Entomological authentication of honey based on a DNA method that distinguishes Apis mellifera mitochondrial C mitotypes: Application to honey produced by A. m. ligustica and A. m. carnica

Honey contains DNA of all organisms that directly or indirectly have been involved in its production, including the DNA of the honey bees. Therefore, using DNA extracted from honey, it is possible to analyse DNA markers useful to authenticate its entomological origin. In this study we developed an assay that can distinguish two mitotypes within the mitochondrial C lineage of Apis mellifera: the C1 mitotype, mainly carried by A. m. ligustica subspecies; the C2 mitotype, that is highly specific for the A. m. carnica subspecies. This method is based on Sanger sequencing of an informative regions of the honey bee mitochondrial DNA. A total of 255 honey samples were analysed. These samples included 157 commercial honey samples produced in three Italian regions of the North of Italy, in which A. m. ligustica is widely spread and 15 honey samples directly obtained from honeycombs. Fifteen of these commercial samples and all samples from honeycombs were known to be produced by A. m. ligustica. Other commercial honey samples, produced in Slovenia (n 38), Croatia (n. 22) and Serbia (n. 23), derived from A. m. carnica. All honey samples produced by A. m. ligustica had only the C1 mitotype whereas all honey samples produced by A. m. carnica had the C2 mitotype. C1 was the most frequent mitotype in Emilia-Romagna region (Italy). This assay can be used to identify honey produced by these two subspecies and for population genetic studies in A. mellifera using the honey as source of DNA.