Shotgun metagenomic analysis of honey DNA at high sequencing depth can describe a multi-kingdom and multi-level honey bee derived environmental DNA signature and the honey bee hologenome complexity

Honey bees are large-scale monitoring tools due to their extensive environmental exploration. In their activities and from the hive ecosystem complex, they get in close contact with many organisms whose traces can be transferred into the honey, which can represent an interesting reservoir of environmental DNA (eDNA) signatures useful to characterize the honey bee hologenome and pathosphere. In this study we applied a deep shotgun metagenomic sequencing approach coupled with a specifically adapted bioinformatic pipeline to characterize honey eDNA. From the analysed honey samples, we detected sequences from 191 organisms spanning different kingdoms or phyla (viruses, bacteria, plants, fungi, protozoans, arthropods, mammals). Bacteria included the largest number of species. These multi-kingdom signatures listed common hive and honey bee gut microorganisms, honey bee pathogens, parasites and pests, which resembled a complex interplay that might provide a general picture of the health state of the colony that produced the analysed honey. Mining the Apis mellifera Filamentous Virus genome (the most abun-dant detected DNA source) we obtained information to define the origin of the honey at the apiary level. Mining Apis mellifera sequences made it possible to identify the honey bee subspecies both at the mitochondrial and nuclear genome levels.