Entomological signatures in honey: combining metabarcoding approaches to provide insights into plant-sucking insects that populate agricultural and forest landscapes

Honeydew, primarily produced by plant-sucking insects of the order Hemiptera, is an important source of carbohydrates that complement the honey bee diet, especially when nectar is unavailable or does not fully meet the bees’ dietary needs. Honeydew honey is a unique type of honey that comes from this prevalent source of sugar components. Honeydew contains valuable environmental DNA (eDNA) signatures that provide insights into the plant-sucking insect communities living in agricultural and forest landscapes. The presence and composition of these insect communities can indirectly indicate the botanical and geographical origin of honey, as many plant-sucking insects have host-plant specialization linked to specific ecosystems. In this study, we used DNA extracted from honey as a source of eDNA to determine its entomological fingerprinting, focusing specifically on honeydew-producing Hemiptera. We designed two primer pairs targeting mitochondrial DNA (mtDNA) genes (cytochrome b and COXI) of Hemiptera species to amplify the DNA extracted from 70 honey samples of various geographic and botanical origins, including several honeydew honey samples. We utilized a next-generation sequencing (NGS) metabarcoding approach, along with specially designed bioinformatics pipelines and a reference sequence database to analyse the amplicons obtained for the two targeted mtDNA regions. On average, 47 and 16.5 taxa per honey sample were identified with the cytochrome b and COXI primer pairs, respectively, demonstrating the high informativeness of this multiple metabarcoding approach. Metcalfa pruinosa was one of the most common hemipters identified, along with several other specialised aphids that provide an indirect signature of the botanical origin based on the specific insect community. Our results emphasize the potential of entomological metabarcoding as a robust tool for honey authentication and landscape characterization. This approach could provide new possibilities for verifying honey authenticity and analyzing the intricate entomological ecosystems of agricultural and forest landscapes, including the geographical identity and origin of honey.