Patrick Maes

Microbial Ecology of the Hive and Pollination Landscape: Bacterial Associates from Floral Nectar, the Alimentary Tract and Stored Food of Honey Bees (Apis mellifera)

Nearly all eukaryotes are host to beneficial or benign bacteria in their gut lumen, either vertically inherited, or acquired from the environment. While bacteria core to the honey bee gut are becoming evident, the influence of the hive and pollination environment on honey bee microbial health is largely unexplored. Here we compare bacteria from floral nectar in the immediate pollination environment, different segments of the honey bee (Apis mellifera) alimentary tract, and food stored in the hive (honey and packed pollen or “beebread”). We used cultivation and sequencing to explore bacterial communities in all sample types, coupled with culture-independent analysis of beebread. We compare our results from the alimentary tract with both culture-dependent and culture-independent analyses from previous studies. Culturing the foregut (crop), midgut and hindgut with standard media produced many identical or highly similar 16S rDNA sequences found with 16S rDNA clone libraries and next generation sequencing of 16S rDNA amplicons. Despite extensive culturing with identical media, our results do not support the core crop bacterial community hypothesized by recent studies. We cultured a wide variety of bacterial strains from 6 of 7 phylogenetic groups considered core to the honey bee hindgut. Our results reveal that many bacteria prevalent in beebread and the crop are also found in floral nectar, suggesting frequent horizontal transmission. From beebread we uncovered a variety of bacterial phylotypes, including many possible pathogens and food spoilage organisms, and potentially beneficial bacteria including Lactobacillus kunkeei, Acetobacteraceae and many different groups of Actinobacteria. Contributions of these bacteria to colony health may include general hygiene, fungal and pathogen inhibition and beebread preservation. Our results are important for understanding the contribution to pollinator health of both environmentally vectored and core microbiota, and the identification of factors that may affect bacterial detection and transmission, colony food storage and disease susceptibility.

The Bacterial Communities Associated with Honey Bee (Apis mellifera) Foragers

The honey bee is a key pollinator species in decline worldwide. As part of a commercial operation, bee colonies are exposed to a variety of agricultural ecosystems throughout the year and a multitude of environmental variables that may affect the microbial balance of individuals and the hive. While many recent studies support the idea of a core microbiota in guts of younger in-hive bees, it is unknown whether this core is present in forager bees or the pollen they carry back to the hive. Additionally, several studies hypothesize that the foregut (crop), a key interface between the pollination environment and hive food stores, contains a set of 13 lactic acid bacteria (LAB) that inoculate collected pollen and act in synergy to preserve pollen stores. Here, we used a combination of 454 based 16S rRNA gene sequencing of the microbial communities of forager guts, crops, and corbicular pollen and crop plate counts to show that (1) despite a very different diet, forager guts contain a core microbiota similar to that found in younger bees, (2) corbicular pollen contains a diverse community dominated by hive-specific, environmental or phyllosphere bacteria that are not prevalent in the gut or crop, and (3) the 13 LAB found in culture-based studies are not specific to the crop but are a small subset of midgut or hindgut specific bacteria identified in many recent 454 amplicon-based studies. The crop is dominated by Lactobacillus kunkeei, and Alpha 2.2 (Acetobacteraceae), highly osmotolerant and acid resistant bacteria found in stored pollen and honey. Crop taxa at low abundance include core hindgut bacteria in transit to their primary niche, and potential pathogens or food spoilage organisms seemingly vectored from the pollination environment. We conclude that the crop microbial environment is influenced by worker task, and may function in both decontamination and inoculation.

Origin and Effect of Alpha 2.2 Acetobacteraceae in Honey Bee Larvae and Description of Parasaccharibacter apium gen. nov., sp. nov.

ABSTRACT The honey bee hive environment contains a rich microbial community that differs according to niche. Acetobacteraceae Alpha 2.2 (Alpha 2.2) bacteria are present in the food stores, the forager crop, and larvae but at negligible levels in the nurse and forager midgut and hindgut. We first sought to determine the source of Alpha 2.2 in young larvae by assaying the diversity of microbes in nurse crops, hypopharyngeal glands (HGs), and royal jelly (RJ). Amplicon-based pyrosequencing showed that Alpha 2.2 bacteria occupy each of these environments along with a variety of other bacteria, including Lactobacillus kunkeei. RJ and the crop contained fewer bacteria than the HGs, suggesting that these tissues are rather selective environments. Phylogenetic analyses showed that honey bee-derived Alpha 2.2 bacteria are specific to bees that “nurse” the hives developing brood with HG secretions and are distinct from the Saccharibacter-type bacteria found in bees that provision their young differently, such as with a pollen ball coated in crop-derived contents. Acetobacteraceae can form symbiotic relationships with insects, so we next tested whether Alpha 2.2 increased larval fitness. We cultured 44 Alpha 2.2 strains from young larvae that grouped into nine distinct clades. Three isolates from these nine clades flourished in royal jelly, and one isolate increased larval survival in vitro. We conclude that Alpha 2.2 bacteria are not gut bacteria but are prolific in the crop-HG-RJ-larva niche, passed to the developing brood through nurse worker feeding behavior. We propose the name Parasaccharibacter apium for this bacterial symbiont of bees in the genus Apis.

