Research Project: The Honey Bee Microbiome in Health and Disease

Location: Carl Hayden Bee Research Center

2022 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 2022-21000-021-000D, "The Honey Bee Microbiome in Health and Disease". In support of Objective 1, we continue to sample and isolate bacteria of larvae from honey bee colonies across multiple apiaries expressing various disease states throughout Michigan. As hives experienced 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 continue to process samples in the microbiology lab to characterize the virus, bacteria and fungi associated with various larval disease states. Recently, ARS scientists in Tucson, Arizona, determined the association of Acute Bee Paralysis Virus with “melty” larval symptomology. This larval symptomology is associated with colony collapse overwinter, common throughout the United States, and often mistaken for European foulbrood, but typically producing a variety of different symptoms, including a variety of odors. Various bacterial opportunists that reach large numbers in older virus-afflicted larvae were associated with odor differences. Because this relationship was discovered in frozen samples and photographs from a broad disease survey, we are presently attempting to cultivate live virus to reproduce larval symptoms in the lab and demonstrate a causal relationship. When the diagnosis of European foulbrood was confirmed by our next generation sequencing approach, we found many other bacteria that could on capitalize on susceptible larvae, including other known enteric pathogens. While most of these bacteria occur in the final stages of disease, some appear able to propagate in younger larvae and may contribute to disease progression. Moreover, many bacterial species native to the adult gut environment behaved as opportunists in the final stages of European foulbrood disease and produced very large populations. We are collating, sequencing and describing these bacteria to better understand opportunism and the rare biosphere of honey bees. Other advances in Objective 1 include significant progress on a subordinate National Institute of Food and Agriculture (NIFA) project; “Using Big Data to Improve Diagnosis of Larval Health and Disease in Honey Bees”. In collaboration with NIFA and scientists in Beltsville, Maryland, scientists in Tucson, Arizona, have optimized protocols and approaches for the study and characterization of brood disease with the end goal of improving diagnostic services and the production of potential remedies. We continue to develop and nurture relationships with beekeepers throughout the United States to correspond about larval disease, including samples and photographic documentation of atypical brood disease. Many of these collections are presently prepped for deep sequencing following a molecular diagnostic protocol that deductively eliminates known causes of brood disease. Based on diseased larvae samples collected from throughout the United States, we recovered a variety of Melissococcus plutonius and other disease-associated bacteria from samples diagnosed as idiopathic. We recovered over 300 isolates via culture-based methods with 65% of these isolates representing disease related M. plutonius strains. To understand microbe interactions with honey bee antimicrobials, we tested a taxonomic variety of disease associated strains against various concentrations of royal jelly and honey in the lab. We tested the ability of various microbes to grow in concentrated and dilute honey and royal jelly, as an indicator of host-microbial co-evolution. In general, royal jelly was more inhibitory than honey. It was determined that most of the disease-associated strains isolated from sick larvae could proliferate in concentrated honey and royal jelly, suggesting they are co-evolved to exploit these environments. Bacterial species related to or classified as core gut bacteria of adults were inhibited to varying degrees in both honey and royal jelly. To understand larval immunity and oxidative stress gene expression, we setup different in lab trials feeding young larvae bacteria and pathogen associated molecular patterns (PAMPs). We used known pathogen strains of M. plutonius and a Serratia strain cultured from diseased larvae as comparative controls. To represent the response to PAMPs, we chose a variety of molecular patterns including mannan, lipopolysaccharide, peptidoglycan, and ß 1-3glucan. We recorded survival and surviving larvae in downstream analysis of gene expression and assayed gene expression to evaluate factors that promote larval survival. These results help differentiate cause from consequence and suggest approaches and compounds to mitigate disease and reduce colony loss. In support of Objective 2, exploration of the queen microbiome, documenting effects of colony assimilation and egg production continues. 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. 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. Newly mated queens were placed in live-storage in small containment cages, or in normal colony life associated with continuous egg laying. Feeding intensity, social context, and metabolic demand differ greatly between the two environments. We performed 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. We found that both social context and queen breeder source affect gut microbiota and associated queen metabolism. In the ileum, upregulation of most immune and oxidative stress genes occurred regardless of treatment conditions, suggesting post-mating effects on gut gene expression. Counterintuitively, queens exposed to the more social colony environment contained significantly less bacterial diversity indicative of social immune factors shaping the queens’ microbiome. Queen bank queens resembled much older queens with decreased beneficial bacteria in the hindgut, and significantly larger ileum microbiotas, dominated by blooms of worker associated gut bacteria. Combined with earlier findings, we conclude that the queen gut microbiota experiences an extended period of microbial succession associated with queen breeder source, post-mating development and colony assimilation. These states may produce signals perceived by the workers and may represent a non-invasive sampling method to assess queen quality. Additionally, methods have been developed to profile chemical emissions and exchanges in worker-queen interactions and techniques to better characterize queen performance. We developed novel visualization techniques (EthoVision queen tracking, fluorescent illumination of eggs and larvae) to separate sub-lethal effects on queen productivity from those on attendant retinue workers. These methods will be applied to assess impacts of various microbiota character and known stressors on queen quality and performance. For Objective 3, the two most popular probiotics on the U.S. market were tested in a large longitudinal study of commercially managed colonies in Grass Valley, California. We examined the effects of antibiotic exposure and probiotic rescue on the size and composition of the honey bee gut microbiome, and prevalent disease agents including virus. Colony metrics were monitored to determine if colony growth was impacted by antibiotic induced imbalances in gut microbiota that contribute to poor health (dysbiosis), and whether probiotic application improves the gut microbiome or rescues negative effects of antibiotic induced dysbiosis. We could not detect the introduced probiotic strains in the gut microbiome, and found no difference in the gut microbiome, disease incidence, or colony size by probiotic application or antibiotic recovery associated with probiotic treatment. Our results show that a colony-level application of the antibiotics oxytetracycline and tylosin produce an immediate decrease in gut microbiome size, and over the longer-term, produce very different and persistent dysbiotic effects on the composition of the hindgut microbiome. Our results demonstrate the lack of probiotic effect or antibiotic rescue, detail the dysbiotic states resulting from different antibiotics, and highlight the importance of the gut microbiome for honeybee health. Further support of Objective 3 looks at the rare biosphere associated with honey bees. Recent honey bee microbiota research has focused on the handful of dominant species core to the worker hindgut. Early culture-based work distinguished over 6000 microorganisms associated with honey bees. Rare biosphere organisms contribute substantial ecological potential and genetic diversity, and include opportunistic pathogens not clearly defined in the honey bee pathosphere. These results represent the first comprehensive review of the gut microbiota in honeybees considering all bacteria present with the reproductive swarm. We established a macro view of species diversity in honey bee microbiota exploring a variety of niches including those associated with social nutrient processing. Additionally, an updated standard was provided for hypothesis generation and consideration of species that typically occur in the gut with low prevalence and abundance. As an example, the worker midgut is a highly diverse bacterial niche that can become vulnerable to pathogen invasion and opportunism as workers age. In future experiments, we will highlight functional characterization, including culture-based approaches to further resolve the role of the extended microbiota in honey bee health.

