Symbiotic bacteria and fungi proliferate in diapause and may enhance overwintering survival in a solitary bee.

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.

Nontarget impacts of neonicotinoids on nectar-inhabiting microbes.

Plant-systemic neonicotinoid (NN) insecticides can exert non-target impacts on organisms like beneficial insects and soil microbes. NNs can affect plant microbiomes, but we know little about their effects on microbial communities that mediate plant-insect interactions, including nectar-inhabiting microbes (NIMs). Here we employed two approaches to assess the impacts of NN exposure on several NIM taxa. First, we assayed the in vitro effects of six NN compounds on NIM growth using plate assays. Second, we inoculated a standardised NIM community into the nectar of NN-treated canola (Brassica napus) and assessed microbial survival and growth after 24 h. With few exceptions, in vitro NN exposure tended to decrease bacterial growth metrics. However, the magnitude of the decrease and the NN concentrations at which effects were observed varied substantially across bacteria. Yeasts showed no consistent in vitro response to NNs. In nectar, we saw no effects of NN treatment on NIM community metrics. Rather, NIM abundance and diversity responded to inherent plant qualities like nectar volume. In conclusion, we found no evidence that NIMs respond to field-relevant NN levels in nectar within 24 h, but our study suggests that context, specifically assay methods, time and plant traits, is important in assaying the effects of NNs on microbial communities.

Plant species within Streptanthoid Complex associate with distinct microbial communities that shift to be more similar under drought.

Prolonged water stress can shift rhizoplane microbial communities, yet whether plant phylogenetic relatedness or drought tolerance predicts microbial responses is poorly understood. To explore this question, eight members of the Streptanthus clade with varying affinity to serpentine soil were subjected to three watering regimes. Rhizoplane bacterial communities were characterized using 16S rRNA gene amplicon sequencing and we compared the impact of watering treatment, soil affinity, and plant species identity on bacterial alpha and diversity. We determined which taxa were enriched among drought treatments using DESeq2 and identified features of soil affinity using random forest analysis. We show that water stress has a greater impact on microbial community structure than soil affinity or plant identity, even within a genus. Drought reduced alpha diversity overall, but plant species did not strongly differentiate alpha diversity. Watering altered the relative abundance of bacterial genera within Proteobacteria, Firmicutes, Bacteroidetes, Planctomycetes, and Acidobacteria, which responded similarly in the rhizoplane of most plant species. In addition, bacterial communities were more similar when plants received less water. Pseudarthrobacter was identified as a feature of affinity to serpentine soil while Bradyrhizobium, Chitinophaga, Rhodanobacter, and Paenibacillus were features associated with affinity to nonserpentine soils among Streptanthus. The homogenizing effect of drought on microbial communities and the increasing prevalence of Gram-negative bacteria across all plant species suggest that effects of water stress on root-associated microbiome structure may be predictable among closely related plant species that inhabit very different soil environments. The functional implications of observed changes in microbiome composition remain to be studied.

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.

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.

Bees just wanna have fungi: a review of bee associations with nonpathogenic fungi.

Bee-fungus associations are common, and while most studies focus on entomopathogens, emerging evidence suggests that bees associate with a variety of symbiotic fungi that can influence bee behavior and health. Here, we review nonpathogenic fungal taxa associated with different bee species and bee-related habitats. We synthesize results of studies examining fungal effects on bee behavior, development, survival, and fitness. We find that fungal communities differ across habitats, with some groups restricted mostly to flowers (Metschnikowia), while others are present almost exclusively in stored provisions (Zygosaccharomyces). Starmerella yeasts are found in multiple habitats in association with many bee species. Bee species differ widely in the abundance and identity of fungi hosted. Functional studies suggest that yeasts affect bee foraging, development, and pathogen interactions, though few bee and fungal taxa have been examined in this context. Rarely, fungi are obligately beneficial symbionts of bees, whereas most are facultative bee associates with unknown or ecologically contextual effects. Fungicides can reduce fungal abundance and alter fungal communities associated with bees, potentially disrupting bee-fungi associations. We recommend that future study focus on fungi associated with non-honeybee species and examine multiple bee life stages to document fungal composition, abundance, and mechanistic effects on bees.

Nectar compounds impact bacterial and fungal growth and shift community dynamics in a nectar analog.

