plant abundance had a positive, direct effect on parasite and pathogen richness in bumble bees, suggesting that resource provisioning amplifies transmission. We also found that parasitism rates in honey bees were positively associated with parasites and pathogens in bumble bees, suggesting spillover between species. Encouragingly, we found evidence that management may mitigate parasitism through indirect effects: garden size had a positive impact on bee diversity, which in turn was negatively associated with parasite and pathogen richness in bumble bees. Unexpectedly, we observed that that parasite and pathogen richness in honey bees had no significant predictors, highlighting the complexity of comparing transmission dynamics between species. Although floral resources provide bees with food, we suggest more research on the tradeoffs between resource provisioning and disease transmission to implement conservation plantings in changing landscapes.

Elevated Temperature May Affect Nectar Microbes, Nectar Sugars, and Bumble Bee Foraging Preference.

Floral nectar, an important resource for pollinators, is inhabited by microbes such as yeasts and bacteria, which have been shown to influence pollinator preference. Dynamic and complex plant-pollinator-microbe interactions are likely to be affected by a rapidly changing climate, as each player has their own optimal growth temperatures and phenological responses to environmental triggers, such as temperature. To understand how warming due to climate change is influencing nectar microbial communities, we incubated a natural nectar microbial community at different temperatures and assessed the subsequent nectar chemistry and preference of the common eastern bumble bee, Bombus impatiens. The microbial community in floral nectar is often species-poor, and the cultured Brassica rapa nectar community was dominated by the bacterium Fructobacillus. Temperature increased the abundance of bacteria in the warmer treatment. Bumble bees preferred nectar inoculated with microbes, but only at the lower, ambient temperature. Warming therefore induced an increase in bacterial abundance which altered nectar sugars and led to significant differences in pollinator preference.

Mass-flowering monoculture attracts bees, amplifying parasite prevalence.

As the global agricultural footprint expands, it is increasingly important to address the link between the resource pulses characteristic of monoculture farming and wildlife epidemiology. To understand how mass-flowering crops impact host communities and subsequently amplify or dilute parasitism, we surveyed wild and managed bees in a monoculture landscape with varying degrees of floral diversification. We screened 1509 bees from 16 genera in sunflower fields and in non-crop flowering habitat across 200 km2 of the California Central Valley. We found that mass-flowering crops increase bee abundance. Wild bee abundance was subsequently associated with higher parasite presence, but only in sites with a low abundance of non-crop flowers. Bee traits related to higher dispersal ability (body size) and diet breadth (pollen lecty) were also positively related to parasite presence. Our results highlight the importance of non-crop flowering habitat for supporting bee communities. We suggest monoculture alone cannot support healthy bees.

Reclassification of seven honey bee symbiont strains as Bombella apis.

Honey bees are important pollinators of many major crops and add billions of dollars annually to the US economy through their services. Recent declines in the health of the honey bee have startled researchers and lay people alike as honey bees are agriculture's most important pollinator. One factor that may influence colony health is the microbial community. Although honey bee worker guts have a characteristic community of bee-specific microbes, the honey bee queen digestive tracts are colonized predominantly by a single acetic acid bacterium tentatively named 'Parasaccharibacter apium'. This bacterium is related to flower-associated microbes such as Saccharibacter floricola, and initial phylogenetic analyses placed it as sister to these environmental bacteria. We used a combination of phylogenetic and sequence identity methods to better resolve evolutionary relationships among 'P. apium', strains in the genus Saccharibacter, and strains in the closely related genus Bombella. Interestingly, measures of genome-wide average nucleotide identity and aligned fraction, coupled with phylogenetic placement, indicate that many strains labelled as 'P. apium' and Saccharibacter species are all the same species as Bombella apis. We propose reclassifying these strains as Bombella apis and outline the data supporting that classification below.

Landscape simplification shapes pathogen prevalence in plant-pollinator networks.

Species interaction networks, which play an important role in determining pathogen transmission and spread in ecological communities, can shift in response to agricultural landscape simplification. However, we know surprisingly little about how landscape simplification-driven changes in network structure impact epidemiological patterns. Here, we combine mathematical modelling and data from eleven bipartite plant-pollinator networks observed along a landscape simplification gradient to elucidate how changes in network structure shape disease dynamics. Our empirical data show that landscape simplification reduces pathogen prevalence in bee communities via increased diet breadth of the dominant species. Furthermore, our empirical data and theoretical model indicate that increased connectance reduces the likelihood of a disease outbreak and decreases variance in prevalence among bee species in the community, resulting in a dilution effect. Because infectious diseases are implicated in pollinator declines worldwide, a better understanding of how land use change impacts species interactions is therefore critical for conserving pollinator health.

