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 and information useful to analyse the honey bee hologenome 
complexity. In this study, we tested a deep shotgun sequencing approach of honey 
DNA coupled with a specifically adapted bioinformatic pipeline. This methodology 
was applied to a few honey samples pointing out DNA 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 honey bee 
pathosphere. Based on the Apis mellifera filamentous virus genome diversity (the 
most abundant detected DNA source) we obtained information that could 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.