Floral nectar is frequently colonised by microbes. However, nectar microbial communities are typically species-poor and dominated by few cosmopolitan genera. One hypothesis is that nectar constituents may act as environmental filters. We tested how five non-sugar nectar compounds as well as elevated sugar impacted the growth of 12 fungal and bacterial species isolated from nectar, pollinators, and the environment. We hypothesised that nectar isolated microbes would have the least growth suppression. Additionally, to test if nectar compounds could affect the outcome of competition between microbes, we grew a subset of microbes in co-culture across a subset of treatments. We found that some compounds such as H2 O2 suppressed microbial growth across many but not all microbes tested. Other compounds were more specialised in the microbes they impacted. As hypothesised, the nectar specialist yeast Metschnikowia reukaufii was unaffected by most nectar compounds assayed. However, many non-nectar specialist microbes remained unaffected by nectar compounds thought to reduce microbial growth. Our results show that nectar chemistry can influence microbial communities but that microbe-specific responses to nectar compounds are common. Nectar chemistry also affected the outcome of species interactions among microbial taxa, suggesting that non-sugar compounds can affect microbial community assembly in flowers.

Incipiently social carpenter bees (Xylocopa) host distinctive gut bacterial communities and display geographical structure as revealed by full-length PacBio 16S rRNA sequencing.

The gut microbiota of bees affects nutrition, immunity and host fitness, yet the roles of diet, sociality and geographical variation in determining microbiome structure, including variant-level diversity and relatedness, remain poorly understood. Here, we use full-length 16S rRNA amplicon sequencing to compare the crop and gut microbiomes of two incipiently social carpenter bee species, Xylocopa sonorina and Xylocopa tabaniformis, from multiple geographical sites within each species' range. We found that Xylocopa species share a set of core taxa consisting of Bombilactobacillus, Bombiscardovia and Lactobacillus, found in >95% of all individual bees sampled, and Gilliamella and Apibacter were also detected in the gut of both species with high frequency. The crop bacterial community of X. sonorina comprised nearly entirely Apilactobacillus with occasionally abundant nectar bacteria. Despite sharing core taxa, Xylocopa species' microbiomes were distinguished by multiple bacterial lineages, including species-specific variants of core taxa. The use of long-read amplicons revealed otherwise cryptic species and population-level differentiation in core microbiome members, which was masked when a shorter fragment of the 16S rRNA (V4) was considered. Of the core taxa, Bombilactobacillus and Bombiscardovia exhibited differentiation in amplicon sequence variants among bee populations, but this was lacking in Lactobacillus, suggesting that some bacterial genera in the gut may be structured by different processes. We conclude that these Xylocopa species host a distinctive microbiome, similar to that of previously characterized social corbiculate apids, which suggests that further investigation to understand the evolution of the bee microbiome and its drivers is warranted.

Thermal sensitivity and seasonal change in the gut microbiome of a desert ant, Cephalotes rohweri.

Microorganisms within ectotherms must withstand the variable body temperatures of their hosts. Shifts in host body temperature resulting from climate change have the potential to shape ectotherm microbiome composition. Microbiome compositional changes occurring in response to temperature in nature have not been frequently examined, restricting our ability to predict microbe-mediated ectotherm responses to climate change. In a set of field-based observations, we characterized gut bacterial communities and thermal exposure across a population of desert arboreal ants (Cephalotes rohweri). In a paired growth chamber experiment, we exposed ant colonies to variable temperature regimes differing by 5°C for three months. We found that the abundance and composition of ant-associated bacteria were sensitive to elevated temperatures in both field and laboratory experiments. We observed a subset of taxa that responded similarly to temperature in the experimental and observational study, suggesting a role of seasonal temperature and local temperature differences amongst nests in shaping microbiomes within the ant population. Bacterial mutualists in the genus Cephaloticoccus (Opitutales: Opitutaceae) were especially sensitive to change in temperature-decreasing in abundance in naturally warm summer nests and warm growth chambers. We also report the discovery of a member of the Candidate Phlya Radiation (Phylum: Gracilibacteria), a suspected epibiont, found in low abundance within the guts of this ant species.

Floral Microbes Suppress Growth of Monilinia laxa with Minimal Effects on Honey Bee Feeding.

