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Abstract Anthropogenic pollution affects environments differently depending on proximity to pollution source, exposure route, and species ecology. Thus, understanding organism’s ecological role and exposure route to contaminants is central to assessing pollution impact. Treated municipal wastewater releases contaminants into waterways and alters microbial communities. Plants absorb contaminants and expose animals through foraging and nest-building activities. Nesting ecology differences of ground vs wood cavity-nesting bees offers insight into niche-specific susceptibility to pollution. Because contaminants bind to soil strongly, ground-nesting bees near wastewater are likely most impacted, while wood cavity-nesting bees likely less impacted since plants’ ability to uptake contaminants are species dependent. We compared gut microbiomes of directly exposed soil-nestingHalictus ligatusand indirectly exposed wood-nestingCeratinaspp. upstream/downstream of wastewater. We collected bees, flowers, and soil, and analyzed their bacteria microbiomes (16S rRNA). Wastewater altered ground-nestingH. ligatusmicrobiome >18 times greater than wood cavity-nestingCeratinaadults.Ceratinalarvae and pollen provisions showed significant but smaller shifts. Conversely, soil and flower microbiomes remained stable, indicating higher resilience. These results demonstrate that exposure routes drive contaminants susceptibility, with animal-associated microbes most vulnerable. Because bees are important pollinators and biodiversity contributors, these disruptions point to broader ecological risks in increasingly contaminated landscapes. Abstract Figure 

Synopsis While social bees are a primary focus of research and monitoring efforts among pollinators, more than 80% of bee species are solitary. For solitary bees, foraging behavior and efficiency directly impact the reproductive outcomes of individuals (fitness), as the consequences of foraging performance are not buffered by a colony. Cavity-nesting bees (e.g., Osmia spp. and Megachile spp. (Megachilidae)) progressively provision pollen in cells within hollow stems and other cavities, with each cell generally containing one offspring, making their reproductive output easily quantifiable. Cavity-nesting bees thus represent an ideal system to study fitness effects of individual variation in foraging niche, including differences in foraging activity and responses to environmental conditions. Understanding the links between individual performance and fitness can improve our understanding of solitary bee behavior, physiology, and ecology, particularly in response to environmental change. However, quantifying both foraging activity and provisioning rates within nests is prohibitively labor-intensive in many cases, especially for the extended time periods (i.e., weeks or months) over which nests are provisioned and under naturalistic ecological conditions. While recent work has established computer vision tools for automated monitoring of foraging activity at solitary bee nests, combining these approaches with in-nest monitoring could help better link foraging to fitness in solitary bees. Here we introduce Osmia Camera Activity Monitoring (osmiaCAM), a low-cost, open-source, automated monitoring system for foraging and nest-provisioning behavior of cavity-nesting bees, including key pollinator and research model organisms, such as Osmia spp. and Megachile spp. We demonstrate the potential of this system in a small validation experiment in Central California, where we quantified foraging transits and nest provisioning rates of Osmia spp. under variable weather conditions. This validation experiment yielded accurate, near-continuous monitoring for several weeks continuously. The osmiaCAM system has broad applications across cavity-nesting solitary bee species, facilitating research on their behavior, ecology, and responses to rapid environmental change. The development of autonomous monitoring systems and associated data analysis pipelines provides an opportunity for open-source ecological methods that would be applicable across a multitude of systems and disciplines. 

ABSTRACT Microbial environmental transmission among individuals plays an important role in shaping the microbiomes of many species. Despite the importance of the microbiome for host fitness, empirical investigations on environmental transmission are scarce, particularly in systems where interactions across multiple trophic levels influence symbiotic dynamics. Here, we explore microbial transmission within insect microbiomes, focusing on solitary bees. Specifically, we investigate the environmental transmission hypothesis, which posits that solitary bees acquire and deposit their associated microbiota from and to their surroundings, especially flowers. Using experimental setups, we examine the transmission dynamics ofApilactobacillus micheneri, a fructophilic and acidophilic bacterium, between the solitary beeOsmia lignaria(Megachilidae) and the plantPhacelia tanacetifolia(Boraginaceae). Our results demonstrate that bees not only acquire bacteria from flowers but also deposit these microbes onto uninoculated flowers for other bees to acquire them, supporting a bidirectional microbial exchange. We therefore find empirical support for the environmental transmission hypothesis, and we discuss the multitrophic dependencies that facilitate microbial transmission between bees and flowers. 

