Establishment of characteristic gut bacteria during development of the honeybee worker

Abstract

Previous surveys have shown that adult honeybee (Apis mellifera) workers harbor a characteristic gut microbiota that may play a significant role in bee health. For three major phylotypes within this microbiota, we have characterized distributions and abundances across the life cycle and among gut organs. These distinctive phylotypes, called Beta, Firm-5, and Gamma-1 (BFG), were assayed using quantitative PCR, fluorescent in situ hybridization (FISH) microscopy, and the experimental manipulation of inoculation routes within developing bees. Adult workers (9 to 30 days posteclosion) contained a large BFG microbiota with a characteristic distribution among gut organs. The crop and midgut were nearly devoid of these phylotypes, while the ileum and rectum together contained more than 95% of the total BFG microbiota. The ileum contained a stratified community in which the Beta and Gamma-1 phylotypes dominated, filling the longitudinal folds of this organ. Deep sequencing of 16S rRNA genes showed clear differences among communities in midgut, ileum, and rectum. In contrast with older workers, larvae and newly emerged workers contain few or no bacteria, and their major food source, bee bread, lacks most characteristic phylotypes. In experiments aimed at determining the route of inoculation, newly emerged workers (NEWs) sometimes acquired the typical phylotypes through contact with older workers, contact with the hive, and emergence from the brood cell; however, transmission was patchy in these assays. Our results outline a colonization pattern for the characteristic phylotypes through A. mellifera ontogeny. We propose the names "Candidatus Snodgrassella alvi" and "Candidatus Gilliamella apicola" for the Beta and Gamma-1 phylotypes, respectively.

Fig 1

Abundances of the Beta, Firm-5, and Gamma-1 phylotypes (BFG) per adult worker, for different ages and gut organs, measured as copies of the 16S rRNA gene. (a) Total BFG abundance in workers at 1, 9, 19, and 30 days postemergence. (b) Average phylotype abundances in adult A. mellifera workers (days 1 to 30). (c) Average BFG abundances of the gut organs in adult A. mellifera workers (days 1 to 30). Letters above confidence intervals (1 standard deviation) represent significance levels (Tukey's HSD).

Fig 2

Comparison of the BFG community parsed by age, gut organ, and bacterial phylotype. (a) Numbers of 16S rRNA gene copies corresponding to BFG phylotypes in A. mellifera gut organs for worker adults of different ages. (b) Mean phylotype abundances relative to total BFG abundance for gut organs of adult workers, excluding day 1 workers. The circle's area is proportional to the organ's total BFG abundance, and the crop chart is expanded. (c) Abundances of phylotypes in each gut organ for adult workers, excluding day 1 workers. Letters above confidence intervals (1 standard deviation) represent significance levels (Tukey's HSD).

Fig 3

Localization of the Beta, Firm-5, and Gamma-1 phylotypes within the crop, midgut, ileum, and rectum of mature adult workers. Confocal microscopic images of phylotype-specific and universal bacterial FISH probes are shown, with the false coloration of specific BFG and universal bacterial probes as listed. Column 1 and frames f and g show composite images for the BFG FISH probes, columns 2 to 4 show hybridization for the individual BFG probes, and column 5 shows hybridization for the universal bacterial probe. Rows represent different gut organs; the boxed area in row c, column 1 is enlarged in row d to show the deep infoldings of the ileum filled with bacterial cells. Abbreviations: L, gut lumen; I, cuticular intima; W, midgut wall; P, partially digested pollen; and M, Malpighian tubules.

Fig 4

Bacterial community profiles in midgut, ileum, and rectum samples from four individual A. mellifera workers (3 day 9 workers and 1 day 30 worker) characterized using abundances of 16S rRNA gene sequences in 454 Pyrotag data. (a) Phylotype frequencies. The dendrogram shows UPGMA clustering of bacterial communities based on the weighted-UniFrac metric; all nodes have >90% bootstrap support. Each phylotype was assigned sequence clusters having top Blastn hits to members of that phylotype in GenBank. No clusters comprising >0.05% of any sample had a top Blastn hit different from that of the characteristic A. mellifera phylotypes (Alpha-2.2, Beta, Bifido, Firm-4, Firm-5, Gamma-1, and Gamma-2), and these low-frequency clusters are not shown. (b) Principal components analysis of the A. mellifera gut organ bacterial communities (day 30 worker samples are labeled). Pyrotag data were analyzed in QIIME (4) using the weighted-UniFrac metric.

Fig 5

Phylogenetic placement of (a) “Candidatus Snodgrassella alvi” (Beta phylotype) within the Neisseriaceae and (b) “Candidatus Gilliamella apicola” (Gamma-1 phylotype) within the Gammaproteobacteria. Numbers on branches represent bootstrap support (RAxML with 100 bootstrap replicates).