Table 1.
Bumble bees used in amplicon sequencing and ARISA, and their geographic origins.
Table 2.
Results of taxonomic classification (Class to Family) conducted on representative sequences of 74 bacterial OTUs.
Fig 1.
Consensus Bayesian inference tree showing phylogenetic relationships of representative sequences of 74 bacterial OTUs, and their classifications at the level of Class (classification bootstrap confidence in parenthesis).
The IDs of the OTUs are abbreviated by only keeping their unique, last five characters (e.g., HUSOE0I01A9P4E to A9P4E). The tree was inferred by combining these sequences with 274 16S rRNA sequences from [42], followed by dropping the latter sequences from the tree using the drop.tip function found in the R package “ape”. Thickened branches indicate posterior probabilities of 90% or higher; stars signify sequences that have classification-phylogeny incompatibility. See S1 Fig. for classifications of the OTUs at various taxonomic levels. See S2 Fig. for the full trees that include sequences from [42].
Fig 2.
Heatmap showing relative abundance of each bacterial OTU (rows) in each of the pyrosequencing sample (columns).
On the left is a cladogram showing phylogenetic relationships of the OTUs (tip labels = representative sequence ID). The IDs of the OTUs are abbreviated by only keeping their unique, last five characters (e.g., HUSOE0I01BDDRB to BDDRB). On the right are classifications of the OTUs at two taxonomic levels, if available. For clarity, only labels for OTUs with relative abundance > 10% are shown. Atop the heatmap, a cladogram indicates phylogenetic relationships of the bee species investigated. The bars at the bottom of the graph indicate the phylogenetic clade in which each bee species falls. MD = Mendacibombus (subgenus). See S4 Fig. for fully labeled tips.
Table 3.
Sequencing depth and number of OTUs detected for each pyrosequencing sample.
Fig 3.
Relative abundance (averaged across 13 pyrosequencing samples) of different bacterial classes.
The average number of sequences obtained from each pyrosequencing sample was 15,170.
Table 4.
Taxonomic assignments of six OTUs based on naïve Bayesian classification of 16S rRNA sequence.
Fig 4.
Plot of pyrosequencing samples in the 2-D nMDS space based on inter-sample UniFrac distances (A).
Minimum stress after 999 iterations is 0.05. The cluster of samples in the lower-left quadrate (ones without labels) is expanded in the top right corner (B).
Table 5.
Significance of UniFrac tests that compared a single pyrosequencing sample against all other samples combined.
Fig 5.
Three-dimensional (DIM1–3) nMDS ordination of ARISA profiles of different bumble bee gut DNA samples.
Corresponding bumble bee species and populations (collection sites) are illustrated by the colors and shapes of the markers on the right. Abbreviations: B.FR = B. fraternus; B.IM = B. impatiens; B.PE = B. pensylvanicus; B.TE = B. terricola; B.TRS = B. terrestris. Abbreviations following species names represent different collection sites, sorted by states in the US (FL, GA, MI, MN, NC, SC, WI, and WV), or across countries (ARG and FRA). ARG: Argentina, FRA: France.
Fig 6.
Images of B. impatiens gut (A) from light (B, C and D) and fluorescent microscopy (E and F).
(A) shows the whole alimentary canal and general locations where sectioning and imaging were made (multiple individuals were used to create the dissected gut and microscopy images). Section made at (1), (2) and (3) were used to produce images (B), (C) and (D), respectively. Fluorescent images (E) and (F) were produced by visualizing a portion of the ileum located between (1) and (2), and between (2) and (3), respectively. (B) shows both yeast and bacterial cells in the lumen, whereas (C) and (D) show only bacterial cells in the lumen. Villi and longitudinal folds composed of epithelial cells can be observed in (C) and (D), respectively. Insert in (C) shows a villus and bacterial cells (black dots) in greater detail. In (E) and (F), each fluorescing green spot represents one bacterial cell. Abbreviations: ba = bacteria; ep = epithelial cells; lu = lumen; mu = transverse muscle; yc = yeast cell.