Theoretical frameworks of terrestrial community assembly often focus on single trophic levels (e.g. plants) without considering how complex interdependencies across different trophic levels influence assembly mechanisms. Yet, when multiple trophic levels are considered (e.g. plant–pollinator, plant–microbe interactions) the focus is typically on network analyses at local spatial scales. As spatial variation in biodiversity (β‐diversity) is increasingly being recognized for its relevance in understanding community assembly and conservation, considering how β‐diversity at one trophic level may be influenced by assembly processes that alter abundance and composition of interacting communities at a different trophic level (multitrophic dependency) is critical. Here, we build on single trophic level community assembly frameworks to explore the assembly processes affecting β‐diversity in multitrophic communities comprising flowering plants, their bee pollinators, and the corresponding bee‐gut microbiota to better understand the importance of multitrophic dependency in community assembly. Using distance‐based redundancy analysis and variation partitioning, we investigated community assembly processes across three interconnected trophic levels in two ecological regions in southern California: the Santa Monica Mountains and three islands of the Channel Island Archipelago. We found that the deterministic effects of multitrophic dependency are stronger on directly connected trophic levels than on indirectly connected trophic levels (i.e. flowers explain bee communities and bees explain bee‐gut bacteria communities, but flowers weakly explain variation in bee‐gut bacteria communities). We also found notable regional variation, where multitrophic dependency was weaker on the Channel Islands as ecological drift was more pronounced. Our results suggest that integrating the influence of multitrophic dependency on community assembly is important for elucidating drivers of β‐diversity and that multitrophic dependency can be determined by the regional context in which β‐diversity is measured. Taken together, our results highlight the importance of considering multiscale perspectives – both multitrophic and multiregional – in community assembly to fully elucidate assembly processes. 

ABSTRACT <p>Social bees have been extensively studied for their gut microbial functions, but the significance of the gut microbiota in solitary bees remains less explored. Solitary bee,<italic>Megachile rotundata</italic>females provision their offspring with pollen from various plant species, harboring a diverse microbial community that colonizes larvae guts. The<italic>Apilactobacillus</italic>is the most abundant microbe, but evidence concerning the effects of<italic>Apilactobacillus</italic>and other provision microbes on growth and survival are lacking. We hypothesized that the presence of<italic>Apilactobacillus</italic>in abundance would enhance larval and prepupal development, weight, and survival, while the absence of intact microbial communities was expected to have a negative impact on bee fitness. We reared larvae on pollen provisions with naturally collected microbial communities (Natural pollen) or devoid of microbial communities (Sterile pollen). We also assessed the impact of introducing<italic>Apilactobacillus micheneri</italic>by adding it to both types of pollen provisions. Feeding larvae with sterile pollen +<italic>A. micheneri</italic>led to the highest mortality rate, followed by natural pollen +<italic>A. micheneri</italic>, and sterile pollen. Larval development was significantly delayed in groups fed with sterile pollen. Interestingly, larval and prepupal weights did not significantly differ across treatments compared to natural pollen-fed larvae. 16S rRNA gene sequencing found a dominance of<italic>Sodalis</italic>, when<italic>A. micheneri</italic>was introduced to natural pollen. The presence of<italic>Sodalis</italic>with abundant<italic>A. michene</italic>ri suggests potential crosstalk between both, shaping bee nutrition and health. Hence, this study highlights that the reliance on nonhost-specific environmental bacteria may not impact fitness of<italic>M. rotundata</italic>.</p><sec><title>IMPORTANCEThis study investigates the impact of environmentally acquired gut microbes of solitary bee fitness with insights into the microbial ecology of bee and their health. While the symbiotic microbiome is well-studied in social bees, the role of environmental acquired microbiota in solitary bees remains unclear. Assessing this relationship in a solitary pollinator, the leaf-cutting bee,Megachile rotundata, we discovered that this bee species does not depend on the diverse environmental bacteria found in pollen for either its larval growth or survival. Surprisingly, high concentrations of the most abundant pollen bacteria, Apilactobacillus micheneridid not consistently benefit bee fitness, but caused larval mortality. Our findings also suggest an interaction betweenApilactobacillusand theSodalisand perhaps their role in bee nutrition. Hence, this study provides significant insights that contribute to understanding the fitness, conservation, and pollination ecology of other solitary bee species in the future. 

