Location: Carl Hayden Bee Research Center
2019 Annual Report
Objectives
Increasing evidence points to a core honey bee gut microbiota, however the distribution and function of peripheral bacterial and fungal communities in honeybees and their food stores are relatively unknown. Our work will focus on bee bread to define the contribution of unknown and seemingly benign microbes to colony health and nutrition. Bee bread provides the bulk of proteins, vitamins and lipids that bees consume. We will determine the microbial succession in bee bread to understand the distribution of these bee bread-associated microbial communities and whether these communities contribute to the nutritive value of pollen, its digestion and storage, and the potential for disease transmission and amplification. We will examine factors associated with beekeeping and agricultural practices that may affect the microbial balance of the honey bee and its stored food, including colony origin, supplemental feeding, nectar source, pollen type, and exposure to biocides. These data will inform our perspective on how microbial communities contribute to colony health.
OBJECTIVES
Our overall goal is to provide beekeepers and growers with practical advice for the maintenance of transitory commercial honey bee populations. Using a combination of laboratory and field approaches we will develop an understanding of the diversity, abundance, persistence and functional capacities of the microorganisms that occur in bees, stored food, the hive, and the general pollination environment. This information will be applied to the management of disease, nutrition, overwintering and biocides in the context of commercial beekeeping.
Objective 1: Determine the core fungal microbiota in bee bread and determine relationships with bacterial communities.
Subobjective 1A: Enumerate, identify, and characterize the core fungal and bacterial microbiota of bee bread.
Objective 2: Characterize microbial succession in bee bread, including core and non- core microbes and their persistence during overwinter pollen storage.
Subobjective 2A: Identify the microbial communities involved in the conversion of corbicular pollen to bee bread.
Subobjective 2B: Determine the impact of overwintering on the microbes in bee bread.
Objective 3: Identify factors affecting a colony's microbial diversity, including plant monocultures, exposure to biocides, and supplemental feeding.
Subobjective 3A: Determine the effect of supplemental feeding on microbial communities.
Subobjective 3B: Determine the effect of monoculture nectar and pollen source on microbial communities.
Subobjective 3C: Determine the effect of fungicides on microbes during bee bread
formation.
Approach
Subobjective 1A: Determine whether bee bread contains a core microbial community.
Bee bread will be sampled from multiple colonies, apiaries, and commercial operations across a variety of locations and seasons. rRNA will be used to
characterize the active microbiota of bee bread. Fungal and bacterial groups
identified at different levels of taxonomic certainty will be examined for
significant co-occurrence using a variety of available metrics (including options
for degenerate matrices) and a null hypothesis of random community assembly.
Subobjective 2A: Determine whether the active microbial (bacterial and fungal)
community remains constant as corbicular pollen becomes bee bread and as bee bread ages. We will detail the active fungal and bacterial communities in multiple colonies, controlling for the source of corbicular pollen and season. Multiple replicates of bee bread at 0.5, 1, 3, 7, 14, and 30 days of age will be sampled and processed for microbial composition. Dependent upon the predictability of a "core" functional set of successional genes in bee bread, we will develop metagenomic profiling methods for a more efficient characterization of microbial function.
Subobjective 2B: Determine whether the microbial communities of overwintered bee
bread sampled from old wax comb differ from those of new wax comb. Bee bread will be collected in RNA later from both old and new wax comb from commercial beekeeping operations. RNA extracted from bee bread will be subject to 454 amplicon sequencing and compared according to overwintering status and comb age. If differences in the microbial communities are discovered, we will determine the nutrients, preservatives and metabolites associated with these changes.
Subobjective 3A: Determine whether supplemental feed affects the active honey bee
gut microbial community. Bees will be fed commonly used brewer's yeast/soy/sucrosebased nutritional supplements containing thymol alone, citric acid alone, thymol and citric acid, honey bee healthy, no additives, and fresh bee bread/honey as a control. RNA will be extracted from these pooled samples and community composition of the gut will be assessed using qRT-PCR targeted to the core gut bacteria.