The direct and indirect effects of environmental toxicants on the health of bumblebees and their microbiomes.

Bumblebees (Bombus spp.) are important and widespread insect pollinators, but the act of foraging on flowers can expose them to harmful pesticides and chemicals such as oxidizers and heavy metals. How these compounds directly influence bee survival and indirectly affect bee health via the gut microbiome is largely unknown. As toxicants in floral nectar and pollen take many forms, we explored the genomes of bee-associated microbes for their potential to detoxify cadmium, copper, selenate, the neonicotinoid pesticide imidacloprid, and hydrogen peroxide-which have all been identified in floral nectar and pollen. We then exposed Bombus impatiens workers to varying concentrations of these chemicals via their diet and assayed direct effects on bee survival. Using field-realistic doses, we further explored the indirect effects on bee microbiomes. We found multiple putative genes in core gut microbes that may aid in detoxifying harmful chemicals. We also found that while the chemicals are largely toxic at levels within and above field-realistic concentrations, the field-realistic concentrations-except for imidacloprid-altered the composition of the bee microbiome, potentially causing gut dysbiosis. Overall, our study shows that chemicals found in floral nectar and pollen can cause bee mortality, and likely have indirect, deleterious effects on bee health via their influence on the bee microbiome.

Environment Shapes the Microbiome of the Blue Orchard Bee, Osmia lignaria : RRH: Environmental Drivers of Bee Microbiome.

Wild bees encounter environmental microbes while foraging. While environmental context affects bee diversity, little is known about it how affects the wild bee microbiome. We used field surveys in 17 urban gardens to examine whether and how variation in local and landscape habitat features shapes the microbiome of the solitary Blue Orchard Bee, Osmia lignaria. We installed O. lignaria cocoons at each site, allowed bees to emerge and forage, then collected them. We measured local features of gardens using vegetation transects and landscape features with GIS. We found that in microbiome composition between bee individuals varied by environmental features such as natural habitat, floral resources, and bee species richness. We also found that environmental features were associated with the abundance of bacterial groups important for bee health, such as Lactobacillus. Our study highlights complex interactions between environment context, bee species diversity, and the bee-associated microbes.

Cross-infectivity of honey and bumble bee-associated parasites across three bee families.

Recent declines of wild pollinators and infections in honey, bumble and other bee species have raised concerns about pathogen spillover from managed honey and bumble bees to other pollinators. Parasites of honey and bumble bees include trypanosomatids and microsporidia that often exhibit low host specificity, suggesting potential for spillover to co-occurring bees via shared floral resources. However, experimental tests of trypanosomatid and microsporidial cross-infectivity outside of managed honey and bumble bees are scarce. To characterize potential cross-infectivity of honey and bumble bee-associated parasites, we inoculated three trypanosomatids and one microsporidian into five potential hosts - including four managed species - from the apid, halictid and megachilid bee families. We found evidence of cross-infection by the trypanosomatids Crithidia bombi and C. mellificae, with evidence for replication in 3/5 and 3/4 host species, respectively. These include the first reports of experimental C. bombi infection in Megachile rotundata and Osmia lignaria, and C. mellificae infection in O. lignaria and Halictus ligatus. Although inability to control amounts inoculated in O. lignaria and H. ligatus hindered estimates of parasite replication, our findings suggest a broad host range in these trypanosomatids, and underscore the need to quantify disease-mediated threats of managed social bees to sympatric pollinators.

Contaminants of emerging concern affect Trichoplusia ni growth and development on artificial diets and a key host plant.

Many countries are utilizing reclaimed wastewater for agriculture because drought, rising temperatures, and expanding human populations are increasing water demands. Unfortunately, wastewater often contains biologically active, pseudopersistent pharmaceuticals, even after treatment. Runoff from farms and output from wastewater treatment plants also contribute high concentrations of pharmaceuticals to the environment. This study assessed the effects of common pharmaceuticals on an agricultural pest, Trichoplusia ni (Lepidoptera: Noctuidae). Larvae were reared on artificial diets spiked with contaminants of emerging concern (CECs) at environmentally relevant concentrations. Trichoplusia ni showed increased developmental time and mortality when reared on artificial diets containing antibiotics, hormones, or a mixture of contaminants. Mortality was also increased when T. ni were reared on tomatoes grown hydroponically with the same concentrations of antibiotics. The antibiotic-treated plants translocated ciprofloxacin through their tissues to roots, shoots, and leaves. Microbial communities of T. ni changed substantially between developmental stages and when exposed to CECs in their diets. Our results suggest that use of reclaimed wastewater for irrigation of crops can affect the developmental biology and microbial communities of an insect of agricultural importance.

