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ARS Home Pacific West Area Tucson, Arizona Carl Hayden Bee Research Center Research





- Anderson, Kirk
- Carroll, Mark
- 2023
- 2022
- 2021
- 2020
- Publications
- Crop Production (305)

Research Project: The Honey Bee Microbiome in Health and Disease **Location:
                            Carl Hayden Bee Research Center** 2020 Annual Report **Objectives**
                                

Our long-term objective is to understand the structure and function of the honey bee microbiome in health and disease. Using a combination of laboratory and field approaches we will further our understanding of the diversity, abundance, persistence and functional capacities of the microorganisms that occur in the hive environment, the alimentary tracts of queens, workers and developing larvae. This information will be applied to the diagnosis and management of disease associated with commercial beekeeping. Industry applications include management strategies to reduce the severity of brood disease, diagnostic tools for queen health and productivity, and a novel context to assess disease prevention and progression.
The studies outlined in this Project Plan are directed at understanding the healthy microbial balance of a honey bee colony, with particular emphasis on dysbiotic states as precursors to disease. In a social insect like the honey bee, disease must be considered at many levels of organization (Evans and Spivak 2010). This rule also applies to beneficial host-microbe associations. We hypothesize that bacteria commonly shared among developmental stages, tissues, and reproductive castes may represent cryptic drivers of disease evolution (Figure 1). The long-term objective of this project is to identify native microbes that promote or discourage disease. Specifically, during the next five years we will focus on the following objectives.
Objective 1: Develop an integrated research approach (e.g. improved sampling and analytical methods) for the understanding and the management of honey bee larval microbiota, immune priming and brood disease. [NP305, Component 2, Problem Statements 2A and 2B] (Anderson)
Sub-objective 1A: Enumerate, identify, and characterize the microbial succession of healthy and diseased larvae. (Anderson)
Sub-objective 1B: Identify the species and interactions that cause or contribute to larval disease and/or affect larval immune response. (Anderson)
Objective 2: Analyze the population dynamics of the adult honey bee gut microbiota, and extended microbiota, with reference to species and strain variation, ecological niches, potential for functional redundancy, and corresponding host responses. [NP305, Component 2, Problem Statement 2B] (Anderson, Carroll)
Sub-objective 2A: Determine gut succession of the queen microbiota with respect to bacterial function, occupied niche, hive environment and host gene expression. (Anderson)
Sub-objective 2B: Determine how worker trophallactic feeding of queens is associated with the microbiota, queen quality, and worker-queen interactions in established queens. (Carroll, Anderson)
Objective 3: Investigate the effects of plant compounds on honey bee microbiota, their contributions to bee immunity, and their detoxification at the individual and colony-levels. [NP305, Component 2, Problem Statements 2A and 2B] (Anderson, Palmer-Young)
Sub-objective 3A: Determine the effect of plant secondary metabolites on microbial health of workers. (Palmer-Young, Anderson)
Sub-objective 3B: Determine the effect of recalcitrant polysaccharides on host-microbial function in workers. (Anderson) **Approach**
                                

Objective 1. Develop an integrated research approach (e.g. improved sampling and
analytical methods) for the understanding and the management of honey bee larval microbiota, immune priming and brood disease. [NP305, Component 2, Problem Statements 2A and 2B] (Anderson)

Sub-objective 1A: Enumerate, identify, and characterize the microbial succession of healthy and diseased larvae.