Hive-stored pollen of honey bees: many lines of evidence are consistent with pollen preservation, not nutrient conversion

Honey bee hives are filled with stored pollen, honey, plant resins and wax, all antimicrobial to differing degrees. Stored pollen is the nutritionally rich currency used for colony growth and consists of 40–50% simple sugars. Many studies speculate that prior to consumption by bees, stored pollen undergoes long‐term nutrient conversion, becoming more nutritious ‘bee bread’ as microbes predigest the pollen. We quantified both structural and functional aspects associated with this hypothesis using behavioural assays, bacterial plate counts, microscopy and 454 amplicon sequencing of the 16S rRNA gene from both newly collected and hive‐stored pollen. We found that bees preferentially consume fresh pollen stored for <3 days. Newly collected pollen contained few bacteria, values which decreased significantly as pollen were stored >96 h. The estimated microbe to pollen grain surface area ratio was 1:1 000 000 indicating a negligible effect of microbial metabolism on hive‐stored pollen. Consistent with these findings, hive‐stored pollen grains did not appear compromised according to microscopy. Based on year round 454 amplicon sequencing, bacterial communities of newly collected and hive‐stored pollen did not differ, indicating the lack of an emergent microbial community co‐evolved to digest stored pollen. In accord with previous culturing and 16S cloning, acid resistant and osmotolerant bacteria like Lactobacillus kunkeei were found in greatest abundance in stored pollen, consistent with the harsh character of this microenvironment. We conclude that stored pollen is not evolved for microbially mediated nutrient conversion, but is a preservative environment due primarily to added honey, nectar, bee secretions and properties of pollen itself.

Diet‐related gut bacterial dysbiosis correlates with impaired development, increased mortality and Nosema disease in the honeybee (Apis mellifera)

Dysbiosis, defined as unhealthy shifts in bacterial community composition, can lower the colonization resistance of the gut to intrinsic pathogens. Here, we determined the effect of diet age and type on the health and bacterial community composition of the honeybee (Apis mellifera). We fed newly emerged bees fresh or aged diets, and then recorded host development and bacterial community composition from four distinct regions of the hosts’ digestive tract. Feeding fresh pollen or fresh substitute, we found no difference in host mortality, diet consumption, development or microbial community composition. In contrast, bees fed aged diets suffered impaired development, increased mortality and developed a significantly dysbiotic microbiome. The consumption of aged diets resulted in a significant reduction in the core ileum bacterium Snodgrassella alvi and a corresponding increase in intrinsic pathogen Frischella perrara. Moreover, the relative abundance of S. alvi in the ileum was positively correlated with host survival and development. The inverse was true for both F. perrara and Parasacharibacter apium. Collectively, our findings suggest that the early establishment of S. alvi is associated with healthy nurse development and potentially excludes F. perrara and P. apium from the ileum. Although at low abundance, establishment of the common midgut pathogen Nosema spp. was significantly associated with ileum dysbiosis and associated host deficiencies. Moreover, dysbiosis in the ileum was reflected in the rectum, mouthparts and hypopharyngeal glands, suggesting a systemic host effect. Our findings demonstrate that typically occurring alterations in diet quality play a significant role in colony health and the establishment of a dysbiotic gut microbiome.

Key Edaphic Properties Largely Explain Temporal and Geographic Variation in Soil Microbial Communities across Four Biomes.

Soil microbial communities play a critical role in nutrient transformation and storage in all ecosystems. Quantifying the seasonal and long-term temporal extent of genetic and functional variation of soil microorganisms in response to biotic and abiotic changes within and across ecosystems will inform our understanding of the effect of climate change on these processes. We examined spatial and seasonal variation in microbial communities based on 16S rRNA gene sequencing and phospholipid fatty acid (PLFA) composition across four biomes: a tropical broadleaf forest (Hawaii), taiga (Alaska), semiarid grassland-shrubland (Utah), and a subtropical coniferous forest (Florida). In this study, we used a team-based instructional approach leveraging the iPlant Collaborative to examine publicly available National Ecological Observatory Network (NEON) 16S gene and PLFA measurements that quantify microbial diversity, composition, and growth. Both profiling techniques revealed that microbial communities grouped strongly by ecosystem and were predominately influenced by three edaphic factors: pH, soil water content, and cation exchange capacity. Temporal variability of microbial communities differed by profiling technique; 16S-based community measurements showed significant temporal variability only in the subtropical coniferous forest communities, specifically through changes within subgroups of Acidobacteria. Conversely, PLFA-based community measurements showed seasonal shifts in taiga and tropical broadleaf forest systems. These differences may be due to the premise that 16S-based measurements are predominantly influenced by large shifts in the abiotic soil environment, while PLFA-based analyses reflect the metabolically active fraction of the microbial community, which is more sensitive to local disturbances and biotic interactions. To address the technical issue of the response of soil microbial communities to sample storage temperature, we compared 16S-based community structure in soils stored at -80°C and -20°C and found no significant differences in community composition based on storage temperature. Free, open access datasets and data sharing platforms are powerful tools for integrating research and teaching in undergraduate and graduate student classrooms. They are a valuable resource for fostering interdisciplinary collaborations, testing ecological theory, model development and validation, and generating novel hypotheses. Training in data analysis and interpretation of large datasets in university classrooms through project-based learning improves the learning experience for students and enables their use of these significant resources throughout their careers.

