Development of the honey bee gut microbiome throughout the queen-rearing process

Abstract

The European honey bee (Apis mellifera) is used extensively to produce hive products and for crop pollination, but pervasive concerns about colony health and population decline have sparked an interest in the microbial communities that are associated with these important insects. Currently, only the microbiome of workers has been characterized, while little to nothing is known about the bacterial communities that are associated with queens, even though their health and proper function are central to colony productivity. Here, we provide a large-scale analysis of the gut microbiome of honey bee queens during their developmental trajectory and through the multiple colonies that host them as part of modern queen-rearing practices. We found that queen microbiomes underwent a dramatic shift in size and composition as they aged and encountered different worker populations and colony environments. Queen microbiomes were dominated by enteric bacteria in early life but were comprised primarily of alphaproteobacteria at maturity. Furthermore, queen gut microbiomes did not reflect those of the workers who tended them and, indeed, they lacked many of the bacteria that are considered vital to workers. While worker gut microbiotas were consistent across the unrelated colony populations sampled, the microbiotas of the related queens were highly variable. Bacterial communities in mature queen guts were similar in size to those of mature workers and were characterized by dominant and specific alphaproteobacterial strains known to be associated with worker hypopharyngeal glands. Our results suggest a model in which queen guts are colonized by bacteria from workers' glands, in contrast to routes of maternal inoculation for other animal microbiomes.

FIG 1

(A) Microbial composition of young larvae (from the grafting source colony, destined to be reared into queens) and queens (newly emerged and mature), and their associated workers, at each stage of queen development. N, number of individuals; the number of colonies is in parentheses. (B) Workers exhibited a relatively consistent microbiome profile, although workers that were the offspring of laying adult queens (workers after) exhibited larger proportions of Alpha-2.1 and smaller proportions of Firm-5 than workers present in final host colonies before queen progeny emerged (workers before). ID, identity. (C) Bray-Curtis dissimilarity metrics for pairwise comparisons between sampled communities were significantly different between workers and all other sampled castes (P < 0.01 by pairwise t tests with Bonferroni correction), supporting the assertion that microbiomes of unrelated worker populations were more consistent across the host colonies than were the microbiomes of the related queens over the course of their development. CI, confidence intervals. (D) Visual depiction of consistency in microbiome composition between workers across eight different colonies compared to those of three mature queens. In this series, all individual queens interacted with the same populations of grafting-source and cell-builder workers, but each was moved to its own mating nucleus and final host colony (sampled before emergence of queens' genetic offspring).

FIG 2

Distribution of the top 20 bacterial operational taxonomic units (OTUs) found across all samples and comprising >96% of the data. The prevalence of 19 of these top 20 OTUs (excluding Lactobacillales incertae sedis) was found to distinguish worker and queen microbiomes (df = 3; P < 0.05 by Kruskal-Wallis test). Libraries from mature queens are characterized by a bacterial community in which alphaproteobacteria predominate. Error bars indicate SE of the means across all sampled libraries.

FIG 3

UniFrac analysis of bacterial communities found across sampled environments, including larvae transferred from the grafting source (GS), newly emerged queens from the cell builder (CB), and mature queens in their mating nucs (MN) or final colonies (FC). Communities from workers interacting with queens at each stage of development did not cluster with their respective queens but instead claded separately (P < 0.001 in all pairwise comparisons between queens, workers, and larvae by Unifrac weighted analysis). Outliers are denoted with an asterisk.

FIG 4

Principal component analysis clustered the microbial communities of workers separately from those of mature queens, while newly emerged queens and larvae clustered together. Ellipses are presented to highlight the visual pattern only. REGR, regression.

FIG 5

Total number of 16S rRNA gene copies (as detected by quantitative PCR using 16S rRNA gene primers) from queens and workers (n = 5 for each caste and developmental stage). Mean total number of bacteria colonizing queens was influenced by age (χ² = 8.0; df = 3; P = 0.046 by Kruskal-Wallis test); larval queens hosted fewer bacteria than final queens (U = 2, Z = −2.2, and P = 0.032 by Mann-Whitney U test). Mean total numbers of bacteria in mature queen digestive tracts did not differ significantly from the mean total number of bacteria colonizing workers (F = 1.5; df = 1.4; P = 0.24). Symbols and numbers represent outliers.