Hypothesis 1A: The microbial communities associated with phenotypically healthy and diseased larvae 
do not differ.
Sub-objective 1B: Identify the species and interactions that cause or contribute to larval disease and/or affect larval immune response (Anderson)
Hypothesis 1B: Larval disease defined phenotypically as EFB or EFB-like is due solely to M. plutonius. Objective 2: Analyze the population dynamics of the adult honey bee gut microbiota, and extended microbiota, with reference to species and strain variation, ecological niches, potential for functional redundancy, and corresponding host responses. [NP305, Component 2, Problem Statement 2B] (Anderson, Carroll)
Sub-objective 2A: Determine gut succession of the queen microbiota with respect to bacterial function, occupied niche, hive environment and host gene expression. (Anderson)
Hypothesis 2A: Microbial succession of queen alimentary tracts and host gene expression does not differ by niche and early hive environment.
Sub-objective 2B: Determine how worker trophallactic feeding of queens is associated with the microbiota, queen quality, and worker-queen interactions in established queens. (Carroll, Anderson)
Hypothesis 2B: Selective trophallactic feeding of queens by workers is associated with the queen or worker microbiota, queen and worker quality, and worker-queen interactions mediated by pheromone exchanges.
Objective 3: Investigate the effects of plant compounds on honey bee microbiota, their contributions to bee immunity, and their detoxification at the individual and colony-levels. [NP305, Component 2, Problem Statements 2A and 2B] (Anderson, Palmer-Young)
Sub-objective 3A: Determine the effect of plant secondary metabolites on microbial health of workers. (Palmer-Young, Anderson)
Hypothesis 3A: Hindgut microbial communities and/or host health metrics are unaffected by plant secondary metabolites in the diet. Sub-objective 3B: Determine the effect of recalcitrant polysaccharides on host-microbial function in workers. (Anderson)
Hypothesis 3B: Hindgut microbial communities and/or host health metrics are unaffected by the addition of recalcitrant polysaccharides in the diet. **Progress Report**
                                

This report documents progress for project 2022-21000-021-00D, "The Honey Bee Microbiome in Health and Disease", which started October 2019 and continues research from project 2022-21000-019-00D, "Understanding Honey Bee Microbiota to Improve Bee Nutrition and Colony Health".

In support of Sub-Objective 1A, larvae from honey bee colonies were sampled from multiple apiaries expressing various forms of brood disease throughout the state of Michigan. As hives moved throughout various pollination routes, shifting disease states collected by longitudinal and replicated sampling were recorded, including covariate hive metrics from both recovered and deceased hives. Concurrently, from the state of Illinois, research continued on molecular exploration of different disease states and larval stages as designated by high-resolution photographs and extension professionals. 850 additional larval disease microbiomes from 18 apiaries in Illinois were prepared, sequenced, and analyzed, bringing our grand sequencing total to 1070 larval microbiomes from 24 Illinois apiaries. 

In support of Sub-Objective 1B, Researchers from Tucson, Arizona, sequenced the bacterial microbiota associated with larval disease transmission, isolated a variety of disease causing bacterial strains, determined the virulence of these bacteria against larvae in the laboratory setting, and explored the potential for probiotic treatment of European foul Brood disease. For the duration of the present project plan, an agreement was established with a prominent pollination specialist to sample major migratory routes of commercial beekeepers subject to larval disease outbreaks throughout the western states and northern grasslands. 

To supplement the funding of Objective 1, an Agriculture and Food Research Initiative, National Institute of Food and Agriculture grant was applied for and awarded. The grant is dedicated primarily to deep sequencing the larval microbiome and associated pathogen genomes, titled, “Using big data to improve diagnosis of larval health and disease in honey bees”. The size of our microbial culture collection was increased, and a number of disease related pathogen strains were genome sequenced to complement our laboratory work. A materials transfer research agreement was established to explore the development of immunity throughout larval stages and early adult growth. These factors will continue to be explored in relationship to the larval, worker and queen microbiome, and the introduction of molecular patterns in the queen diet.

In support of Sub-Objective 2A, work continued on the early queen microbiome exploring gene expression and microbial changes in early life associated with age, queen breeder, and environmental context. The analysis is ongoing and thus far, confirms previous hypothesis that a major unnamed bacterial species is abundant in early life, but gradually replaced by core Lactobacillus species as queens age. The microbiome structure varied by both breeder and queen age, suggesting that early age related changes in the queen’s microbiome are associated with health and colony establishment, and are very different from those of workers, but share similarities with larvae. Analyses of microbiomes from the queen mouth, midgut, ileum and rectum, has been completed and the relationship of age and social context with immune gene expression, nutritional state, and microbiome variation is currently being explored. 