Management of Monilinia laxa, the causal agent of brown rot blossom blight in almond (Prunus dulcis), relies heavily on the use of chemical fungicides during bloom. However, chemical fungicides can have nontarget effects on beneficial arthropods, including pollinators, and select for resistance in the pathogen of concern. Almond yield is heavily reliant on successful pollination by healthy honey bees (Apis mellifera); thus, identifying sustainable, effective, and pollinator-friendly control methods for blossom blight during bloom is desirable. Flower-inhabiting microbes could provide a natural, sustainable form of biocontrol for M. laxa, while potentially minimizing costly nontarget effects on almond pollinators and the services they provide. As pollinators are sensitive to floral microbes and their associated taste and scent cues, assessing effects of prospective biocontrol species on pollinator attraction is also necessary. Here, our objective was to isolate and identify potential biocontrol microbes from an array of agricultural and natural flowering hosts and test their efficacy in suppressing M. laxa growth in culture. Out of an initial 287 bacterial and fungal isolates identified, 56 were screened using a dual culture plate assay. Most strains reduced M. laxa growth in vitro. Ten particularly effective candidate microbes were further screened for their effect on honey bee feeding. Of the 10, nine were found to both strongly suppress M. laxa growth in culture and not reduce honey bee feeding. These promising results suggest a number of strong candidates for augmentative microbial biocontrol of brown rot blossom blight in almond with potentially minimal effects on honey bee pollination.

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.

Plant phenology influences rhizosphere microbial community and is accelerated by serpentine microorganisms in Plantago erecta.

Serpentine soils are drought-prone and rich in heavy metals, and plants growing on serpentine soils host distinct microbial communities that may affect plant survival and phenotype. However, whether the rhizosphere communities of plants from different soil chemistries are initially distinct or diverge over time may help us understand drivers of microbial community structure and function in stressful soils. Here, we test the hypothesis that rhizosphere microbial communities will converge over time (plant development), independent of soil chemistry and microbial source. We grew Plantago erecta in serpentine or nonserpentine soil, with serpentine or nonserpentine microbes and tracked plant growth and root phenotypes. We used 16S rRNA gene barcoding to compare bacterial species composition at seedling, vegetative, early- and late-flowering phases. Plant phenotype and rhizosphere bacterial communities were mainly structured by soil type, with minor contributions by plant development, microbe source and their interactions. Serpentine microorganisms promoted early flowering in plants on nonserpentine soils. Despite strong effects of soil chemistry, the convergence in bacterial community composition across development demonstrates the importance of the plant-microbe interactions in shaping microbial assembly processes across soil types.

Acinetobacter pollinis sp. nov., Acinetobacter baretiae sp. nov. and Acinetobacter rathckeae sp. nov., isolated from floral nectar and honey bees.

A detailed evaluation of eight bacterial isolates from floral nectar and animal visitors to flowers shows evidence that they represent three novel species in the genus Acinetobacter. Phylogenomic analysis shows the closest relatives of these new isolates are Acinetobacter apis, Acinetobacter boissieri and Acinetobacter nectaris, previously described species associated with floral nectar and bees, but high genome-wide sequence divergence defines these isolates as novel species. Pairwise comparisons of the average nucleotide identity of the new isolates compared to known species is extremely low (<83 %), thus confirming that these samples are representative of three novel Acinetobacter species, for which the names Acinetobacter pollinis sp. nov., Acinetobacter baretiae sp. nov. and Acinetobacter rathckeae sp. nov. are proposed. The respective type strains are SCC477T (=TSD-214T=LMG 31655T), B10AT (=TSD-213T=LMG 31702T) and EC24T (=TSD-215T=LMG 31703T=DSM 111781T).

Orchard Management and Landscape Context Mediate the Pear Floral Microbiome.

