Honey bee colonies can buffer short-term stressor effects of pollen restriction and fungicide exposure on colony development and the microbiome.

Honey bees (Apis mellifera) have to withstand various environmental stressors alone or in combination in agriculture settings. Plant protection products are applied to achieve high crop yield, but residues of their active substances are frequently detected in bee matrices and could affect honey bee colonies. In addition, intensified agriculture could lead to resource limitation for honey bees. This study aimed to compare the response of full-sized and nucleus colonies to the combined stressors of fungicide exposure and resource limitation. A large-scale field study was conducted simultaneously at five different locations across Germany, starting in spring 2022 and continuing through spring 2023. The fungicide formulation Pictor® Active (active ingredients boscalid and pyraclostrobin) was applied according to label instructions at the maximum recommended rate on oil seed rape crops. Resource limitation was ensured by pollen restriction using a pollen trap and stressor responses were evaluated by assessing colony development, brood development, and core gut microbiome alterations. Furthermore, effects on the plant nectar microbiome were assessed since nectar inhabiting yeast are beneficial for pollination. We showed, that honey bee colonies were able to compensate for the combined stressor effects within six weeks. Nucleus colonies exposed to the combined stressors showed a short-term response with a less favorable brood to bee ratio and reduced colony development in May. No further impacts were observed in either the nucleus colonies or the full-sized colonies from July until the following spring. In addition, no fungicide-dependent differences were found in core gut and nectar microbiomes, and these differences were not distinguishable from local or environmental effects. Therefore, the provision of sufficient resources is important to increase the resilience of honey bees to a combination of stressors.

Intraspecific variation in realized dispersal probability and host quality shape nectar microbiomes.

Epiphytic microbes frequently affect plant phenotype and fitness, but their effects depend on microbe abundance and community composition. Filtering by plant traits and deterministic dispersal-mediated processes can affect microbiome assembly, yet their relative contribution to predictable variation in microbiome is poorly understood. We compared the effects of host-plant filtering and dispersal on nectar microbiome presence, abundance, and composition. We inoculated representative bacteria and yeast into 30 plants across four phenotypically distinct cultivars of Epilobium canum. We compared the growth of inoculated communities to openly visited flowers from a subset of the same plants. There was clear evidence of host selection when we inoculated flowers with synthetic communities. However, plants with the highest microbial densities when inoculated did not have the highest microbial densities when openly visited. Instead, plants predictably varied in the presence of bacteria, which was correlated with pollen receipt and floral traits, suggesting a role for deterministic dispersal. These findings suggest that host filtering could drive plant microbiome assembly in tissues where species pools are large and dispersal is high. However, deterministic differences in microbial dispersal to hosts may be equally or more important when microbes rely on an animal vector, dispersal is low, or arrival order is important.

Culturable bacteria are more common than fungi in floral nectar and are more easily dispersed by thrips, a ubiquitous flower visitor.

Variation in dispersal ability among taxa affects community assembly and biodiversity maintenance within metacommunities. Although fungi and bacteria frequently coexist, their relative dispersal abilities are poorly understood. Nectar-inhabiting microbial communities affect plant reproduction and pollinator behavior, and are excellent models for studying dispersal of bacteria and fungi in a metacommunity framework. Here, we assay dispersal ability of common nectar bacteria and fungi in an insect-based dispersal experiment. We then compare these results with the incidence and abundance of culturable flower-inhabiting bacteria and fungi within naturally occurring flowers across two coflowering communities in California across two flowering seasons. Our microbial dispersal experiment demonstrates that bacteria disperse via thrips among artificial habitat patches more readily than fungi. In the field, incidence and abundance of culturable bacteria and fungi were positively correlated, but bacteria were much more widespread. These patterns suggest shared dispersal routes or habitat requirements among culturable bacteria and fungi, but differences in dispersal or colonization frequency by thrips, common flower visitors. The finding that culturable bacteria are more common among nectar sampled here, in part due to superior thrips-mediated dispersal, may have relevance for microbial life history, community assembly of microbes, and plant-pollinator interactions.

Nectar bacteria stimulate pollen germination and bursting to enhance microbial fitness.

Many organisms consume pollen, yet mechanisms of its digestion remain a fundamental enigma in pollination biology,1-3 as pollen is protected by a recalcitrant outer shell.4-8 Pollen is commonly found in floral nectar,9,10 as are nectar microbes, which are nearly ubiquitous among flowers.11-13 Nectar specialist bacteria, like Acinetobacter, can reach high densities (up to 109 cells/mL), despite the fact that floral nectar is nitrogen poor.14-17 Here, we show evidence that the genus Acinetobacter, prevalent nectar- and bee-associated bacteria,12,18-20 can induce pollen germination and bursting, gain access to protoplasm nutrients, and thereby grow to higher densities. Although induced germination had been suggested as a potential method in macroscopic pollen consumers,2,21-23 and fungal inhibition of pollen germination has been shown,24-27 direct biological induction of germination has not been empirically documented outside of plants.28-32Acinetobacter pollinis SCC47719 induced over 5× greater pollen germination and 20× greater pollen bursting than that of uninoculated pollen by 45 min. When provided with germinable pollen, A. pollinis stimulates protein release and grows to nearly twice the density compared to growth with ungerminable pollen, indicating that stimulation of germination benefits bacterial fitness. In contrast, a common nectar-inhabiting yeast (Metschnikowia)33 neither induced nor benefited from pollen germination. We conclude that Acinetobacter both specifically causes and benefits from inducing pollen germination and bursting. Further study of microbe-pollen interactions may inform many aspects of pollination ecology, including floral microbial ecology,34,35 pollinator nutrient acquisition from pollen,2,3,21,36 and cues of pollen germination for plant reproduction.37-39.

