## 4 Discussion
Erosion is the phenomenon by which a given species loses races and populations, but in this case, the term refers to the loss of _N. ceranae_ alleles [75; 76]. Factors promoting HGT events include an organism's ability to acquire exogenous DNA, accessibility to its \"germline\" and the frequency of exogenous DNA \"donors\" in the environment [74]. From this point of view, _N. ceranae_ and other microsporidia, as a unicellular eukaryotic parasite, are candidates particularly prone to acquire exogenous DNA. Each _N. ceranae_ spore represents a single germline and the organ in which it stabilizes in _A. mellifera_, the ventricle, as a part of the digestive system is colonized by an extensive bacterial flora, consisting of at least 9 different species [77]. To this adds the environmental microbial contamination. Therefore, microsporidia like _N. ceranae_, when penetrating the host, may come into contact with exogenous bacterial DNA dispersed in the environment, that may subsequently acquired [73]. Such a mechanism might have occurred with _16s rRNA_ bacterial genes, which may have been repeatedly incorporated independently into the different genetic variants of _N. ceranae_, further expanding the observed variability of the number of _16S rRNA_ copies. It is, therefore, clear that qPCR methods based on the amplification of _16S rRNA_ are not accurate in precisely defining the extent of the infection. Doubts about the use of this gene in diagnostics have also been previously raised. In a comparative study, it has been indicated that primer-based PCR assays capable of amplifying a sequence belonging to _16S rRNA_ can produce false negatives or, in the case of mixed infections of _N. apis_ and _N. ceranae_, overestimate the infection of the latter pathogen [78]. Further evidence is provided reporting false negative results using assays based on _16S rRNA_[79], and indicating that assays based on this gene can amplify sequences belonging to _N. apis_, so altering the analytical result [80]. It is true that both studies base on End Point PCR assays; however, those primers can be used in Real Time PCR assays also and, as the low reliability has to be ascribed to the molecular characteristics of _16S rRNA_, those statements can also be extended to the latter technique. The other assay considered in the study is based on the amplification of a highly conserved sequence belonging to the _Hsp70 gene_. This gene is present in a single copy within the _N. ceranae_ genome [49; 52] and no evidence of intraspecific genetic variability has been reported so far [50]. Furthermore, it was shown to be specific for _N. ceranae_, generating no reaction with DNA from _N. apis_[50]. For these characteristics, this assay represents a valid and reliable alternative in the quantification of the number of _N. ceranae_ copies in a honey bee sample. The regression analysis highlighted an anomaly in the distribution corresponding to a number of copies close to the inoculated dose (187,500 spores/bee), indicating an increased _16S rRNA_ polymorphism. Given the specific experimental conditions, the bees were unable to defecate, so unloading the intestinal spores. Likely, in that area of the distribution concentrated spores that were killed or made quiescent by the experimental treatmentsin progress, whereas vegetative forms of the pathogen were prevailing elsewhere. The coincidence of the high concentration of spores in an area of high polymorphism of _16S rRNA_ would therefore be compatible with a potential accentuation of this phenomenon in the forms of resistance compared to the vegetative ones. Alternatively, the dead or quiescent spores could undergo degradation within the infected tissues, thus releasing nucleic acids in the cellular matrix. Fragments of dispersed _16S rRNA_ could therefore be acquired from viable spores or vegetative forms of _N. ceranae_ in a similar way to what happens with other microorganisms [73], thus altering the quantification and thus also increasing the eventual _16S rRNA/Hsp70_ ratio.