## 3 Results
### Next-Generation Sequencing Analysis
#### 3.2.2 OTU Identification and Taxonomic Annotation  
In honey bees, the most abundant genera were _Lactobacillus_, _Gilliamella_, _Snodgrassella_, _Bartonella_ and _Bifidobacterium_. _Bartonella_ displayed high variability among the three biological replicates in group A (1-22%). _Friscella_, _Commensalibacter_ and _Escherichia-Shigella_ were also identified in all samples but in significantly lower relative abundance (roughly 3-4% in total). Two genera, _Serratia_ and _Thiodhrix_, demonstrated \(>\)1% relative abundance  
Figure 2: OTU annotation tree construct for each sample group (A: bee samples demonstrating low _V. ceranae_ spore count; B: bee samples demonstrating high _V. ceranae_ spore count; C: bee bread collected from colonies with low _V. ceranae_ spore count and D: bee bread collected from colonies with high _V. ceranae_ spore count), generated by V3–V4 16S rRNA region, in (**a–d**), respectively. Different taxonomic ranks range from inside out. Size of circles stands for abundance of species. Different colors stand for different phyla. Solid circles stand for the top 40 species in high abundance. Full-size tree constructs are provided as Supplementary Material. only in sample A2 (1.2% and 1.5%, respectively). No bacterial genus could be identified as group-specific (present in one group and absent in the other), thus differentiating bee samples. Similarly, no fungal genus seemed to be group-specific (present in one group and absent in the other) among honey bee samples (samples A1-3 and B1-3). The fungal genera _Lachancea_, _Alternaria_, _Mycosphaerella_, _Stemplybium_ and _Chaetonium_ displayed high variability regarding relative abundance among the three biological replicates of group A (bees with low _V. ceranae_ spore count), ranging between 4-95%, 0.13-19.8%, 0.3-17.48%, 0.03-17.45% and 0.01-10.59%, respectively. It was observed that the _Lactobacillus_, _Acidothermus_ and _Buchrera_ genera displayed high variability regarding relative abundance among the three biological replicates in group C, ranging between 0.69-24.5%, 0-28.45% and 0.01-32.38%, respectively. Similarly, in group D, _Arsenophanus_' relative abundance was highly variable, ranging from 1.75% to 26.79%. Interestingly, _Rosenbergiella_ spp. were relatively abundant (reaching up to 10%) in bee bread samples collected from infected colonies (group D) as compared with non-infected (0-0.77% in group C). Bioinformatics analysis of fungal ITS sequences revealed that _Podosphaera_ spp. were present only in group C and absent in group D. Furthermore, a _t_-test of each OTU was performed separately against the two groups, and it was demonstrated that _Blumeria_ spp. were significantly (_p_-value < 0.05) more abundant in group C (1.48-3.45%) as compared with group D (0-1.03%). The fungal genera _Mycosphaerella_ and _Saccharomyces_ displayed high variability regarding relative abundance among the three biological replicates in group C, ranging between 22.37-55.57% and 0.38-21.69%, respectively. _Thecaphora_ displayed high variability among the replicates in group D, with relative abundance ranging between 0-41.67%. Overall, variability of relative abundance of ITS sequences was significantly (_t_-test _p_-value < 0.003) higher than variability of relative abundance of V3-V4 16S rRNA sequences, as demonstrated in Table 2.  
OTU annotation tree constructs for group A and B, generated by 16S rRNA region, displayed no significant differences (Figure 2a,b). On the other hand, OTU annotation tree constructs for group C and D, generated by 16S rRNA region, demonstrated that Bacteroidetes were abundant in group D but not in group C. On the contrary, Acidobacteriota and Crenarchaeota were abundant in group C but not in group D (Figure 2c,d). OTU annotation tree constructs for group A and B, generated by ITS fungal region, showed that the phyla Ascomycota and Basidiomycota were abundant in group A. Glomerycota were also identified in group A.