Abstract 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. 

Host–microbe interactions underlie the development and fitness of many macroorganisms, including bees. Whereas many social bees benefit from vertically transmitted gut bacteria, current data suggests that solitary bees, which comprise the vast majority of species diversity within bees, lack a highly specialized gut microbiome. Here, we examine the composition and abundance of bacteria and fungi throughout the complete life cycle of the ground-nesting solitary bee Anthophora bomboides standfordiana. In contrast to expectations, immature bee stages maintain a distinct core microbiome consisting of Actinobacterial genera (Streptomyces, Nocardiodes) and the fungus Moniliella spathulata. Dormant (diapausing) larval bees hosted the most abundant and distinctive bacteria and fungi, attaining 33 and 52 times their initial copy number, respectively. We tested two adaptive hypotheses regarding microbial functions for diapausing bees. First, using isolated bacteria and fungi, we found that Streptomyces from brood cells inhibited the growth of multiple pathogenic filamentous fungi, suggesting a role in pathogen protection during overwintering, when bees face high pathogen pressure. Second, sugar alcohol composition changed in tandem with major changes in fungal abundance, suggesting links with bee cold tolerance or overwintering biology. We find that A. bomboides hosts a conserved core microbiome that may provide key fitness advantages through larval development and diapause, which raises the question of how this microbiome is maintained and faithfully transmitted between generations. Our results suggest that focus on microbiomes of mature or active insect developmental stages may overlook stage-specific symbionts and microbial fitness contributions during host dormancy. 

Pathogens and parasites of solitary bees have been studied for decades, but the microbiome as a whole is poorly understood for most taxa. Comparative analyses of microbiome features such as composition, abundance, and specificity, can shed light on bee ecology and the evolution of host–microbe interactions. Here we study microbiomes of ground-nesting cellophane bees (Colletidae: Diphaglossinae). From a microbial point of view, the diphaglossine genus Ptiloglossa is particularly remarkable: their larval provisions are liquid and smell consistently of fermentation. We sampled larval provisions and various life stages from wild nests of Ptiloglossa arizonensis and two species of closely related genera: Caupolicana yarrowi and Crawfordapis luctuosa . We also sampled nectar collected by P. arizonensis . Using 16S rRNA gene sequencing, we find that larval provisions of all three bee species are near-monocultures of lactobacilli. Nectar communities are more diverse, suggesting ecological filtering. Shotgun metagenomic and phylogenetic data indicate that Ptiloglossa culture multiple species and strains of Apilactobacillus , which circulate among bees and flowers. Larval lactobacilli disappear before pupation, and hence are likely not vertically transmitted, but rather reacquired from flowers as adults. Thus, brood cell microbiomes are qualitatively similar between diphaglossine bees and other solitary bees: lactobacilli-dominated, environmentally acquired, and non-species-specific. However, shotgun metagenomes provide evidence of a shift in bacterial abundance. As compared with several other bee species, Ptiloglossa have much higher ratios of bacterial to plant biomass in larval provisions, matching the unusually fermentative smell of their brood cells. Overall, Ptiloglossa illustrate a path by which hosts can evolve quantitatively novel symbioses: not by acquiring or domesticating novel symbionts, but by altering the microenvironment to favor growth of already widespread and generalist microbes. 

Despite 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 the bee Ceratina australensis from three different wild 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.