The impact of pollen consumption on honey bee (Apis mellifera) digestive physiology and carbohydrate metabolism

Carbohydrate-active enzymes play an important role in the honey bee (Apis mellifera) due to its dietary specialization on plant-based nutrition. Secretory glycoside hydrolases (GHs) produced in worker head glands aid in the processing of floral nectar into honey and are expressed in accordance with age-based division of labor. Pollen utilization by the honey bee has been investigated in considerable detail, but little is known about the metabolic fate of indigestible carbohydrates and glycosides in pollen biomass. Here, we demonstrate that pollen consumption stimulates the hydrolysis of sugars that are toxic to the bee (xylose, arabinose, mannose). GHs produced in the head accumulate in the midgut and persist in the hindgut that harbors a core microbial community composed of approximately 108 bacterial cells. Pollen consumption significantly impacted total and specific bacterial abundance in the digestive tract. Bacterial isolates representing major fermentative gut phylotypes exhibited primarily membrane-bound GH activities that may function in tandem with soluble host enzymes retained in the hindgut. Additionally, we found that plant-originating β-galactosidase activity in pollen may be sufficient, in some cases, for probable physiological activity in the gut. These findings emphasize the potential relative contributions of host, bacteria, and pollen enzyme activities to carbohydrate breakdown, which may be tied to gut microbiome dynamics and associated host nutrition.

Internal hive temperature as a means of monitoring honey bee colony health in a migratory beekeeping operation before and during winter

Internal temperatures of honey bee hives kept at different sites in North Dakota were monitored before and during winter to evaluate the effects of treatment, in the form of exposure to commercial pollination, and location on colony health. In October, hives exposed to commercial pollination during the summer had fewer adult bees and less brood than hives kept near natural forage, as well as lower average temperatures throughout winter. Within-day temperature variability was higher among hives exposed to commercial agriculture than for those kept near natural forage, indicating reduced temperature control. Fungicides, insecticides, varroacides, and an herbicide were detected in bee bread and wax samples; no major differences were observed either in the diversity or in the concentrations of agrochemicals with the exception of chlorpyrifos at one site. Varroa and Nosema densities were low overall. Data from the same site used in successive years showed significantly more brood the first year, as well as lower temperature variability; high levels of chlorpyrifos were detected in bee bread of colonies in the second year. Colony average temperature and temperature variability were informative with respect to colony phenology and post-winter status.

The queen’s gut refines with age: longevity phenotypes in a social insect model

BackgroundIn social insects, identical genotypes can show extreme lifespan variation providing a unique perspective on age-associated microbial succession. In honey bees, short- and long-lived host phenotypes are polarized by a suite of age-associated factors including hormones, nutrition, immune senescence, and oxidative stress. Similar to other model organisms, the aging gut microbiota of short-lived (worker) honey bees accrue Proteobacteria and are depleted of Lactobacillus and Bifidobacterium, consistent with a suite of host senescence markers. In contrast, long-lived (queen) honey bees maintain youthful cellular function with much lower expression of oxidative stress genes, suggesting a very different host environment for age-associated microbial succession.ResultsWe sequenced the microbiota of 63 honey bee queens exploring two chronological ages and four alimentary tract niches. To control for genetic and environmental variation, we quantified carbonyl accumulation in queen fat body tissue as a proxy for biological aging. We compared our results to the age-specific microbial succession of worker guts. Accounting for queen source variation, two or more bacterial species per niche differed significantly by queen age. Biological aging in queens was correlated with microbiota composition highlighting the relationship of microbiota with oxidative stress. Queens and workers shared many major gut bacterial species, but differ markedly in community structure and age succession. In stark contrast to aging workers, carbonyl accumulation in queens was significantly associated with increased Lactobacillus and Bifidobacterium and depletion of various Proteobacteria.ConclusionsWe present a model system linking changes in gut microbiota to diet and longevity, two of the most confounding variables in human microbiota research. The pattern of age-associated succession in the queen microbiota is largely the reverse of that demonstrated for workers. The guts of short-lived worker phenotypes are progressively dominated by three major Proteobacteria, but these same species were sparse or significantly depleted in long-lived queen phenotypes. More broadly, age-related changes in the honey bee microbiota reflect the regulatory anatomy of reproductive host metabolism. Our synthesis suggests that the evolution of colony-level reproductive physiology formed the context for host-microbial interactions and age-related succession of honey bee microbiota.