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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** 2021 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. 

In support of Objective 1 a larval experiment was performed, examining the effect of transgenerational immune priming on queens. According to this theory, molecular patterns from queens are transmitted to her eggs, and are hypothesized to affect the immune system development of larvae and adults. A series of complicated experimental steps are required to test this hypothesis, including queen inoculation, in vitro larval rearing, pathogen quantification and pathogen challenge. Initial results were promising but inconclusive and indicate that further internal controls are necessary to validate distinct stages of the experimental process. Potential industry application is vaccinated queens producing disease resistant larvae. 

In a separate endeavor, we continue to sample larvae from honey bee colonies from multiple apiaries expressing various forms of brood disease throughout the state of Michigan. As hives moved throughout various pollination routes, we recorded shifting disease states collected by longitudinal and replicated sampling, including covariate hive metrics from both recovered and deceased hives. We have also formalized methods for partitioning samples of putative larval disease sent for diagnostic services to the Beltsville, Maryland, lab. Since May 2021, we have processed many samples in the microbiology lab to characterize the bacteria and fungi associated with various disease states. 

In support of Objective 2, we continue our exploration of the queen microbiome. Early queen death or rejection by the colony has become more common among commercial beekeepers and defining compromised or incipient disease states in queens will rely in part on the structure and function of native gut bacteria. Past and present ARS research on the queen’s microbiome indicates an age-associated microbial succession that may signal queen quality. The pattern of microbial succession is largely the reverse of that demonstrated for workers. A marker of stress and biological aging, carbonyl accumulation in the queen’s fat body, is significantly associated with increased Lactobacillus and Bifidobacterium and depletion of various Proteobacteria in the gut.  

The gut microbiome is intimately associated with host metabolism. To understand how the age of the queen microbiome alters during a time of decreased metabolic demand (egg laying), we sampled first and second year queens from colonies throughout the winter dearth in Tucson Arizona. We are presently performing microbiome analysis examining the queens’ mouthparts, midguts, ileums, and rectums, and associated host-gene expression analysis exploring the queen fat body, midgut, and Malpighian tubules. 

In a second experiment with queens, we investigated the entire gut microbiota and host gene expression associated with early life environments comparing two very different metabolic states experienced by fertilized commercial queens: 1) newly mated queens were placed in live-storage in small containment cages, or in normal colony life associated with continuous egg laying. Early results suggest that distinct microbiomes are associated with the onset of egg laying; and 2) we suggest that these may be signals perceived by the workers and may represent a non-invasive sampling method to assess queen quality. 

In support of Objectives 1 and 2, queen and worker samples associated with trophallactic feeding were collected. Samples are being assessed using biochemical, semiochemical, and microbial analyses. Pilot studies are being conducted to characterize putative chemical cues involved in trophallactic feeding between queens and workers. Additionally, new analytical techniques are being developed  to characterize minute amounts of short chain fatty acids. To provide an accurate biochemical context for the discovery of new semiochemical cues, we are characterizing odor and contact chemical profiles of workers and larvae experiencing a variety of common colony stressors including starvation, pathogens, gut dysbiosis, temperature extremes, and extended dearth. 

In support of Objective 3, an experiment introducing the two most popular probiotics to commercially managed hives in California resulted in no apparent benefit to colony strength (number of bees) or colony weight. Molecular and microbial analyses are underway to determine the ability of probiotics to protect from pathogens or hasten recovery from antibiotic treatment. A separate experiment is underway locally examining a marked cohort of known age worker bees to assess the effects of antibiotics/probiotics on workers at different stages of gut microbial succession. 

Additional research in support of Objective 3 included lining colonies with propolis (plant resin), which resulted in a 10X reduction of fungi in the hindgut, but produced no change in fat body expression for genes associated with immunity and oxidative state. Deep sequenced hindgut microbial communities are presently under bioinformatic analysis. To further explore changes in the social environment involved with these samples, we have processed gene expression of the social nurse gland and are awaiting raw sequencing results of midguts from the local next generation sequencing facility. 

