Microbial ecology of the hive and pollination landscape: bacterial associates from floral nectar, the alimentary tract and stored food of honey bees (Apis mellifera)

Abstract

Nearly all eukaryotes are host to beneficial or benign bacteria in their gut lumen, either vertically inherited, or acquired from the environment. While bacteria core to the honey bee gut are becoming evident, the influence of the hive and pollination environment on honey bee microbial health is largely unexplored. Here we compare bacteria from floral nectar in the immediate pollination environment, different segments of the honey bee (Apis mellifera) alimentary tract, and food stored in the hive (honey and packed pollen or "beebread"). We used cultivation and sequencing to explore bacterial communities in all sample types, coupled with culture-independent analysis of beebread. We compare our results from the alimentary tract with both culture-dependent and culture-independent analyses from previous studies. Culturing the foregut (crop), midgut and hindgut with standard media produced many identical or highly similar 16S rDNA sequences found with 16S rDNA clone libraries and next generation sequencing of 16S rDNA amplicons. Despite extensive culturing with identical media, our results do not support the core crop bacterial community hypothesized by recent studies. We cultured a wide variety of bacterial strains from 6 of 7 phylogenetic groups considered core to the honey bee hindgut. Our results reveal that many bacteria prevalent in beebread and the crop are also found in floral nectar, suggesting frequent horizontal transmission. From beebread we uncovered a variety of bacterial phylotypes, including many possible pathogens and food spoilage organisms, and potentially beneficial bacteria including Lactobacillus kunkeei, Acetobacteraceae and many different groups of Actinobacteria. Contributions of these bacteria to colony health may include general hygiene, fungal and pathogen inhibition and beebread preservation. Our results are important for understanding the contribution to pollinator health of both environmentally vectored and core microbiota, and the identification of factors that may affect bacterial detection and transmission, colony food storage and disease susceptibility.

Figure 1. PCoA analysis of bacterial communities from Apis mellifera associated niches.

Analysis based on unweighted UniFrac distances. Positions of the bacterial communities for each sampled niche along the first three principal coordinate axes are illustrated, along with the percentage of variation explained by each axis. The simulated gut community was composed of cloned sequences specific to the midgut and hindgut. Symbols are colored by general niche; yellow: alimentary tract, red: food stores and black: flowers. Sample size shown in parentheses.

Figure 2. Bacterial communities in the alimentary tract.

Results based on sequenced isolates from multiple growth media. Number of sequences for each niche is shown in parentheses. See methods for the determination of the simulated core gut community, and Tables S2 and S3 for detailed taxonomy.

Figure 3. Rarefaction curves according to media pH.

Genera as determined by the RDPII classifier were regarded as OTU's. Acidic media ranged in pH from 5.6–6.1, and neutral media from 7.0–7.4.

Figure 4. Bacteria cultured from the crop.

Pie chart at bottom right shows the proportional grand total. In the upper row, bacteria were cultured on MRS (deMan Rosaga Sharp) media over a yearly time course. The middle row is a month long time-course, sampling only newly emerged bees (NEB's) that had no contact with older siblings, but were allowed to feed on food stores present in the wax comb. The lower row shows culturing results from three standard media types: Blood Heart Infusion; BHI, Sabaroud dextrose agar; SDA, and Tryptic soy agar; TSA. Note that the top right pie chart corresponds to the same time period and pool of sampled individuals as does the lower row. See Table S2 for detailed taxonomy.

Figure 5. Bacterial communities found in food stores.

Communities based on either 16S cloning or sequenced isolates from multiple growth media. Number of sequences for each niche is shown in parentheses. Each bar is an independent sampling event. See Table S4 for detailed taxonomy.

Figure 6. Venn diagrams depicting unique and shared OTU's.

Diagrams comparing cultured isolates and cloned sequences derived from beebread and the gut (mid and hind gut). Operational taxonomic units (OTU's) are defined at 99% and 97%. Percent relative abundance of shared OTUs across all libraries is shown in parentheses.

Figure 7. Neighbor joining phylogenetic tree of Firmicutes.

Tree based on 1076 positions of the 16S-rDNA bacterial sequence from beebread, honey, alimentary tract and flowers visited by A. mellifera, and comparison with related microorganisms (GenBank accessions). Abbreviated taxon labels refer to clones (C), or isolates (I), and symbols mapped to the right of the topology represent the sampled niche. Bootstrap values (n = 1000) are given at the branching points.

Figure 8. Neighbor-joining phylogenetic tree of Acetobacteraceae (Alpha 2.2).

Tree based on 1024 positions of the 16S-rDNA bacterial sequence from a variety of A. mellifera associated microenvironments. Sampled niche is mapped to the right of each sequence label (see color key). All mapped sequences are unique according to at least one of the following: sampled niche, culture media, or DNA sequence. Abbreviated taxon labels begin with a letter designating clones downloaded from GenBank (C), clones produced from different colonies (libraries) in this study (C1 = 19, C2 = 20), or isolates (I). Isolates from this study are labeled according to growth media. Isolates from larval guts are according to . Numbers following LV designate the stage of larval instar.