In support of Sub-objective 3A, researchers in Tucson, Arizona, explored the mouth microbiomes of worker bees exposed to plant resins (propolis), performing 16S rRNA gene sequencing, bioinformatics, and associated statistical analyses. The results showed a significant positive effect on social hygiene, and this effect will be explored in the context of adult and larval disease transmission. Introduced probiotics may also possess the potential to control disease. A research collaboration was established to sequence the microbiomes and associated host health metrics of honey bee colonies exposed to two commercial probiotic formulations that claim to improve honey bee health. One major output from the gut microbiota with a major effect on honey bee health are Short Chain Fatty Acids (SCFAs). The introduction of multiple short-chain fatty acids, an expected result of consuming plant recalcitrant molecules like pollen, was explored in the honey bee diet. This research is fundamental for the improvement of honey bee supplemental diets. **Accomplishments**
                                

1. Honey bee social hygiene is enhanced by a propolis envelope. Honey bee social hygiene is enhanced by a propolis envelope, a layer of plant resin distributed throughout the hive. Honey bee colony loss is often attributed to bacterial pathogens, but overuse and misuse of antibiotics has spurred development of alternative treatments. Scientists in Tucson, Arizona, hypothesized that the antimicrobial activity of an experimentally applied propolis envelope would influence the bacterial diversity and abundance associated with social disease transmission. We found that mouthparts of worker bees in colonies with a propolis envelope had significantly lower bacterial diversity and significantly higher bacterial abundance compared to the mouthparts of bees in colonies without a propolis envelope. The propolis envelope improved social hygiene by reducing pathogenic and opportunistic microbes and promoting the proliferation of putatively beneficial microbes on the honey bee mouthparts. This technology can be applied by commercial beekeeping to reduce the need for antibiotic application. 2. Prospective diet additives rescue effects of pollen restriction. Microbial metabolites produced by gut bacteria are strongly influenced by diet and considered important drivers of host health. Popular commercial diets used to supplement honey bees during forage dearth were not designed with a consideration of microbiome health. To evaluate the effects of fermentative metabolites on host endocrine function, pollen-restricted bees were fed an equimolar mixture of organic acid sodium salts (acetate, lactate, butyrate, formate, and succinate). Dietary pollen restriction markedly reduced total and specific bacterial abundance in the anterior rectum but not in the ileum. Organic acid feeding rescued some effects of pollen restriction, altering gene expression implicated in energy metabolism and feeding behaviors. Our findings provide new insights into the diet-microbiota-host axis in honey bees and may inform future efforts to improve bee health through diet-based microbiota manipulations. 3. The larval microbiome associated with idiopathic brood disease. Honey bee larval disease is strongly associated with colony mortality, and on the rise throughout the beekeeping industry. Researchers in Tucson, Arizona, sampled many apiaries throughout the state of Illinois classified as different forms of brood disease. Microbial abundance and diversity were analyzed from six sites, revealing a variety of disease related microbiome structures. At all but one site, larval disease was likely due to European Foul Brood, but one site characterized as idiopathic brood disease was not associated with established pathogens.  At this site, the guts of diseased larvae revealed a microbiome structure suggesting a disease caused by multiple microbial factors. This discovery provides a novel molecular diagnostic tool for beekeepers in the management of larval disease. 4. The virulence of European foul brood is dose dependent and varies by strain. European foulbrood is a larval disease that cost beekeepers millions of dollars each year, and standard antibiotic treatments are detrimental to the microbiome, and destined for failure. Researchers in Tucson, Arizona, have improved the experimental and informational context of larval disease with the end goal of developing a management strategy to control larval disease. They sequenced the bacterial microbiota associated with larval disease transmission, isolated a variety of disease causing bacterial strains and determined their virulence against larvae in the lab setting. The larval microbiota was a low diversity environment similar to honey, while worker mouthparts and stored pollen contained significantly greater bacterial diversity. Virulence of bacterial pathogens against larvae varied markedly by strain and inoculant concentration. Results are important for understanding the spread and cause of larval disease, a widespread economic problem for commercial beekeepers. **Review Publications**
                            *Ricigliano, V.A., Anderson, K.E. 2020. Probing the honey bee diet-microbiota-host axis using pollen restriction and organic acid feeding. Insects. 11(5):1-14. https://doi.org/10.3390/insects11050291.*
                                *Floyd, A.S., Mott, B.M., Maes, P., Copeland, D., McFrederick, Q., Anderson, K.E. 2020. Microbial ecology of European foul brood disease in the honey bee (Apis mellifera): towards a microbiome understanding of disease susceptibility. Insects. 11(9):555. https://doi.org/10.3390/insects11090555.*
                                *Dalenberg, H., Maes, P., Mott, B.M., Anderson, K.E., Spivak, M. 2020. Propolis envelope promotes beneficial bacteria in the honey bee (Apis mellifera) mouthpart microbiome. Insects. 11(7):453. https://doi.org/10.3390/insects11070453.*


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