DNA can make the difference: the two sides of the entomological authentication of the honey.

Honey is one of the most frauded animal-derived food products: the most common frauds are derived from the adulteration of the sugar components by intentional dilution with cheap syrups, and the masking and mislabelling of the geographic, botanical, and entomological origin of the honey. The entomological origin of the honey can be considered from two different perspectives: i) the subspecies of Apis mellifera that has produced the honey; ii) the footprints derived from plant-suckling insects that produce honeydew, which are always present in authentic honey. The declaration of the A. mellifera subspecies is relevant in some protected designation of origin (PDO) honey, providing an important link to boost the conservation and integrity of honey bee genetic resources. The identification of the A. mellifera subspecies can also provide an indirect information on the geographic origin of the honey, according to the natural geographic distribution of the different subspecies. Plant-suckling insects provide multidimensional information related to the plant origin of the honeydew, according to the botanical specialization of some of these plant parasites, also providing indirectly information on the botanical and geographic origin of the honey. For the identification of the honey bee subspecies, we set up two assays to analyse DNA extracted from the honey and that targeted two regions of the A. mellifera mitochondrial DNA (mtDNA). The assays can distinguish between different subspecies carrying the mtDNA lineages A (e.g. A. m. siciliana), C1 (A. m. ligustica), C2 (A. m. carnica) and M (e.g. A. m. mellifera). Another assay that is able to identify the A. mellifera subspecies, was designed to genotype about 100 subspecies-informative honey bee single nucleotide polymorphisms (SNPs) using the DNA extracted from the honey and a genotyping-by-sequencing approach. The identification of plant-suckling insects is based on a targeted metabarcoding analysis on Rhynchota mtDNA conserved regions using next generation sequencing approaches, coupled with bioinformatic pipelines to interpret sequenced reads. These different methods are highly informative and are routinely applied in our lab to authenticate the honey based on their entomological origin.