Accomplishments
1. Idiopathic brood disease symptoms are linked to the incidence of Acute bee paralysis virus. Brood disease has been implicated repeatedly as a precursor to colony collapse in commercial operations throughout the United States. There are only three known causes of larval disease, but over 50% of honey bee larval disease remains undiagnosed. In two major and widespread larval diseases, referred to as idiopathic, larvae appear melted at the bottom of cells due to an unknown cause. ARS scientists in Tucson, Arizona, provide the first evidence linking “melty” larval symptomology to Acute Bee Paralysis Virus in honeybee colonies. Various bacteria act as secondary invaders, producing a variety of odors unassociated with known brood disease. This diagnosis informs beekeepers, scientists and apiary inspectors concerning the application of antibiotics to control brood disease.

2. Discovery and description of a social microbiota shared by queens, workers, and larvae. ARS scientists in Tucson, Arizona, performed a meta-analysis of 26 publicly available Illumina datasets (over 3000 individual gut libraries) exploring distinct alimentary tract tissues, defining the social microbiota and rare biosphere. We standardized these studies using the BEExact database to classify sequences at the lowest taxonomic level and test the hypothesis of a social microbiota. Worker mouthparts share similar species with queen mouthparts and midguts, stored food, secreted jelly, and developing larvae, highlighting the social microbiota. It is likely that studies designed to sample individual tissues may be more appropriate for revealing significant host-microbe interactions over traditional whole gut samples. Our results contribute novel insights into strain diversity, niche partitioning in the gut, the rare biosphere, and the social microbiota.

3. Two major probiotics have no effect over the long term or when applied for antibiotic recovery. Antibiotic treatments can greatly distort the honey bee gut microbiome, reducing its protective abilities and facilitating the growth of antibiotic resistant pathogens. Commercial beekeepers regularly apply antibiotics to combat bacterial infections, often followed by an application of probiotics advertised to ease the impact of antibiotic-induced imbalances in gut microbiota that contribute to poor health (dysbiosis). ARS researchers in Tucson, Arizona, performed a large longitudinal study of commercial honey bee colonies overwinter to explore the effects of probiotics and antibiotics. The two most popular honey bee probiotics have no discernable effect over the long-term, and do not rescue antibiotic treatment. The researchers found no difference in the gut microbiome or disease incidence by probiotic application or by probiotic treatment associated with antibiotic recovery. Thier results demonstrate the lack of probiotic effect or antibiotic rescue, detail the dysbiotic states resulting from different antibiotics and highlight the importance of the gut microbiome for honeybee health.

4. Detection of odors associated with a model disease state. Honey bee workers use pathogen-associated odors to detect and hygienically remove diseased individuals from the colony. ARS researchers in Tucson, Arizona, characterized odors emitted in active colonies during progression of chalkbrood (caused by the fungus Ascosphaera apis) infection in larvae and adult workers. They detected four compounds emitted by chalkbrood-infected brood and workers from early asymptomatic stages (brood and workers) to late stage desiccated corpses (brood only). Odor compounds were emitted by both inoculated and uninoculated brood including three compounds (phenyl acetate, 2-phenylethanol, and benzyl alcohol) previously thought to be limited to chalkbrood-infected brood. The volatile pheromone E-B-ocimene was emitted by chalkbrood-infected larvae from early stages to late stage desiccated remains. The lack of volatile specificity during early stages of A. apis infection suggests that more generalized stress odors may also function as cues to allow hygienic workers to locate affected brood. Sampling in active colonies provides a less disturbed and broader volatile characterization technique for identifying potential hygienic cues.

Review Publications
Anderson, K.E., Maes, P. 2022. Social microbiota and social gland gene expression of worker honey bees by age and climate. Scientific Reports. 12. Article 10690. https://doi.org/10.1038/s41598-022-14442-0.
Copeland, D.C., Anderson, K.E., Mott, B.M. 2022. Early queen development in honey bees: Social context and queen breeder source affect gut microbiota and associated metabolism. Microbiology Spectrum. Article 00383-22. https://doi.org/10.1128/spectrum.00383-22.