Variation in honey bee gut microbial diversity affected by ontogenetic stage, age and geographic location

Social honey bees, Apis mellifera, host a set of distinct microbiota, which is similar across the continents and various honey bee species. Some of these bacteria, such as lactobacilli, have been linked to immunity and defence against pathogens. Pathogen defence is crucial, particularly in larval stages, as many pathogens affect the brood. However, information on larval microbiota is conflicting. Seven developmental stages and drones were sampled from 3 colonies at each of the 4 geographic locations of A. mellifera carnica, and the samples were maintained separately for analysis. We analysed the variation and abundance of important bacterial groups and taxa in the collected bees. Major bacterial groups were evaluated over the entire life of honey bee individuals, where digestive tracts of same aged bees were sampled in the course of time. The results showed that the microbial tract of 6-day-old 5th instar larvae were nearly equally rich in total microbial counts per total digestive tract weight as foraging bees, showing a high percentage of various lactobacilli (Firmicutes) and Gilliamella apicola (Gammaproteobacteria 1). However, during pupation, microbial counts were significantly reduced but recovered quickly by 6 days post-emergence. Between emergence and day 6, imago reached the highest counts of Firmicutes and Gammaproteobacteria, which then gradually declined with bee age. Redundancy analysis conducted using denaturing gradient gel electrophoresis identified bacterial species that were characteristic of each developmental stage. The results suggest that 3-day 4th instar larvae contain low microbial counts that increase 2-fold by day 6 and then decrease during pupation. Microbial succession of the imago begins soon after emergence. We found that bacterial counts do not show only yearly cycles within a colony, but vary on the individual level. Sampling and pooling adult bees or 6th day larvae may lead to high errors and variability, as both of these stages may be undergoing dynamic succession

Heatmap summarising the relative density of dominant denaturing electrophoresis bands of the 16S rRNA amplicon profiles of the total gastrointestinal tract contents of several honey bee ontogenetic stages.

<p>1-, 3- and 6-day old larvae (L1, L3 and L6, respectively), white and black pupae (PW, PB), young bees, drones and flying bees (BY, DR and BF, respectively) collected in 4 different locations (Dol, Postrizin, Ustrasice, Hostice). Samples are sorted by ontogenetic stage (A) and location (B). The colours refer to relative band strength according to the colour key.</p

Dynamics of selected bacterial groups in the total gastrointestinal tract during development and aging of a “single” honey bee.

<p>Data were obtained by collecting pooled samples of sister honey bees from the eggs of the same oviposition. Young bees were marked by paint shortly after emergence. Legend in the grey field provides a link to the first experiment EXP2 described here and shows at which approximate time points the samples for EXP1 were collected.</p

Boxplot of quantitative real-time PCR (qRT-PCR) data of the abundance of selected bacterial groups in pooled samples of total gastrointestinal tract of each honey bee ontogenetic stage.

<p>The Y axis shows log-transformed copies of the 16S rRNA gene per gram of honey bee gastrointestinal tract. Boxes show pooled data from 4 locations and 3 hives at each location. 1-, 3- and 6-day old larvae (L1, L3 and L6, respectively), white and black pupae (PW, PB), young bees, drones and flying bees (BY, DR and BF, respectively). The codes of the outliers refer to the location (Pos: Postrizin, Hos: Hostice, Ust: Ustrasice) and colony number (1, 2, 3).</p

Biplot from redundancy analysis (RDA) explaining the distribution of honey bee ontogenetic stages according to major bacterial strain abundance.

<p>Test of interactions between factors location and ontogenetic stage: A, crude data considering absolute DGGE band intensities; B, centred and standardized data considering relative band intensities. Abbreviations: Gil, <i>Gilliamella apicola</i>; Sno, <i>Snodgrassella alvi</i>; Lac, <i>Lactobacillus</i> sp.; Rhi, <i>Rhizobiales</i> bacterium; Fri, <i>Frischella perrara</i>, UM, unknown multiple—probable DNA heterodimer. For further strain descriptions, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118707#pone.0118707.s005" target="_blank">S5 Fig.</a> Dotted shapes surrounding each ontogenetic stage were created as an aid in visualization. Eighteen bacterial strains occurring as major 16S rDNA DGGE bands were used for statistical analysis, while only selected strains are plotted as arrows. Same descriptions are for bands of the same sequence occurring at multiple locations of the line. Blue arrows show hypothetical developmental timeline. Its dotted part is ambiguous.</p