## 4 Discussion  
Nosemosis is a widespread disease in adult honey bees, caused by microsporidia _Vairinorpha_ (_Nosema_) _apis_ ([PERSON]) and _Vairomorpha_ (_Nosema_) _ceranae_ ([PERSON]) [32]. Recently, [PERSON] et al. [33] proposed a redefinition of the _Nosema_ genus (now designated _Vairinorpha_) based on molecular phylogenetics. _Vairinorpha_ epis is a common parasite in the European honey bee (_Apis mellifera_) while _Vairinorpha_ (_Nosema_) _ceranae_ was introduced to _A. mellifera_ recently, originating from the Asian honey bee (_Apis cerana_). It was detected for the first time in Europe in 2004 [34] and has spread to South and North America [35; 36], Africa and Asia [37]. Nosemosis is associated with various predisposing or synergistic factors ranging from climate crisis to exposure to neonicotinoid pesticide [38; 39; 40; 41]. Spore transmission that spreads Nosema infection takes place among adult bees during feeding (trophallaxis) or comb-cleaning. Furthermore, stored honey in the hive, water and beekeeping equipment contribute to the transmission of Nosemosis. Severity of _Nosema_ spp. infection in a colony is inferred by most researchers through the number of spores per bee in the sample at hand [42]. Still, it should be noted that high spore counts of _V. ceranae_ are often found in asymptomatic colonies, and measured spores cannot constitute on their own a criterion of a colony health or survival [43]. Diagnosis of Nosemosis, especially that caused by _V. ceranae_, is of critical importance both for bee health and beekeeping sustainability. Figure 3: OTU annotation tree construct for each sample group (A–D), generated by ITS fungal 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 constructs are provided as Supplementary Material. The clinical symptoms of Nosemosis caused by _V. ceranae_ are highly variable, ranging from delayed colony growth or slow progressive depopulation to sudden colony collapse. An increased number of spores is found in collapsed colonies; however, it has not been established whether _V. ceranae_ is the main cause of collapse or one among other stressors that synergistically lead to colony collapse. _V. ceranae_ infestation provokes intestinal epithelial cell destruction, poor nutrition, reduced lifespan of adult bees and weaker immune response [7]. A possible cause of these findings could be an imbalance (dysbiosis) in the adult bee gut microbiome and/or BB microbiome even in the early stages of Nosemosis, when spore counts are low to medium, as in our study. Alpha-diversity analysis revealed a clear trend of reduction in number of observed species (unique OTUs) of both bacterial and fungal sequences in honey bee and BB samples derived from colonies with high _V. ceranae_ spore count as compared with those with low _V. ceranae_ spore count (Figures 1 and 2). One notable exception was sample A3, whose bacterial diversity was the lowest out of all bee samples (Figure 1). Overall, this observed reduction was not statistically significant, and a larger number of honey bee and BB samples should be analyzed in order to confirm reduced microbial diversity. The mechanism explaining this interesting finding has not been elucidated. However, reduced microbial diversity may lead to dysbiosis, which in turn might facilitate _V. ceranae_ infection, according to [PERSON] et al. [44]. Therefore, it could affect the development of the disease and serve as a prognostic marker for the survival of the colony. A significant impact of _V. ceranae_ infection on bacterial diversity was also revealed by alpha-diversity analysis. We hypothesized that this could partly be ascribed to reactive oxygen species (ROS) production, which is known to modulate microbiota within the insect gut lumen of _V. ceranae_-infected bees [45].