ENVIRONMENTAL DNA IN HONEY: GENOMIC TOOLS FOR ENTOMOLOGICAL AUTHENTICATION OF THIS PRODUCT.

Honey is one of the most commonly frauded food products, with the most frequent frauds involving adulteration and mislabeling of its origin. The entomological origin of honey can be considered from two perspectives: i) the subspecies of Apis mellifera that produced the honey; and ii) the footprints derived from plant-suckling insects that produce honeydew, which are always present in authentic honey. Identifying the A. mellifera subspecies provides an important link to boost the conservation and integrity of honeybee populations and also offer indirect information on the geographic origin of the honey, based on the natural distribution of the different subspecies. Plant-suckling insects belonging to the Rhynchota order provide multidimensional information related to the plant origin of honeydew, based on the botanical specialization of these plant parasites, indirectly indicating the geographic origin of the honey. To identify the honey bee subspecies, we set up assays to analyse DNA extracted from honey that targeted regions of the A. mellifera mitochondrial DNA (mtDNA). These regions are informative to distinguishing different subspecies carrying the mtDNA lineages A (e.g. A. m. siciliana) and M (e.g. A. m. mellifera), as well haplotypes C1 (A. m. ligustica) and C2 (A. m. carnica). Another assay was designed to genotype approximately100 subspecies-informative honey bee single nucleotide polymorphisms (SNPs) using DNA extracted from honey and a genotyping-by-sequencing approach. For identifying plantsuckling insects, we designed a targeted metabarcoding assay based on two conserved regions of Rhynchota mtDNA using a next generation sequencing approach, along with bioinformatic pipelines to interpret sequenced reads and assign them to Rhynchota families. These approaches were applied to more than 150 honey samples, revealing that these methods are highly informative for authenticating the honey based on their entomological origin.