Monitoring the distribution of Apis mellifera genetic resources in Italy using mtDNA information.

The genetic integrity of Apis mellifera subspecies and populations is a matter of concern all over Europe. Honey contains environmental DNA (eDNA) traces from all organisms that directly or indirectly were involved in its production, including the DNA of the honey bees that produced it. Specific mitochondrial DNA (mtDNA) lineages (i.e. mitotypes) characterize several A. mellifera subspecies. Different mitotypes can be detected using honey as source of honey bee DNA, providing approximate population genetic information useful to estimate the diffusion and frequency of honey bee mitotypes. In this study, we combined two approaches based on the analysis of: (1) honey eDNA; and (2) DNA extracted from honey bee workers of many different colonies to monitor the distribution of A. mellifera mtDNA lineages all over Italy. DNA was extracted from a total of about 2,300 honey samples produced over five years (2018-2022) in several regions of the Italian peninsula and Sicily. In addition, worker bees from 1,100 colonies were analysed to compare the distribution map of the mtDNA lineages in a region of the North of Italy (Emilia-Romagna). PCR products were analysed using a fragment size-based assay followed by Sanger sequencing. Results from the two approaches confirmed that the C lineage was the most frequent mitotype all over Italy except in Sicily where the A lineage was highly represented across all years. Other mtDNA haplotypes (A and M lineages) were present in almost all Italian regions. The obtained results are useful for designing conservation strategies for A. mellifera genetic resources in Italy and evaluating the efficacy of previously adopted measures. Moreover, we demonstrated that eDNA from honey can be exploited to design cost-effective non-invasive and simple methods to obtain information on the genetic distribution of honey bee mitotypes at large geographic scale.