## 4 Discussion  
In this investigation we wanted to compare two different methods of qPCR, in use to quantify the number of _N. ceranae_ copies present in infected _A. mellifera_ samples. Those methods differ mainly for the target sequence in the microsporidian DNA, in that they respectively amplify sequences belonging to the genes coding for _16S rRNA_ (SSU-rDNA) and _Hsp70_ (Heat Shock Protein). The comparison indicates that the abundance obtained with the qPCR technique based on _16S rRNA_ is systematically higher. This observation is in line with what reported in other comparative studies considering the same methods [8, 69], where the authors detected instable ratios between the data obtained on different samples. This variability was also observed in the present study, with values of the _16S rRNA/Hsp70_ ratio oscillating between 1.01 and 18.68. Those values are in line with previous observations [8], where the ratio varied within a range of 1.35 and 16.53. Besides, the distribution of the _16S rRNA/Hsp70_ ratio did not appear evenly distributed, as the frequency peaked between 10.00 and 12.00, with more than 50% of all observed values included in that range and 91.5% of all values in the range 8.00-13.00. This indicates that the observed large variability was concentrated  
Figure 4: Scatterplot showing the relationship between the error variance of the 16S rRNA/Hsp70 ratio, and the abundance measured with the Hsp70 method (N = 306). Legend-blue crosses: individual standardized residuals, red dashed line: inoculated dose, dotted black lines: 95% interval of the normally distributed data (z = \(\pm\)1.96). within a specific range. The average ratio was 10.80, which is higher but still in line with the previously observed value of 8.59 [8]. Since in the _N. ceranae_ genome the _Hsp70_ gene is present as a single copy [49; 52], whereas the _16S rRNA_ gene is polymorphic, with high variability, and presenting an undefined number of copies in its genome [34; 37], we can assume that the _16S rRNA/Hsp70_ ratio reflects the number of _16S rRNA_ copies present in the _N._[34; 37], it we can be assumed that the _16S rRNA/Hsp70_ ratio reflects the copy number of _16S rRNA_ copies present in the _N. ceranae_ genome of the considered samples. Based on both observed data and literature, it is possible to hypothesize that _16S rRNA_ is generally present in _N. ceranae_ in an average number of copies oscillating between 8 and 14. This hypothesis is in agreement with what reported by [PERSON] et al. (2010), who suggest that approximately 10 copies of the gene are present [70]. However, it is necessary to consider that the qPCR analysis was performed on the total DNA extracted from a single individual of _A. mellifera_, which can contain millions of _N. ceranae_ copies [71]. As all the data obtained, including the _16S rRNA/Hsp70_ ratio, refer to a given infecting _N. ceranae_ population, the number of _16S rRNA_ copies represent the average for the population found within the host bees in the sample. As indicated, the calculated ratio largely falls within a specific range. However, the lowest (e.g., 1.12; 1.73; 2.01) and highest (e.g., 16.61; 17.78; 18.68) values (Supplementary Table S1) account for high genetic variability in _N. ceranae_[6; 49; 64; 67]. To explain the genetic variability observed in this microsporidium, two main explanations were proposed. The first hypothesis suggests the presence of a single clonal _N. ceranae_ population, with dikaryotic and polyploid spores [6; 67]. Although this hypothesis partly clarifies the extensive genetic variability in _N. ceranae_, it does not appear explanatory of the observed values. Considering, as suggested, a tetraploid genome, it would be possible to have four variants of ribosomal genes in each spore [67]. These variants could differ from each other both in terms of repeated number of copies and nucleotide sequences which, taken together, would determine the average copy number of _16S rRNA_ for a given _N. ceranae_ spore.