Honey environmental DNA as a non‑invasive tool for parasite detection in honey bees: a case study of Lotmaria passim in Northern Italian apiaries

Environmental DNA (eDNA) contained in honey derives from organisms that have been directly and indirectly involved in the production process of this matrix and that have played a role in the hive ecosystems where the honey has been produced. This matrix therefore represents a valuable source of information for the non-invasive monitoring of honey bee pathogens and parasites at the colony level. In this study, we evaluated the use of honey-derived eDNA for the detection and surveillance of the trypanosomatid parasite Lotmaria passim, an emerging gut parasite of Apis mellifera. An axenic culture of a trypanosomatid isolate was first established and morphologically characterised. Molecular cofirmation was achieved through PCR amplification and sequencing using three primer pairs specifically designed for L. passim. DNA obtained from the confirmed isolate was subsequently used to validate PCR assays targeting parasite DNA traces recovered from honey. A total of 164 honey samples collected from apiaries distributed across Northern Italy were analysed. Successful amplification of honey bee DNA confirmed adequate DNA preservation in all samples. Overall, 78% of honey samples tested positive for L. passim. These results demonstrate that honey-derived eDNA provides an effective, non-invasive approach for pathogen surveillance and represents a promising tool for large-scale monitoring of honey bee health. Expanding the number of sampled regions and analysed honey samples will enable retrospective and longitudinal analyses, allowing the assessment of parasite and pathogen distribution across different regions and the monitoring of temporal trends over multiple years throughout Italy.