The bumble bee microbiome increases survival of bees exposed to selenate toxicity.

Bumble bees are important and widespread insect pollinators who face many environmental challenges. For example, bees are exposed to the metalloid selenate when foraging on pollen and nectar from plants growing in contaminated soils. As it has been shown that the microbiome of animals reduces metalloid toxicity, we assayed the ability of the bee microbiome to increase survivorship against selenate challenge. We exposed uninoculated or microbiota-inoculated Bombus impatiens workers to a field-realistic dose of 0.75 mg l-1 selenate and found that microbiota-inoculated bees survive slightly but significantly longer than uninoculated bees. Using 16S rRNA gene sequencing, we found that selenate exposure altered gut microbial community composition and relative abundance of specific core bacteria. We also grew two core bumble bee microbes - Snodgrassella alvi and Lactobacillus bombicola - in selenate-spiked media and found that these bacteria grew in the tested concentrations of 0.001-10 mg l-1 selenate. Furthermore, the genomes of these microbes harbour genes involved in selenate detoxification. The bumble bee microbiome slightly increases survivorship when the host is exposed to selenate, but the specific mechanisms and colony-level benefits under natural settings require further study.

Temperature dependence of parasitic infection and gut bacterial communities in bumble bees.

High temperatures (e.g., fever) and gut microbiota can both influence host resistance to infection. However, effects of temperature-driven changes in gut microbiota on resistance to parasites remain unexplored. We examined the temperature dependence of infection and gut bacterial communities in bumble bees infected with the trypanosomatid parasite Crithidia bombi. Infection intensity decreased by over 80% between 21 and 37°C. Temperatures of peak infection were lower than predicted based on parasite growth in vitro, consistent with mismatches in thermal performance curves of hosts, parasites and gut symbionts. Gut bacterial community size and composition exhibited slight but significant, non-linear, and taxon-specific responses to temperature. Abundance of total gut bacteria and of Orbaceae, both negatively correlated with infection in previous studies, were positively correlated with infection here. Prevalence of the bee pathogen-containing family Enterobacteriaceae declined with temperature, suggesting that high temperature may confer protection against diverse gut pathogens. Our results indicate that resistance to infection reflects not only the temperature dependence of host and parasite performance, but also temperature-dependent activity of gut bacteria. The thermal ecology of gut parasite-symbiont interactions may be broadly relevant to infectious disease, both in ectothermic organisms that inhabit changing climates, and in endotherms that exhibit fever-based immunity.

Cadmium and Selenate Exposure Affects the Honey Bee Microbiome and Metabolome, and Bee-Associated Bacteria Show Potential for Bioaccumulation.

Honey bees are important insect pollinators used heavily in agriculture and can be found in diverse environments. Bees may encounter toxicants such as cadmium and selenate by foraging on plants growing in contaminated areas, which can result in negative health effects. Honey bees are known to have a simple and consistent microbiome that conveys many benefits to the host, and toxicant exposure may impact this symbiotic microbial community. We used 16S rRNA gene sequencing to assay the effects that sublethal cadmium and selenate treatments had over 7 days and found that both treatments significantly but subtly altered the composition of the bee microbiome. Next, we exposed bees to cadmium and selenate and then used untargeted liquid chromatography-mass spectrometry (LC-MS) metabolomics to show that chemical exposure changed the bees' metabolite profiles and that compounds which may be involved in detoxification, proteolysis, and lipolysis were more abundant in treatments. Finally, we exposed several strains of bee-associated bacteria in liquid culture and found that each strain removed cadmium from its medium but that only Lactobacillus Firm-5 microbes assimilated selenate, indicating the possibility that these microbes may reduce the metal and metalloid burden on their host. Overall, our report shows that metal and metalloid exposure can affect the honey bee microbiome and metabolome and that strains of bee-associated bacteria can bioaccumulate these toxicants.IMPORTANCE Bees are important insect pollinators that may encounter environmental pollution when foraging upon plants grown in contaminated areas. Despite the pervasiveness of pollution, little is known about the effects of these toxicants on honey bee metabolism and their symbiotic microbiomes. Here, we investigated the impact of selenate and cadmium exposure on the gut microbiome and metabolome of honey bees. We found that exposure to these chemicals subtly altered the overall composition of the bees' microbiome and metabolome and that exposure to toxicants may negatively impact both host and microbe. As the microbiome of animals can reduce mortality upon metal or metalloid challenge, we grew bee-associated bacteria in media spiked with selenate or cadmium. We show that some bacteria can remove these toxicants from their media in vitro and suggest that bacteria may reduce metal burden in their hosts.