Worker age is associated with physiological and behavioral roles in the colony. Commercial beekeepers split their colonies or subject them to conditions that disrupt the age demographics within the colony. We performed an experiment to disrupt colony age structure, then examined the effect on the hindgut microbiome and host gene expression. The hindgut microbiota of foragers that reverted to nursing behavior remains stable in size, structure and abundance and is resistant to fungal proliferation. Foragers that reverted to nursing duties expressed significantly greater vitellogenin in the fat body and decreased expression of immune genes and antimicrobial peptides. Taken collectively our results demonstrate that the honey bee hindgut microbiome is robust to social and physiological reversion under the tested conditions, and likely reflects a major selection pressure at the colony level. **Accomplishments**
                                

1. Colony and gut microbiota are mitigated by pro and antioxidant secretions produced in honey bee head glands. The honey bee is host to variety of opportunistic disease states. As part of a larger overwintering study, ARS scientists in Tucson, Arizona, sequenced the mouthparts and midguts of overwintering bees and characterized gene expression of their head gland secretions. They discovered that long-lived overwintering bees express pro and antioxidant genes that are associated with alterations in microbial communities throughout the entire gut. The relationship of colony and gut microbiota with host social-immune function highlights the range of host-microbial interaction associated with the honey bee superorganism, and its potential influence on colony health, disease resistance and gut integrity.  Results are important for understanding the fundamental role of the colony and gut microbiota in various opportunistic disease states. 2. The midgut is a target of opportunistic microbial growth. The adult honey bee becomes more vulnerable to disease during winter and following the age-related shift to foraging. Understanding which tissues become vulnerable under various conditions is important for the design of artificial diets and beekeeping in general. ARS researchers in Tucson, Arizona, characterized distinct microbiotas throughout the alimentary tract to examine concurrent changes in various gut sections associated with aging. Fungal load increased significantly in the midgut and hindgut overwinter with increases of bacterial opportunists in the midgut, but the mouthparts decreased in fungal load, and distinct hindgut sections remained relatively unperturbed for core gut bacterial structure and abundance. This information provides the beekeeping and scientific community with a target tissue for host health, a critical distinction because researchers are fixated on bacteria in the hindgut as the primary marker. 3. Queen productivity is associated with a distinct hindgut microbiome. Increased queen loss has become a major concern for the beekeeping industry. ARS researchers in Tucson, Arizona, investigated the entire queen gut microbiota and host gene expression associated with early life environments. They placed newly mated queens in live-storage in small containment cages, or in normal colony life associated with continuous egg laying. Concurrent with reproductive host metabolism, results suggest that distinct microbiomes are associated with egg laying behavior. We suggest that these microbiotas may be signals perceived by the workers and may serve as a basis for a non-invasive sampling method to assess queen quality. 4. The hindgut microbiome and associated host physiology adjusts to age related role reversal. Worker age is associated with physiological and behavioral roles in the colony. Commercial beekeepers split their colonies yearly subjecting them to conditions that disrupt colony age demographics. ARS researchers  in Tucson, Arizona, performed an experiment to disrupt colony age structure, then examined the effect on the hindgut microbiome and host gene expression. The hindgut microbiota of foragers that reverted to nursing behavior remains stable in size, structure and abundance and is resistant to fungal proliferation. Foragers that reverted to nursing duties expressed significantly greater vitellogenin in the fat body and decreased expression of immune genes and antimicrobial peptides. Taken collectively, our results demonstrate that the honey bee hindgut microbiome is robust to social and physiological reversion under the tested conditions, and likely reflects a major selection pressure at the colony level. **Review Publications**
                            *Maes, P., Floyd, A., Mott, B.M., Anderson, K.E. 2021. Overwintering honey bee colonies: Effect of worker age and climate on the Hindgut Microbiota. Insects. 12(3). Article 224. https://doi.org/10.3390/insects12030224.*


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