Location: Carl Hayden Bee Research Center
2024 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, titled "The Honey Bee Microbiome in Health and Disease", which started in March 2020.
In support of Objective 1, research continued on brood disease with a focus on virulence factors. ARS researchers in Tucson, Arizona, confirmed that colonies with European foulbrook (EFB) symptoms were infected with M. plutonius and revealed the sequence type distribution for various sites across Michigan. Four previously reported sequence types were found at these sites, each of which had at least one reported incidence in North America. While some are unique many sequence types have a broad distribution range, also having been commonly found in the United Kingdom, Europe, and Asia. Five sequence types were novel, which was unique to Michigan isolates. Two of these novel sequence types were defined by a new allele at locus gbpB. The marker gbpB is part of the coding sequence of a putative secreted antigen that may be an important virulence factor in other well-known and virulent cocci. It has the greatest number of reported alleles, and the coding sequence contains a variable number tandem repeat (VNTR). The concurrent presence of multiple sequence types within individual larvae combined with tentative evidence of local recombination suggests a potential for the movement of virulence factors and antibiotic resistance genes within M. plutonius populations. An example of one such virulence factor, the pMP19 plasmid was found with high prevalence across Michigan isolates, with CC12 strains more likely to carry the plasmid. The pMP19 plasmid is easily lost during cell culture, and CC3 strains are in general more fastidious, less virulent and more difficult to grow in culture.
For Objective 2, as part of an ongoing study on commercial queen and colony health, ARS researchers performed RNA-Seq on the guts and fat-bodies of variably aged queens. Pacbio sequence full length 16S reads and meta-transcriptomes of variably aged queen hindguts were also performed. Differentially expressed genes were identified that are involved in cellular components, biological processes, and molecular function related to both chronological and molecular (carbonyl accumulation) age and the relative abundance of a key hindgut bacterium associated with queen fecundity. These findings offer insights into the relationships among queen and worker health, pathogen loads, metabolism, and immune responses in honey bee queens. This knowledge contributes to the understanding of colony and queen health and resilience, crucial for maintaining healthy populations of honey bees.
In support of Sub-objective 2B, worker-queen interactions were assessed over three seasonal time points in mid-western commercial colonies. Queens showed a marked preference to engage in trophallactic feeding exchanges with certain workers over others. Queen microbiota, semiochemical, reproductive quality, and physiological metrics are currently being analyzed and compared against local queens. Both workers that fed queens and ones that tried but failed to feed queens have been sampled and will be analyzed for microbiota and semiochemical contents. Worker trophallactic interactions with queens are the source of both microbe exposures and nutrients and likely central to queen support and microbial health. Analyses will be completed by fall 2024.
For Objective 3, ARS researchers determined the impact of antibiotics on the assembly of the gut microbiome of newly emerged worker bees. Immediately after eclosion from the pupa, newly emerged workers have little to no bacterial abundance in the gut, but through exposure to nestmates and colony materials, the microbiome quickly assembles. The gut microbiome is essential to worker health and immunity and may even be an important determinant for behavior and learning. Dysbiotic bees are more susceptible to infection, and tetracycline, a broad-spectrum veterinary antibiotic, can make bees less able to survive pathogen challenge. As a macrolide antibiotic, Tylosin targets primarily Gram-positive bacteria, but affects some Gram-negative species as well. Since the honey bee gut microbiota consists of both Gram-positive and Gram-negative core members, expected unequal impacts of treatment on the community were anticipated. This disproportionate effect on some members of the gut community may then break mutualist interactions or allow opportunist patterns of overgrowth among less affected microbiota. ARS researchers in Tucson, Arizona, have demonstrated that a common veterinary antibiotic application interferes with the normal acquisition of the honey bee worker gut microbiome. Tylosin-exposed NEWs develop a putatively dysbiotic microbiome deficient in certain core members and with a lower total bacterial abundance. Microbiome community size and structure started out similar in initial measurements but developed differently in exposed bees. As predicted, some microbiome members were affected disproportionately by Tylosin treatment. Lactobacillus apis is a major early colonizer of the hindgut, quickly followed by the other core microbiome members. ARS researchers found that Tylosin-treated bees in the first day post-eclosion establish an abundance of L. apis similar to control bees, but that other species failed to establish at their usual rate, creating a smaller, less even, and less diverse community by the fourth day. Bifidobacterium asteroides failed to establish in Tylosin treated bees despite making up around 10% of the relative abundance of hindgut bacteria on days three and four for controls. Bifidobacteria provide protective effects in the microbiomes of bees and other animals. Bombilactobacillus mellifer also failed to establish in treated bees, but B. mellis proportional abundance remained about the same, possibly revealing differences in antibiotic resistance between these species. More Alpha 2.1 appeared in Tylosin treated bees on the third and fourth days. Alpha 2.1 is gram-negative, and a dominant core microbiome member in queen guts, correlated with markers of queen health and fecundity. Its function in the worker microbiome is unknown but its presence is more sporadic, increasing in prevalence as workers age. The bloom of this taxon in the early worker gut may be an example of metabolic niche opportunism, as it seemed to increase in relative abundance as groups associated with fermentation of complex sugars decreased.
