Location: Honey Bee Research
Project Number: 2022-21000-019-00-D
Project Type: In-House Appropriated
Start Date: Feb 7, 2019
End Date: Feb 6, 2020
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.
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.