Gut microbiota structure differs between honeybees in winter and summer

Abstract

Adult honeybees harbor a specialized gut microbiota of relatively low complexity. While seasonal differences in community composition have been reported, previous studies have focused on compositional changes rather than differences in absolute bacterial loads. Moreover, little is known about the gut microbiota of winter bees, which live much longer than bees during the foraging season, and which are critical for colony survival. We quantified seven core members of the bee gut microbiota in a single colony over 2 years and characterized the community composition in 14 colonies during summer and winter. Our data show that total bacterial loads substantially differ between foragers, nurses, and winter bees. Long-lived winter bees had the highest bacterial loads and the lowest community α-diversity, with a characteristic shift toward high levels of Bartonella and Commensalibacter, and a reduction of opportunistic colonizers. Using gnotobiotic bee experiments, we show that diet is a major contributor to the observed differences in bacterial loads. Overall, our study reveals that the gut microbiota of winter bees is remarkably different from foragers and nurses. Considering the importance of winter bees for colony survival, future work should focus on the role of the gut microbiota in winter bee health and disease.

Fig. 1. Gut bacterial communities differ between foraging and winter season in a single colony monitored over 2 years.

a Monthly changes in the absolute abundance assessed by qPCR, as determined by the number of genome equivalents per sample, of seven phylotypes monitored monthly, depicted as mean values (±SE) of the analyzed bees. The number of bees per month is indicated at the bottom of the plot. Asterisk indicates missing data for July 2015 due to DNA extraction failure. Double asterisk indicates that the queen of the colony was replaced in the corresponding month. b Relative community composition of the gut microbiota of bees sampled in each month as based on the seven monitored phylotypes. c Absolute bacterial abundance of each phylotype per gut in foragers (F) and winter bees (W), as determined by the number of genome equivalents. The sum of the abundances of the seven monitored phylotypes is also plotted. Mean values are shown as black horizontal lines. Only bees with detectable levels were plotted (the number of bees is given at the bottom of the plot; for prevalence see Supplementary Fig. S1). d Copy number of the 16S rRNA gene in gut samples of a subset of the analyzed months (Apr 2015, Aug 2015, Oct 2015, Jan 2016, Apr 2016, Jul 2016, Oct 2016, Jan 2017, and Apr 2017). e Effective number of species calculated from cell numbers of different bacterial phylotypes in foragers (F) and winter bees (W). f Projection of the abundances of monitored phylotypes into the first and second principal components in all analyzed bees, together with correlation vectors representing variables driving the separation on both axes. Permutation T-Test was used for pairwise comparisons. ns, nonsignificant; ***P < 0.001.

Fig. 2. Bacterial load and community composition differ between foragers, nurses, and winter bees across 14 colonies.

a Average 16S rRNA gene copy number per gut in foragers, nurses, and winter bees as determined from the pooled gut samples from 14 different colonies. b Differences in α-diversity, i.e., effective number of species, in the gut microbiota of foragers, nurses, and winter bees based on 16S rRNA gene amplicon sequencing. c NMDS based on Bray–Curtis dissimilarities on the gut communities of foragers, nurses, and winter bees based on 16S rRNA gene amplicon sequencing. d Relative community composition of the gut microbiota as determined by 16S rRNA gene amplicon sequencing. The seven phylotypes monitored by qPCR (see Fig. 1 and Supplementary Fig. S4), making up the vast majority of the community, are highlighted by a gray box in the legend. Capital letters below the stacked bars indicate the hive of origin. e Absolute abundance of each of the seven major phylotypes in foragers (F), nurses (N), and winter bees (W) across hives, as determined based on the number of genome equivalents per gut calculated by multiplying the relative abundance of each phylotype by the total 16S rRNA gene copy number. f Absolute abundance of a subset of the minor community members in foragers (F), nurses (N), and winter bees (W) across hives, as determined based on the number of genome equivalents per gut calculated by multiplying the relative abundance of each phylotype by the total 16S rRNA gene copy number. <LOD, below limit of detection of the 16S rRNA amplicon sequencing, i.e., no reads were obtained for that particular taxa in the respective sample. Mean values for the samples above the LOD are shown as black horizontal lines. Absolute abundance of the remaining phylotypes is depicted in Supplementary Fig. S4b. In a, b, e, f levels (bee types) not connected by the same letter are significantly different as based on ANOVA followed by Tukey’s HSD test (see Supplementary Table S3).

Fig. 3. Diet is a major factor shaping the gut microbiota of honeybees.

a–c Dissected guts of foragers (a), nurses (b), and winter bees (c) consisting of the crop (c) (missing in some samples), the midgut (m), the ileum (i), and the rectum (r) with attached stinger and the last abdominal tergite. d Wet gut weight of individual foragers (F), nurses (N), and winter bees (W). Different letters indicate groups that are significantly different from each other based on ANOVA followed by Tukey’s HSD test (see Supplementary Table S4 for details). e Spearman correlation between gut weight and bacterial loads (assessed with universal bacterial 16S rRNA primers) across all bee types. The gray area indicates the 95% confidence interval. f Wet gut weight of experimentally colonized bees that were fed either sugar water (SW) or sugar water and pollen (SW+P). g Spearman correlation between gut weight and bacterial loads (assessed with universal bacterial 16S rRNA gene primers) across the two treatment groups of colonized bees. h Total abundances of the seven monitored phylotypes in the two treatment groups of colonized bees, as determined by genome equivalents per gut using phylotype-specific qPCR primers. The sum of the abundances of the seven monitored phylotypes is also depicted. Bees with bacterial loads below the limit of detection (<LOD) of the qPCR method are shown below the cut of the axis at 10⁴. Two-group comparisons were done by Permutation T-Test. ns, nonsignificant; ***P < 0.001.