From hive to lab: molecular detection of Lotmaria passim and Crithidia mellificae in Australian honey bees using honey-derived eDNA

• First nationwide screening of Lotmaria passim and Crithidia mellificae using honey-derived eDNA. • Species-specific PCR assays enabled accurate discrimination between the two trypanosmatid protists. • L. passim was the dominant species (47%), followed by C. mellificae (29%). • Sequenced amplicons showed 90–100% identity with global trypanosmatid lineages Gut parasites from the trypanosomatid family , Crithidia mellificae , and the recently identified Lotmaria passim , have emerged as potential contributors to colony losses and a serious threat to the honey bee ( Apis mellifera ) population globally. These parasites have not been studied in Australia’s managed bee populations due to limited surveillance efforts and focus on monitoring established pests and pathogens. In this study, we have optimised an existing end-point polymerase chain reaction (PCR) assay for detecting L. passim and C. mellificae from honey-derived environmental DNA . We screened 135 honey samples collected from all Australian states between 2022 and 2024. Our results showed that 47% of samples were positive for L. passim , 29% for C. mellificae , 24% had mixed presence of both parasites, and 48% tested negative. Sequence analysis of positive samples, followed by phylogenetic comparison with related species, confirmed a similarity range of 90–100%. This study provides the first comprehensive assessment of the molecular detection, distribution, and phylogenetic relationship of honey bee trypanosomatids, highlighting their widespread presence in Australia. These findings highlight the need to investigate further hive-level presence and assess the potential impacts of these parasites on honey bee health and colony performance.