Microbial abundance, composition, and function in nectar are shaped by flower visitor identity.

Microbial dispersal is essential for establishment in new habitats, but the role of vector identity is poorly understood in community assembly and function. Here, we compared microbial assembly and function in floral nectar visited by legitimate pollinators (hummingbirds) and nectar robbers (carpenter bees). We assessed effects of visitation on the abundance and composition of culturable bacteria and fungi and their taxonomy and function using shotgun metagenomics and nectar chemistry. We also compared metagenome-assembled genomes (MAGs) of Acinetobacter, a common and highly abundant nectar bacterium, among visitor treatments. Visitation increased microbial abundance, but robbing resulted in 10× higher microbial abundance than pollination. Microbial communities differed among visitor treatments: robbed flowers were characterized by predominant nectar specialists within Acetobacteraceae and Metschnikowiaceae, with a concurrent loss of rare taxa, and these resulting communities harbored genes relating to osmotic stress, saccharide metabolism and specialized transporters. Gene differences were mirrored in function: robbed nectar contained a higher percentage of monosaccharides. Draft genomes of Acinetobacter revealed distinct amino acid and saccharide utilization pathways in strains isolated from robbed versus pollinated flowers. Our results suggest an unrecognized cost of nectar robbing for pollination and distinct effects of visitor type on interactions between plants and pollinators. Overall, these results suggest vector identity is an underappreciated factor structuring microbial community assembly and function.

Gazing into the anthosphere: considering how microbes influence floral evolution.

The flower is the hallmark of angiosperms and its evolution is key to their diversification. As knowledge of ecological interactions between flowers and their microbial communities (the anthosphere) expands, it becomes increasingly important to consider the evolutionary impacts of these associations and their potential eco-evolutionary dynamics. In this Viewpoint we synthesize current knowledge of the anthosphere within a multilevel selection framework and illustrate the potential for the extended floral phenotype (the phenotype expressed from the genes of the plant and its associated flower microbes) to evolve. We argue that flower microbes are an important, but understudied, axis of variation that shape floral trait evolution and angiosperm reproductive ecology. We highlight knowledge gaps and discuss approaches that are critical for gaining a deeper understanding of the role microbes play in mediating plant reproduction, ecology, and evolution.

Community-wide consequences of sexual dimorphism: evidence from nectar microbes in dioecious plants.

Intraspecific trait variation is receiving renewed interest as a factor affecting the structure of multi-species communities within and across trophic levels. One pervasive form of intraspecific trait variation is sexual dimorphism in animals and plants, which might exert large effects particularly on the communities of host-associated organisms, but the extent of these effects is not well understood. We investigated whether host-associated microbial communities developed differently in the floral nectar of female and male individuals of the dioecious shrubs, Eurya emarginata and E. japonica. We found that nectar-colonizing microbes such as bacteria and fungi were more than twice as prevalent and, overall, more than 10 times as abundant in male flowers as in female flowers. Microbial species composition also differed between flower sexes. To examine potential mechanisms behind these differences, we manipulated the frequency of flower visitation by animals and the order of arrival of microbial species to nectar. Animal visitation frequency affected microbial communities more greatly in male flowers, while arrival order affected them more in female flowers. These sex-specific effects appeared attributable to differences in how animals and microbes altered the chemical characteristics of nectar that limited microbial growth. Taken together, our results provide evidence that sexual dimorphism can have large effects on the structure of host-associated communities.

Solving the puzzle of yeast survival in ephemeral nectar systems: exponential growth is not enough.

Flower nectar is a sugar-rich ephemeral habitat for microorganisms. Nectar-borne yeasts are part of the microbial community and can affect pollination by changing nectar chemistry, attractiveness to pollinators or flower temperature if yeast population densities are high. Pollinators act as dispersal agents in this system; however, pollination events lead potentially to shrinking nectar yeast populations. We here examine how sufficiently high cell densities of nectar yeast can develop in a flower. In laboratory experiments, we determined the remaining fraction of nectar yeast cells after nectar removal, and used honeybees to determine the number of transmitted yeast cells from one flower to the next. The results of these experiments directly fed into a simulation model providing an insight into movement and colonization ecology of nectar yeasts. We found that cell densities only reached an ecologically relevant size for an intermediate pollination probability. Too few pollination events reduce yeast inoculation rate and too many reduce yeast population size strongly. In addition, nectar yeasts need a trait combination of at least an intermediate growth rate and an intermediate remaining fraction to compensate for highly frequent decimations. Our results can be used to predict nectar yeast dispersal, growth and consequently their ecological effects.

Historical contingency in species interactions: towards niche-based predictions.

The way species affect one another in ecological communities often depends on the order of species arrival. The magnitude of such historical contingency, known as priority effects, varies across species and environments, but this variation has proven difficult to predict, presenting a major challenge in understanding species interactions and consequences for community structure and function. Here, we argue that improved predictions can be achieved by decomposing species' niches into three components: overlap, impact and requirement. Based on classic theories of community assembly, three hypotheses that emphasise related, but distinct influences of the niche components are proposed: priority effects are stronger among species with higher resource use overlap; species that impact the environment to a greater extent exert stronger priority effects; and species whose growth rate is more sensitive to changes in the environment experience stronger priority effects. Using nectar-inhabiting microorganisms as a model system, we present evidence that these hypotheses complement the conventional hypothesis that focuses on the role of environmental harshness, and show that niches can be twice as predictive when separated into components. Taken together, our hypotheses provide a basis for developing a general framework within which the magnitude of historical contingency in species interactions can be predicted.