Non-invasive Investigation of Polychromophilus Parasite Infections in Bat Populations in Serbia Using Bat Flies

Background Haemosporidian parasites of the genus Polychromophilus infect bats worldwide. They are vectored by obligate ectoparasitic bat flies of the family Nycteribiidae. Despite their global distribution, only five Polychromophilus morphospecies have been described to date. The two predominant species, Polychromophilus melanipherus and Polychromophilus murinus , are broadly distributed and mainly infect miniopterid and vespertilionid bats, respectively. In areas where species from different bat families aggregate together, the infection dynamics and ability of either Polychromophilus species to infect other host families is poorly characterized. Methods We collected 215 bat flies from two bat species, Miniopterus schreibersii and Rhinolophus ferrumequinum , which sometimes form mixed clusters in Serbia. Miniopterus schreibersii is known to be frequently infected with P. melanipherus , whereas R. ferrumequinum has been observed to be incidentally infected with both Polychromophilus species. All flies were screened for Polychromophilus infections using a PCR targeting the haemosporidian cytb gene. Positive samples were subsequently sequenced for 579 bp of cytochrome b ( cytb ) and 945 bp of cytochrome oxidase subunit 1 ( cox1 ). Results Polychromophilus melanipherus DNA was detected at six out of nine sampling locations and in all three examined bat fly species collected from M. schreibersii ( Nycteribia schmidlii , n = 21; Penicillidia conspicua , n = 8; Penicillidia dufourii , n = 3). Four and five haplotypes were found for cytb and cox1 , respectively. Evidence for multiple Polychromophilus haplotypes was found in 15 individual flies. These results point to a high diversity of P. melanipherus parasites in Miniopterus hosts and efficient transmission throughout the study area. A single Phthiridium biarticulatum bat fly collected from R. ferrumequinum screened positive for P. melanipherus , but only yielded a partial cox1 sequence fragment. Nevertheless, this result suggests that secondary hosts (both bat and fly species) are regularly confronted with this parasite. Conclusions The results of this study provide new insights into the prevalence and distribution of Polychromophilus parasites in European bats and their nycteribiid vectors. The use of bat flies for the non-invasive investigation of Polychromophilus infections in bat populations has proven to be efficient and thus represents an alternative for large-scale studies of infections in bat populations without the need to invasively collect blood from bats. Graphical Abstract