Microbes, the 'silent third partners' of bee-angiosperm mutualisms.

While bee-angiosperm mutualisms are widely recognized as foundational partnerships that have shaped the diversity and structure of terrestrial ecosystems, these ancient mutualisms have been underpinned by 'silent third partners': microbes. Here, we propose reframing the canonical bee-angiosperm partnership as a three-way mutualism between bees, microbes, and angiosperms. This new conceptualization casts microbes as active symbionts, processing and protecting pollen-nectar provisions, consolidating nutrients for bee larvae, enhancing floral attractancy, facilitating plant fertilization, and defending bees and plants from pathogens. In exchange, bees and angiosperms provide their microbial associates with food, shelter, and transportation. Such microbial communities represent co-equal partners in tripartite mutualisms with bees and angiosperms, facilitating one of the most important ecological partnerships on land.

Omnivory in Bees: Elevated Trophic Positions among All Major Bee Families.

As pollen and nectar foragers, bees have long been considered strictly herbivorous. Their pollen provisions, however, are host to abundant microbial communities, which feed on the pollen before and/or while it is consumed by bee larvae. In the process, microbes convert pollen into a complex of plant and microbial components. Since microbes are analogous to metazoan consumers within trophic hierarchies, the pollen-eating microbes are, functionally, herbivores. When bee larvae consume a microbe-rich pollen complex, they ingest proteins from plant and microbial sources and thus should register as omnivores on the trophic "ladder." We tested this hypothesis by examining the isotopic compositions of amino acids extracted from native bees collected in North America over multiple years. We measured bee trophic position across the six major bee families. Our findings indicate that bee trophic identity was consistently and significantly higher than that of strict herbivores, providing the first evidence that omnivory is ubiquitous among bee fauna. Such omnivory suggests that pollen-borne microbes represent an important protein source for larval bees, which introduces new questions as to the link between floral fungicide residues and bee development.

Pollen-borne microbes shape bee fitness.

Teeming within pollen provisions are diverse communities of symbiotic microbes, which provide a variety of benefits to bees. Microbes themselves may represent a major dietary resource for developing bee larvae. Despite their apparent importance in sustaining bee health, evidence linking pollen-borne microbes to larval health is currently lacking. We examined the effects of microbe-deficient diets on the fitness of larval mason bees. In a series of diet manipulations, microbe-rich maternally collected pollen provisions were replaced with increasing fractions of sterilized, microbe-deficient pollen provisions before being fed to developing larvae. Convergent findings from amino acid and fatty acid trophic biomarker analyses revealed that larvae derived a substantial amount of nutrition from microbial prey and occupied a significantly higher trophic position than that of strict herbivores. Larvae feeding on increasingly sterile diets experienced significant adverse effects on growth rates, biomass and survivorship. When completely deprived of pollen-borne microbes, larvae consistently exhibited marked decline in fitness. We conclude that microbes associated with aged pollen provisions are central to bee health, not only as nutritional mutualists, but also as a major dietary component. In an era of global bee decline, the conservation of such bee-microbe interactions may represent an important facet of pollinator protection strategies.

Empirical, Metagenomic, and Computational Techniques Illuminate the Mechanisms by which Fungicides Compromise Bee Health.

Growers often use fungicide sprays during bloom to protect crops against disease, which exposes bees to fungicide residues. Although considered "bee-safe," there is mounting evidence that fungicide residues in pollen are associated with bee declines (for both honey and bumble bee species). While the mechanisms remain relatively unknown, researchers have speculated that bee-microbe symbioses are involved. Microbes play a pivotal role in the preservation and/or processing of pollen, which serves as nutrition for larval bees. By altering the microbial community, it is likely that fungicides disrupt these microbe-mediated services, and thereby compromise bee health. This manuscript describes the protocols used to investigate the indirect mechanism(s) by which fungicides may be causing colony decline. Cage experiments exposing bees to fungicide-treated flowers have already provided the first evidence that fungicides cause profound colony losses in a native bumble bee (Bombus impatiens). Using field-relevant doses of fungicides, a series of experiments have been developed to provide a finer description of microbial community dynamics of fungicide-exposed pollen. Shifts in the structural composition of fungal and bacterial assemblages within the pollen microbiome are investigated by next-generation sequencing and metagenomic analysis. Experiments developed herein have been designed to provide a mechanistic understanding of how fungicides affect the microbiome of pollen-provisions. Ultimately, these findings should shed light on the indirect pathway through which fungicides may be causing colony declines.