Crop-associated microbiota are a key factor affecting host health and productivity. Most crops are grown within heterogeneous landscapes, and interactions between management practices and landscape context often affect plant and animal biodiversity in agroecosystems. However, whether these same factors typically affect crop-associated microbiota is less clear. Here, we assessed whether orchard management strategies and landscape context affected bacterial and fungal communities in pear (Pyrus communis) flowers. We found that bacteria and fungi responded differently to management schemes. Organically certified orchards had higher fungal diversity in flowers than conventional or bio-based integrated pest management (IPM) orchards, but organic orchards had the lowest bacterial diversity. Orchard management scheme also best predicted the distribution of several important bacterial and fungal genera that either cause or suppress disease; organic and bio-based IPM best explained the distributions of bacterial and fungal genera, respectively. Moreover, patterns of bacterial and fungal diversity were affected by interactions between management, landscape context, and climate. When examining the similarity of bacterial and fungal communities across sites, both abundance- and taxon-related turnovers were mediated primarily by orchard management scheme and landscape context and, specifically, the amount of land in cultivation. Our study reveals local- and landscape-level drivers of floral microbiome structure in a major fruit crop, providing insights that can inform microbiome management to promote host health and high-yielding quality fruit. IMPORTANCE Proper crop management during bloom is essential for producing disease-free tree fruit. Tree fruits are often grown in heterogeneous landscapes; however, few studies have assessed whether landscape context and crop management affect the floral microbiome, which plays a critical role in shaping plant health and disease tolerance. Such work is key for identification of tactics and/or contexts where beneficial microbes proliferate and pathogenic microbes are limited. Here, we characterize the floral microbiome of pear crops in Washington State, where major production occurs in intermountain valleys and basins with variable elevation and microclimates. Our results show that both local-level (crop management) and landscape-level (habitat types and climate) factors affect floral microbiota but in disparate ways for each kingdom. More broadly, these findings can potentially inform microbiome management in orchards for promotion of host health and high-quality yields.

Nitrogen Assimilation Varies Among Clades of Nectar- and Insect-Associated Acinetobacters.

Floral nectar is commonly colonized by yeasts and bacteria, whose growth largely depends on their capacity to assimilate nutrient resources, withstand high osmotic pressures, and cope with unbalanced carbon-to-nitrogen ratios. Although the basis of the ecological success of these microbes in the harsh environment of nectar is still poorly understood, it is reasonable to assume that they are efficient nitrogen scavengers that can consume a wide range of nitrogen sources in nectar. Furthermore, it can be hypothesized that phylogenetically closely related strains have more similar phenotypic characteristics than distant relatives. We tested these hypotheses by investigating the growth performance on different nitrogen-rich substrates of a collection of 82 acinetobacters isolated from nectar and honeybees, representing members of five species (Acinetobacter nectaris, A. boissieri, A. apis, and the recently described taxa A. bareti and A. pollinis). We also analyzed possible links between growth performance and phylogenetic affiliation of the isolates, while taking into account their geographical origin. Results demonstrated that the studied isolates could utilize a wide variety of nitrogen sources, including common metabolic by-products of yeasts (e.g., ammonium and urea), and that phylogenetic relatedness was associated with the variation in nitrogen assimilation among the studied acinetobacters. Finally, nutrient source and the origin (sample type and country) of isolates also predicted the ability of the acinetobacters to assimilate nitrogen-rich compounds. Overall, these results demonstrate inter-clade variation in the potential of the acinetobacters as nitrogen scavengers and suggest that nutritional dependences might influence interactions between bacteria and yeasts in floral nectar.

Floral traits affecting the transmission of beneficial and pathogenic pollinator-associated microbes.

Flowers provide resources for pollinators, and can also be transmission venues for beneficial or pathogenic pollinator-associated microbes. Floral traits could mediate transmission similarly for beneficial and pathogenic microbes, although some beneficial microbes can grow in flowers while pathogenic microbes may only survive until acquired by a new host. In spite of conceptual similarities, research on beneficial and pathogenic pollinator-associated microbes has progressed mostly independently. Recent advances demonstrate that floral traits are associated with transmission of beneficial and pathogenic microbes, with consequences for pollinator populations and communities. However, there is a near-absence of experimental manipulations of floral traits to determine causal effects on transmission, and a need to understand how floral, microbe and host traits interact to mediate transmission.

Volatile microbial semiochemicals and insect perception at flowers.

Many plant-associated microbial communities produce volatile signals that influence insect responses, yet the impact of floral microorganisms has received less attention than other plant microbiomes. Floral microorganisms alter plant and floral odors by adding their own emissions or modifying plant volatiles. These contextual and microbe species-specific changes in floral signaling are detectable by insects and can modify their behavior. Opportunities for future work in floral systems include identifying specific microbial semiochemicals that underlie insect behavioral responses and examining if insect species vary in their responses to microbial volatiles. Examining if documented patterns are consistent across diverse plant-microbe-insect interactions and in realistic plant-based studies will improve our understanding of how microbes mediate pollination interactions in complex system.