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.

Exosymbiotic microbes within fermented pollen provisions are as important for the development of solitary bees as the pollen itself.

Developing bees derive significant benefits from the microbes present within their guts and fermenting pollen provisions. External microbial symbionts (exosymbionts) associated with larval diets may be particularly important for solitary bees that suffer reduced fitness when denied microbe-colonized pollen.To investigate whether this phenomenon is generalizable across foraging strategy, we examined the effects of exosymbiont presence/absence across two solitary bee species, a pollen specialist and generalist. Larvae from each species were reared on either microbe-rich natural or microbe-deficient sterilized pollen provisions allocated by a female forager belonging to their own species (conspecific-sourced pollen) or that of another species (heterospecific-sourced pollen). Our results reveal that the presence of pollen-associated microbes was critical for the survival of both the generalist and specialist larvae, regardless of whether the pollen was sourced from a conspecific or heterospecific forager.Given the positive effects of exosymbiotic microbes for larval fitness, we then examined if the magnitude of this benefit varied based on whether the microbes were provisioned by a conspecific forager (the mother bee) or a heterospecific forager. In this second study, generalist larvae were reared only on microbe-rich pollen provisions, but importantly, the sources (conspecific versus heterospecific) of the microbes and pollen were experimentally manipulated.Bee fitness metrics indicated that microbial and pollen sourcing both had significant impacts on larval performance, and the effect sizes of each were similar. Moreover, the effects of conspecific-sourced microbes and conspecific-sourced pollen were strongly positive, while that of heterospecific-sourced microbes and heterospecific-sourced pollen, strongly negative.Our findings imply that not only is the presence of exosymbionts critical for both specialist and generalist solitary bees, but more notably, that the composition of the specific microbial community within larval pollen provisions may be as critical for bee development as the composition of the pollen itself.

Microbial Diversity Associated with the Pollen Stores of Captive-Bred Bumble Bee Colonies.

The pollen stores of bumble bees host diverse microbiota that influence overall colony fitness. Yet, the taxonomic identity of these symbiotic microbes is relatively unknown. In this descriptive study, we characterized the microbial community of pollen provisions within captive-bred bumble bee hives obtained from two commercial suppliers located in North America. Findings from 16S rRNA and ITS gene-based analyses revealed that pollen provisions from the captive-bred hives shared several microbial taxa that have been previously detected among wild populations. While diverse microbes across phyla Firmicutes, Proteobacteria, Bacteroidetes, Actinobacteria, and Ascomycota were detected in all commercial hives, significant differences were detected at finer-scale taxonomic resolution based on the supplier source. The causative agent of chalkbrood disease in honey bees, Ascosphaera apis, was detected in all hives obtained from one supplier source, although none of the hives showed symptoms of infection. The shared core microbiota across both commercial supplier sources consisted of two ubiquitous bee-associated groups, Lactobacillus and Wickerhamiella/Starmerella clade yeasts that potentially contribute to the beneficial function of the microbiome of bumble bee pollen provisions.

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.

In Vitro Rearing of Solitary Bees: A Tool for Assessing Larval Risk Factors.

Although solitary bees provide crucial pollination services for wild and managed crops, this species-rich group has been largely overlooked in pesticide regulation studies. The risk of exposure to fungicide residues is likely to be especially high if the spray occurs on, or near host plants while the bees are collecting pollen to provision their nests. For species of Osmia that consume pollen from a select group of plants (oligolecty), the inability to use pollen from non-host plants can increase their risk factor for fungicide-related toxicity. This manuscript describes protocols used to successfully rear oligolectic mason bees, Osmia ribifloris sensu lato, from egg to prepupal stage within cell culture plates under standardized laboratory conditions. The in vitro-reared bees are subsequently used to investigate the effects of fungicide exposure and pollen source on bee fitness. Based on a 2 × 2 fully crossed factorial design, the experiment examines the main and interactive effects of fungicide exposure and pollen source on larval fitness, quantified by prepupal biomass, larval developmental time, and survivorship. A major advantage of this technique is that using in vitro-reared bees reduces natural background variability and allows the simultaneous manipulation of multiple experimental parameters. The described protocol presents a versatile tool for hypotheses testing involving the suite of factors affecting bee health. For conservation efforts to be met with significant, lasting success, such insights into the complex interplay of physiological and environmental factors driving bee declines will prove to be critical.

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.

Intra-trophic isotopic discrimination of 15N/14N for amino acids in autotrophs: Implications for nitrogen dynamics in ecological studies.

