The effects of V. ceranae infection and the prebiotic isomaltooligosaccharide (IMO) on the honey bee hindgut microbiota were investigated in this study. IMO alone (no infection) did not significantly affect mortality and sugar water consumption. However, mortality in infected bees fed IMO was not different from the controls while mortality in infected bees fed sucrose was higher. Feeding IMO significantly increased V. ceranae infection intensities but did not significantly alter bacteria population ratios in either qPCR or microbiome analyses; nonetheless, feeding IMO significantly reduced microbiome alpha diversity and unevenness in the fecal microbiome of infected bees. These subtle changes in IMO-fed bees appeared to reduce mortality caused by V. ceranae infection. These results support our hypothesis that the specific gut bacteria were enhanced by the infection to maintain the host homeostasis.
Sugar consumption and mortality of the caged bees
No significant difference in consumption of sugar water was found between IMO and sucrose fed groups but V. ceranae-inoculated groups consumed significantly more sugar-water, both IMO and sucrose (P = 0.034, Fig. 1), than the uninfected controls. There was no difference in mortality between control groups fed the two sugars, however, mortality was significantly lower (P < 0.001) in the infected group fed IMO compared to the infected group fed only sucrose (Fig. 2). Mortality was not different for V. ceranae-inoculated bees fed IMO and uninfected control bees fed either solution.
Differences in V. ceranae infection intensities
To evaluate infection intensities in the midgut, five bees were analyzed by microscopy from each cage at 12 dpi and then every other day. The trial was terminated at 20 dpi because fewer than five bees survived in the cages. The inoculation resulted in a 100% infection rate in the collected bees, and the uninoculated bees were free of spores. Midguts of the same group had similar infection intensities collected in 12–20 dpi. V. ceranae infection intensities were slightly, but significantly, higher in IMO groups (Fig. 3). Individual differences and variation were found among the samples, and the bees fed IMO tended to have higher spore counts in the dissected midguts. Because we were investigating fully developed infections, infected bees with less than 107 spores were not included in the following analyses.
Quantification PCR results
To investigate bacterial microbiota alterations of hindgut linings and feces, we used qPCR to quantify the core-bacteria species with higher populations. Lactobacillus spp., Bifidobacterium spp., Snodgrassella alvi, and Gilliamella apicola, were included in the qPCR. We attempted to normalize the results using the honey bee Beta-actin gene and the universal bacteria primer set; however, both primer sets were problematic as the references for the bacteria qPCR results. Fecal samples yielded low (> 35) or no Ct values using the actin gene primer set. The universal bacteria primer set yielded slightly but significantly lower (P < 0.001) values in V. ceranae infected bees, both hindgut and fecal samples, whereas IMO feeding did not alter the values. The results suggested V. ceranae infection significantly increases bacteria populations, but the prebiotic, IMO, did not. Because this study aimed to investigate the microbiota alterations caused by the infection, we decided to present the hindgut results (Fig. 4A) normalized by the host Beta-actin to show the bacterial population alterations and the fecal results (Fig. 4B) normalized by the universal bacteria primer set to show the bacteria ratio alterations. Hindgut sample results normalized with the universal bacteria primer set are provided in supplementary materials. In addition, we noted that not all fecal samples generated detectable Ct values within 40 cycles, especially in the qPCR of S. alvi and G. apicola. The correlation analysis suggested that detectable S. alvi and G. apicola Ct values in fecal samples were significantly correlated with V. ceranae infection (P < 0.001). To further clarify the alterations in fecal samples, we submitted randomly selected fecal samples for 16S rDNA sequencing.
Vairimorpha ceranae infection alone enhanced all quantified core-bacteria populations and ratios in hindgut and feces (Fig. 4A and B), except Lactobacillus. The hindgut samples used for DNA extraction and qPCR included no visible feces or food debris; therefore, Snodgrassellla and Gilliamella that attached to the ileum generated much lower values as expected (Fig. 4A). Overall, Bifidobacterium, Snodgrassella and Gilliamella were higher in infected bees; all alterations are significant in hindgut samples (Fig. 4A), and some in fecal samples (Fig. 4B).
Feeding IMO alone significantly increased the Snodgrassella population in hindgut samples and significantly decreased the Lactobacillus population ratio in fecal samples, which resulted in a marginally significant difference (P = 0.053) between the control and the infected groups fed IMO. For infected groups, IMO did not significantly alter bacteria populations in hindgut and fecal samples according to the qPCR results.
16S rDNA sequencing of fecal samples
The sequencing results of the fecal samples from the four groups showed significant differences. In the alpha diversity analyses (Fig. 5), the infected groups showed higher Shannon index values and lower Simpson index values, which suggested that V. ceranae infection significantly reduces the alpha diversity of the fecal microbiota. In addition, the infected group fed IMO showed the most distinctive changes in the analyses (Fig. 5). Feeding IMO alone did not result in any significant alteration in the microbiota (alpha diversity analyses), but the within-group differences were much higher than those in other groups (Fig. 5). Infected bees fed IMO showed the opposite results, the lowest within-group differences. Beta diversity analyses suggested that all the infected samples were grouped together in hierarchical clustering and Principal Co-ordinates Analysis (PCoA). In permutational multivariate analysis of variance (PERMANOVA) with Bray-Curtis method, V. ceranae infection alone significantly affected the microbiota composition (P = 0.002), and IMO feeding alone did not (P = 0.488).
Bacterial population analysis at the genus level indicated that Lactobacillus, Commensalibacter, Snodgrassella, and Bartonella populations were significantly different among the four groups; Bifidobacterium and Gilliamella were marginally significantly different (Fig. 6). Comparing the infected and control groups fed IMO, all the core bacteria species were significantly altered. Similar to the qPCR results, there were no significantly altered bacterial populations between the infected groups fed sucrose and IMO. Neither was there a significant alteration in bacterial population ratios between control groups fed sucrose and IMO, although the mean value was different (Fig. 6), possibly because of the high diversity and deviation of the control group fed IMO, shown in Fig. 5.