Subobjective 3B: Determine whether the active microbial communities in bee bread
differ by pollination environment. We will sample both corbicular pollen and bee
bread microbial communities of colonies actively pollinating two distantly located monocultures and two distantly located plant polycultures. Samples will be subject to qRT-PCR targeting specific genera, and also pooled by colony and subject to 454 amplicon sequencing for comparative purposes. If species-specific qRT-PCR primers prove overly time consuming or inefficient, we will rely on the sequencing of major functional COGs.
Subobjective 3C: Determine whether the microbes and nutrition in bee bread are
affected by fungicide. Endura fungicide will be applied at field concentrations to a Brassica mix grown in greenhouses. Bees will be allowed to forage on fungicide sprayed and non-fungicide controls. Bee bread will be examined for microbial communities, fungicide concentrations and nutritional analysis.
Progress Report
This report documents progress for bridging project 2022-21000-019-00D, which continues research from expired project 2022-21000-017-00D, currently undergoing NP305 Office of Scientific Quality Review.
Research continued on the microbiome of honey bees addressing hypotheses associated with worker homeostasis overwinter, bacterial colonization in queens, larval health and disease, and the development of a bacterial biocide to combat perhaps the most damaging of all honey bee afflictions: an ecto-parasitic mite known as Varroa destructor, a known vector of virus and likely vector of fungal and bacterial pathogens.
Protection from pathogens is considered the primary function of the eukaryotic microbiome, and changes to the normal gut microbiota can range from mildly anticommensal to pathogenic. Similar to other model organisms, the aging gut microbiota of short-lived (worker) honey bees becomes depleted of fermentative bacteria and Firmicutes while the abundance of Proteobacteria increases. The gut microbiota of winter honey bees was sequenced from three locations and two alimentary tract niches. A known age cohort was sampled congruently to act as a control for chronological age. We found no discernable differences in the gut microbiota of aging worker bees based on chronological age or climate.
Additionally, there were no major differences in community size across treatment groups considering both bacteria and fungus. These results complete the spectrum of aging microbiota across three honey bee longevity phenotypes. The guts of short-lived worker phenotypes are progressively dominated by three major Proteobacteria, long lived reproductive phenotypes become enriched with probiotic species, while the long-lived worker phenotype maintains a remarkably static hindgut microbiome. Stability of the gut microbiota over extended time periods provides a novel tool to investigate host-microbial interactions. The gut microbial succession documented here highlights the honey bee as a model for understanding the contribution of diet and microbiota in the aging process.
Early queen health is critical for colony longevity and performance. We continued our work on the queen microbiome, exploring gene expression and microbial changes in early life associated with queen breeder and environmental context. The analysis is ongoing but confirms previous hypothesis that an unnamed Acetobacteraceae (Alpha 2.1) species is abundant in early life, but gradually replaced by Lactobacillus species as queens age. The microbiome structure varied by both breeder and queen age, suggesting that queen microbial succession is very different from that of workers.
Research continued on the development of a potential Varroa biocide, testing CRY (crystal) proteins and spores produced by the bacterium Bacillus thuriengensis (Bt) for their efficacy killing the Varroa mite. Using clay as a medium to carry and spread the Bt derived spores and CRY proteins, we examined the effect of three treatment conditions on Varroa mortality using eleven colonies per treatment; clay without Bt, clay with Bt, and Amitraz as a positive control.
Accomplishments
1. Access to U.S. Conservation Reserve Program (CRP) lands greatly improves honey bee colony health. The nutritional landscape is critical to the sustainability of commercial beekeeping. Exposure of colonies to either U.S. Conservation Reserve Program (CRP) lands or intensively cultivated landscapes revealed that nutritional state, oxidative stress resistance, and immunity were significantly improved by habitat conservation efforts. Colonies exposed to CRP landscapes showed markedly improved performance and higher mRNA levels of vitellogenin (vg), a nutritionally regulated protein with central storage and regulatory functions. Mirroring vg levels, gene transcripts encoding antioxidant enzymes and immune-related proteins were significantly higher in colonies exposed to CRP landscapes compared to those exposed to intensive agriculture. The study confirms the overwhelming utility of U.S. conservation lands for commercial beekeepers and agricultural pollination services.