Wild Bee Pollen Usage and Microbial Communities Co-vary Across Landscapes.

Bees forage for pollen and nectar at flowers but simultaneously acquire pathogenic, commensal, and likely beneficial microbes from these same flowers. Characterizing pollen usage of wild bees is therefore crucial to their conservation yet remains a challenging task. To understand pollen usage across landscapes and how this affects microbial communities found in the pollen provisions collected from flowers, we studied the generalist small carpenter bee Ceratina australensis. We collected C. australensis nests from three different climatic zones across eastern and southern Australia. To characterize the plant, fungal, and bacterial composition of these pollen provisions, we used a metabarcoding and next-generation sequencing approach. We found that the species richness of plant types, fungi, and bacteria was highest in a subtropical zone compared to a temperate or a grassland zone. The composition of these communities also differentiated by zone, particularly in pollen composition and fungal communities. Moreover, pollen composition strongly correlated with fungal community composition, suggesting that variation in pollen usage across landscapes results in variation in microbial communities. While how these pollen usage and microbial community patterns affect bee health merits additional work, these data further our understanding of how flowering plant community composition affects not only the pollen usage of a generalist bee but also its associated microbial communities.

Floral and Foliar Source Affect the Bee Nest Microbial Community.

Managed pollinators such as the alfalfa leafcutting bee, Megachile rotundata, are essential to the production of a wide variety of agricultural crops. These pollinators encounter a diverse array of microbes when foraging for food and nest-building materials on various plants. To test the hypothesis that food and nest-building source affects the composition of the bee-nest microbiome, we exposed M. rotundata adults to treatments that varied both floral and foliar source in a 2 × 2 factorial design. We used 16S rRNA gene and internal transcribed spacer (ITS) sequencing to capture the bacterial and fungal diversity of the bee nests. We found that nest microbial communities were significantly different between treatments, indicating that bee nests become inoculated with environmentally derived microbes. We did not find evidence of interactions between the fungi and bacteria within our samples. Furthermore, both the bacterial and fungal communities were quite diverse and contained numerous exact sequence variants (ESVs) of known plant and bee pathogens that differed based on treatment. Our research indicates that bees deposit plant-associated microbes into their nests, including multiple plant pathogens such as smut fungi and bacteria that cause blight and wilt. The presence of plant pathogens in larval pollen provisions highlights the potential for bee nests to act as disease reservoirs across seasons. We therefore suggest that future research should investigate the ability of bees to transmit pathogens from nest to host plant.

pH-mediated inhibition of a bumble bee parasite by an intestinal symbiont.

Gut symbionts can augment resistance to pathogens by stimulating host-immune responses, competing for space and nutrients, or producing antimicrobial metabolites. Gut microbiota of social bees, which pollinate many crops and wildflowers, protect hosts against diverse infections and might counteract pathogen-related bee declines. Bumble bee gut microbiota, and specifically abundance of Lactobacillus 'Firm-5' bacteria, can enhance resistance to the trypanosomatid parasite Crithidia bombi. However, the mechanism underlying this effect remains unknown. We hypothesized that the Firm-5 bacterium Lactobacillus bombicola, which produces lactic acid, inhibits C. bombi via pH-mediated effects. Consistent with our hypothesis, L. bombicola spent medium inhibited C. bombi growth via reduction in pH that was both necessary and sufficient for inhibition. Inhibition of all parasite strains occurred within the pH range documented in honey bees, though sensitivity to acidity varied among strains. Spent medium was slightly more potent than HCl, d- and l-lactic acids for a given pH, suggesting that other metabolites also contribute to inhibition. Results implicate symbiont-mediated reduction in gut pH as a key determinant of trypanosomatid infection in bees. Future investigation into in vivo effects of gut microbiota on pH and infection intensity would test the relevance of these findings for bees threatened by trypanosomatids.