The differential discrimination of nitrogen isotopes (15N/14N) within amino acids in consumers and their diets has been routinely used to estimate organismal tropic position (TP). Analogous isotopic discrimination can occur within plants, particularly in organs lacking chloroplasts. Such discrimination likely arises from the catabolic deamination of amino acids, resulting in a numerical elevation of estimated TP, within newly synthesized biomass. To investigate this phenomenon, we examined the 15N/14N of amino acids (δ15 NAA) in spring leaves and flowers from eight deciduous and two annual plants. These plants were classified on the basis of their time of bloom, plants that bloomed when their leaves were absent (Type I) versus plants that bloomed while leaves were already present (Type II). Based on the δ15 NAA values from leaves, both plant types occupied comparable and ecologically realistic mean TPs (=1.0 ± 0.1, mean ± 1σ). However, the estimated TPs of flowers varied significantly (Type I: 2.2 ± 0.2; Type II: 1.0 ± 0.1). We hypothesize that these results can be interpreted by the following sequence of events: (1) Type I floral biomass is synthesized in absence of active photosynthesis; (2) the catabolic deamination of amino acids in particular, leaves behind 15N in the residual pool of amino acids; and (3) the incorporation of these 15N-enriched amino acids within the biomass of Type I flowers results in the numerical elevation of the TPs. In contrast, the actively photosynthesizing Type II leaves energetically sustain the synthesis of Type II flower biomass, precluding any reliance on catabolic deamination of amino acids. Amino acids within Type II flowers are therefore isotopically comparable to the Type II leaves. These findings demonstrate the idiosyncratic nature of the δ15 NAA values within autotrophic organs and have implications for interpreting trophic hierarchies using primary producers and their consumers.

Unpacking brown food-webs: Animal trophic identity reflects rampant microbivory.

Detritivory is the dominant trophic paradigm in most terrestrial, aquatic, and marine ecosystems, yet accurate measurement of consumer trophic position within detrital (="brown") food webs has remained unresolved. Measurement of detritivore trophic position is complicated by the fact that detritus is suffused with microbes, creating a detrital complex of living and nonliving biomass. Given that microbes and metazoans are trophic analogues of each other, animals feeding on detrital complexes are ingesting other detritivores (microbes), which should elevate metazoan trophic position and should be rampant within brown food webs. We tested these hypotheses using isotopic (15N) analyses of amino acids extracted from wild and laboratory-cultured consumers. Vertebrate (fish) and invertebrate detritivores (beetles and moths) were reared on detritus, with and without microbial colonization. In the field, detritivorous animal specimens were collected and analyzed to compare trophic identities among laboratory-reared and free-roaming detritivores. When colonized by bacteria or fungi, the trophic positions of detrital complexes increased significantly over time. The magnitude of trophic inflation was mediated by the extent of microbial consumption of detrital substrates. When detrital complexes were fed to vertebrate and invertebrate animals, the consumers registered similar degrees of trophic inflation, albeit one trophic level higher than their diets. The wild-collected detritivore fauna in our study exhibited significantly elevated trophic positions. Our findings suggest that the trophic positions of detrital complexes rise predictably as microbes convert nonliving organic matter into living microbial biomass. Animals consuming such detrital complexes exhibit similar trophic inflation, directly attributable to the assimilation of microbe-derived amino acids. Our data demonstrate that detritivorous microbes elevate metazoan trophic position, suggesting that detritivory among animals is, functionally, omnivory. By quantifying the impacts of microbivory on the trophic positions of detritivorous animals and then tracking how these effects propagate "up" food chains, we reveal the degree to which microbes influence consumer groups within trophic hierarchies. The trophic inflation observed among our field-collected fauna further suggests that microbial proteins represent an immense contribution to metazoan biomass. Collectively, these findings provide an empirical basis to interpret detritivore trophic identity, and further illuminate the magnitude of microbial contributions to food webs.

Mercury levels in largemouth bass (Micropterus salmoides) from regulated and unregulated rivers.

Within areas of comparable atmospheric mercury deposition rates methylmercury burden in largemouth bass populations vary significantly between regulated and unregulated rivers. To investigate if trophic dynamics strongly influenced pollutant body load, we sampled largemouth bass from two adjacent rivers, one regulated and one unregulated, and applied a suite of biochemical and stable isotope assays to compare their trophic dynamics. Total mercury burden in the bass from the unregulated Sipsey River (Elrod, AL, USA) and the regulated Black Warrior River (Demopolis, AL, USA) averaged 0.87 mg kg-1 and 0.19 mg kg-1 wet weight, respectively. For both populations, age, weight, and length were positively correlated with muscle mercury concentration. Compound specific stable isotope analysis of amino acids showed the trophic position of both populations was just under four. Quantitative and isotopic analysis of neutral lipid fatty acid of Sipsey River bass indicated a greater reliance upon the detrital component of the food web compared to Demopolis Reservoir bass which fed within the autochthonous, pelagic component of the food web. Since the close proximity of the rivers makes differences in atmospheric deposition unlikely and both populations had similar trophic position, our findings indicate that food web dynamics should be included among the factors that can strongly influence mercury concentration in fish.