NGS analysis of DNA extracted from Honeydew honey and blossom honey to identify entomological signatures.

Honeydew produced from the excretion of plant-sucking insects (order Hemiptera) is a carbohydrate-rich material that is foraged by honey bees to integrate their diets. In this study, we used DNA extracted from honey as a source of environmental DNA to disclose its entomological signature determined by honeydew producing Hemiptera that was recovered not only from honeydew honey but also from blossom honey. We designed PCR primers that amplified a fragment of mitochondrial cytochrome c oxidase subunit 1 (COI) gene of Hemiptera species using DNA isolated from unifloral, polyfloral and honeydew honeys. Ion Torrent next generation sequencing metabarcoding data analysis assigned Hemiptera species using a customized bioinformatic pipeline. The forest honeydew honeys reported the presence of high abundance of Cinara pectinatae DNA, confirming their silver fir forest origin. In all other honeys, most of the sequenced reads were from the planthopper Metcalfa pruinosa for which it was possible to evaluate the frequency of different mitotypes. Aphids of other species were identified from honeys of different geographical and botanical origins. This unique entomological signature derived by environmental DNA contained in honey opens new applications for honey authentication and to disclose and monitor the ecology of plant-sucking insects in agricultural and forest landscapes.