Trypanosomatids form a group of high prevalence protozoa that parasitise honey bees, with Lotmaria passim as the predominant
species worldwide. However, the knowledge about the ecology of trypanosomatids in isolated areas is limited. The Portuguese
archipelagos of Madeira and Azores provide an interesting setting to investigate these parasites because of their geographic
isolation, and because they harbour honey bee populations devoid of two major enemies: Varroa destructor and Nosema ceranae.
Hence, a total of 661 honey bee colonies from Madeira and the Azores were analysed using different molecular techniques,
through which we found a high prevalence of trypanosomatids despite the isolation of these islands. L. passim was the predominant
species and, in most colonies, was the only one found, even on islands free of V. destructor and/or N. ceranae with
severe restrictions on colony movements to prevent the spread of them. However, islands with V. destructor had a significantly
higher prevalence of L. passim and, conversely, islands with N. ceranae did not shown any significant correlation with the
trypanosomatid. Crithidia bombi was detected in Madeira and on three islands of the Azores, almost always coincident with L.
passim. By contrast, Crithidia mellificae was not detected in any sample. A high-throughput sequencing analysis distinguished
two main haplotypes of L. passim, which accounted for 98% of the total sequence reads. This work suggests that L. passim and
C. bombi are parasites that have been associated with honey bees predating the spread of V. destructor and N. ceranae.
