DISCLOSING PHENOLOGICAL INTERACTIONS BETWEEN PLANTS AND INSECTS THROUGH ENVIRONMENTAL DNA ANALYSIS FROM HONEY SAMPLES COLLECTED THROUGHOUT THE SEASON

Honey contains DNA traces from all organisms that directly or indirectly contributed to its production and that have been encountered by honey bees during their foraging activities. This honey-derived environmental DNA (eDNA) can provide insights into the ecological, climatic and seasonal changes of all these organisms. Botanical fingerprints present in honey primarily derive from the pollen content, while entomological fingerprints, excluding those from the honey bees, are primarily constituted by plant-sucking insects that produce honeydew. Honeydew is collected by honey bees as a sweet feed source and all honey samples may be derived from a fraction of this component. In this study, we investigated the botanical and entomological DNA fingerprints from honey samples collected from three different apiaries at three different time points throughout the beekeeping season (end of June, end of July and end of October). The three apiaries were located in three different agroecological spots in the province of Bologna, Italy. From each apiary, honey samples were collected from three different colonies at all three time points. Metabarcoding analysis was based on using five different metabarcoding systems, including three plant universal primer pairs (matK, rbcl and trnL_UAA) and two universal entomological primer pairs targeting the COX1 and CYTB mitochondrial DNA genes, specifically designed to capture hemipters. Next generation sequencing data analysis assigned approximately 50% of produced reads to botanical or insect taxa. Complementary information was obtained through the multiple barcoding analyses applied. Differences between apiaries and within apiaries across the three sampling time points were observed with more frequently recurrent entomological profiles providing interesting information on the different botanical and entomological associations. These findings demonstrate that honey-derived eDNA can be useful to dissect phenological interactions between plants and their pests, specifically their plant-sucking insects, potentially capturing ecological and climate-derived changes.