Also, in support of Objective 3, ARS researchers examined the effect of popular commercial and non-native probiotics on honey bees. This effort reflects a significant investment by beekeepers. Probiotics were applied as directed, at a stated concentration of five billion bacterial cells per colony. Their double-blind test of probiotic effectiveness examined a large cohort of commercial colonies (60 commercial colonies and two different apiaries) under two distinct circumstances; (1) Following prophylactic probiotic application every month for seven months, and (2) The application of probiotics following antibiotic induced gut dysbiosis. Consistent with our molecular results, a colony-level analysis of this same sample set revealed no effect of probiotic treatment on colony weight or number of worker bees. High-throughput DNA sequencing resulted in 14 million DNA sequences representing the hindgut microbiomes of 233 workers, but ARS researchers found that only twenty-three of 14 million DNA signatures may have originated with probiotic application. Collectively this provides overwhelming evidence that non-native probiotics simply do not survive in the honey bee colony or gut. Additionally, non-native probiotics had no effect on pathogen prevalence. In total, ARS researchers assayed seven common pathogens, but found no meaningful differences in pathogen abundance or prevalence associated with probiotic application. They concluded that the introduced probiotic microbes have no effect upon the host organism, primarily because they could not survive or effectively propagate in the colony or gut environment.
Accomplishments
1. The honey bee social resource niche hosts a consistent, co-evolved microbiota that functions in pathogen protection. The microbiome of the honey bee worker hindgut has been explored thoroughly, but less effort has been devoted to the aerobic or microaerophilic social niches associated with colony process. ARS researchers in Tucson, Arizona, performed a broad review and meta-analysis to consolidate separate studies and reveal a social resource microbiota associated with nine distinct niches. Characterized by dominance environments and rapid growth, this shared social microbiota functions primarily in disease prevention at both the individual and colony level. These aerobic microbes may also function in gut microbiome resilience under conditions that alter worker hindgut physiology. Defining the microbiota of social function contributes to a systems-based understanding of opportunistic disease, colony hygiene, and gut microbiome resilience in honey bees.
2. Popular and non-native probiotics do not rescue antibiotic treatment, and are generally not beneficial. Probiotics are widely used in agriculture including commercial beekeeping, but there is little evidence supporting their effectiveness. ARS researchers in Tucson, Arizona, tested whether non-native probiotics affect the gut microbiome or disease prevalence or rescue the negative effects of antibiotic induced gut dysbiosis. They found no difference in the gut microbiome or disease markers by probiotic application or antibiotic recovery associated with probiotic treatment. Their results demonstrate the lack of probiotic effect or antibiotic rescue, detail the duration and character of dysbiotic states resulting from different antibiotics, and highlight the importance of the gut microbiome for honeybee health. This finding provides beekeepers with informed management decisions concerning the health of their colonies.
3. A holistic colony-level approach provides context for seasonal pathogen pressure affecting honey bee queens. The health and continued productivity of honey bee queens is crucial for colony success, particularly during stressful periods like overwintering. As part of an ongoing longitudinal study of colony and queen health throughout the winter dearth, ARS researchers in Tucson, Arizona, explored niche-specific gut microbiota, host gene expression, pathogen prevalence, queen pheromone signaling, queen reproductive quality, and worker care of honey bee queens. Queens and workers had similar pathogen prevalence by season, while high viral titers in queens were associated with fungal and bacterial pathogen prevalence and decreased vitellogenin expression, detailing a trade-off between immune function and reproductive capacity. Throughout this period, workers fed and supported queens at similar levels despite indications of decreased fecundity suggesting worker flexibility in maintaining a viable egg-laying queen. Collectively, the multifaceted interactions between the microbiota, pathogens, immune responses, metabolic status and colony behavior presented here highlight the utility of a holistic colony-level approach to understanding queen health and resilience.
Review Publications
Anderson, K.E., Allen, N., Copeland, D.C., Kortenkamp, O., Erickson, R.J., Mott, B.M., Oliver, R. 2024. A longitudinal field study of commercial honey bees shows that non-native probiotics do not rescue antibiotic treatment, and are generally not beneficial. Scientific Reports. 14. Article 1954. https://doi.org/10.1038/s41598-024-52118-z.
Carroll, M.J., Brown, N.J., Ruetz, Z.J., Ricigliano, V.A., Anderson, K.E. 2023. Honey bee retinue workers respond similarly to queens despite seasonal differences in Queen Mandibular Pheromone (QMP) signaling. PLOS ONE. 18(9). Article e0291710. https://doi.org/10.1371/journal.pone.0291710.
Copeland, D.C., Ricigliano, V.A., Mott, B.M., Kortenkamp, O.L., Erickson, R.J., Gorrochategui-Ortega, J., Anderson, K.E. 2024. A longitudinal study of queen health in honey bees reveals tissue specific response to seasonal changes and pathogen pressure. Scientific Reports. 14. Article 8963. https://doi.org/10.1038/s41598-024-58883-1.
Anderson, K.E., Copeland, D.C. 2024. The honey bee "hive" microbiota: Meta-analysis reveals a native and aerobic microbiota prevalent throughout the social resource niche. Frontiers in Bee Science. 2. Article 1410331. https://doi.org/10.3389/frbee.2024.1410331.
