## 4 Discussion
However, assuming clonal reproduction, all spores would maintain a high degree of genetic similarity and, therefore, the values observed in different isolates should be characterized by constancy or little variability, making it unlikely to observe the ratios mentioned above. The second hypothesis includes the possibility that every single infected individual of _A. mellifera_ contains a heterogeneous population made of a number of _N. ceranae_ genetic variants [7; 34; 49; 61; 64]. Those variants, would be polymorphic for a certain number of sequences in their genome, including the _16S rRNA_ gene, that would be present in multiple copies. Therefore, the average number of _16S rRNA_ copies for a given _N. ceranae_ population infecting an individual _A. mellifera_, would depend on the specific genetic variants present within the host. This second hypothesis appears to account for a larger variability of the _16S rRNA/Hsp70_ ratio [7; 34; 49; 61; 64]. Theose genetic variants, would in addition to being polymorphic for a certain number of sequences placed alongin their genome, including would also be polymorphic for the _16S rRNA_ gene, as well asthat would be presenting a different number of repetitions for this genein multiple copies. Therefore, the average number of copies of _16S rRNA_ copies for a given _N. ceranae_ population of _N. ceranae_ infecting an individual of _A. mellifera_, wouldoll depend on the specific genetic variants present within the host. TThrough this second hypothesis, therefore, appears to account for a larger it is possible to include greater variability of the _16S rRNA/Hsp70_ ratio. However, the aspects above provide only a limited perspective. Indeed, there is clear evidence of recombination in _N. ceranae_, both in sequences coding for specific proteins [7; 49; 61; 62; 63] and in ribosomal genes [6; 34; 37]. Recombination requires fusion of two nuclei followed by meiosis, which allows to obtain new variants. This could happen both between two nuclei present within the same spore and between nuclei coming from different spores, admitting, in this case, the presence of a cryptic sexual phase in an organism that has been so far considered asexual [64]. The occurrence of recombinant events could therefore lead to the generation of variants with a new number of copies of the _16S rRNA_ gene. Recombination events could also generate new nucleotide sequenceson that gene. If the new sequences were formed at the pairing sites of the primers used in the qPCR assay, the primers would not pair, preventing amplification and leading to a low _16S rRNA/Hsp70_ ratio value. It is then necessary to consider that in the specific case of rDNA further mechanisms, in addition to meiosis, could lead to recombination. One of them is the Sister Chromatid Exchange (SCE) implying that, during DNA replication, two identical chromosomes pair up and exchange portions [72]. Considering that the ribosomal genes of _N. ceranae_ are present at least in tandem and as variably repeated number of copies within the same genome [26; 34], new sequences of this gene could be generated through the recombination of the different repetitions [34], without the need of a fusion between two nuclei. However, this mechanism would not be effective in the case of the _Hsp70_ gene, as it represents a highly conserved portion present in a single copy in the _N. ceranae_ genome [49; 52]. For this reason, the _16S rRNA_ gene can be considered much more prone to recombination than _Hsp70_. The picture may be completed considering that a number of microsporidian genes derives from the horizontal genetic transfer (HGT), to which other organisms, mainly bacteria, concur [73]. In _N. ceranae_, the 0.44 to 1.31% of the genes result from HGT events [73]. The occurrence of such events between prokaryotes and eukaryotes are not rare and, in most cases, they end in the functional loss of the newly acquired gene or in its more or less rapid erosion [74].